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Sample records for activity lactate dehydrogenase

  1. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

    PubMed

    Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

    2006-04-01

    Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

  2. Lactate dehydrogenase test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003471.htm Lactate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Lactate dehydrogenase (LDH) is a protein that helps produce energy ...

  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. Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

    PubMed Central

    Peter, A T; Bosu, W T; MacWilliams, P; Gallagher, S

    1987-01-01

    Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum. PMID:3453274

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

    PubMed Central

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

    2011-01-01

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(−)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L-1 of optically pure D(−)-lactic acid from glucose in < 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

  6. 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.

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

    PubMed

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

    2013-11-01

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

  8. Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice.

    PubMed

    Purkey, Hans E; Robarge, Kirk; Chen, Jinhua; Chen, Zhongguo; Corson, Laura B; Ding, Charles Z; DiPasquale, Antonio G; Dragovich, Peter S; Eigenbrot, Charles; Evangelista, Marie; Fauber, Benjamin P; Gao, Zhenting; Ge, Hongxiu; Hitz, Anna; Ho, Qunh; Labadie, Sharada S; Lai, Kwong Wah; Liu, Wenfeng; Liu, Yajing; Li, Chiho; Ma, Shuguang; Malek, Shiva; O'Brien, Thomas; Pang, Jodie; Peterson, David; Salphati, Laurent; Sideris, Steve; Ultsch, Mark; Wei, BinQing; Yen, Ivana; Yue, Qin; Zhang, Huihui; Zhou, Aihe

    2016-10-13

    A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 μM lactate IC50 in a MiaPaca2 cell line. Optimization of the series led to 29, a potent cell active molecule (MiaPaca2 IC50 = 0.67 μM) that also possessed good exposure when dosed orally to mice.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. ACTIVITY AND ISOZYME CONTENT OF LACTATE DEHYDROGENASE UNDER LONG-TERM ORAL TAURINE ADMINISTRATION TO RATS.

    PubMed

    Ostapiv, R D; Humenyuk, S L; Manko, V V

    2015-01-01

    The effect of long-term oral taurine administration to rats on activity of lactate dehydrogenase (LDH), its isozyme content and activity in the whole blood, liver, thigh muscle, brain and testes tissues were studied in the present work. For this purpose male Wistar rats with body weight 190-220 g were randomly divided into three groups, they were orally administered drinking water (control group) or taurine solution 40 and 100 mg per kg of body weight ( groups I and II, respectively). The total lactate dehydrogenase activity was measured spectrophotometrically, the percentage content of isozymes was determined by electrophoresis in 7.5% poliacrylamide gel withfurther staining according to J. Garbus. It was found that the total lactate dehydrogenase activity increased in all studied tissues. In testes of animals of both groups and in brain of group I animals, the total percentage contents of isozymes that are responsible for lactate production (LDH4+LDH5) increased. In liver of animals of both groups and in whole blood of group II animals, the total percentage content of isozymes that produce pyruvate (LDH1+LDH2) increased. In thigh muscle of both groups and in brain of group II animals the balance between LDH1+LDH2 and LDH4+LDH5 content did not differ from control values, though total lactate dehydrogenase activity was significantly higher, than that in the control group. Thus, the increase in the lactate dehydrogenase activity under long-term oral taurine administration in different rat tissues was found to be tissue- and dose-dependent and was caused by the increase in the content of different isozymes. Such increase in group I animals might be explained by adaptive mechanisms to hypoxia caused by high doses of taurine. For group II animals high doses of taurine were toxic and directly affected metabolic processes in the animal bodies.

  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, 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...

  13. 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...

  14. 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...

  15. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    SciTech Connect

    Ma, Lianjia

    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.

  16. Lactate dehydrogenase-elevating virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Van Noorden, C J; Vogels, I M

    1989-01-01

    Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.

  18. Genistein inhibits activities of methylenetetrahydrofolate reductase and lactate dehydrogenase, enzymes which use NADH as a substrate.

    PubMed

    Grabowski, Michał; Banecki, Bogdan; Kadziński, Leszek; Jakóbkiewicz-Banecka, Joanna; Kaźmierkiewicz, Rajmund; Gabig-Cimińska, Magdalena; Węgrzyn, Grzegorz; Węgrzyn, Alicja; Banecka-Majkutewicz, Zyta

    2015-09-25

    Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a natural isoflavone revealing many biological activities. Thus, it is considered as a therapeutic compound in as various disorders as cancer, infections and genetic diseases. Here, we demonstrate for the first time that genistein inhibits activities of bacterial methylenetetrahydrofolate reductase (MetF) and lactate dehydrogenase (LDH). Both enzymes use NADH as a substrate, and results of biochemical as well as molecular modeling studies with MetF suggest that genistein may interfere with binding of this dinucleotide to the enzyme. These results have implications for our understanding of biological functions of genistein and its effects on cellular metabolism.

  19. 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.

  20. Physiological and fermentation properties of Bacillus coagulans and a mutant lacking fermentative lactate dehydrogenase activity.

    PubMed

    Su, Yue; Rhee, Mun Su; Ingram, Lonnie O; Shanmugam, K T

    2011-03-01

    Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.

  1. 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.

  2. 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.

  3. Thermal activation of 'allosteric-like' large-scale motions in a eukaryotic Lactate Dehydrogenase.

    PubMed

    Katava, Marina; Maccarini, Marco; Villain, Guillaume; Paciaroni, Alessandro; Sztucki, Michael; Ivanova, Oxana; Madern, Dominique; Sterpone, Fabio

    2017-01-23

    Conformational changes occurring during the enzymatic turnover are essential for the regulation of protein functionality. Individuating the protein regions involved in these changes and the associated mechanical modes is still a challenge at both experimental and theoretical levels. We present here a detailed investigation of the thermal activation of the functional modes and conformational changes in a eukaryotic Lactate Dehydrogenase enzyme (LDH). Neutron Spin Echo spectroscopy and Molecular Dynamics simulations were used to uncover the characteristic length- and timescales of the LDH nanoscale motions in the apo state. The modes involving the catalytic loop and the mobile region around the binding site are activated at room temperature, and match the allosteric reorganisation of bacterial LDHs. In a temperature window of about 15 degrees, these modes render the protein flexible enough and capable of reorganising the active site toward reactive configurations. On the other hand an excess of thermal excitation leads to the distortion of the protein matrix with a possible anti-catalytic effect. Thus, the temperature activates eukaryotic LDHs via the same conformational changes observed in the allosteric bacterial LDHs. Our investigation provides an extended molecular picture of eukaryotic LDH's conformational landscape that enriches the static view based on crystallographic studies alone.

  4. Thermal activation of ‘allosteric-like’ large-scale motions in a eukaryotic Lactate Dehydrogenase

    PubMed Central

    Katava, Marina; Maccarini, Marco; Villain, Guillaume; Paciaroni, Alessandro; Sztucki, Michael; Ivanova, Oxana; Madern, Dominique; Sterpone, Fabio

    2017-01-01

    Conformational changes occurring during the enzymatic turnover are essential for the regulation of protein functionality. Individuating the protein regions involved in these changes and the associated mechanical modes is still a challenge at both experimental and theoretical levels. We present here a detailed investigation of the thermal activation of the functional modes and conformational changes in a eukaryotic Lactate Dehydrogenase enzyme (LDH). Neutron Spin Echo spectroscopy and Molecular Dynamics simulations were used to uncover the characteristic length- and timescales of the LDH nanoscale motions in the apo state. The modes involving the catalytic loop and the mobile region around the binding site are activated at room temperature, and match the allosteric reorganisation of bacterial LDHs. In a temperature window of about 15 degrees, these modes render the protein flexible enough and capable of reorganising the active site toward reactive configurations. On the other hand an excess of thermal excitation leads to the distortion of the protein matrix with a possible anti-catalytic effect. Thus, the temperature activates eukaryotic LDHs via the same conformational changes observed in the allosteric bacterial LDHs. Our investigation provides an extended molecular picture of eukaryotic LDH’s conformational landscape that enriches the static view based on crystallographic studies alone. PMID:28112231

  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. 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 ...

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

    PubMed Central

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

    2017-01-01

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

  8. Catecholamine regulation of lactate dehydrogenase in rat brain cell culture

    SciTech Connect

    Kumar, S.; McGinnis, J.F.; de Vellis, J.

    1980-03-25

    The mechanism of catecholamine induction of the soluble cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27) was studied in the rat glial tumor cell line, C6. Lactate dehydrogenase was partially purified from extracts of (/sup 3/H)leucine-labeled cells by affinity gel chromatography and quantitatively immunoprecipitated with anti-lactate dehydrogenase-5 IgG and with antilactate dehydrogenase-1 IgG. The immunoprecipitates were dissociated and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Using this methodology, the increased enzyme activity of lactate dehydrogenase in norepinephrine-treated C6 cells was observed to be concomitant with the increased synthesis of enzyme molecules. Despite the continued presence of norepinephrine, the specific increase in the rate of synthesis of lactate dehydrogenase was transient. It was first detected at 4 h, was maximum at 9 h, and returned to basal levels by 24 h. The half-life of lactate dehydrogenase enzyme activity was 36 h during the induction and 40 h during deinduction. The half-life for decay of /sup 3/H-labeled lactate dehydrogenase was 41 h. These observations suggest that the increase in lactate dehydrogenase activity in norepinephrine-treated cells does not involve any change in the rate of degradation. Norepinephrine increased the specific rate of synthesis of both lactate dehydrogenase-5 (a tetramer of four M subunits) and lactate dehydrogenase-1 (a tetramer of four H subunits), although to different extents. Since these subunits are coded for by two separate genes on separate chromosomes, it suggests that the regulatory mechanism involves at least two separate sites of action.

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

    PubMed

    Ponc, R H; Carriazo, C S; Vermouth, N T

    2001-01-01

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

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

    PubMed

    Skory, C D

    2004-04-01

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

  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.

  13. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of lactate dehydrogenase isoenzymes are used in the diagnosis and treatment of liver diseases, such as viral hepatitis,...

  14. Effect of sex and age on the activities of lactate dehydrogenase and alkaline phosphatase in the lungs of rats.

    PubMed Central

    Lopez, A; Yong, S; Sharma, A; Morwood-Clark, M; Lillie, L E; Albassam, M

    1986-01-01

    Since toxicity studies among different laboratories generally involve rats of different sex and age, this study was conducted to investigate the effect of sex, age and animal to animal variation in the activities of lactate dehydrogenase and alkaline phosphatase from bronchoalveolar lavage fluid, bronchoalveolar cell lysate and lung homogenate. Correlation between numbers of bronchoalveolar cells recovered from lungs and enzyme activity in bronchoalveolar cell lysate or lung homogenate supernatant were also investigated. Male rats showed significantly (p less than 0.05) higher activities of alkaline phosphatase in the bronchoalveolar lavage fluid and lung homogenate. Animal to animal variation for lactate dehydrogenase and alkaline phosphatase was higher in lungs than in serum. The number of bronchoalveolar cells recovered from lungs revealed a significant (p less than 0.01) positive correlation with the activities of both enzymes in the supernatant of cell lysates but not in the bronchoalveolar fluid. These results indicated that in an inhalation study interindividual variation in the levels of pulmonary enzymes should be considered in order to minimize the numerous possible sources of experimental error. PMID:3742377

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

    PubMed

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

    2005-12-01

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

  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. [Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels].

    PubMed

    Kulis, Iu Iu

    1979-03-01

    The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.

  18. 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.

  19. 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.

  20. Differences and similarities in binding of pyruvate and L-lactate in the active site of M4 and H4 isoforms of human lactate dehydrogenase.

    PubMed

    Swiderek, Katarzyna; Paneth, Piotr

    2011-01-01

    We present QM/MM calculations that show differences in geometries of active sites of M(4) and H(4) isoforms of human LDH ligated with oxamate, pyruvate or L-lactate. As the consequence of these differences, binding isotope effects of the methyl hydrogen atoms of pyruvate and l-lactate may be used to experimentally distinguish these isoforms. Based on the FEP calculations we argue that L-lactate is a better candidate for the experimental studies. Our calculations of energies of interactions of ligands with the active site residues provide explanation for the observed experimentally sensitivity to inhibition of the M(4) isoenzyme isoform and pinpoint the differences to interactions of the ligand with the histidine residue. We conclude that pyruvate interacts much stronger in the active site of H(4) than M(4) isoform and that the latter interactions are weaker than with water molecules in the aqueous solution.

  1. Re-evaluation of the glycerol-3-phosphate dehydrogenase/L-lactate dehydrogenase enzyme system. Evidence against the direct transfer of NADH between active sites.

    PubMed Central

    Brooks, S P; Storey, K B

    1991-01-01

    An investigation of the direct transfer of metabolites from rabbit muscle L-lactate dehydrogenase (LDH, EC 1.1.1.27) to glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) revealed discrepancies between theoretical predictions and experimental results. Measurements of the GPDH reaction rate at a fixed NADH concentration and in the presence of increasing LDH concentrations gave experimental results similar to those previously obtained by Srivastava, Smolen, Betts, Fukushima, Spivey & Bernhard [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6464-6468]. However, a mathematical solution of the direct-transfer-mechanism equations as described by Srivastava et al. (1989) showed that the direct-transfer model did not adequately describe the experimental behaviour of the reaction rate at increasing LDH concentrations. In addition, experiments designed to measure the formation of an LDH4.NADH.GPDH2 complex, predicted by the direct-transfer model, indicated that no significant formation of tertiary complex occurred. An examination of other kinetic models, developed to describe the LDH/GPDH/NADH system better, revealed that the experimental results may be best explained by assuming that free NADH, and not E1.NADH, is the sole substrate for GPDH. These results suggest that direct transfer of NADH between rabbit muscle LDH and GPDH does not occur in vitro. PMID:1898374

  2. NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

    PubMed Central

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Ma, Yanhe

    2015-01-01

    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 Asn174 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. PMID:26150461

  3. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    PubMed

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

  4. Changes in lactate dehydrogenase and 3-hydroxyacetyl-CoA dehydrogenase activities in rat skeletal muscle by the administration of Eucommia ulmoides OLIVER leaf with spontaneous running-training.

    PubMed

    Li, Y; Koike, K; Che, Q; Yamaguchi, M; Takahashi, S

    1999-09-01

    We examined the effect of Eucommia ulmoides OLIVER leaf on rat skeletal muscles together with spontaneous running-training in terms of the isozyme profile and specific activity of lactate dehydrogenase (LDH; EC 1.1.1.27) and 3-hydroxyacetyl-CoA dehydrogenase (HAD; EC 1.1.1.35). On the twenty-ninth day of the experimental period, a mandatory endurance running exercise (treadmill, 7 degrees grade) was conducted. Twenty-four hours later, the rats were sacrificed and the skeletal muscles and other organs were dissected. Due to the training, the HAD specific activity in the skeletal muscles had increased and a more oxidative metabolism had developed, which was further enhanced by the administration of the leaf. In soleus (SOL) muscle in the Eucommia leaf treated running-training group (ET), the LDH specific activity in the skeletal muscle was significantly higher than in the sedentary control group (SC). The isozyme profile of the group ET was significantly different when compared with the group SC. The changes in the LDH isozyme profile were larger in the SOL than that in extensor digitorum longus (EDL) muscle. The results show that mechanical training and the use of the leaf cooperatively increase the ability to avoid lactate accumulation in skeletal muscle. This effect is supported by the group where 67% of rats accomplished the endurance running exercise. Theses results suggest that the administration of Eucommia ulmoides OLIVER leaf along with light intensity training enhances the ability of a muscle to resist fatigue.

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

    PubMed

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

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

  6. Modulation in the activity of lactate dehydrogenase and level of c-Myc and c-Fos by modified base queuine in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-01-01

    Cancer is characterized by uncontrolled cell growth, which results from unlimited proliferation and disturbs various cellular activities. Queuine is a highly modified base analogue of guanine found at first anti-codon position of specific tRNAs i.e. tRNA(Tyr), tRNA(His), tRNA(Asp) and tRNA(Asn). These tRNAs are known as Q-family of tRNA. The tRNAs of Q-family are completely modified to Q-tRNAs in terminally differentiated somatic cells, however hypomodification of Q-tRNA is closely associated with cell proliferation and malignancy. Queuosine modification of tRNAs may be essential for normal development, differentiation and cellular functions. Physiological role of queuine remains ill defined but direct or indirect evidences suggest that queuine or Q-tRNA participates in many cellular functions such as regulation of cell proliferation, control of glycolytic metabolism, alteration in expression of proto-oncogenes, modulation of signal transduction pathways but the mechanism is not well known. Increase in LDH-A expression regulated by c-myc is well documented in a variety of tumor cells. Overexpression of proto-oncogenes cause deregulated cellular responses which may lead to development of cancer. The cellular proto-oncogenes like c-myc and c-fos have important role in cell growth, proliferation and differentiation. The present study is aimed to investigate queuine mediated modulation in the activity of lactate dehydrogenase and expression of proto-oncogenes like c-myc and c-fos in T-cell lymphoma (DLAT) induced cancerous mouse. The results indicate that elevated lactate dehydrogenase activity is brought down by queuine treatments and the elevated levels of c-Myc and c-Fos in DLAT cancerous mouse are down-regulated, suggesting that queuine inhibits anaerobic metabolism and cell proliferation.

  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.

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

    PubMed

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

    2015-12-01

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

  10. Radial immunodiffusion and immunoelectrophoresis compared for identifying autoantibodies to lactate dehydrogenase in human serum.

    PubMed

    Harff, G A; Backer, E T

    1990-12-14

    Variant electrophoretic patterns of lactate dehydrogenase isoenzymes were studied. By radial immunodiffusion and immunoelectrophoresis, immunoglobulin and light chain class of autoantibodies to lactate dehydrogenase were identified in nine sera: seven of these sera demonstrated IgG (5 lambda, 2 kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase. We conclude that radial immunodiffusion and immunoelectrophoresis are equally effective for identifying auto-antibodies to lactate dehydrogenase in serum. Radial immunodiffusion, however, is easier to perform than immunoelectrophoresis.

  11. Separation of turkey lactate dehydrogenase isoenzymes using isoelectric focusing technique.

    PubMed

    Heinová, Dagmar; Kostecká, Zuzana; Csank, Tomáš

    2016-01-01

    Native polyacrylamide gel electrophoresis at pH 8.8 did not allow to separate lactate dehydrogenase (LDH) isoenzymes of turkey origin. Five electrophoretically distinguishable forms of the enzyme were detected in serum and tissues of turkey using IEF technique in a pH range of 3-9. Generally, three different groups were seen: (i) those having an anodic domination (heart, kidney, pancreas, and erythrocytes) with mainly LDH-1 fraction, (ii) those having a cathodic domination (breast muscle and serum) with prevalence of LDH-5, and (iii) those with a more uniform distribution (liver, spleen, lung, and brain). The specific enzyme activity was the highest in the breast muscle, followed by heart muscle, and brain. Low activities were detected in serum, kidney, and liver.

  12. The significance of matrix effects on the measurement of lactate dehydrogenase (LD) activity using Kodak dry slide technology in the Ontario Laboratory Proficiency Testing Program.

    PubMed

    Hill, S A; Heathcote, J C; McQueen, M J

    1990-04-01

    A recent lactate dehydrogenase (LD) survey of the Laboratory Proficiency Testing Program (LPTP) of Ontario showed interlaboratory coefficients of variation ranging from 6.5% to 40% for five lyophilized vials on the 12 Kodak analyzers. All the LPTP survey samples had similar protein and LD isoenzyme electrophoretic patterns which remained unchanged after reconstitution and storage for 5 days at 4 degrees C, although the total LD activities fell. Four Ektachem 700 analyzers were subsequently tested using LPTP material and no difference in LD activity between instruments or between two LD slide lot numbers was shown. Generation 9 slides gave higher LD activities than generation 10 on all the reconstituted lyophilized proficiency testing samples. There was no significant difference between slide generations when 19 liquid human sera were analyzed, indicating that the variability on LPTP samples was due to a matrix effect. Definition of the matrix effect of lyophilized proficiency testing material is essential before any proficiency testing program can use such material to reflect analytical performance on patient specimens.

  13. 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.

  14. Effects of Metmyoglobin Reducing Activity and Thermal Stability of NADH-Dependent Reductase and Lactate Dehydrogenase on Premature Browning in Ground Beef.

    PubMed

    Djimsa, Blanchefort A; Abraham, Anupam; Mafi, Gretchen G; VanOverbeke, Deborah L; Ramanathan, Ranjith

    2017-02-01

    Premature browning is a condition wherein ground beef exhibits a well-done appearance before reaching the USDA recommended internal cooked meat temperature of 71.1 °C; however, the mechanism is unclear. The objectives of this study were: (1) to determine the effects of packaging and temperature on metmyoglobin reducing activity (MRA) of cooked ground beef patties and (2) to assess the effects of temperature and pH on thermal stability of NADH-dependent reductase, lactate dehydrogenase (LDH), and oxymyoglobin (OxyMb) in-vitro. Beef patties (lean: fat = 85:15) were packaged in high-oxygen modified atmosphere (HiOX-MAP) or vacuum (VP) and cooked to either 65 or 71 °C. Internal meat color and MRA of both raw and cooked patties were determined. Purified NADH-dependent reductase and LDH were used to determine the effects of pH and temperature on enzyme activity. MRA of cooked patties was temperature and packaging dependent (P < 0.05). Vacuum packaged patties cooked to 71 °C had greater (P < 0.05) MRA than HiOX-MAP counterparts. Thermal stability of OxyMb, NADH-dependent reductase, and LDH were different and pH-dependent. LDH was able to generate NADH at 84 °C; whereas NADH-dependent reductase was least stable to heat. The results suggest that patties have MRA at cooking temperatures, which can influence cooked meat color.

  15. Thermostability of lactate dehydrogenase LDH-A4 isoenzyme: effect of heat shock protein DnaK on the enzyme activity.

    PubMed

    Zietara, M S; Skorkowski, E F

    1995-11-01

    Cells exposed to temperature a few degrees higher than their growth temperature synthesize heat shock proteins (hsp) which may then compose even 20% of total protein content. This paper examined the in vitro protective effect of heat shock protein DnaK (70 kDa) from Escherichia coli against the heat inactivation of lactate dehydrogenase isoenzyme LDH-A4. The LDH-A4 isoenzyme was purified from fish skeletal muscle using the affinity chromatography on Oxamate-agarose. The enzyme was then heated in the absence and the presence of DnaK protein in a water bath at either 51 or 55 degrees C. The LDH activity was determined by measuring the change in absorbency at 340 nm min-1 at 30 degrees C. The addition of DnaK protein to the LDH-A4 isoenzyme before heat treatment can protect enzyme activity against mild thermal inactivation. Incubation of the LDH-A4 isoenzyme at 51 degrees C in the presence of DnaK protein stimulates its activity by about 30%. The presence of 2 mM ATP can raise LDH activity by another 10%. No significant recovery was observed when DnaK protein was added to LDH at 25 degrees C following earlier inactivation. The maximal activities (Vmax) in the presence of DnaK protein are almost twice those without DnaK protein in the case of heat-treated LDH-A4 isoenzyme at 51 degrees C. The observed protection of LDH-A4 activity increased with the increasing DnaK protein concentration in the incubation medium. Results suggested that the presence of DnaK protein can protect LDH-A4 from heat inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Expression of lactate dehydrogenase C correlates with poor prognosis in renal cell carcinoma.

    PubMed

    Hua, Yibo; Liang, Chao; Zhu, Jundong; Miao, Chenkui; Yu, Yajie; Xu, Aimin; Zhang, Jianzhong; Li, Pu; Li, Shuang; Bao, Meiling; Yang, Jie; Qin, Chao; Wang, Zengjun

    2017-03-01

    Lactate dehydrogenase C is an isoenzyme of lactate dehydrogenase and a member of the cancer-testis antigens family. In this study, we aimed to investigate the expression and functional role of lactate dehydrogenase C and its basic mechanisms in renal cell carcinoma. First, a total of 133 cases of renal cell carcinoma samples were analysed in a tissue microarray, and Kaplan-Meier survival curve analyses were performed to investigate the correlation between lactate dehydrogenase C expression and renal cell carcinoma progression. Lactate dehydrogenase C protein levels and messenger RNA levels were significantly upregulated in renal cell carcinoma tissues, and the patients with positive lactate dehydrogenase C expression had a shorter progression-free survival, indicating the oncogenic role of lactate dehydrogenase C in renal cell carcinoma. In addition, further cytological experiments demonstrated that lactate dehydrogenase C could prompt renal cell carcinoma cells to produce lactate, and increase metastatic and invasive potential of renal cell carcinoma cells. Furthermore, lactate dehydrogenase C could induce the epithelial-mesenchymal transition process and matrix metalloproteinase-9 expression. In summary, these findings showed lactate dehydrogenase C was associated with poor prognosis in renal cell carcinoma and played a pivotal role in the migration and invasion of renal cell carcinoma cells. Lactate dehydrogenase C may act as a novel biomarker for renal cell carcinoma progression and a potential therapeutic target for the treatment of renal cell carcinoma.

  17. 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.

  18. Increasing the Heme-Dependent Respiratory Efficiency of Lactococcus lactis by Inhibition of Lactate Dehydrogenase

    PubMed Central

    Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B.; Pedersen, Per Dedenroth; Dal Bello, Fabio

    2013-01-01

    The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

  19. Parasite Lactate Dehydrogenase for Diagnosis of Plasmodium Falciparum. Phase II.

    DTIC Science & Technology

    1997-04-01

    Diagnosis of Plasmodium Falciparum PRINCIPAL INVESTIGATOR: Robert C. Piper, Ph.D. CONTRACTING ORGANIZATION: Flow, Incorporated Portland, Oregon 97201...Phase 11 (24 Mar 95 - 23 Mar 97) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Parasite Lactate Dehydrogenase for Diagnosis of Plasmodium Falciparum DAMD...that infected patients become ill. Four species of Plasmodium infect humans. P. falciparum accounts for -85 % of the world’s malaria. P. falciparum is

  20. Reappraisal of the Regulation of Lactococcal l-Lactate Dehydrogenase

    PubMed Central

    van Niel, Ed W. J.; Palmfeldt, Johan; Martin, Rani; Paese, Marco; Hahn-Hägerdal, Bärbel

    2004-01-01

    Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed. PMID:15006814

  1. Modulation of lactate dehydrogenase isozymes by modified base queuine.

    PubMed

    Pathak, C; Vinayak, Manjula

    2005-09-01

    The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA(GUN)) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.

  2. Salivary lactate dehydrogenase and aminotransferases in diabetic patients

    PubMed Central

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

    2016-01-01

    Abstract Diabetes mellitus (DM) is a group of metabolic diseases resulting from impaired insulin secretion and/or action. DM is characterized by hyperglycemia that can lead to the dysfunction or damage of organs, including the salivary glands. The aim of this study was to compare the levels of salivary lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in diabetic patients. The study was approved by the Bioethics Committee of Wroclaw Medical University (Poland). The study comprised 90 adults of both sexes, aged 21 to 57 years. The patients were divided into 3 groups: type 1 diabetics (D1), type 2 diabetics (D2), and a healthy control group (C). Each group consisted of 30 age- and sex-matched subjects. Total protein (P, by Lowry method), LDH, AST, ALT (with Alpha Diagnostics kits), and salivary flow rate were measured in unstimulated mixed saliva. The level of glycosylated hemoglobin (HbA1c) was measured with DCA 2000 Reagent Kit. The obtained data were analyzed using the Mann–Whitney U test and the Spearman rank at a significance level of P < 0.05 with the use of STATISTICA 9.0 software. In comparison with C, D1 presented a significantly higher activity of LDH (P < 0.001), AST (P < 0.001), and ALT (P < 0.01), whereas D2 indicated higher levels of LDH (P < 0.001) and ALT (P < 0.05) compared with C. Comparing D1 to D2, approximately 3-fold higher activity of AST (P < 0.01) and approximately 4.5-fold higher activity of ALT (P < 0.01) was observed. Higher levels of salivary LDH, AST, and ALT in D1 compared with D2 and C confirm that salivary glands of D1 might be attributed to autoimmunological damage associated with the pathomechanism of DM. PMID:27893660

  3. Mechanism of Thermal Adaptation in the Lactate Dehydrogenases.

    PubMed

    Peng, Huo-Lei; Egawa, Tsuyoshi; Chang, Eric; Deng, Hua; Callender, Robert

    2015-12-10

    The mechanism of thermal adaptation of enzyme function at the molecular level is poorly understood but is thought to lie within the structure of the protein or its dynamics. Our previous work on pig heart lactate dehydrogenase (phLDH) has determined very high resolution structures of the active site, via isotope edited IR studies, and has characterized its dynamical nature, via laser-induced temperature jump (T-jump) relaxation spectroscopy on the Michaelis complex. These particular probes are quite powerful at getting at the interplay between structure and dynamics in adaptation. Hence, we extend these studies to the psychrophilic protein cgLDH (Champsocephalus gunnari; 0 °C) and the extreme thermophile tmLDH (Thermotoga maritima LDH; 80 °C) for comparison to the mesophile phLDH (38-39 °C). Instead of the native substrate pyruvate, we utilize oxamate as a nonreactive substrate mimic for experimental reasons. Using isotope edited IR spectroscopy, we find small differences in the substate composition that arise from the detailed bonding patterns of oxamate within the active site of the three proteins; however, we find these differences insufficient to explain the mechanism of thermal adaptation. On the other hand, T-jump studies of reduced β-nicotinamide adenine dinucleotide (NADH) emission reveal that the most important parameter affecting thermal adaptation appears to be enzyme control of the specific kinetics and dynamics of protein motions that lie along the catalytic pathway. The relaxation rate of the motions scale as cgLDH > phLDH > tmLDH in a way that faithfully matches kcat of the three isozymes.

  4. Phosphatase-like activity, DNA binding, DNA hydrolysis, anticancer and lactate dehydrogenase inhibition activity promoting by a new bis-phenanthroline dicopper(II) complex.

    PubMed

    Anbu, Sellamuthu; Kandaswamy, Muthusamy; Kamalraj, Subban; Muthumarry, Johnpaul; Varghese, Babu

    2011-07-28

    A new bis-phenanthroline dicopper(II) complex has been synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the dinuclear Cu(II) complex [Cu(2)(μ-CH(3)COO)(μ-H(2)O)(μ-OH)(phen)(2)](2+) (phen = 1,10-phenanthroline) (1) was determined by single crystal X-ray diffraction technique. The coordination environment around each Cu(II) ion in complex 1 can be described as slightly distorted square pyramidal geometry. The distance between the CuCu centers in the complex is found to be 2.987 Å. The electronic, redox, phosphate hydrolysis, DNA binding and DNA cleavage have been studied. The antiproliferative effect of complex 1 was confirmed by the lactate dehydrogenase (LDH) enzyme level in MCF-7 cancer cell lysate and content media. The dicopper(II) complex inhibited the LDH enzyme as well as the growth of the human breast cancer MCF7 cell line at an IC(50) value of 0.011 μg ml(-1). The results strongly suggest that complex 1 is a good cancer therapeutic agent. Electrochemical studies of complex 1 showed an irreversible, followed by a quasi-reversible, one electron reduction processes between -0.20 to -0.8 V. Michaelis-Menten kinetic parameters for the hydrolysis of 4-nitrophenyl phosphate by complex 1 are k(cat) = 3.56 × 10(-2) s(-1) and K(M) = 4.3 × 10(-2) M. Complex 1 shows good binding propensity to calf thymus DNA, with a binding constant value of 1.3 (±0.13) × 10(5) M(-1) (s = 2.1). The size of the binding site and viscosity data suggest a DNA intercalative binding nature of the complex. Complex 1 shows efficient hydrolytic cleavage of supercoiled pBR322-DNA in the dark and in the absence of any external reagents, as demonstrated by the T4 ligase experiment. The pseudo-Michaelis-Menten kinetic parameters for DNA hydrolysis by complex 1 are k(cat) = 1.27 ± 0.4 h(-1) and K(M) = 7.7 × 10(-2) M.

  5. [Effective method of isolating M4-lactate dehydrogenase from rat liver].

    PubMed

    Gorbach, Z V; Maglysh, S S; Konovalenko, O V

    1984-01-01

    Lactate dehydrogenase M4-isoform in the homogeneous state was isolated from the rat liver by successive application of sulphate-ammonium fractionation, phosphocellulose ion-exchange chromatography with high-affinity elution of 1 mM NADH and subsequent hydroxyl apatite fractionation. The method permits obtaining the preparation amounts of the enzymic protein with yield 37.5%, specific activity 386.8 units per 1 mg of protein. It is established that 1 mM NAD+, 10 mM pyruvate and 100 mM lactate are also effective as agents of the selective enzyme elution.

  6. Lactate Dehydrogenase Catalysis: Roles of Keto, Hydrated, and Enol Pyruvate

    NASA Astrophysics Data System (ADS)

    Meany, J. E.

    2007-09-01

    Many carbonyl substrates of oxidoreductase enzymes undergo hydration and enolization so that these substrate systems are partitioned between keto, hydrated (gem-diol), and enol forms in aqueous solution. Some oxidoreductase enzymes are subject to inhibition by high concentrations of substrate. For such enzymes, two questions arise pertaining to enzyme "substrate" interactions: (i) which form of the substrate system serves as the preferential substrate and (ii) which form acts to inhibit the enzyme? Thus the relative concentrations of the forms of these substrate systems (keto, hydrated, enol) may provide a form of metabolic control. In this light, the present article considers the reduction of pyruvate by lactate dehydrogenase in the presence of NADH. This reaction is inhibited by relatively high concentrations of pyruvate and the physiological significance of this inhibition has been a subject of controversy for many years. Summarized in this article are data from the literature pertaining to the interactions of keto, hydrated, and enol pyruvate with lactate dehydrogenase. Biochemistry instructors and their students are invited to review such pertinent articles so that they also may evaluate the possibility that the "substrate" inhibition of the isoenzymes in the heart muscle may be, under certain conditions, relevant as a form of metabolic control.

  7. Lactate dehydrogenase inhibitors can reverse inflammation induced changes in colon cancer cells.

    PubMed

    Manerba, Marcella; Di Ianni, Lorenza; Govoni, Marzia; Roberti, Marinella; Recanatini, Maurizio; Di Stefano, Giuseppina

    2017-01-01

    The inflammatory microenvironment is an essential component of neoplastic lesions and can significantly impact on tumor progression. Besides facilitating invasive growth, inflammatory cytokines were also found to reprogram cancer cell metabolism and to induce aerobic glycolysis. Previous studies did not consider the possible contribution played in these changes by lactate dehydrogenase (LDH). The A isoform of LDH (LDH-A) is the master regulator of aerobic glycolysis; it actively reduces pyruvate and causes enhanced lactate levels in tumor tissues. In cancer cells, lactate was recently found to directly increase migration ability; moreover, when released in the microenvironment, it can facilitate matrix remodeling. In this paper, we illustrate that treatment of human colon adenocarcinoma cells with TNF-α and IL-17, two pro-inflammatory cytokines, modifies LDH activity, causing a shift toward the A isoform which results in increased lactate production. At the same time, the two cytokines appeared to induce features of epithelial-mesenchymal transition in the treated cells, such as reduction of E-cadherin levels and increased secretion of metalloproteinases. Noteworthy, oxamate and galloflavin, two inhibitors of LDH activity which reduce lactate production in cells, were found to relieve the inflammation-induced effects. These results suggest LDH-A and/or lactate as common elements at the cross-road between cancer cell metabolism, tumor progression and inflammation. At present, LDH inhibitors suitable for clinical use are actively searched as possible anti-proliferative agents; our data lead to hypothesize for these compounds a wider potential in anticancer treatment.

  8. Identification of New Structural Fragments for the Design of Lactate Dehydrogenase A Inhibitors

    PubMed Central

    Nilov, D.K.; Kulikov, A.V.; Prokhorova, E.A.; Švedas, V.K.

    2016-01-01

    Human lactate dehydrogenase A plays an important role in the glucose metabolism of tumor cells and constitutes an attractive target for chemotherapy. Molecular fragments able to bind in the active site of this enzyme and form hydrogen bonds with the Arg168 guanidinium group, as well as additional interactions with the loop 96–111 in the closed conformation, have been identified by virtual screening of sulfonates and experimental testing of their inhibitory effect. The sulfo group can occupy a similar position as the carboxyl group of the substrate and its structural analogs, whereas the benzothiazole group attached via a linker can be located in the coenzyme (NADH) binding site. Thus, the value of merging individual structural elements of the inhibitor by a linker was demonstrated and ways of further structural modification for the design of more effective inhibitors of lactate dehydrogenase A were established. PMID:27795851

  9. 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

  10. Fabrication of lactate biosensor based on lactate dehydrogenase immobilized on cerium oxide nanoparticles.

    PubMed

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

    2013-11-15

    An electrochemical biosensor was developed to determine lactate that plays an important role in clinical diagnosis, fermentation and food quality analysis. Abnormal concentration of lactate has been related to diseases such as hypoxia, acute heart disorders, lactic acidosis, muscle fatigue and meningitis. Also, lactate concentration in blood helps to evaluate the athletic performance in sports. The main aim of the work is to fabricate NADH/LDH/Nano-CeO2/GCE bio-electrode for sensing lactate in human blood samples. Toward this, CeO2 nanoparticles were synthesized by a hydroxide mediated approach using cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and NaOH as precursors. X-ray diffraction (XRD) and Field Emission Scanning Electron Microscopy (FE-SEM) studies were carried out to determine the structural and morphological characteristics of CeO2 nanoparticles. XRD pattern indicated the formation of highly crystalline CeO2 nanoparticles with face centered cubic structure. The FE-SEM studies revealed the formation of nanospherical particles of size 29.73±2.59 nm. The working electrode was fabricated by immobilizing nicotinamide adenine dinucleotide (NADH) and lactate dehydrogenase (LDH) on GCE surface with CeO2 nanoparticles as an interface. Electrochemical studies were carried out through cyclic voltammetry using a three electrode system with NADH/LDH/NanoCeO2/GCE as a working electrode, Ag/AgCl saturated with 0.1M KCl as a reference electrode and Pt wire as a counter electrode. From the amperometric study, the linearity was found to be in the range of 0.2-2 mM with the response time of less than 4s.

  11. Lactate dehydrogenase A silencing in IDH mutant gliomas

    PubMed Central

    Chesnelong, Charles; Chaumeil, Myriam M.; Blough, Michael D.; Al-Najjar, Mohammad; Stechishin, Owen D.; Chan, Jennifer A.; Pieper, Russell O.; Ronen, Sabrina M.; Weiss, Samuel; Luchman, H. Artee; Cairncross, J. Gregory

    2014-01-01

    Background Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. Methods We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant (mt) and IDH wild-type (wt) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. Results We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDHmt gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDHmt derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDHwt), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDHmt glioblastomas. Conclusion To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDHmt gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis. PMID:24366912

  12. LDHk, an unusual oxygen-sensitive lactate dehydrogenase expressed in human cancer.

    PubMed Central

    Anderson, G R; Kovacik, W P

    1981-01-01

    An unusual isozyme of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27), LDHk, has been described in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme appears to contain one or more subunits encoded by the transforming gene of KiMSV and is readily distinguished from other isozymes of LDH. Specifically, it is more basic than other LDH isozymes, has an apparent subunit structure of (35,000)4(22,000)1, is essentially inactive if assayed under a normal atmosphere, and is strongly inhibited by GTP and various related compounds. We have examined human cancer and normal tissue controls for expression of an activity like LDHk. In 11 out of 16 human carcinomas, LDHk activity was increased 10- to 500-fold over the level seen in adjoining nontumor tissue. In contrast, other LDH isozymes were increased by only 2- to 5-fold. Images PMID:6942426

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

    PubMed

    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.

  14. 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.

  15. 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

  16. Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood.

    PubMed

    Gaspar, Paula; Al-Bayati, Firas A Y; Andrew, Peter W; Neves, Ana Rute; Yesilkaya, Hasan

    2014-12-01

    Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.

  17. Identification of 3,6-disubstituted dihydropyrones as inhibitors of human lactate dehydrogenase.

    PubMed

    Fauber, Benjamin P; Dragovich, Peter S; Chen, Jinhua; Corson, Laura B; Ding, Charles Z; Eigenbrot, Charles; Labadie, Sharada; Malek, Shiva; Peterson, David; Purkey, Hans E; Robarge, Kirk; Sideris, Steve; Ultsch, Mark; Wei, BinQing; Yen, Ivana; Yue, Qin; Zhou, Aihe

    2014-12-15

    A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC50=30 nM).

  18. Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors.

    PubMed

    Vanderporten, Erica; Frick, Lauren; Turincio, Rebecca; Thana, Peter; Lamarr, William; Liu, Yichin

    2013-10-15

    Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD⁺) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD⁺. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z' scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.

  19. 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.

  20. 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

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

    PubMed

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

    2009-12-01

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

  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. Metabolic Control of Anaerobic Glycolysis (Overexpression of Lactate Dehydrogenase in Transgenic Tomato Roots Supports the Davies-Roberts Hypothesis and Points to a Critical Role for Lactate Secretion.

    PubMed

    Rivoal, J.; Hanson, A. D.

    1994-11-01

    Roots of all plants examined so far have the potential for both ethanol and lactate fermentation. A short burst of lactate fermentation usually occurs when plant tissues are transferred from normoxic to anoxic conditions. According to the Davies-Roberts hypothesis, the consequent pH drop both initiates ethanol fermentation and blocks further production of lactate by inhibiting lactate dehydrogenase (LDH). However, the role of LDH in this pH control mechanism is still a matter of debate. To perturb the control system in a defined way, a barley LDH cDNA under the control of the cauliflower mosaic virus 35S promoter was introduced into tomato (Lycopersicon esculentum Mill. cv VFMT) using Agrobacterium rhizogenes. The transgenic root clones expressed up to 50 times the LDH activity of controls. The fermentative metabolism of these clones was compared using roots grown previously in normoxic conditions or roots given a 3-d hypoxic pretreatment. During the transition from normoxia to anoxia, lactate accumulation was no faster and no more extensive in transgenic roots than in controls. Similarly, during prolonged anoxia the flux of 14C from [U-14C] glucose to lactate and ethanol was not modified by the expression of the transgene. However, in both transgenic and control roots, hypoxic pretreatment increased the flux to lactate and promoted lactate export to the medium. These results show that LDH has a very low flux control coefficient for lactate fermentation, consistent with the Davies-Roberts hypothesis. Moreover, they suggest that lactate secretion exerts major control over long-term lactate glycolysis in vivo.

  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.

  6. Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

    PubMed

    Srivastava, D K; Smolen, P; Betts, G F; Fukushima, T; Spivey, H O; Bernhard, S A

    1989-09-01

    Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that

  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. Antiplasmodial studies of Eurycoma longifolia Jack using the lactate dehydrogenase assay of Plasmodium falciparum.

    PubMed

    Chan, Kit-Lam; Choo, Chee-Yan; Abdullah, Noor Rain; Ismail, Zakiah

    2004-06-01

    The roots of Eurycoma longifolia Jack have been used as traditional medicine to treat malaria. A systematic bioactivity-guided fractionation of this plant was conducted involving the determination of the effect of its various extracts and their chemical constituents on the lactate dehydrogenase activity of in vitro chloroquine-resistant Gombak A isolate and chloroquine-sensitive D10 strain of Plasmodium falciparum parasites. Their antiplasmodial activity was also compared with their known in vitro cytotoxicity against KB cells. Four quassinoids, eurycomanone (1), 13,21-dihydroeurycomanone (3), 13 alpha(21)-epoxyeurycomanone (4), eurycomalactone (6) and an alkaloid, 9-methoxycanthin-6-one (7), displayed higher antiplasmodial activity against Gombak A isolate but were less active against the D10 strain when compared with chloroquine. Amongst the compounds tested, 1 and 3 showed higher selectivity indices obtained for the cytotoxicity to antiplasmodial activity ratio than 14,15 beta-dihydroxyklaineanone (2), eurycomanol (5), 6 and 7.

  9. 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

  10. Impact of high pyruvate concentration on kinetics of rabbit muscle lactate dehydrogenase.

    PubMed

    Eggert, Matthew Warren; Byrne, Mark E; Chambers, Robert P

    2011-09-01

    In order to evaluate the effectiveness of L: -lactate dehydrogenase (LDH) from rabbit muscle as a regenerative catalyst of the biologically important cofactor nicotinamide adenine dinucleotide (NAD), the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression. Despite robust literature describing the intricate complexations, the mammalian rabbit muscle LDH lacks a quantitative kinetic rate expression accounting for simultaneous inhibition parameters, specifically at high pyruvate concentrations. Product inhibition by L: -lactate was observed to reduce activity at concentrations greater than 25 mM, while expected substrate inhibition by pyruvate was significant above 4.3 mM concentration. The combined effect of ternary and binary complexes of pyruvate and the coenzymes led to experimental rates as little as a third of expected activity. The convenience of the statistical software package JMP allowed for effective determination of experimental kinetic constants and simplification to a suitable rate expression: [formula: see text] where the last three terms represent the inhibition complex terms for lactate, pyruvate, and pyruvate-NAD, respectively. The corresponding values of K (I-Lac), K (I-Pyr), and K (I-Pyr-NAD) for rabbit muscle LDH are 487.33 mM(-1) and 29.91 mM and 97.47 mM at 22 °C and pH 7.8.

  11. Probing the Role of Dynamics in Hydride Transfer Catalyzed by Lactate Dehydrogenase

    PubMed Central

    Zhadin, Nickolay; Gulotta, Miriam; Callender, Robert

    2008-01-01

    The dynamic nature of the interconversion of pyruvate to lactate as catalyzed by lactate dehydrogenase (LDH) is characterized by laser-induced temperature jump relaxation spectroscopy with a resolution of 20 ns. An equilibrium system of LDH·NADH plus pyruvate and LDH·NAD+ plus lactate is perturbed by a sudden T-jump, and the relaxation of the system is monitored by NADH emission and absorption changes. The substrate binding pathway is observed to be similar, although not identical, to previous work on substrate mimics: an encounter complex is formed between LDH·NADH and pyruvate, which collapses to the active Michaelis complex. The previously unresolved hydride transfer event is characterized and separated from other unimolecular isomerizations of the protein important for the catalytic mechanism, such as loop closure, a slower step, and faster events on the nanosecond-microsecond timescales whose structural basis is not understood. The results of this study show that this approach can be applied quite generally to enzyme systems and report on the dynamic nature of proteins over a very wide time range. PMID:18487309

  12. Probing the role of dynamics in hydride transfer catalyzed by lactate dehydrogenase.

    PubMed

    Zhadin, Nickolay; Gulotta, Miriam; Callender, Robert

    2008-08-01

    The dynamic nature of the interconversion of pyruvate to lactate as catalyzed by lactate dehydrogenase (LDH) is characterized by laser-induced temperature jump relaxation spectroscopy with a resolution of 20 ns. An equilibrium system of LDH.NADH plus pyruvate and LDH.NAD+ plus lactate is perturbed by a sudden T-jump, and the relaxation of the system is monitored by NADH emission and absorption changes. The substrate binding pathway is observed to be similar, although not identical, to previous work on substrate mimics: an encounter complex is formed between LDH.NADH and pyruvate, which collapses to the active Michaelis complex. The previously unresolved hydride transfer event is characterized and separated from other unimolecular isomerizations of the protein important for the catalytic mechanism, such as loop closure, a slower step, and faster events on the nanosecond-microsecond timescales whose structural basis is not understood. The results of this study show that this approach can be applied quite generally to enzyme systems and report on the dynamic nature of proteins over a very wide time range.

  13. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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...

  14. 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...

  15. 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...

  16. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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...

  17. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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...

  18. Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

    PubMed

    Gordon, G L; Doelle, H W

    1975-02-01

    Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.

  19. Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase.

    PubMed

    Dragovich, Peter S; Fauber, Benjamin P; Boggs, Jason; Chen, Jinhua; Corson, Laura B; Ding, Charles Z; Eigenbrot, Charles; Ge, HongXiu; Giannetti, Anthony M; Hunsaker, Thomas; Labadie, Sharada; Li, Chiho; Liu, Yichin; Liu, Yingchun; Ma, Shuguang; Malek, Shiva; Peterson, David; Pitts, Keith E; Purkey, Hans E; Robarge, Kirk; Salphati, Laurent; Sideris, Steve; Ultsch, Mark; VanderPorten, Erica; Wang, Jing; Wei, BinQing; Xu, Qing; Yen, Ivana; Yue, Qin; Zhang, Huihui; Zhang, Xuying; Zhou, Aihe

    2014-08-15

    A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).

  20. Biorhythms of activities of liver and blood dehydrogenases and changes in body weight of the rats feeding normal diet or excess of sugar substitutes.

    PubMed

    Petrovich, Yu A; Volozhin, A I; Zubtsov, V A; Kichenko, S M

    2007-12-01

    Biorhythms with higher levels of activity of sorbitol dehydrogenase and lactate dehydrogenase in blood plasma, specific activity of sorbitol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase in the liver, and body weight of rats were more pronounced in the spring-summer period than in the autumn-winter period. These specific features were revealed in animals feeding a normal diet or food with 54 and 27% sugar substitute sorbitol. However, specific activity of glucose-6-phosphate dehydrogenase in the liver was higher in the autumn-winter period. Activity of sorbitol dehydrogenase in blood plasma increased by tens of times due to induction of sorbitol synthesis (substrate) in the liver. Sugar substitute xylitol is structurally similar to sorbitol, but is not the substrate for sorbitol dehydrogenase. However, the effect of xylitol on activities of lactate dehydrogenase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase in the spring-summer period was similar to that of sorbitol.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. Electrophoretic variation in muscle lactate dehydrogenase in Snake Valley cutthroat trout, Salmo clarki subsp.

    PubMed

    Klar, G T; Stalnaker, C B

    1979-01-01

    1. Electrophoretic variation observed in muscle A group lactate dehydrogenase in Snake Valley cutthroat trout (Salmo clarki subsp.) suggested the presence of two variant alleles at the A1 locus and a null allele at the A2 locus. 2. The taxonomic status of the Snake Valley cutthroat trout was reviewed.

  7. Modification of Rhizopus lactate dehydrogenase for improved resistance to fructose 1,6-bisphosphate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is frequently used for fermentative production of lactic acid. We determined that one of the key enzymes, lactate dehydrogenase (LDH), involved in synthesis of lactic acid by R. oryzae was significantly inhibited by fructose 1,6-bisphosphate (FBP) at physiological concentrations. Thi...

  8. Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase.

    PubMed

    Kaushal, Nuzhat A; Kaushal, Deep C

    2014-01-01

    Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been shown to be biochemically, immunologically and structurally different from the mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydrogenase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug targeting. In the present study, we have produced six monoclonal antibodies, which are against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and 10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff 0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be utilized for identification of compounds inhibitory to PfLDH (binding to substrate specific loop region of parasite LDH) from combinatory chemical libraries or medicinal plants extracts. The Microplate Sandwich ELISA has also shown potential for specific diagnosis of malaria using finger prick blood samples.

  9. Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site.

    PubMed

    Holland, L Z; McFall-Ngai, M; Somero, G N

    1997-03-18

    Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 1.1.1.27; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active

  10. 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.

  11. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    PubMed

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast.

  12. Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions.

    PubMed

    Ramljak, Sanja; Schmitz, Matthias; Zafar, Saima; Wrede, Arne; Schenkel, Sara; Asif, Abdul R; Carimalo, Julie; Doeppner, Thorsten R; Schulz-Schaeffer, Walter J; Weise, Jens; Zerr, Inga

    2015-09-01

    Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified, a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion protein knockout mice (Prnp(0/0)) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrP(c). Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp(0/0) knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp(0/0) primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp(0/0) mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrP(c) show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrP(c) was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrP(c)-overexpression under normoxic conditions. Our data suggest LDH as a direct PrP(c) interactor with possible physiological relevance under low oxygen conditions.

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

    PubMed

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

    2016-01-07

    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.

  14. Identification of substituted 2-thio-6-oxo-1,6-dihydropyrimidines as inhibitors of human lactate dehydrogenase.

    PubMed

    Dragovich, Peter S; Fauber, Benjamin P; Corson, Laura B; Ding, Charles Z; Eigenbrot, Charles; Ge, HongXiu; Giannetti, Anthony M; Hunsaker, Thomas; Labadie, Sharada; Liu, Yichin; Malek, Shiva; Pan, Borlan; Peterson, David; Pitts, Keith; Purkey, Hans E; Sideris, Steve; Ultsch, Mark; VanderPorten, Erica; Wei, BinQing; Xu, Qing; Yen, Ivana; Yue, Qin; Zhang, Huihui; Zhang, Xuying

    2013-06-01

    A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships.

  15. Increase in lactate dehydrogenase isoenzyme-4 and splenocyte toxicity in methomyl-treated rats.

    PubMed

    Lohitnavy, O; Sinhaseni, P

    1998-09-01

    The toxic effect of methomyl was studied in rats after a single or repeated oral administration. Rats treated with a single dose of methomyl (3, 5, or 7 mg/kg) showed significant increase (P < 0.05) in total lactate dehydrogenase (LDH) activity on day 1. The highest level of LDH activity was observed on day 3 in rats receiving 7 mg/kg of methomyl. The total LDH activity returned to normal on day 7 after dosing. Specific increases in LDH-3 and LDH-4 isoenzyme activities were observed. In rats treated with a single dose of 6 and 8 mg/kg of methomyl, spleen weight and splenocyte viability significantly dropped (P < 0.05) on days 1 and 3, respectively. Splenotoxicity was prevented by pretreatment with 60 mg/kg of N-acetylcysteine. The results suggest that the splenotoxic effect of methomyl is more likely directly related to oxidative cell injury than to cholinesterase inhibition. The significance of cytotoxic effects and the nature of cytotoxicity in relation to reactive oxidative damage deserve further investigation.

  16. 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

  17. A novel flavin adenine dinucleotide (FAD) containing d-lactate dehydrogenase from the thermoacidophilic crenarchaeota Sulfolobus tokodaii strain 7: purification, characterization and expression in Escherichia coli.

    PubMed

    Satomura, Takenori; Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2008-07-01

    Dye-linked D-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80 degrees C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50 degrees C. The preferred substrate for this enzyme was D-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme's prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked D-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to D-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked D-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing D-lactate dehydrogenase.

  18. The core of allosteric motion in Thermus caldophilus L-lactate dehydrogenase.

    PubMed

    Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

    2014-11-07

    For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S(0.5) value 10(3)-fold and increased the V(max) value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus L-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V(max) 4-fold but reduced pyruvate S(0.5) only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V(max), but 10(2)-reduced pyruvate S(0.5), and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.

  19. Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria.

    PubMed

    Elustondo, Pia A; White, Adrienne E; Hughes, Meghan E; Brebner, Karen; Pavlov, Evgeny; Kane, Daniel A

    2013-08-30

    The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

  20. 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.

  1. Extraction and separation of lactate dehydrogenase inhibitors from Poria cocos (Schw.) Wolf based on a hyphenated technique and in vitro methods.

    PubMed

    Li, Sainan; Zhang, Jianxu; Li, Senlin; Liu, Chunming; Liu, Shu; Liu, Zhiqiang

    2017-02-20

    Stroke is one of the most common diseases worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke, with natural products considered a promising source of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Poria cocos. Five lactate dehydrogenase inhibitors were selected: dehydropachymic acid, pachymic acid, dehydrotrametenolic acid, trametenolic acid, and eburicoic acid. The inhibitors were extracted and isolated with purities of 96.75, 98.15, 97.25, 95.46, and 94.88%, respectively, by using a new "hyphenated" strategy of microwave-assisted extraction coupled with counter-current chromatography and centrifugal partition chromatography by a two-phase solvent system of n-hexane/ethyl acetate/ethanol/water at the volume ratio 0.965:1.000:0.936:0.826 v/v/v/v. The bioactivity of the isolated compounds was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in PC12 cells. The results also showed that the hyphenated technique of microwave-assisted extraction coupled with counter-current chromatography and centrifugal partition chromatography was an efficient method for the continuous extraction and online isolation of chemical constituents from medicinal herbs. Furthermore, the research route based on the activity screening, extraction, separation, and activity verification of the compounds offered advantages of efficiency, orientation, and objectivity.

  2. 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.

  3. Cloning and characterization of l-lactate dehydrogenase gene of Staphylococcus aureus.

    PubMed

    Yeswanth, Sthanikam; Nanda Kumar, Yellapu; Venkateswara Prasad, Uppu; Swarupa, Vimjam; Koteswara rao, Valasani; Venkata Gurunadha Krishna Sarma, Potukuchi

    2013-12-01

    Staphylococcus aureus a natural inhabitant of nasopharyngeal tract survives in the host as biofilms. In the present study S. aureus ATCC12600 grown under anaerobic conditions showed biofilm units of 0.086 as compared to 0.07 when this pathogen grown in aerobic conditions with elevated lactate formation and the same was also observed with increased biofilm units of 0.06, 0.084 and 0.167 under 0.05%, 0.1% and 0.15% glucose supplementation in BHI broth. The lactate dehydrogenase (LDH) gene which catalyzes the formation of lactate was cloned, sequenced (Accession Numbers: JN645813) and expressed in Escherichia coli DH5α. The pure recombinant LDH exhibited molecular weight of 34 kDa in SDS-PAGE and the enzyme kinetics of recombinant enzyme was found to be in the direction of lactate to pyruvate Km of 2.03 ± 0.025 μM and Kcat of 1.69 ± 0.03/min and from pyruvate to lactate Km of 1.62 ± 0.10 μM and Kcat of 1.75 ± 0.03/min. In the LDH gene sequence "LKDIMA" was found to be conserved in all Gram positive bacteria and in all human LDH isoforms even though only 39% sequence homology was observed with all human LDH isoforms. However, 92% structural homology was observed with all human LDH isoforms. The molecular docking of pyruvate and lactate to the LDH structure showed -10.298 for pyruvate while -9.297 for lactate indicating higher affinity of pyruvate compared to lactate which concurred with the elevated LDH kinetics and rate of biofilm units in anaerobic conditions.

  4. 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.

  5. 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

  6. Chemosensitivity of MCF-7 cells to eugenol: release of cytochrome-c and lactate dehydrogenase.

    PubMed

    Al Wafai, Rana; El-Rabih, Warde; Katerji, Meghri; Safi, Remi; El Sabban, Marwan; El-Rifai, Omar; Usta, Julnar

    2017-03-08

    Phytochemicals have been extensively researched for their potential anticancer effects. In previous study, direct exposure of rat liver mitochondria to eugenol main ingredient of clove, uncoupled mitochondria and increased F0F1ATPase activity. In the present study, we further investigated the effects of eugenol on MCF-7 cells in culture. Eugenol demonstrated: a dose-dependent decrease in viability (MTT assay), and proliferation (real time cell analysis) of MCF-7 cells, (EC50: 0.9 mM); an increase in reactive oxygen species; a decrease in ATP level and mitochondrial membrane potential (MitoPT JC-1 assay); and a release of cytochrome-c and lactate dehydrogenase (Cytotoxicity Detection Kit (PLUS)) into culture media at eugenol concentration >EC50. Pretreatment with the antioxidants Trolox and N-acetyl cysteine partially restored cell viability and decreased ROS, with Trolox being more potent. Expression levels of both anti- and pro-apoptotic markers (Bcl-2 and Bax, respectively) decreased with increasing eugenol concentration, with no variation in their relative ratios. Eugenol-treated MCF-7 cells overexpressing Bcl-2 exhibited results similar to those of MCF-7. Our findings indicate that eugenol toxicity is non-apoptotic Bcl-2 independent, affecting mitochondrial function and plasma membrane integrity with no effect on migration or invasion. We report here the chemo-sensitivity of MCF-7 cells to eugenol, a phytochemical with anticancer potential.

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

    NASA Astrophysics Data System (ADS)

    Masterson, Jean E.; Schwartz, Steven D.

    2014-10-01

    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.

  8. Cerebrospinal fluid lactate dehydrogenase isoenzymes in children with bacterial and aseptic meningitis.

    PubMed

    Nussinovitch, Moshe; Finkelstein, Yaron; Elishkevitz, Keren Politi; Volovitz, Benjamin; Harel, Daniella; Klinger, Gil; Razon, Yaron; Nussinovitch, Udi; Nussinovitch, Naomi

    2009-10-01

    Differentiation of bacterial from aseptic meningitis may be difficult. Our aim was to determine the pattern of distribution of lactate dehydrogenase (LDH) isoenzymes in the cerebrospinal fluid (CSF) of patients with bacterial and aseptic meningitis. One hundred and fifty-seven patients with suspected meningitis were enrolled in the study. They were divided into 3 groups according to the culture- or bacterial antigen assay-proven diagnosis and CSF findings: bacterial meningitis (n = 31), aseptic meningitis (n = 65), and non-meningitis (n = 61). Total LDH level and percentages of LDH isoenzymes in the CSF were measured in each patient. Each group showed a distinct LDH isoenzyme distribution pattern, with a statistically significant difference among the groups in the percentages of the various isoenzymes. Compared with the non-meningitis group, total LDH activity in the CSF was high in the aseptic meningitis group (49.82+/-35.59 U/L, P < 0.001) and exaggerated in the bacterial meningitis group (944.53+/-112.3 U/L, P < 0.001). Low LDH-2 levels were unique to bacterial meningitis (P < 0.01), whereas high LDH-3 levels were characteristic of aseptic meningitis (P < 0.05). Both groups had low levels of LDH-1 and high levels of LDH-4 and LDH-5. In conclusion, the LDH isoenzyme pattern may be of clinical diagnostic value in meningitis, particularly when culture results are pending.

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

    PubMed

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

    2012-09-01

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

  10. Chemosensitivity of MCF-7 cells to eugenol: release of cytochrome-c and lactate dehydrogenase

    PubMed Central

    Al Wafai, Rana; El-Rabih, Warde; Katerji, Meghri; Safi, Remi; El Sabban, Marwan; El-Rifai, Omar; Usta, Julnar

    2017-01-01

    Phytochemicals have been extensively researched for their potential anticancer effects. In previous study, direct exposure of rat liver mitochondria to eugenol main ingredient of clove, uncoupled mitochondria and increased F0F1ATPase activity. In the present study, we further investigated the effects of eugenol on MCF-7 cells in culture. Eugenol demonstrated: a dose-dependent decrease in viability (MTT assay), and proliferation (real time cell analysis) of MCF-7 cells, (EC50: 0.9 mM); an increase in reactive oxygen species; a decrease in ATP level and mitochondrial membrane potential (MitoPT JC-1 assay); and a release of cytochrome-c and lactate dehydrogenase (Cytotoxicity Detection Kit PLUS) into culture media at eugenol concentration >EC50. Pretreatment with the antioxidants Trolox and N-acetyl cysteine partially restored cell viability and decreased ROS, with Trolox being more potent. Expression levels of both anti- and pro-apoptotic markers (Bcl-2 and Bax, respectively) decreased with increasing eugenol concentration, with no variation in their relative ratios. Eugenol-treated MCF-7 cells overexpressing Bcl-2 exhibited results similar to those of MCF-7. Our findings indicate that eugenol toxicity is non-apoptotic Bcl-2 independent, affecting mitochondrial function and plasma membrane integrity with no effect on migration or invasion. We report here the chemo-sensitivity of MCF-7 cells to eugenol, a phytochemical with anticancer potential. PMID:28272477

  11. Efficient production of enantiomerically pure D-phenyllactate from phenylpyruvate by structure-guided design of an engineered D-lactate dehydrogenase.

    PubMed

    Wang, Min; Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Yin, Ruochun; Yu, Bo

    2016-09-01

    3-Phenyllactic acid (PLA) is an antimicrobial compound with broad-spectrum activity against bacteria and fungi that could be widely used in the food industry and livestock feeds. Notably, D-PLA exhibits higher antibacterial activity, which gains more attention than L-PLA. In this report, the D-lactate dehydrogenase DLDH744 from Sporolactobacillus inulinus CASD was engineered to increase the enzymatic activities toward phenylpyruvate by protein structure-guided modeling analysis. The phenylpyruvate molecule was first docked in the active center of DLDH744. The residues that might tightly pack around the benzene ring of phenylpyruvate were all selected for mutation. The single site mutant M307L showed the highest increased activity toward bulkier substrate phenylpyruvate than the wild type. By using the engineered D-lactate dehydrogenase M307L expressed in Escherichia coli strains, without coexpression of the cofactor regeneration system, 21.43 g/L D-PLA was produced from phenylpyruvate with a productivity of 1.58 g/L/h in the fed-batch biotransformation process, which ranked in the list as the highest production titer of D-PLA by D-lactate dehydrogenase. The enantiomeric excess value of produced D-PLA in the broth was higher than 99.7 %. Additionally, the structure-guided design of this enzyme will also provide referential information for further engineering other 2-hydroxyacid dehydrogenases, which are useful for a wide range of fine chemical synthesis.

  12. 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.

  13. The influence of oxygen on radiation-induced structural and functional changes in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Bubinski, Michal; Krokosz, Anita; Puchala, Mieczyslaw

    2012-07-01

    Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure. Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (-SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

  14. The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli.

    PubMed

    Berríos-Rivera, Susana J; San, Ka-Yiu; Bennett, George N

    2003-01-01

    In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh(-) strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh(-) mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal.

  15. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN EWING'S SARCOMA

    PubMed Central

    Baptista, André Mathias; Zumárraga, Juan Pablo; dos Santos, Renan Pires Negrão; Haubert, Guilherme de Oliveira; de Camargo, Olavo Pires

    2016-01-01

    ABSTRACT Objective: To study the relationship between the serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) in patients with Ewing´s Sarcoma (ES). Methods: This is a case series with retrospective evaluation of patients with diagnosis of ES divided into 2 groups: Group 1, patients whose serum levels of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were obtained in the staging phase before preoperative chemotherapy (CT), and Group 2, patients whose values were measured after completion of the preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens extracted in surgery was also evaluated. Results: Eighty four medical records from 1995 to 2015 were included. Both AP as LDH decreased in the patients studied, the pre CT value being higher than the post CT value. The average decrease of LHD was 272.95 U/L and AP was 10.17 U/L. The average tumor necrosis was 65.12 %. There was no statistical correlation between serums levels and the tumor necrosis percentage. Conclusion: The serum levels values of AP and LDH are not predictors for chemotherapy-induced necrosis in patients with ES. Level of Evidence IV, Case Series. PMID:28243173

  16. 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.

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

    PubMed

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

    2015-08-10

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

  18. Parasite-specific lactate dehydrogenase for the diagnosis of Plasmodium falciparum infection in an endemic area in west Uganda.

    PubMed

    Jelinek, T; Kilian, A H; Henk, M; Mughusu, E B; Nothdurft, H D; Löscher, T; Knobloch, J; Van Sonnenburg, F

    1996-04-01

    The measurement of parasite lactate dehydrogenase (pLDH) has been presented as an easy and rapid method for the diagnosis of malaria in humans. In order to evaluate the sensitivity and specificity of such a test we examined blood samples from 429 Ugandan patients. While pLDH activity was significantly linked to parasitaemia, sensitivity and specificity were found to be rather low at 58.8 and 62.2% respectively. The positive and negative predictive values failed to meet necessary standards. We conclude that the methods of measurement of pLDH activity in malaria infection, although potentially useful for the fast diagnosis of malaria, need to be improved to be of true value in endemic areas.

  19. Lactate dehydrogenase in Toxoplasma gondii controls virulence, bradyzoite differentiation, and chronic infection.

    PubMed

    Abdelbaset, Abdelbaset E; Fox, Barbara A; Karram, Mohamed H; Abd Ellah, Mahmoud R; Bzik, David J; Igarashi, Makoto

    2017-01-01

    In the asexual stages, Toxoplasma gondii stage converts between acute phase rapidly replicating tachyzoites and chronic phase slowly dividing bradyzoites. Correspondingly, T. gondii differentially expresses two distinct genes and isoforms of the lactate dehydrogenase enzyme, expressing LDH1 exclusively in the tachyzoite stage and LDH2 preferentially in the bradyzoite stage. LDH catalyzes the interconversion of pyruvate and lactate in anaerobic growth conditions and is utilized for energy supply, however, the precise role of LDH1 and LDH2 in parasite biology in the asexual stages is still unclear. Here, we investigated the biological role of LDH1 and LDH2 in the asexual stages, and the vaccine strain potential of deletion mutants lacking LDH1, LDH2, or both genes (Δldh1, Δldh2 and Δldh1/2). Deletion of LDH1 reduced acute parasite virulence, impaired bradyzoite differentiation in vitro, and markedly reduced chronic stage cyst burdens in vivo. In contrast, deletion of LDH2 impaired chronic stage cyst burdens without affecting virulence or bradyzoite differentiation. Deletion of both LDH1 and LDH2 induced a more severe defect in chronic stage cyst burdens. These LDH mutant phenotypes were not associated with any growth defect. Vaccination of mice with a low dose of mutants deleted for LDH elicited effective protective immunity to lethal challenge infection, demonstrating the vaccine potential of LDH deletion mutants. These results suggest that lactate dehydrogenase in T. gondii controls virulence, bradyzoite differentiation, and chronic infection and reveals the potential of LDH mutants as vaccine strains.

  20. Lactate dehydrogenase in Toxoplasma gondii controls virulence, bradyzoite differentiation, and chronic infection

    PubMed Central

    Abdelbaset, Abdelbaset E.; Fox, Barbara A.; Karram, Mohamed H.; Abd Ellah, Mahmoud R.; Bzik, David J.; Igarashi, Makoto

    2017-01-01

    In the asexual stages, Toxoplasma gondii stage converts between acute phase rapidly replicating tachyzoites and chronic phase slowly dividing bradyzoites. Correspondingly, T. gondii differentially expresses two distinct genes and isoforms of the lactate dehydrogenase enzyme, expressing LDH1 exclusively in the tachyzoite stage and LDH2 preferentially in the bradyzoite stage. LDH catalyzes the interconversion of pyruvate and lactate in anaerobic growth conditions and is utilized for energy supply, however, the precise role of LDH1 and LDH2 in parasite biology in the asexual stages is still unclear. Here, we investigated the biological role of LDH1 and LDH2 in the asexual stages, and the vaccine strain potential of deletion mutants lacking LDH1, LDH2, or both genes (Δldh1, Δldh2 and Δldh1/2). Deletion of LDH1 reduced acute parasite virulence, impaired bradyzoite differentiation in vitro, and markedly reduced chronic stage cyst burdens in vivo. In contrast, deletion of LDH2 impaired chronic stage cyst burdens without affecting virulence or bradyzoite differentiation. Deletion of both LDH1 and LDH2 induced a more severe defect in chronic stage cyst burdens. These LDH mutant phenotypes were not associated with any growth defect. Vaccination of mice with a low dose of mutants deleted for LDH elicited effective protective immunity to lethal challenge infection, demonstrating the vaccine potential of LDH deletion mutants. These results suggest that lactate dehydrogenase in T. gondii controls virulence, bradyzoite differentiation, and chronic infection and reveals the potential of LDH mutants as vaccine strains. PMID:28323833

  1. 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.

  2. [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.

  3. Novel control of lactate dehydrogenase from the freeze tolerant wood frog: role of posttranslational modifications

    PubMed Central

    Abboud, Jean

    2013-01-01

    Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, plays a crucial role both in sustaining glycolytic ATP production under oxygen-limiting conditions and in facilitating the catabolism of accumulated lactate when stress conditions are relieved. In this study, the effects on LDH of in vivo freezing and dehydration stresses (both of which impose hypoxia/anoxia stress on tissues) were examined in skeletal muscle of the freeze-tolerant wood frog, Rana sylvatica. LDH from muscle of control, frozen and dehydrated wood frogs was purified to homogeneity in a two-step process. The kinetic properties and stability of purified LDH were analyzed, revealing no significant differences in Vmax, Km and I50 values between control and frozen LDH. However, control and dehydrated LDH differed significantly in Km values for pyruvate, lactate, and NAD, I50 urea, and in temperature, glucose, and urea effects on these parameters. The possibility that posttranslational modification of LDH was responsible for the stable differences in enzyme behavior between control and dehydrated states was assessed using ProQ diamond staining to detect phosphorylation and immunoblotting to detect acetylation, methylation, ubiquitination, SUMOylation and nitrosylation of the enzyme. LDH from muscle of dehydrated wood frogs showed significantly lower levels of acetylation, providing one of the first demonstrations of a potential role for protein acetylation in the stress-responsive control of a metabolic enzyme. PMID:23638346

  4. Novel control of lactate dehydrogenase from the freeze tolerant wood frog: role of posttranslational modifications.

    PubMed

    Abboud, Jean; Storey, Kenneth B

    2013-01-01

    Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, plays a crucial role both in sustaining glycolytic ATP production under oxygen-limiting conditions and in facilitating the catabolism of accumulated lactate when stress conditions are relieved. In this study, the effects on LDH of in vivo freezing and dehydration stresses (both of which impose hypoxia/anoxia stress on tissues) were examined in skeletal muscle of the freeze-tolerant wood frog, Rana sylvatica. LDH from muscle of control, frozen and dehydrated wood frogs was purified to homogeneity in a two-step process. The kinetic properties and stability of purified LDH were analyzed, revealing no significant differences in V max, K m and I 50 values between control and frozen LDH. However, control and dehydrated LDH differed significantly in K m values for pyruvate, lactate, and NAD, I 50 urea, and in temperature, glucose, and urea effects on these parameters. The possibility that posttranslational modification of LDH was responsible for the stable differences in enzyme behavior between control and dehydrated states was assessed using ProQ diamond staining to detect phosphorylation and immunoblotting to detect acetylation, methylation, ubiquitination, SUMOylation and nitrosylation of the enzyme. LDH from muscle of dehydrated wood frogs showed significantly lower levels of acetylation, providing one of the first demonstrations of a potential role for protein acetylation in the stress-responsive control of a metabolic enzyme.

  5. Evidence supporting the existence of an activity-dependent astrocyte-neuron lactate shuttle.

    PubMed

    Pellerin, L; Pellegri, G; Bittar, P G; Charnay, Y; Bouras, C; Martin, J L; Stella, N; Magistretti, P J

    1998-01-01

    Mounting evidence from in vitro experiments indicates that lactate is an efficient energy substrate for neurons and that it may significantly contribute to maintain synaptic transmission, particularly during periods of intense activity. Since lactate does not cross the blood-brain barrier easily, blood-borne lactate cannot be a significant source. In vitro studies by several laboratories indicate that astrocytes release large amounts of lactate. In 1994, we proposed a mechanism whereby lactate could be produced by astrocytes in an activity-dependent, glutamate-mediated manner. Over the last 2 years we have obtained further evidence supporting the notion that a transfer of lactate from astrocytes to neurons might indeed take place. In this article, we first review data showing the presence of mRNA encoding for two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Second, by using monoclonal antibodies selectively directed against the two distinct lactate dehydrogenase isoforms, LDH1 and LDH5, a specific cellular distribution between neurons and astrocytes is revealed which suggests that a population of astrocytes is a lactate 'source' while neurons may be a lactate 'sink'. Third, we provide biochemical evidence that lactate is interchangeable with glucose to support oxidative metabolism in cortical neurons. This set of data is consistent with the existence of an activity-dependent astrocyte-neuron lactate shuttle for the supply of energy substrates to neurons.

  6. 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.

  7. Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.

    PubMed

    Kim, Sung Hyun; Lee, Gyun Min

    2007-02-01

    Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75-89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54-87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45-79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.

  8. Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

    PubMed Central

    Rule, G S; Pratt, E A; Chin, C C; Wold, F; Ho, C

    1985-01-01

    Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH. Images PMID:3882663

  9. 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…

  10. Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme.

    PubMed

    Dabrowska, A; Terlecki, G; Gutowicz, J

    1989-04-28

    The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H4 from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M4, and NADH favors the dissociation.

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

    PubMed Central

    MACK, NZINGA; MAZZIO, ELIZABETH A; BAUER, DAVID; ROZAS, HERNAN FLORES; SOLIMAN, KARAM F.A.

    2017-01-01

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

  12. Testis-specific lactate dehydrogenase is expressed in somatic tissues of plateau pikas☆

    PubMed Central

    Wang, Duowei; Wei, Lian; Wei, Dengbang; Rao, Xinfeng; Qi, Xinzhang; Wang, Xiaojun; Ma, Benyuan

    2013-01-01

    LDH-C4 is a lactate dehydrogenase that catalyzes the interconversion of pyruvate with lactate. In mammals the, Ldh-c gene was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), belonging to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living at 3000–5000 m above sea levelon the Qinghai-Tibet Plateau. We found that the expression pattern of six LDH isoenzymes in the somatic tissues of female and male plateau pikas to be the same as those in testis and sperm, suggesting that LDH-C4 was expressed in somatic tissues of plateau pika. Here we report the detection of LDHC in the somatic tissues of plateau pika using RT-PCR, Western blotting and immunohistochemistry. Our results indicate that Ldh-c mRNA is transcribed in the heart, liver, lung, kidney, brain, skeletal muscle and testis. In somatic tissues LDHC was translated in the cytoplasm, while in testis it was expressed in both cytoplasm and mitochondria. The third band from cathode to anode in LDH isoenzymes was identified as LDH-C4. The finding that Ldh-c is expressed in both somatic tissues and testis of plateau pika provides important implications for more in-depth research into the Ldh-c function in mammals. PMID:23772382

  13. 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.

  14. The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase.

    PubMed Central

    Yamagata, M.; Hasuda, K.; Stamato, T.; Tannock, I. F.

    1998-01-01

    Solid tumours develop an acidic extracellular environment with high concentration of lactic acid, and lactic acid produced by glycolysis has been assumed to be the major cause of tumour acidity. Experiments using lactate dehydrogenase (LDH)-deficient ras-transfected Chinese hamster ovarian cells have been undertaken to address directly the hypothesis that lactic acid production is responsible for tumour acidification. The variant cells produce negligible quantities of lactic acid and consume minimal amounts of glucose compared with parental cells. Lactate-producing parental cells acidified lightly-buffered medium but variant cells did not. Tumours derived from parental and variant cells implanted into nude mice were found to have mean values of extracellular pH (pHe) of 7.03 +/- 0.03 and 7.03 +/- 0.05, respectively, both of which were significantly lower than that of normal muscle (pHe = 7.43 +/- 0.03; P < 0.001). Lactic acid concentration in variant tumours (450 +/- 90 microg g(-1) wet weight) was much lower than that in parental tumours (1880 +/- 140 microg/g(-1)) and similar to that in serum (400 +/- 35 microg/g(-1)). These data show discordance between mean levels of pHe and lactate content in tumours; the results support those of Newell et al (1993) and suggest that the production of lactic acid via glycolysis causes acidification of culture medium, but is not the only mechanism, and is probably not the major mechanism responsible for the development of an acidic environment within solid tumours. PMID:9667639

  15. Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases.

    PubMed

    Lee, B I; Chang, C; Cho, S J; Eom, S H; Kim, K K; Yu, Y G; Suh, S W

    2001-04-13

    The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Molecular cloning and characterization of lactate dehydrogenase gene from Eimeria tenella.

    PubMed

    Dong, Hui; Wang, Yange; Zhao, Qiping; Han, Hongyu; Zhu, Shunhai; Li, Liujia; Wu, Youling; Huang, Bing

    2014-08-01

    Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway and is crucial for parasite survival. In this study, we cloned and expressed the LDH of Eimeria tenella (EtLDH). Real-time polymerase chain reaction and Western blot analysis revealed that the expression of EtLDH was developmentally regulated at the messenger RNA (mRNA) and protein levels. EtLDH mRNA levels were higher in second-generation merozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and sporozoites). EtLDH protein expression levels were most prominent in second-generation merozoites, moderately expressed in unsporulated oocysts and sporulated oocysts, and weakly detected in sporozoites. Immunostaining with anti-recombinant EtLDH (rEtLDH) antibody indicated that EtLDH was mainly located in the anterior region in free sporozoites and became concentrated in the anterior region of intracellular sporozoites except for the apex after invasion into DF-1 cells. Specific staining of EtLDH protein was more intense in trophozoites and immature first-generation schizonts, but decreased in mature first-generation schizonts. Inhibition of EtLDH function using specific antibodies cannot efficiently reduce the ability of E. tenella sporozoites to invade host cells. These results suggest that EtLDH may be involved in glycolysis during the first-generation merogony stage in E. tenella and has little role in host invasion.

  3. Prognostic significance of serum lactate dehydrogenase levels in Ewing's sarcoma: A meta-analysis

    PubMed Central

    Li, Suoyuan; Yang, Qing; Wang, Hongsheng; Wang, Zhuoying; Zuo, Dongqing; Cai, Zhengdong; Hua, Yingqi

    2016-01-01

    A number of studies have investigated the role of serum lactate dehydrogenase (LDH) levels in patients with Ewing's sarcoma, although these have yielded inconsistent and inconclusive results. Therefore, the present study aimed to systematically review the published studies and conduct a meta-analysis to assess its prognostic value more precisely. Cohort studies assessing the prognostic role of LDH levels in patients with Ewing's sarcoma were included. A pooled hazard ratio (HR) with 95% confidence intervals (CIs) of overall survival (OS) or 5-year disease-free survival (DFS) was used to assess the prognostic role of the levels of serum LDH. Nine studies published between 1980 and 2014, with a total of 1,412 patients with Ewing's sarcoma, were included. Six studies, with a total of 644 patients, used OS as the primary endpoint and four studies, with 795 patients, used 5-year DFS. Overall, the pooled HR evaluating high LDH levels was 2.90 (95% CI: 2.09–4.04) for OS and 2.40 (95% CI: 1.93–2.98) for 5-year DFS. This meta-analysis demonstrates that high levels of serum LDH are associated with lower OS and 5-year DFS rates in patients with Ewing's sarcoma. Therefore, serum LDH levels are an effective biomarker of Ewing's sarcoma prognosis. PMID:28105365

  4. Validity of a New Kit Measuring Salivary Lactate Dehydrogenase Level for Screening Gingivitis

    PubMed Central

    Ekuni, Daisuke; Kataoka, Kota; Yokoi, Aya; Taniguchi-Tabata, Ayano; Mizuno, Hirofumi; Miyai, Hisataka; Uchida, Yoko; Fukuhara, Daiki; Sugiura, Yoshio; Morita, Manabu

    2017-01-01

    Aim. The aim of this study was to determine the usefulness of a new kit that can evaluate salivary lactate dehydrogenase (LD) level in real time for screening gingivitis. Materials and Methods. The study included 70 systemic healthy volunteers [29 males and 41 females; mean age ± SD: 24.1 ± 2.6 years]. Resting saliva was collected from each participant and LD level was evaluated in real time using the kit (a color-changing sheet with an integer scale ranging from 1 to 10). A dentist measured probing pocket depth, clinical attachment level, and the proportion of sites with bleeding on probing (% BOP) at six sites on all teeth. Gingivitis was diagnosed when the BOP value was ≥20%. Results. Salivary LD level was positively correlated with mean % BOP (odds ratio: 1.47, 95% confidence interval: 1.132–1.916, and P < 0.001) in a logistic regression model. The sensitivity and specificity of the kit were 0.89 and 0.98, respectively, at a cut-off value of 8.0 for LD level. Conclusions. The new kit for measurement of salivary LD level may be a useful tool to screen for gingivitis in young adults, which contributes to early detection of future periodontitis. PMID:28337048

  5. A highly sensitive aptasensor towards Plasmodium lactate dehydrogenase for the diagnosis of malaria.

    PubMed

    Lee, Seonghwan; Song, Kyung-Mi; Jeon, Weejeong; Jo, Hunho; Shim, Yoon-Bo; Ban, Changill

    2012-05-15

    Finding a highly sensitive diagnostic technique for malaria has challenged scientists for the last century. In the present study, we identified versatile single-strand DNA aptamers for Plasmodium lactate dehydrogenase (pLDH), a biomarker for malaria, via the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The pLDH aptamers selectively bound to the target proteins with high sensitivity (K(d)=16.8-49.6 nM). The selected aptamers were characterized using an electrophoretic mobility shift assay, a quartz crystal microbalance, a fluorescence assay, and circular dichroism spectroscopy. We also designed a simple aptasensor using electrochemical impedance spectroscopy; both Plasmodium vivax LDH and Plasmodium falciparum LDH were selectively detected with a detection limit of 1 pM. Furthermore, the pLDH aptasensor clearly distinguished between malaria-positive blood samples of two major species (P. vivax and P. falciparum) and a negative control, indicating that it may be a useful tool for the diagnosis, monitoring, and surveillance of malaria.

  6. 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

  7. 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.

  8. Characterization and Inhibitor Screening of Plateau Zokor Lactate Dehydrogenase C4.

    PubMed

    He, Qinghua; Zhang, Qinglian; Huang, Lin; Ma, Jinhu

    2016-07-01

    Lactate dehydrogenase C4 (LDH-C4) is considered to be a target protein for the development of contraceptives. In this work, the characterization of plateau zokor LDH-C4 and the screening of a series of N-substituted oxamic acids as inhibitors against zokor LDH-C4 were reported. The cDNA of zokor LDH-C gene was cloned and expressed in Escherichia coli, from which the protein was purified and further characterized. The protein was a tetramer (LDH-C4) and thermally stable up to 62 °C with a K m of 63.9 μM for pyruvate and with optimal pH values of 7.95 and 10.1 for the forward and backward reactions respectively. Virtual and in vitro screening against zokor LDH-C4 revealed eight N-substituted oxamic acids with IC50s ranging from 198 to 2513 μM, higher than that of oxamic acid (150 μM) and (ethylamino)(oxo)acetic acid (59 μM). The inhibition potencies of N-substituted oxamic acids tested are in the micromolar range, and the increase in the length of substituting chain seems not to increase inhibition potency.

  9. [C-reactive protein and lactate dehydrogenase as single prognostic factors of severity in acute pancreatitis].

    PubMed

    Zrnić, Irena Krznarić; Milić, Sandra; Fisić, Elizabeta; Radić, Mladen; Stimac, Davor

    2007-01-01

    Ranson and Glasgow scores are routinely used for prediction of severity in acute pancreatitis. We undertook a prospective study to investigate the role of lactate dehydrogenase (LDH) and C-reactive protein (CRP) as potential single predictors of severity in acute pancreatitis. In our study we included 100 patients with diagnosis of acute pancreatitis admitted to our hospital during last two years. The inclusion criteria consisted of a combination of clinical features, a typical case history, elevation of serum pancreatic enzymes and diagnosis confirmed by imaging studies (ultrasound or computerised tomography). We used Ranson score for assesment of severity and compared it with single parameters as LDH and CRP on the first and the third day after admission. Cut off values for predicting local and systemic complications were > or =3 for Ranson score, 320 IU for LDH and 5 mg/L for CRP. Ranson score showed highest sensitivity in the prediction of local and systemic complication of acute pancreatitis. Specificity and diagnostic accuracy were highest for LDH on the first day (67.74; 57%). Diagnostic accuracy for Ranson score and CRP on the third day after admission was around 50%. We can conclude that LDH and CRP are available, simple and economical biochemical parameters that can help us predict complications of acute pancreatitis in the early phase of the disease. They showed similar diagnostic accuracy as the far more clinically used Ranson score.

  10. Prognostic value of preoperative serum lactate dehydrogenase levels for resectable gastric cancer and prognostic nomograms

    PubMed Central

    Zhou, Yi-Xin; Wang, Feng; Zhang, Dong-Sheng; Wang, Feng-Hua; Li, Yu-Hong; Xu, Rui-Hua

    2016-01-01

    The present study aimed to evaluate the prognostic significance of preoperative serum lactate dehydrogenase (SLDH) levels for resected gastric cancer and construct prognostic nomograms for risk prediction. The study cohort consisted of 619 patients with D2-resected gastric cancer. The relationship of SLDH levels with clinicopathological features and clinical outcomes was evaluated. Prognostic nomograms were created using identified prognosticators to predict 3-year overall survival (OS) and 3-year disease-free survival (DFS), and bootstrap validation was performed. High SLDH levels were correlated with old age but not depth of invasion or lymph node metastasis. When assessed as a continuous variable, high SLDH levels were independently associated with poor OS and DFS. Internal validation of the developed nomograms revealed good predictive accuracy (bootstrap-corrected concordance indices: 0.77 and 0.75, respectively for prediction of OS and DFS). The preoperative SLDH levels, an identified unfavorable prognosticator, were incorporated into nomograms along with other clinicopathological features to refine the prediction of clinical outcomes for patients with D2-resected gastric cancer. PMID:27223065

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

    PubMed

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

    2017-03-01

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

  12. 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

  13. 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.

  14. 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

    2015-05-20

    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.

  15. Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells.

    PubMed

    Fu, Tuo; Zhang, Cunchao; Jing, Yu; Jiang, Cheng; Li, Zhenhua; Wang, Shengyu; Ma, Kai; Zhang, Dapeng; Hou, Sheng; Dai, Jianxin; Kou, Geng; Wang, Hao

    2016-06-01

    Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins.

  16. Lactate dehydrogenase inhibitors sensitize lymphoma cells to cisplatin without enhancing the drug effects on immortalized normal lymphocytes.

    PubMed

    Manerba, Marcella; Di Ianni, Lorenza; Fiume, Luigi; Roberti, Marinella; Recanatini, Maurizio; Di Stefano, Giuseppina

    2015-07-10

    Up-regulation of glycolysis, a well recognized hallmark of cancer cells, was also found to be predictive of poor chemotherapy response. This observation suggested the attempt of sensitizing cancer cells to conventional chemotherapeutic agents by inhibiting glucose metabolism. Lactate dehydrogenase (LDH) inhibition can be a way to hinder glycolysis of cancer cells without affecting the metabolism of normal tissues, which usually does not require this enzymatic activity. In this paper, we showed that two LDH inhibitors (oxamate and galloflavin) can increase the efficacy of cisplatin in cultured Burkitt's lymphoma (BL) cells and that this potentiating effect is not exerted in proliferating normal lymphocytes. This result was explained by the finding that in BL cells LDH inhibition induced reactive oxygen species (ROS) generation, which was not evidenced in proliferating normal lymphocytes. In BL cells treated with the association of cisplatin and LDH inhibitors, these ROS can be a further cause of DNA damage, to be added to that produced by cisplatin, leading to the failure of the response repair. At present LDH inhibitors suitable for clinical use are actively searched; our results can allow a better understanding of the potentiality of LDH as a possible target to develop innovative anticancer treatments.

  17. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements.

    PubMed

    Firth, J D; Ebert, B L; Ratcliffe, P J

    1995-09-08

    The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, we have studied the 5' flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between -41 and -84 base pairs upstream of the transcription initiation site, which were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, had the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competed for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain was positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3' hypoxic enhancer. The third domain had the motif of a cAMP response element (CRE). Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnified response to hypoxia that was dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites.

  18. Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

    PubMed

    Noh, Soo Min; Park, Jin Hyoung; Lim, Myung Sin; Kim, Jong Won; Lee, Gyun Min

    2017-02-01

    Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

  19. Proteomic identification of the lactate dehydrogenase A in a radioresistant prostate cancer xenograft mouse model for improving radiotherapy

    PubMed Central

    Hao, Jingli; Graham, Peter; Chang, Lei; Ni, Jie; Wasinger, Valerie; Beretov, Julia; Deng, Junli; Duan, Wei; Bucci, Joseph; Malouf, David; Gillatt, David; Li, Yong

    2016-01-01

    Radioresistance is a major challenge for prostate cancer (CaP) metastasis and recurrence after radiotherapy. This study aimed to identify potential protein markers and signaling pathways associated with radioresistance using a PC-3 radioresistant (RR) subcutaneous xenograft mouse model and verify the radiosensitization effect from a selected potential candidate. PC-3RR and PC-3 xenograft tumors were established and differential protein expression profiles from two groups of xenografts were analyzed using liquid chromatography tandem-mass spectrometry. One selected glycolysis marker, lactate dehydrogenase A (LDHA) was validated, and further investigated for its role in CaP radioresistance. We found that 378 proteins and 51 pathways were significantly differentially expressed between PC-3RR and PC-3 xenograft tumors, and that the glycolysis pathway is closely linked with CaP radioresistance. In addition, we also demonstrated that knock down of LDHA with siRNA or inhibition of LDHA activity with a LDHA specific inhibitor (FX-11), could sensitize PC-3RR cells to radiotherapy with reduced epithelial-mesenchymal transition, hypoxia, DNA repair ability and autophagy, as well as increased DNA double strand breaks and apoptosis. In summary, we identified a list of potential RR protein markers and important signaling pathways from a PC-3RR xenograft mouse model, and demonstrate that targeting LDHA combined with radiotherapy could increase radiosensitivity in RR CaP cells, suggesting that LDHA is an ideal therapeutic target to develop combination therapy for overcoming CaP radioresistance. PMID:27708237

  20. Effect of varying rest intervals between sets of assistance exercises on creatine kinase and lactate dehydrogenase responses.

    PubMed

    Machado, Marco; Koch, Alexander J; Willardson, Jeffrey M; Pereira, Luis S; Cardoso, M Isabel; Motta, Michela K S; Pereira, Rafael; Monteiro, André N

    2011-05-01

    To examine the effects of different rest intervals between sets on serum creatine kinase (CK) and lactate dehydrogenase (LDH) activity, 10 men (age = 25.6 ± 2.2 years, height = 173.1 ± 7.1 cm, and body mass = 75.9 ± 10.0 kg) participated in a randomized within-subject design that involved 4 resistance exercise sessions. Each session consisted of 4 sets of 10 repetitions with 10 repetition maximum loads for the chest press, pullover, biceps curl, triceps extension, leg extension, and prone leg curl. The sessions differed only in the length of the rest interval between sets and exercises, specifically: 60, 90, 120, 180 seconds. Serum CK and LDH were significantly (p < 0.05) elevated 24-72 hours after each session, with no significant differences between rest intervals (p = 0.94 and p = 0.99, respectively). The mechanical stress imposed by the 4 resistance exercise sessions invoked similar damage to the muscle fibers independent of the rest interval between sets. These data indicate that the accumulated volume of work is the primary determinant of muscle damage in trained subjects who are accustomed to resistance exercise with short rest intervals.

  1. Varying postresection lactate dehydrogenase with overall survival of early stage pancreatic cancer patients

    PubMed Central

    Xiao, Yuanyuan; Xie, Zhihui; Shao, Zhenyi; Chen, Wen; Xie, Hua; Qin, Guoyou; Zhao, Naiqing

    2017-01-01

    Abstract Several previously published studies revealed a hazardous role of pretreatment lactate dehydrogenase (LDH) in survival of advanced or metastatic pancreatic cancer (PC) patients. Nevertheless, in early stage PC patients who are eligible for curative resection, the prognostic role of postresection LDH has never been discussed. In this study, we aimed to explore the prognostic significance of varying postresection LDH among early stage PC patients. In total, 80 PC patients who received curative resection were retrospectively selected from a population-based electronic inpatients database which originated from Shanghai, China. A dynamic survival analysis method, counting process approach in combination with the multiple failure-time Cox model, was applied to evaluate the association between postresection LDH and OS. The multiple failure-time Cox model found that age, resection modality, and postresection LDH were significantly associated with OS: an elevated LDH (defined as > 250 U/L) was related to 2.93 (95% CI: 1.26–6.79) folds of death hazard. Further analysis disclosed an identifiable dose–response association between LDH and OS: compared with LDH≤155 U/L, the HRs for 155 U/L < LDH < 196 U/L, and LDH≥196 U/L were 2.07 (95% CI: 0.88–4.88) and 3.15 (95% CI: 1.30–7.59), respectively. Our study results suggest that postresection LDH is a prominent prognostic factor in this group of early stage PC patients. Maintaining normally ranged LDH after resection might bring about survival benefit in early stage PC patients. PMID:28328834

  2. Varying postresection lactate dehydrogenase with overall survival of early stage pancreatic cancer patients: A retrospective study.

    PubMed

    Xiao, Yuanyuan; Xie, Zhihui; Shao, Zhenyi; Chen, Wen; Xie, Hua; Qin, Guoyou; Zhao, Naiqing

    2017-03-01

    Several previously published studies revealed a hazardous role of pretreatment lactate dehydrogenase (LDH) in survival of advanced or metastatic pancreatic cancer (PC) patients. Nevertheless, in early stage PC patients who are eligible for curative resection, the prognostic role of postresection LDH has never been discussed. In this study, we aimed to explore the prognostic significance of varying postresection LDH among early stage PC patients. In total, 80 PC patients who received curative resection were retrospectively selected from a population-based electronic inpatients database which originated from Shanghai, China. A dynamic survival analysis method, counting process approach in combination with the multiple failure-time Cox model, was applied to evaluate the association between postresection LDH and OS. The multiple failure-time Cox model found that age, resection modality, and postresection LDH were significantly associated with OS: an elevated LDH (defined as > 250 U/L) was related to 2.93 (95% CI: 1.26-6.79) folds of death hazard. Further analysis disclosed an identifiable dose-response association between LDH and OS: compared with LDH≤155 U/L, the HRs for 155 U/L < LDH < 196 U/L, and LDH≥196 U/L were 2.07 (95% CI: 0.88-4.88) and 3.15 (95% CI: 1.30-7.59), respectively. Our study results suggest that postresection LDH is a prominent prognostic factor in this group of early stage PC patients. Maintaining normally ranged LDH after resection might bring about survival benefit in early stage PC patients.

  3. Effect of oligomer procyanidins on reperfusion arrhythmias and lactate dehydrogenase release in the isolated rat heart.

    PubMed

    Al-Makdessi, Samar; Sweidan, Hicham; Jacob, Ruthard

    2006-01-01

    The antiarrhythmic effect of an oral 3-week-pretreatment with oligomer procyanidins derived from Vitis vinifera was investigated on the isolated perfused heart after global no-flow ischemia (procyanidin-treated group: n = 9, control group: n = 13). Hearts were perfused with a modified Krebs-Henseleit solution in which the K+ content was reduced to 3.0 mmol/l in order to lower the fibrillation threshold. Monophasic action potentials in addition to ECG were recorded. The durations of ischemia and reperfusion were 20 and 30 min, respectively. Arrhythmias including ventricular fibrillation (VF), ventricular tachycardia (VT), flutter (Fl) and bradycardia were evaluated. During the reperfusion, irreversible VF occurred in most of control hearts. The incidence of VF (percentage of the hearts in which VF occurred) was lowered by oligomer procyanidins from 84.6 to 55.6 %, and the duration of the episodes of VF (expressed as percentage relative to the total duration) was significantly shortened from 76.1 +/- 27.9 % to 36.6 +/- 40.6 % (p = 0.036). Simultaneously, the percentage of duration of normal sinus rhythm (NSR) increased from 19.5 +/- 30.3 % to 46.2 +/- 35.9 % (n.s.). VF occuring in the procyanidin-treated hearts could be reversed in two hearts within few minutes to a stage of "reversible arrhythmias" consisting of short episodes (1 to 60 s) of either Fl or VT or bradycardia or NSR alternating with each other. LDH (lactate dehydrogenase) release in the first drops appearing from the reperfused heart was significantly reduced in the procyanidin-treated rats (66.7 +/- 36.2 mU/min, n = 8) in comparison to controls (159.7 +/- 79.0 mU/min, n = 10; p = 0.010). These results demonstrate an antiarrhythmic and cytoprotective effect of oral pretreatment with oligomer procyanidins under the given experimental conditions.

  4. 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.

  5. 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.

  6. Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids

    PubMed Central

    Whitehouse, Sue; Cooper, Ronald H.; Randle, Philip J.

    1974-01-01

    1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2′-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100μm for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2′-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca2+ and Mg2+. 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2′-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads

  7. Lactation

    PubMed Central

    1989-01-01

    Lactation is the most energy-efficient way to provide for the dietary needs of young mammals, their mother's milk being actively protective, immunomodulatory, and ideal for their needs. Intrauterine mammary gland development in the human female is already apparent by the end of the sixth week of gestation. During puberty and adolescence secretions of the anterior pituitary stimulate the maturation of the graafian follicles in the ovaries and stimulate the secretion of follicular estrogens, which stimulate development of the mammary ducts. Pregnancy has the most dramatic effect on the breast, but development of the glandular breast tissue and deposition of fat and connective tissue continue under the influence of cyclic sex-hormone stimulation. Many changes occur in the nipple and breast during pregnancy and at delivery as a prelude to lactation. Preparation of the breasts is so effective that lactation could commence even if pregnancy were discontinued at 16 weeks. Following birth, placental inhibition of milk synthesis is removed, and a woman's progesterone blood levels decline rapidly. The breasts fill with milk, which is a high-density, low-volume feed called colostrum until about 30 hours after birth. Because it is not the level of maternal hormones, but the efficiency of infant suckling and/or milk removal that governs the volume of milk produced in each breast, mothers who permit their infants to feed ad libitum commonly observe that they have large volumes of milk 24-48 hours after birth. The two maternal reflexes involved in lactation are the milk-production and milk-ejection reflex. A number of complementary reflexes are involved when the infant feeds: the rooting reflex (which programmes the infant to search for the nipple), the sucking reflex (rhythmic jaw action creating negative pressure and a peristaltic action of the tongue), and the swallowing reflex. The infant's instinctive actions need to be consolidated into learned behaviour in the postpartum

  8. Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes

    PubMed Central

    Fields, Peter A.; Somero, George N.

    1998-01-01

    To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (−1.86 to 1°C) and three South American (4 to 10°C) notothenioid teleosts. Higher Michaelis–Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs. PMID:9736762

  9. 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.

  10. Pretreatment elevated serum lactate dehydrogenase as a significant prognostic factor in malignant mesothelioma

    PubMed Central

    Zhuo, Yi; Lin, Lanying; Wei, Shushan; Zhang, Mingwei

    2016-01-01

    Abstract Background: Lactate dehydrogenase (LDH) as a hypoxia-regulator plays a vital role in alternative metabolic pathways of cancer cells. Numerous studies have assessed the prognostic value of elevated pretreatment LDH in malignant mesothelioma (MM). However, the results have been largely inconsistent. Hence, the aim of current study was to investigate the prognostic value of pretreatment LDH levels in patients with MM by performing a meta-analysis of relevant studies. Methods: A literature search for English language studies, which investigated the association of LDH levels with overall survival (OS) in malignant mesothelioma, was performed in the electronic databases, PubMed, Medline, Embase, and Web of Science. Pooled hazard ratios (HRs) and their 95% confidence intervals (95% CIs) were calculated. Heterogeneity was assessed using Cochran Q and I2 statistics. Sensitivity analysis, meta-regression model, and subgroup analysis were performed to trace the source of heterogeneity, if applicable. Results: A total of 9 studies with a combined study population of 1977 patients came within the purview of this meta analysis. Pooled HR for OS in patients with high LDH level was 1.68 (95% CI = 1.36–2.00). Significant heterogeneity was observed in the included studies (I2 = 54.1%, P = 0.026). Sensitivity analysis after sequential exclusion of 1 study at a time, and meta-regression with inclusion of 6 confounding factors failed to identify the source of heterogeneity. However, in the subgroup analysis, it was found that the publication of Nojiri et al was the origin of heterogeneity. When omitted the publication of Nojiri et al, the pooled HR of the rest 8 studies was 1.83 (95% CI = 1.45–2.20, I2 = 0.0%, P = 0.723). Egger test and funnel plots excluded the possibility of publication bias affecting the results of the current meta-analysis. Conclusion: A negative association was observed between high LDH levels and poor overall survival in the

  11. Pre-treatment serum lactate dehydrogenase and alkaline phosphatase as predictors of metastases in extremity osteosarcoma

    PubMed Central

    Marais, Leonard C.; Bertie, Julia; Rodseth, Reitze; Sartorius, Benn; Ferreira, Nando

    2015-01-01

    Background The prognosis of patients with metastatic osteosarcoma remains poor. However, the chance of survival can be improved by surgical resection of all metastases. In this study we investigate the value of serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in predicting the presence of metastatic disease at time of diagnosis. Methods Sixty-one patients with histologically confirmed conventional osteosarcoma of the extremity were included in the study. Only 19.7% of cases presented without evidence of systemic spread of the disease. Pre-treatment serum ALP and LDH were analysed in patients with and without skeletal or pulmonary metastases. Results Serum LDH and ALP levels were not significantly different in patients with or without pulmonary metastases (p=0.88 and p=0.47, respectively). The serum LDH and ALP levels did however differ significantly in patients with or without skeletal metastases (p<0.001 and p=0.02, respectively). The optimal breakpoint for serum LDH as a marker of skeletal metastases was 849 IU/L (AUC 0.839; Sensitivity=0.88; Specificity=0.73). LDH >454 IU/L equated to 100% sensitivity for detected bone metastases (positive diagnostic likelihood ratio (DLR)=1.32). With a cut-off of 76 IU/L a sensitivity of 100% was reached for serum ALP predicting the presence of skeletal metastases (positive DLR=1.1). In a multivariate analysis both LDH ≥850 IU/L (odds ratio [OR]=9; 95% confidence interval (CI) 1.8–44.3) and ALP ≥280 IU/L (OR=10.3; 95% CI 2.1–50.5) were predictive of skeletal metastases. LDH however lost its significance in a multivariate model which included pre-treatment tumour volume. Conclusion In cases of osteosarcoma with LDH >850 IU/L and/or ALP >280 IU/L it may be prudent to consider more sensitive staging investigations for detection of skeletal metastases. Further research is required to determine the value and the most sensitive cut-off points of serum ALP and LDH in the prediction of skeletal metastases. PMID

  12. 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.

  13. 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.

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

    DTIC Science & Technology

    2016-06-07

    dehydrogenase (IIB-HSD) were measured in tissue fragment cultures on day 75 of gestation. Dehydrogenase activity was over fivefold greater than oxoreductase...oxoreductase activities in porcine placentae under physiological conditions using placental explant culture and endogenous concentrations of coenzymes and...f!M range). In human placental tissue fragments at midterm and late pregnancy ( 12, 18) and in trophoblast cell cultures from term placentae ( 41

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

    PubMed

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

    2017-01-10

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

  16. Effect of the inactivation of lactate dehydrogenase, ethanol dehydrogenase, and phosphotransacetylase on 2,3-butanediol production in Klebsiella pneumoniae strain

    PubMed Central

    2014-01-01

    Background 2,3-Butanediol (2,3-BD) is a high-value chemical usually produced petrochemically but which can also be synthesized by some bacteria. To date, Klebsiella pneumoniae is the most powerful 2,3-BD producer which can utilize a wide range of substrates. However, many by-products are also produced by K. pneumoniae, such as ethanol, lactate, and acetate, which negatively regulate the 2,3-BD yield and increase the costs of downstream separation and purification. Results In this study, we constructed K. pneumoniae mutants with lactate dehydrogenase (LDH), acetaldehyde dehydrogenase (ADH), and phosphotransacetylase (PTA) deletion individually by suicide vector conjugation. These mutants showed different behavior of production formation. Knock out of ldhA had little influence on the yield of 2,3-BD, whereas knock out of adhE or pta significantly improved the formation of 2,3-BD. The accumulation of the intermediate of 2,3-BD biosynthesis, acetoin, was decreased in all the mutants. The mutants were then tested in five different carbon sources and increased 2,3-BD was observed. Also a double mutant strain with deletion of adhE and ldhA was constructed which resulted in accelerated fermentation and higher 2,3-BD production. In fed-batch culture this strain achieved more than 100 g/L 2,3-BD from glucose with a relatively high yield of 0.49 g/g. Conclusion 2,3-BD production was dramatically improved with the inactivation of adhE and pta. The inactivation of ldhA could advance faster cell growth and shorter fermentation time. The double mutant strain with deletion of adhE and ldhA resulted in accelerated fermentation and higher 2,3-BD production. These results provide new insights for industrial production of 2,3-BD by K. pneumoniae. PMID:24669952

  17. Nutrient deprivation induces the Warburg effect through ROS/AMPK-dependent activation of pyruvate dehydrogenase kinase.

    PubMed

    Wu, Ching-An; Chao, Yee; Shiah, Shine-Gwo; Lin, Wan-Wan

    2013-05-01

    The Warburg effect is known to be crucial for cancer cells to acquire energy. Nutrient deficiencies are an important phenomenon in solid tumors, but the effect on cancer cell metabolism is not yet clear. In this study, we demonstrate that starvation of HeLa cells by incubation with Hank's buffered salt solution (HBSS) induced cell apoptosis, which was accompanied by the induction of reactive oxygen species (ROS) production and AMP-activated protein kinase (AMPK) phosphorylation. Notably, HBSS starvation increased lactate production, cytoplasmic pyruvate content and decreased oxygen consumption, but failed to change the lactate dehydrogenase (LDH) activity or the glucose uptake. We found that HBSS starvation rapidly induced pyruvate dehydrogenase kinase (PDK) activation and pyruvate dehydrogenase (PDH) phosphorylation, both of which were inhibited by compound C (an AMPK inhibitor), NAC (a ROS scavenger), and the dominant negative mutant of AMPK. Our data further revealed the involvement of ROS production in AMPK activation. Moreover, DCA (a PDK inhibitor), NAC, and compound C all significantly decreased HBSS starvation-induced lactate production accompanied by enhancement of HBSS starvation-induced cell apoptosis. Not only in HeLa cells, HBSS-induced lactate production and PDH phosphorylation were also observed in CL1.5, A431 and human umbilical vein endothelial cells. Taken together, we for the first time demonstrated that a low-nutrient condition drives cancer cells to utilize glycolysis to produce ATP, and this increases the Warburg effect through a novel mechanism involving ROS/AMPK-dependent activation of PDK. Such an event contributes to protecting cells from apoptosis upon nutrient deprivation.

  18. Specific and sensitive detection of Plasmodium falciparum lactate dehydrogenase by DNA-scaffolded silver nanoclusters combined with an aptamer.

    PubMed

    Wang, Wei-Xian; Cheung, Yee-Wai; Dirkzwager, Roderick M; Wong, Wai-Chung; Tanner, Julian A; Li, Hong-Wei; Wu, Yuqing

    2017-02-27

    Innovative nanomaterials offer significant potential for diagnosis of severe diseases of the developing world such as malaria. Small sized silver nanoclusters have shown promise for diagnostics due to their intense fluorescence emission and photo-stabilities. Here, double-stranded DNA-scaffolded silver nanoclusters (AgNCs-dsDNA) were prepared to detect the established malaria biomarker, Plasmodium falciparum lactate dehydrogenase (PfLDH). Significant luminescence enhancement over a wide concentration range of PfLDH was demonstrated. In addition, a low limit of detection at 0.20 nM (7.4 pg μL(-1)) was achieved for PfLDH in buffer solution, sensitive enough for practical use correlating with the clinical level of PfLDH in plasma from malaria-infected patients. Unique specificity was observed towards Plasmodium falciparum over Plasmodium vivax and human lactate dehydrogenase, as well as other non-specific proteins, by combining the use of AgNCs-dsDNA with a DNA aptamer against PfLDH. Moreover, the intrinsic mechanism was revealed in detail for the two-step luminescence response. The combination of DNA-scaffolded silver nanoclusters coupled to a selective single-stranded DNA aptamer allows for a highly specific and sensitive detection of PfLDH with significant promise for malaria diagnosis in future.

  19. Inhibitory effect of disulfiram (Antabuse) on alcohol dehydrogenase activity.

    PubMed

    Carper, W R; Dorey, R C; Beber, J H

    1987-10-01

    We investigated the effect of disulfiram (Antabuse) on the activity of alcohol dehydrogenase (EC 1.1.1.1) in vitro. We observed a time-dependent inhibition of this dehydrogenase by disulfiram and diethyldithiocarbamate similar to that obtained for aldehyde dehydrogenase (EC 1.2.1.3). These results suggest a possible explanation for various side effects observed in the clinical use of Antabuse.

  20. LACTIC DEHYDROGENASES OF PSEUDOMONAS NATRIEGENS.

    PubMed

    WALKER, H; EAGON, R G

    1964-07-01

    Walker, Hazel (University of Georgia, Athens), and R. G. Eagon. Lactic dehydrogenases of Pseudomonas natriegens. J. Bacteriol. 88:25-30. 1964.-Lactic dehydrogenases specific for d- and l-lactate were demonstrated in Pseudomonas natriegens. The l-lactic dehydrogenase showed considerable heat stability, and 40% of the activity remained in extracts after heating at 60 C for 10 min. An essential thiol group for enzyme activity was noted. The results of these experiments were consistent with the view that lactate was dehydrogenated initially by a flavin cofactor and that electrons were transported through a complete terminal oxidase system to oxygen. The intracellular site of these lactic dehydrogenases was shown to be the cell membrane. It was suggested that the main physiological role of these lactic dehydrogenases is that of lactate utilization.

  1. Herbicidal Activity of an Isopropylmalate Dehydrogenase Inhibitor.

    PubMed Central

    Wittenbach, V. A.; Teaney, P. W.; Hanna, W. S.; Rayner, D. R.; Schloss, J. V.

    1994-01-01

    Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis. It catalyzes the oxidative decarboxylation of 3-isopropylmalate (3-IPM) to 2-ketoisocaproic acid. The partially purified enzyme from pea (Pisum sativum L.) shows a broad pH optimum of 7.8 to 9.1 and has Km values for 3-IPM and NAD of 18 and 40 [mu]M, respectively. O-Isobutenyl oxalylhydroxamate (O-IbOHA) has been discovered to be an excellent inhibitor of the pea IPMDH, with an apparent inhibitor constant of 5 nM. As an herbicide, O-IbOHA showed only moderate activity on a variety of broadleaf and grass species. We characterized the herbicidal activity of O-IbOHA on corn (Zea mays L.), a sensitive species; giant foxtail (Setaria faberi) and morning glory (Ipomoea purpurea [L.] Roth), moderately tolerant species; and soybean [Glycine max L. Merr.), a tolerant species. Differences in tolerance among the species were not due to differences in the sensitivity of IPMDH. Studies with [14C]O-IbOHA suggested that uptake and translocation were not major limitations for herbicidal activity, nor were they determinants of tolerance. Moreover, metabolism could not account for the difference in tolerance of corn, foxtail, and morning glory, although it might account for the tolerance of soybean. Herbicidal activity on all four species was correlated with the accumulation of 3-IPM in the plants. PMID:12232331

  2. 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.

  3. 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.

  4. Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum

    PubMed Central

    Lorite, María J.; Tachil, Jörg; Sanjuán, Juán; Meyer, Ortwin; Bedmar, Eulogio J.

    2000-01-01

    Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)2 subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein. PMID:10788353

  5. Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum.

    PubMed

    Lorite, M J; Tachil, J; Sanjuán, J; Meyer, O; Bedmar, E J

    2000-05-01

    Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.

  6. 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

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

    PubMed

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

    2017-03-06

    Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.Oncogene advance online publication, 6 March 2017; doi:10.1038/onc.2017.36.

  8. The Conformation of NAD+ Bound to Lactate Dehydrogenase Determined by Nuclear Magnetic Resonance with Suppression of Spin Diffusion

    NASA Astrophysics Data System (ADS)

    Vincent, Sebastien J. F.; Zwahlen, Catherine; Post, Carol Beth; Burgner, John W.; Bodenhausen, Geoffrey

    1997-04-01

    We have reinvestigated the conformation of NAD+ bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD+ is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD+ that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both syn and anti conformations, but that binding interactions in the syn conformation are not catalytically productive.

  9. 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

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

    PubMed

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

    2013-12-02

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

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

    PubMed

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

    1966-05-01

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

  12. 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.

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

    PubMed Central

    Gupta, S; Clark, D P

    1989-01-01

    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 in terms of redox balance. PMID:2661531

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

    PubMed

    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 in terms of redox balance.

  15. 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.

  16. 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

  17. Characterisation of the interaction of lactate dehydrogenase with Tween-20 using isothermal titration calorimetry, interfacial rheometry and surface tension measurements.

    PubMed

    McAuley, William J; Jones, David S; Kett, Vicky L

    2009-08-01

    In this study the nature of the interaction between Tween-20 and lactate dehydrogenase (LDH) was investigated using isothermal titration calorimetry (ITC). In addition the effects of the protein and surfactant on the interfacial properties were followed with interfacial rheology and surface tension measurements in order to understand the mechanism by which the surfactant prevents protein adsorption to the air-water interface. Comparisons were made with Tween-40 and Tween-80 in order to further investigate the mechanism. ITC measurements indicated a weak, probably hydrophobic, interaction between Tween-20 and LDH. Prevention of LDH adsorption to the air-water interface by the Tween surfactants was correlated with surface energy rather than surfactant CMC. While surface pressure appears to be the main driving force for the displacement of LDH from the air-water interface by Tween-20 a solubilisation mechanism may exist for other protein molecules. More generally the results of this study highlight the value of the use of ITC and interfacial measurements in characterising the surface behaviour of mixed surfactant and protein systems.

  18. Molecular basis of evolutionary adaptation at the lactate dehydrogenase-B locus in the fish Fundulus heteroclitus.

    PubMed Central

    Crawford, D L; Powers, D A

    1989-01-01

    At the extremes of its natural distribution, populations of the common killifish Fundulus heteroclitus experience a difference of more than 15 degrees C in mean annual temperature. These populations are virtually fixed for two different codominant alleles at the heart-type lactate dehydrogenase locus (Ldh-B) which code for allozymes with different and adaptive kinetic responses to temperature. Two populations near the extremes of the species range (i.e., Maine and Georgia) were further studied for thermal adaptation at this locus. In the absence of any kinetic differences one would predict that to maintain a constant reaction velocity, 2 to 3 times as much enzyme would be required for each 10 degrees C decrease in environmental temperature. Consistent with this adaptive strategy and in addition to the adaptive kinetic characteristics, the LDH-B4 enzyme (EC 1.1.1.27) concentration and its mRNA concentration were approximately twice as great in the northern population as in the southern population. Acclimation experiments allow us to conclude that these differences are due to a combination of fixed genetic traits (evolutionary adaptation) and plastic responses to temperature (physiological acclimation). Furthermore, our calculations show that the LDH-B4 reaction velocities are essentially equivalent for these two populations, even though they live in significantly different thermal environments. PMID:2594773

  19. Towards improved prognostic scores predicting survival in patients with brain metastases: a pilot study of serum lactate dehydrogenase levels.

    PubMed

    Nieder, Carsten; Marienhagen, Kirsten; Dalhaug, Astrid; Norum, Jan

    2012-01-01

    Accurate prognostic information is desirable when counselling patients with brain metastases regarding their therapeutic options and life expectancy. Based on previous studies, we selected serum lactate dehydrogenase (LDH) as a promising factor on which we perform a pilot study investigating methodological aspects of biomarker studies in patients with brain metastases, before embarking on large-scale studies that will look at a larger number of candidate markers in an expanded patient cohort. For this retrospective analysis, 100 patients with available information on LDH treated with palliative whole-brain radiotherapy were selected. A comprehensive evaluation of different LDH-based variables was performed in uni- and multivariate tests. Probably, the most intriguing finding was that LDH kinetics might be more important, or at least complement, information obtained from a single measurement immediately before radiotherapy. LDH and performance status outperformed several other variables that are part of prognostic models such as recursive partitioning analyses classes and graded prognostic assessment score. LDH kinetics might reflect disease behaviour in extracranial metastatic and primary sites without need for comprehensive imaging studies and is a quite inexpensive diagnostic test. Based on these encouraging results, confirmatory studies in a larger cohort of patients are warranted.

  20. Assessment of three new parasite lactate dehydrogenase (pan-pLDH) tests for diagnosis of uncomplicated malaria.

    PubMed

    Fogg, Carole; Twesigye, Rogers; Batwala, Vincent; Piola, Patrice; Nabasumba, Carolyn; Kiguli, James; Mutebi, Frederick; Hook, Christa; Guillerm, Martine; Moody, Anthony; Guthmann, Jean-Paul

    2008-01-01

    A study to assess the diagnostic capabilities of three parasite lactate dehydrogenase (pan-pLDH) tests, Vistapan), Carestart and Parabank), was conducted in Uganda. An HRP2 test, Paracheck-Pf), and a Giemsa-stained blood film were performed with the pLDH tests for outpatients with suspected malaria. In total, 460 subjects were recruited: 248 with positive blood films and 212 with negative blood films. Plasmodium falciparum was present in 95% of infections. Sensitivity above 90% was shown by two pLDH tests, Carestart (95.6%) and Vistapan (91.9%), and specificity above 90% by Parabank (94.3%) and Carestart (91.5%). Sensitivity decreased with low parasitaemia (chi(2) trend, P<0.001); however, all tests achieved sensitivity >90% with parasitaemia > or =100/microl. All tests had good inter-reader reliability (kappa>0.95). Two weeks after diagnosis, 4-10% of pLDH tests were still positive compared with 69.7% of the HRP2 tests. All tests had similar ease of use. In conclusion, two pLDH tests performed well in diagnosing P. falciparum malaria, and all pLDH tests became negative after treatment more quickly than the HRP2. Therefore the rapid test of choice for use with artemisinin-combination therapies in this area would be one of these new pLDH tests.

  1. Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans

    PubMed Central

    Sun, Lifan; Zhang, Caili; Lyu, Pengcheng; Wang, Yanping; Wang, Limin; Yu, Bo

    2016-01-01

    Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer. PMID:27885267

  2. Markedly elevated serum lactate dehydrogenase levels are a clue to the diagnosis of disseminated histoplasmosis in patients with AIDS.

    PubMed

    Corcoran, G R; Al-Abdely, H; Flanders, C D; Geimer, J; Patterson, T F

    1997-05-01

    Disseminated histoplasmosis is a common late manifestation of AIDS, but the diagnosis may be unsuspected in some patients because the clinical presentation of histoplasmosis may mimic other opportunistic infections. High serum lactate dehydrogenase (LDH) levels have been associated with disseminated histoplasmosis. We therefore evaluated whether markedly increased LDH levels were useful for making a diagnosis of disseminated histoplasmosis by comparing admission LDH levels for 15 patients with culture-proven disseminated histoplasmosis with those for 30 patients with advanced AIDS who were admitted to the hospital for evaluation of pulmonary infiltrates and fever. The mean admission LDH level in patients with disseminated histoplasmosis was 1,356 IU/L (range, 145-5,410 IU) whereas it was 332 (range, 77-832 IU) in the patients with other pulmonary processes. Admission LDH levels were >600 IU in 11 (73%) of the 15 patients with disseminated histoplasmosis vs. 3 (10%) of controls (P < .001). We conclude that markedly elevated admission LDH levels may be a clinical clue to the diagnosis of disseminated histoplasmosis in patients with AIDS.

  3. Venous incompetence, poverty and lactate dehydrogenase in Jamaica are important predictors of leg ulceration in sickle cell anaemia.

    PubMed

    Cumming, V; King, L; Fraser, R; Serjeant, G; Reid, M

    2008-07-01

    Clinical features and potential risk factors for chronic leg ulceration (duration >6 months) in homozygous sickle cell (SS) disease were examined in 225 subjects in the Jamaican Cohort Study. Potential risk factors included the number of HBA genes, steady state haematology, serum lactate dehydrogenase (LDH), venous incompetence, and socio-economic status. Chronic ulcers occurred in 53 subjects with the highest risk of ulcer development at 18 years. The prevalence was 29.5% and cumulative incidence 16.7%. Gender or alpha-thalassaemia trait did not affect the incidence of leg ulcer. Ulceration was associated with lower haemoglobin, red cell count, fetal haemoglobin, and socio-economic status and higher reticulocyte count, platelet count, serum LDH and venous incompetence in univariate analyses. Venous incompetence [Hazard Ratio (HR) 3.0-4.0] and socio-economic status (HR 0.8) were most consistently associated with leg ulceration on multivariate analysis. Regression models incorporating serum LDH suggested this to be a stronger predictor than haematological indices. The prevalence of ulcers at 30% is less than previous estimates in Jamaica, probably reflecting the lack of ascertainment bias in the Cohort Study, and also a real secular decline. In Jamaica, venous incompetence, low socio-economic status, and high serum LDH were the strongest predictors of chronic ulceration.

  4. Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results for the cytotoxicity of these nanomaterials.

    PubMed

    Forest, Valérie; Figarol, Agathe; Boudard, Delphine; Cottier, Michèle; Grosseau, Philippe; Pourchez, Jérémie

    2015-03-31

    Carbon nanotube (CNT) cytotoxicity is frequently investigated using in vitro classical toxicology assays. However, these cellular tests, usually based on the use of colorimetric or fluorimetric dyes, were designed for chemicals and may not be suitable for nanosized materials. Indeed, because of their unique physicochemical properties CNT can interfere with the assays and bias the results. To get accurate data and draw reliable conclusions, these artifacts should be carefully taken into account. The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between CNT and the commonly used lactate dehydrogenase (LDH) assay. Experiments under cell-free conditions were performed, and it was clearly demonstrated that artifacts occurred. They were due to the intrinsic absorbance of CNT on one hand and the adsorption of LDH at the CNT surface on the other hand. The adsorption of LDH on CNT was modeled and was found to fit the Langmuir model. The K(ads) and n(eq) constants were defined, allowing the correction of results obtained from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT.

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

    PubMed Central

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

    2014-01-01

    The thermostable d-lactate dehydrogenase from Lactobacillus jensenii (Lj d-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 Lj d-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 (V M) is 2.58 Å3 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

  6. Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans.

    PubMed

    Sun, Lifan; Zhang, Caili; Lyu, Pengcheng; Wang, Yanping; Wang, Limin; Yu, Bo

    2016-11-25

    Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer.

  7. Conserved and unique thermodynamic properties of lactate dehydrogenases in an ectothermic organism, the teleost Microstomus achne, and an endothermic organism, bovine.

    PubMed

    Yonezawa, Mika; Nakagawa, Mizuki; Nakamura, Shigeyoshi; Goto, Tatsufumi; Sugawara, Kotomi; Kidokoro, Shun-Ichi; Wakui, Hideki; Nunomura, Wataru

    2016-11-01

    It is widely believed that enzymatic activities in ectothermic organisms adapt to environmental temperatures. However, to date, no study has thoroughly compared multiple thermodynamic enzymatic characteristics across species living in dramatically different environments. To start to address this gap, we compared the characteristics of lactate dehydrogenase (LDH) purified from the muscles from slime flounder Microstomus achne white muscle and bovine skeletal muscle (bM4) and heart. The K m and V max for pyruvate reduction were about three times higher for M. achne LDH than bM4 Surprisingly, maximum LDH activity was observed at ∼30 °C and ∼50 °C for M. achne and bovine LDHs, respectively, suggesting that the maximum enzymatic activity of LDH is set at a temperature ∼20 °C higher than environmental or body temperature across species. Although K m and V max values of these LDHs increased with temperature, the V max/K m ratio for M. achne LDH and bM4 was independent. Differential scanning calorimetry and enthalpy change measurements confirmed that M. achne and bovine muscle-specific LDHs shared similar properties. Based on the present findings and previous reports, we hypothesize that the function and thermodynamic properties of muscle LDH are highly conserved between a teleost adapted to cold, M. achne, and bovine.

  8. Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid β and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production*

    PubMed Central

    Newington, Jordan T.; Rappon, Tim; Albers, Shawn; Wong, Daisy Y.; Rylett, R. Jane; Cumming, Robert C.

    2012-01-01

    We previously demonstrated that nerve cell lines selected for resistance to amyloid β (Aβ) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aβ and other neurotoxins. Treatment of Aβ-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aβ treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aβ or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αβ or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aβ deposition without developing Alzheimer disease. PMID:22948140

  9. Group II intron-mediated deletion of lactate dehydrogenase gene in an isolated 1,3-propanediol producer Hafnia alvei AD27.

    PubMed

    Celińska, Ewelina; Drożdżyńska, Agnieszka; Wita, Agnieszka; Juzwa, Wojciech; Białas, Wojciech; Czaczyk, Katarzyna; Grajek, Włodzimierz

    2016-03-03

    Our previous studies showed that glycerol fermentation by Hafnia alvei AD27 strain was accompanied by formation of high quantities of lactate. The ultimate aim of this work was the elimination of excessive lactate production in the 1,3-propanediol producer cultures. Group II intron-mediated deletion of ldh (lactate dehydrogenase) gene in an environmental isolate of H. alvei AD27 strain was conducted. The effect of the Δldh genotype in H. alvei AD27 strain varied depending on the culture medium applied. Under lower initial glycerol concentration (20 gL(-1)), lactate and 1,3-propanediol production was fully abolished, and the main carbon flux was directed to ethanol synthesis. On the other hand, at higher initial glycerol concentrations (40 gL(-1)), 1,3-propanediol and lactate production was recovered in the recombinant strain. The final titers of 1,3-propanediol and ethanol were similar for the recombinant and the WT strains, while the Δldh genotype displayed significantly decreased lactate titer. The by-products profile was altered upon ldh gene deletion, while glycerol utilization and biomass accumulation remained unaltered. As indicated by flow-cytometry analyses, the internal pH was not different for the WT and the recombinant Δldh strains over the culture duration, however, the WT strain was characterized by higher redox potential.

  10. 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.

  11. Sustained translational repression of lactate dehydrogenase 1 in Toxoplasma gondii bradyzoites is conferred by a small regulatory RNA hairpin.

    PubMed

    Holmes, Michael; Itaas, Vaunell; Ananvoranich, Sirinart

    2014-11-01

    In response to environmental stresses, Toxoplasma gondii induces a global translational repression which allows for the remodeling of its transcriptome. While some transcripts are preferentially translated, another subset is translationally repressed and maintained in bradyzoites. Although little is known of how transcripts are targeted for sustained translational repression, the targeting probably operates through an RNA-centric mechanism relying on the recognition of cis-acting elements. In this study, we sought to determine if the targeting of transcripts through recognizable cis-acting elements could be responsible for the transcript-specific sustained translational repression displayed by Toxoplasma bradyzoites. We examined the UTRs of a translationally repressed gene, lactate dehydrogenase 1, and found a 40 nucleotide regulatory element in its 5'UTR. This element specifically induces translational repression in otherwise constitutively expressed transcripts. Mutational studies revealed that the formation of a small 16 nucleotide regulatory RNA hairpin is essential for this activity. We suggest that this hairpin may act as the nucleation site for the binding of an as yet to be identified trans-acting factor that allows for the transcript to be targeted for translational repression removal from the active translational pool. To our knowledge, this is the first report characterizing a specific cis-acting element contributing to post-transcriptional gene regulation in Toxoplasma and suggests the presence of a pathway by which the parasites can recognize, identify and specifically target transcripts for sustained translational repression under stressful conditions.

  12. Function of muscle-type lactate dehydrogenase and citrate synthase of the Galápagos marine iguana, Amblyrhynchus cristatus, in relation to temperature.

    PubMed

    Fields, Peter A; Strothers, Chad M; Mitchell, Mark A

    2008-05-01

    The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures ( approximately 14-40 degrees C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A(4)-LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 degrees C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K(m) of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A(4)-LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus --> I. iguana) individually have no effect on kinetics, but together significantly decrease the K(m) of pyruvate and catalytic rate constant (k(cat)) of the A. cristatus ortholog. Thus, our data show that A. cristatus A(4)-LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog.

  13. 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

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

    PubMed Central

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

    2016-01-01

    Abstract 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

  15. Procalcitonin, C-reactive protein and serum lactate dehydrogenase in the diagnosis of bacterial sepsis, SIRS and systemic candidiasis.

    PubMed

    Miglietta, Fabio; Faneschi, Maria Letizia; Lobreglio, Giambattista; Palumbo, Claudio; Rizzo, Adriana; Cucurachi, Marco; Portaccio, Gerolamo; Guerra, Francesco; Pizzolante, Maria

    2015-09-01

    The aim of this study was to evaluate procalcitonin (PCT), C-reactive protein (CRP), platelet count (PLT) and serum lactate dehydrogenase (LDH) as early markers for diagnosis of SIRS, bacterial sepsis and systemic candidiasis in intensive care unit (ICU) patients. Based on blood culture results, the patients were divided into a sepsis group (70 patients), a SIRS group (42 patients) and a systemic candidiasis group (33 patients). PCT, CRP, LDH and PLT levels were measured on day 0 and on day 2 from the sepsis symptom onset. PCT levels were higher in Gram negative sepsis than those in Gram positive sepsis, although the P value between the two subgroups is not significant (P=0.095). Bacterial sepsis group had higher PCT and CRP levels compared with the systemic candidiasis group, whereas PLT and LDH levels showed similar levels in these two subgroups. The AUC for PCT (AUC: 0.892, P <0.001) was larger than for CRP (AUC: 0.738, P <0.001). The best cut-off values for PCT and CRP were 0.99 ng/mL and 76.2 mg/L, respectively. Diagnostic sensitivity and specificity for PCT were 84.3% and 81.8% whereas CRP showed a sensitivity of 77.2% and a specificity of 63.6%. However, PCT was unable to discriminate between SIRS and systemic candidiasis groups (P=0.093 N.S.). In conclusion, PCT can be used as a preliminary marker in the event of clinical suspicion of systemic candidiasis; however, low PCT levels (<0.99 ng/mL) necessarily require the use of other specific markers of candidaemia to confirm the diagnosis, due to great uniformity of PCT levels in systemic candidiasis and SIRS groups.

  16. Renal Cortical Lactate Dehydrogenase: A Useful, Accurate, Quantitative Marker of In Vivo Tubular Injury and Acute Renal Failure

    PubMed Central

    Zager, Richard A.; Johnson, Ali C. M.; Becker, Kirsten

    2013-01-01

    Studies of experimental acute kidney injury (AKI) are critically dependent on having precise methods for assessing the extent of tubular cell death. However, the most widely used techniques either provide indirect assessments (e.g., BUN, creatinine), suffer from the need for semi-quantitative grading (renal histology), or reflect the status of residual viable, not the number of lost, renal tubular cells (e.g., NGAL content). Lactate dehydrogenase (LDH) release is a highly reliable test for assessing degrees of in vitro cell death. However, its utility as an in vivo AKI marker has not been defined. Towards this end, CD-1 mice were subjected to graded renal ischemia (0, 15, 22, 30, 40, or 60 min) or to nephrotoxic (glycerol; maleate) AKI. Sham operated mice, or mice with AKI in the absence of acute tubular necrosis (ureteral obstruction; endotoxemia), served as negative controls. Renal cortical LDH or NGAL levels were assayed 2 or 24 hrs later. Ischemic, glycerol, and maleate-induced AKI were each associated with striking, steep, inverse correlations (r, −0.89) between renal injury severity and renal LDH content. With severe AKI, >65% LDH declines were observed. Corresponding prompt plasma and urinary LDH increases were observed. These observations, coupled with the maintenance of normal cortical LDH mRNA levels, indicated the renal LDH efflux, not decreased LDH synthesis, caused the falling cortical LDH levels. Renal LDH content was well maintained with sham surgery, ureteral obstruction or endotoxemic AKI. In contrast to LDH, renal cortical NGAL levels did not correlate with AKI severity. In sum, the above results indicate that renal cortical LDH assay is a highly accurate quantitative technique for gauging the extent of experimental acute ischemic and toxic renal injury. That it avoids the limitations of more traditional AKI markers implies great potential utility in experimental studies that require precise quantitation of tubule cell death. PMID:23825563

  17. Pre-treatment serum alkaline phosphatase and lactate dehydrogenase as prognostic factors in triple negative breast cancer

    PubMed Central

    Chen, Bo; Dai, Danian; Tang, Hailin; Chen, Xi; Ai, Xiaohong; Huang, Xiaojia; Wei, Weidong; Xie, Xiaoming

    2016-01-01

    Background: Serum parameters as prognostic parameters are studied widely. We aim to examine the prognostic significance of the serum alkaline phosphatase (ALP) level and lactate dehydrogenase (LDH) level in triple negative breast cancer (TNBC). Methods: Total of 253 TNBC patients from Sun Yat-sen University Cancer Center who underwent treatment between January 2004 and December 2009 was conducted in this retrospective study. Before treatment serum ALP and LDH levels were routinely measured. We use the receiver operating characteristic (ROC) curve analysis to estimate the cutoff value of serum ALP and LDH levels. The Kaplan-Meier method and multivariable Cox regression analysis were used for Disease free survival (DFS) and overall survival (OS) assessed. Results: The ROC curves determined that the optimum cutoff point for ALP and LDH were 66.5u/L and 160.5u/L, respectively. The elevated ALP and LDH were both significantly associated with decreased DFS and OS (both P < 0.001). In addition, the entire cohort was stratified into three subgroups basis of ALP levels and LDH levels. TNBC Patients who with ALP >66.5 u/L and LDH >160.5u/L had the worst DFS and OS (both P < 0.001). In TNBC patients, univariate and multivariate Cox regression analyses conformed ALP and LDH were independent unfavorable prognostic factors for DFS and OS. Conclusions: The serum levels of ALP and LDH before treatment are independent prognostic parameters and may serve as complement to help predict survival in TNBC. PMID:27994669

  18. 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.

  19. Prognostic Value of Serum Lactate Dehydrogenase in Renal Cell Carcinoma: A Systematic Review and Meta-Analysis

    PubMed Central

    Zhuang, Qianfeng; Fan, Min; Ding, Tao; Lu, Hao; He, Xiaozhou

    2016-01-01

    Background Recently, many studies have shown that the serum lactate dehydrogenase (LDH) level is related to the prognosis of renal cell carcinoma (RCC). We launched this meta-analysis to assess the prognostic value of serum LDH in patients with RCC. Methods We searched PubMed, Embase and Web of Science for information on serum LDH and the outcome of RCC through June 14, 2016. The hazard ratio (HR) and its 95% confidence interval (CI) for overall survival (OS) and progression-free survival (PFS) were extracted and integrated from the matching studies. Results A total of 29 studies including 6629 patients with RCC were incorporated in this meta-analysis. Patients whose serum LDH levels were elevated had a lower OS (HR = 2.13, 95% CI = 1.69–2.69, P < 0.001). Meanwhile, the pooled data showed that a higher serum LDH level was a negative prognostic factor for PFS (HR = 1.74, 95% CI = 1.48–2.04, P < 0.001). Furthermore, subgroup analyses indicated elevated serum LDH was associated with poor survival in different tumor types. Elevated serum LDH was significantly associated with worse prognosis for patients with all stages of RCC (OS, HR = 2.41, 95% CI = 1.09–5.33), metastatic RCC (OS, HR = 2.62, 95% CI 1.57–2.59; CSS, HR = 1.79, 95% CI 1.49–2.15), and non-metastatic RCC (OS, HR = 3.67, CI = 1.33–10.13). Besides, elevated serum LDH also indicated a worse prognosis in subgroups of cut-off values, analysis types and ethnicity. Conclusions Our results show that serum LDH levels are associated with the outcomes of RCC and can be used as a valuable biomarker for monitoring prognoses. PMID:27861542

  20. 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

  1. 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

  2. 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.

  3. 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…

  4. Extremely thermostable L(+)-lactate dehydrogenase from Thermotoga maritima: cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state.

    PubMed Central

    Ostendorp, R.; Auerbach, G.; Jaenicke, R.

    1996-01-01

    L(+)-lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195-201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 degrees C, i.e., ca. 60 degrees below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 degrees C show satisfactory diffraction suitable for X-ray analysis up to a resolution of 2.8 A. As shown by gel-permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near- and far-UV CD) of the two species are indistinguishable. The calculated alpha-helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unalatered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 degrees C; irreversible thermal denaturation becomes significant at approximately 95 degrees C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat. PMID:8732758

  5. 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.

  6. 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.

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

    PubMed Central

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

    2016-01-01

    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

  8. MiR-34b-3 and miR-449a inhibit malignant progression of nasopharyngeal carcinoma by targeting lactate dehydrogenase A

    PubMed Central

    Li, Huiling; Li, Xiaoling; Ge, Xiaolu; Jia, Liqing; Zhang, Zhezhe; Fang, Renpeng; Yang, Jing; Liu, Jianpin; Peng, Shuping; Zhou, Ming; Xiang, Juanjuan; Zeng, Zhaoyang; Zhou, Wen; Xiong, Wei; Xiao, Gaoming; Fang, Li; Li, Gui-yuan; Li, Zheng

    2016-01-01

    MicroRNA expression profiling assays have shown that miR-34b/c and miR-449a are down-regulated in nasopharyngeal carcinoma (NPC); however, the targets and functions of miR-34b/c and miR-449a in the pathologenesis of NPC remain elusive. In this study, we verified miR-34b/c and miR-449a were significantly reduced with the advance of NPC. Overexpression of miR-34b-3 and miR-449a suppressed the growth of NPC cells in culture and mouse tumor xenografts. Using tandem mass tags for quantitative labeling and LC-MS/MS analysis to investigate protein changes after restoring expression of miR-34b-3, 251 proteins were found to be down-regulated after miR-34b-3 transfection. Through 3 replicate experiments, we found that miR-34b-3 regulated the expression of 15 potential targeted genes mainly clustered in the key enzymes of glycolysis metabolism, including lactate dehydrogenase A (LDHA). Further investigation revealed that miR-34b-3 and miR-449a negatively regulated LDHA by binding to the 3′ untranslated regions of LDHA. Furthermore, LDHA overexpression rescued the miR-34b-3 and miR-449a induced tumor inhibition effect in CNE2 cells. In addition, miR-34b-3 and miR-449a suppressed LDH activity and reduced LD content, which were directly induced by downregulation of the LDHA. Our findings suggest that miR-34b-3 and miR-449a suppress the development of NPC through regulation of glycolysis via targeting LDHA and may be potential therapeutic targets for the treatment of NPC. PMID:27458165

  9. 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.

  10. 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

  11. Highly elevated serum lactate dehydrogenase is associated with central nervous system relapse in patients with diffuse large B-cell lymphoma: Results of a multicenter prospective cohort study.

    PubMed

    Kim, Seok Jin; Hong, Jun Sik; Chang, Myung Hee; Kim, Jeong-A; Kwak, Jae-Yong; Kim, Jin Seok; Yoon, Dok Hyun; Lee, Won Sik; Do, Young Rok; Kang, Hye Jin; Eom, Hyeon-Seok; Park, Yong; Won, Jong-Ho; Mun, Yeung-Chul; Kim, Hyo Jung; Kwon, Jung Hye; Kong, Jee Hyun; Oh, Sung Yong; Lee, Sunah; Bae, Sung Hwa; Yang, Deok-Hwan; Jun, Hyun Jung; Kim, Yang Soo; Yun, Hwan Jung; Lee, Soon Il; Kim, Min Kyoung; Park, Eun Kyung; Kim, Won Seog; Suh, Cheolwon

    2016-11-01

    Central nervous system involvement remains a challenging issue in the treatment of patients with diffuse large B-cell lymphoma. We conducted a prospective cohort study with newly diagnosed diffuse large B-cell lymphoma patients receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone to identify incidence and risk factors for central nervous system involvement. Among 595 patients, 279 patients received pre-treatment central nervous system evaluation, and 14 patients had central nervous system involvement at diagnosis (2.3% out of entire patients and 5.0% out of the 279 patients). For those patients, median follow-up duration was 38.2 months and some of them achieved long-term survival. Out of 581 patients who did not have central nervous system involvement at diagnosis, 26 patients underwent secondary central nervous system relapse with a median follow-up of 35 months, and the median time to central nervous system involvement was 10.4 months (range: 3.4-29.2). Serum lactate dehydrogenase > ×3 upper limit of normal range, the Eastern Cooperative Oncology Group performance status ≥ 2, and involvement of sinonasal tract or testis, were independent risk factors for central nervous system relapse in multivariate analysis. Our study suggests that enhanced stratification of serum lactate dehydrogenase according to the National Comprehensive Cancer Network-International Prognostic Index may contribute to better prediction for central nervous system relapse in patients with diffuse large B-cell lymphoma. This trial was registered at clinicaltrials.gov identifier: 01202448.

  12. [Effects of H2-blockers on alcohol dehydrogenase (ADH) activity].

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Szmitkowski, Maciej

    2008-12-01

    First-pass metabolism (FPM) of alcohol is demonstrated by lower blood alcohol concentrations after oral than intravenous administration of the same dose. FPM occurs predominantly in the stomach and has been attributed to class IV of alcohol dehydrogenase (ADH) isoenzyme localizated in the gastric mucosa. A number of factors that influence on gastric ADH activity and thereby modulate FPM have been identified. These include age, sex, ethnicity, concentrations and amounts of alcohol consumed and drugs. Several H2-receptor antagonists, including cimetidine and ranitidine, inhibit gastric ADH activity and reduce FPM, resulting in higher blood alcohol concentrations after H2-blockers administration.

  13. Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria

    PubMed Central

    Ashley, Elizabeth A; Touabi, Malek; Ahrer, Margareta; Hutagalung, Robert; Htun, Khayae; Luchavez, Jennifer; Dureza, Christine; Proux, Stephane; Leimanis, Mara; Lwin, Myo Min; Koscalova, Alena; Comte, Eric; Hamade, Prudence; Page, Anne-Laure; Nosten, François; Guerin, Philippe J

    2009-01-01

    Background In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). Methods In Dawei, southern Myanmar, three pLDH based RDTs (CareStart™ Malaria pLDH (Pan), CareStart™ Malaria pLDH (Pan, Pf) and OptiMAL-IT®)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. Results Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria™ pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4] OptiMal-IT®: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7] CareStart Malaria™ pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI9585.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI9571.1-84.4], spec 97.8% [CI95 96.3-98.7] Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart™ Malaria tests and seven days for OptiMAL-IT®. Tests were heat stable up to 90 days except for OptiMAL-IT® (Pf specific pLDH stable to day 20 at 35°C). Conclusion None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low

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

    DOEpatents

    Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

    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.

  15. Regulation of crayfish, Orconectes virilis, tail muscle lactate dehydrogenase (LDH) in response to anoxic conditions is associated with alterations in phosphorylation patterns.

    PubMed

    Green, Stuart R; Storey, Kenneth B

    2016-12-01

    Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, has a crucial role in sustaining ATP production by glycolysis during periods of anoxia via regenerating NAD(+) through the production of lactate. The present study examined the effects of prolonged (20h) anoxic submergence on LDH from the tail muscle of an anoxia-tolerant crayfish (Orconectes virilis). LDH was purified to homogeneity from tail muscle of both aerobic control and anoxic crayfish in a three step process. Analysis of the kinetic parameters and the stability of LDH showed that the Vmax in the pyruvate-reducing direction was significantly higher for the enzyme from anoxic crayfish whereas in the lactate-oxidizing direction the Vmax was significantly higher for the control enzyme. Differential scanning fluorimetry was used to assess thermal unfolding of crayfish LDH. The results showed that the enzyme from control muscle had a significantly higher melting temperature (greater thermal stability) than the anoxic enzyme form, suggesting that there was a structural difference between the two enzyme forms. Immunoblotting of purified LDH implicated post-translational modification as the reason for this difference; purified LDH from aerobic control crayfish showed significantly higher amounts of serine/threonine phosphorylation than did the anoxic enzyme form. This study provides evidence for anoxia-induced modifications of crayfish muscle LDH that may contribute significantly to modulating enzyme function under anoxic conditions.

  16. Pyruvate Dehydrogenase Complex Activity in Normal and Deficient Fibroblasts

    PubMed Central

    Sheu, Kwan-Fu Rex; Hu, Chii-Whei C.; Utter, Merton F.

    1981-01-01

    Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37°C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-14C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of 14CO2 production on the presence of thiamin-PP, coenzyme A (CoA), Mg++, and NAD+. Also, it has been shown that acetyl-CoA and 14CO2 are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg++ and Ca++, or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency

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

    PubMed

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

    2016-01-01

    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.

  18. Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity.

    PubMed

    Bernocchi, G; Barni, S

    1983-12-01

    Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.

  19. 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.

  20. Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.

    PubMed

    Robertson, D; Smith, C; Bolwell, G P

    1996-01-01

    UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40,000. Km values were measured for UDP-glucose as 5.5 +/- 1.4 mM and for NAD+ as 20 +/- 3 microM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

  1. 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.

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

    PubMed

    Li, Chao; Tao, Fei; Xu, Ping

    2016-08-17

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

  3. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.

  4. 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.

  5. Highly elevated serum lactate dehydrogenase is associated with central nervous system relapse in patients with diffuse large B-cell lymphoma: Results of a multicenter prospective cohort study

    PubMed Central

    Kim, Seok Jin; Hong, Jun Sik; Chang, Myung Hee; Kim, Jeong-A; Kwak, Jae-Yong; Kim, Jin Seok; Yoon, Dok Hyun; Lee, Won Sik; Do, Young Rok; Kang, Hye Jin; Eom, Hyeon-Seok; Park, Yong; Won, Jong-Ho; Mun, Yeung-Chul; Kim, Hyo Jung; Kwon, Jung Hye; Kong, Jee Hyun; Oh, Sung Yong; Lee, Sunah; Bae, Sung Hwa; Yang, Deok-Hwan; Jun, Hyun Jung; Kim, Yang Soo; Yun, Hwan Jung; Il Lee, Soon; Kim, Min Kyoung; Park, Eun Kyung; Kim, Won Seog; Suh, Cheolwon

    2016-01-01

    Central nervous system involvement remains a challenging issue in the treatment of patients with diffuse large B-cell lymphoma. We conducted a prospective cohort study with newly diagnosed diffuse large B-cell lymphoma patients receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone to identify incidence and risk factors for central nervous system involvement. Among 595 patients, 279 patients received pre-treatment central nervous system evaluation, and 14 patients had central nervous system involvement at diagnosis (2.3% out of entire patients and 5.0% out of the 279 patients). For those patients, median follow-up duration was 38.2 months and some of them achieved long-term survival. Out of 581 patients who did not have central nervous system involvement at diagnosis, 26 patients underwent secondary central nervous system relapse with a median follow-up of 35 months, and the median time to central nervous system involvement was 10.4 months (range: 3.4–29.2). Serum lactate dehydrogenase > ×3 upper limit of normal range, the Eastern Cooperative Oncology Group performance status ≥ 2, and involvement of sinonasal tract or testis, were independent risk factors for central nervous system relapse in multivariate analysis. Our study suggests that enhanced stratification of serum lactate dehydrogenase according to the National Comprehensive Cancer Network-International Prognostic Index may contribute to better prediction for central nervous system relapse in patients with diffuse large B-cell lymphoma. This trial was registered at clinicaltrials.gov identifier: 01202448. PMID:27713132

  6. 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

  7. Changes in pyruvate dehydrogenase complex activity during and following severe insulin-induced hypoglycemia.

    PubMed

    Cardell, M; Siesjö, B K; Wieloch, T

    1991-01-01

    The effect of severe insulin-induced hypoglycemia on the activity of the pyruvate dehydrogenase enzyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex during burst suppression EEG, after 10, 30, and 60 min of isoelectric EEG, and after 30 and 180 min and 24 h of recovery following 30 min of hypoglycemic coma. Changes in PDHC activity were correlated to levels of labile organic phosphates and glycolytic metabolites. In cortex from control animals, the rate of [1-14C]pyruvate decarboxylation was 7.1 +/- 1.3 U/mg of protein, or 35% of the total PDHC activity. The activity was unchanged during burst suppression EEG whereas the active fraction increased to 81-87% during hypoglycemic coma. Thirty minutes after glucose-induced recovery, the PDHC activity had decreased by 33% compared to control levels, and remained significantly depressed after 3 h of recovery. This decrease in activity was not due to a decrease in the total PDHC activity. At 24 h of recovery, PDHC activity had returned to control levels. We conclude that the activation of PDHC during hypoglycemic coma is probably the result of an increased PDH phosphatase activity following depolarization and calcium influx, and allosteric inhibition of PDH kinase due to increased ADP/ATP ratio. The depression of PDHC activity following hypoglycemic coma is probably due to an increased phosphorylation of the enzyme, as a consequence of an imbalance between PDH phosphatase and kinase activities. Since some reduction of the ATP/ADP ratio persisted and since the lactate/pyruvate ratio had normalized by 3 h of recovery, the depression of PDHC most likely reflects a decrease in PDH phosphatase activity, probably due to a decrease in intramitochondrial Ca2+.

  8. The crystal structure of 1-D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis reveals a zinc hydrolase with a lactate dehydrogenase fold.

    PubMed

    Maynes, Jason T; Garen, Craig; Cherney, Maia M; Newton, Gerald; Arad, Dorit; Av-Gay, Yossef; Fahey, Robert C; James, Michael N G

    2003-11-21

    Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.

  9. Skeletal muscle PGC-1α controls whole-body lactate homeostasis through estrogen-related receptor α-dependent activation of LDH B and repression of LDH A.

    PubMed

    Summermatter, Serge; Santos, Gesa; Pérez-Schindler, Joaquín; Handschin, Christoph

    2013-05-21

    The peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) controls metabolic adaptations. We now show that PGC-1α in skeletal muscle drives the expression of lactate dehydrogenase (LDH) B in an estrogen-related receptor-α-dependent manner. Concomitantly, PGC-1α reduces the expression of LDH A and one of its regulators, the transcription factor myelocytomatosis oncogene. PGC-1α thereby coordinately alters the composition of the LDH complex and prevents the increase in blood lactate during exercise. Our results show how PGC-1α actively coordinates lactate homeostasis and provide a unique molecular explanation for PGC-1α-mediated muscle adaptations to training that ultimately enhance exercise performance and improve metabolic health.

  10. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of L-lactate dehydrogenase and its H171C mutant from Bacillus subtilis

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli

    2012-08-31

    L-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to L-lactate with the simultaneous oxidation of NADH to NAD{sup +}. In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD{sup +} and the crystal diffracted to 2.38 {angstrom} resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 {angstrom}. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD{sup +}, and data sets were collected to 2.20 and 2.49 {angstrom} resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 {angstrom} and a = b = 133.43, c = 99.09 {angstrom}, respectively. Tetramers were observed in the asymmetric units of all three crystals.

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

    PubMed

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

    2005-04-01

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

  12. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  13. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    SciTech Connect

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

    1988-04-15

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

  14. [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.

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

    PubMed Central

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

    2015-01-01

    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

  16. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    PubMed

    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.

  17. 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

  18. Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity

    PubMed Central

    Mathias, Rommel A.; Greco, Todd M.; Oberstein, Adam; Budayeva, Hanna G.; Chakrabarti, Rumela; Rowland, Elizabeth A.; Kang, Yibin; Shenk, Thomas; Cristea, Ileana M.

    2014-01-01

    Summary Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism. PMID:25525879

  19. 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

  20. 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

  1. Pyruvate dehydrogenase activity in the rat cerebral cortex following cerebral ischemia.

    PubMed

    Cardell, M; Koide, T; Wieloch, T

    1989-06-01

    The effect of cerebral ischemia on the activity of pyruvate dehydrogenase (PDH) enzyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex following 15 min of bilateral common carotid occlusion ischemia and following 15 min, 60 min, and 6 h of recirculation after 15 min of ischemia. In frozen cortical tissue from the same animals, the levels of labile phosphate compounds, glucose, glycogen, lactate, and pyruvate was determined. In cortex from control animals, the rate of [1(-14)C]pyruvate decarboxylation was 9.6 +/- 0.5 nmol CO2/(min-mg protein) or 40% of the total PDHC activity. This fraction increased to 89% at the end of 15 min of ischemia. At 15 min of recirculation following 15 min of ischemia, the PDHC activity decreased to 50% of control levels and was depressed for up to 6 h post ischemia. This decrease in activity was not due to a decrease in total PDHC activity. Apart from a reduction in ATP levels, the acute changes in the levels of energy metabolites were essentially normalized at 6 h of recovery. Dichloroacetate (DCA), an inhibitor of PDH kinase, given to rats at 250 mg/kg i.p. four times over 2 h, significantly decreased blood glucose levels from 7.4 +/- 0.6 to 5.1 +/- 0.3 mmol/L and fully activated PDHC. In animals in which the plasma glucose level was maintained at control levels of 8.3 +/- 0.5 mumol/g by intravenous infusion of glucose, the active portion of PDHC increased to 95 +/- 4%. In contrast, the depressed PDHC activity at 15 min following ischemia was not affected by the DCA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Mechanism of pyruvate dehydrogenase activation by increased cardiac work.

    PubMed

    Kobayashi, K; Neely, J R

    1983-06-01

    The effects of increased cardiac work, pyruvate and insulin on the state of pyruvate dehydrogenase (PDH) activation and rate of pyruvate decarboxylation was studied in the isolated perfused rat heart. At low levels of cardiac work, 61% of PDH was present in the active form when glucose was the only substrate provided. The actual rate of pyruvate decarboxylation was only 5% of the available capacity calculated from the percent of active PDH. Under this condition, the rate of pyruvate decarboxylation was restricted by the slow rate of pyruvate production from glycolysis. Increasing cardiac work accelerated glycolysis, but production of pyruvate remained rate limiting for pyruvate oxidation and only 40% of the maximal active PDH capacity was used. Addition of insulin along with glucose reduced the percent of active PDH to 16% of the total at low cardiac work. This effect of insulin was associated with increased mitochondria NADH/NAD and acetyl CoA/CoA ratios. With both glucose and insulin the calculated maximum capacity of active PDH was about the same as measured rates of pyruvate oxidation indicating that pyruvate oxidation was limited by the activation state of PDH. In this case, raising the level of cardiac work increased the active PDH to 85% and although pyruvate oxidation was accelerated, measured flux through PDH was only 73% of the maximal activity of active PDH. With pyruvate as added exogenous substrate, PDH was 82% of active at low cardiac work probably due to pyruvate inhibition of PDH kinase. In this case, the measured rate of pyruvate oxidation was 64% of the capacity of active PDH. However, increased cardiac work still caused further activation of PDH to 96% active. Thus, actual rates of pyruvate oxidation in the intact tissue were determined by (1) the supply of pyruvate in hearts receiving glucose alone, (2) by the percent of active PDH in hearts receiving both glucose and insulin at low work and (3) by end-product inhibition in hearts receiving

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

    PubMed

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

    2009-08-01

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

  4. 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

  5. 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.

  6. 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

    2015-01-02

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

  7. A series of oxyimine-based macrocyclic dinuclear zinc(II) complexes enhances phosphate ester hydrolysis, DNA binding, DNA hydrolysis, and lactate dehydrogenase inhibition and induces apoptosis.

    PubMed

    Anbu, Sellamuthu; Kamalraj, Subban; Varghese, Babu; Muthumary, Johnpaul; Kandaswamy, Muthusamy

    2012-05-21

    that the DNA cleavage acceleration promoted by 1-6 are due to the efficient cooperative catalysis of the two close proximate zinc(II) cation centers. The ligand L(1), dizinc(II) complexes 1, 3, and 6 showed cytotoxicity in human hepatoma HepG2 cancer cells, giving IC(50) values of 117, 37.1, 16.5, and 8.32 μM, respectively. The results demonstrated that 6, a dizinc(II) complex with potent antiproliferative activity, is able to induce caspase-dependent apoptosis in human cancer cells. Cytotoxicity of the complexes was further confirmed by the lactate dehydrogenase enzyme level in HepG2 cell lysate and content media.

  8. [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.

  9. Regulation of pyruvate dehydrogenase kinase activity from pig kidney cortex.

    PubMed Central

    Pawelczyk, T; Olson, M S

    1992-01-01

    The activity of pyruvate dehydrogenase (PDH) kinase in the purified PDH complex from pig kidney is sensitive to changes in ionic strength. The enzyme has optimum activity within a small range of ionic strength (0.03-0.05 M). An increase in ionic strength from 0.04 M to 0.2 M lowers the activity of PDH kinase by 32% and decreases the Km for ATP from 25 microM to 10 microM. At constant ionic strength (0.15 M) the enzyme has optimum activity over a broad pH range (7.2-8.0). The PDH kinase is stimulated 2.2-fold by 20 mM-K+, whereas Na+ even at high concentration (80 mM) has no effect on the enzyme activity. The stimulation of PDH kinase by K+ is not dependent on pH and ionic strength. PDH kinase is inhibited by HPO4(2-) in the presence of K+, whereas HPO4(2-) has no effect on the activity of this enzyme in the absence of K+. HPO4(2-) at concentrations of 2 and 10 mM inhibits PDH kinase by 28% and 55% respectively. The magnitude of this inhibition is not dependent on the ATP/ADP ratio. Inhibition by HPO4(2-) in the concentration range 0-10 mM is non-competitive with respect to ATP, and becomes mixed-type at concentrations over 10 mM. The Ki for HPO4(2-) is 10 mM. When HPO4(2-) is replaced by SO4(2-), the same effects on the activity of PDH kinase are observed. PDH kinase is also inhibited by Cl-. In the presence of 80 mM-Cl- the PDH kinase is inhibited by 40%. The inhibition by Cl- is not dependent on K+. In conclusion, we postulate that changes in phosphate concentrations may play a significant role in the regulation of PDH kinase activity in vivo. PMID:1463442

  10. 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.

  11. Diagnostic value of serum lactate dehydrogenase isoenzyme and amino acid patterns in several schistosomal and non-schistosomal disorders as compared to other biochemical parameters.

    PubMed

    Ahmed, S A; Gad, M Z

    1996-08-01

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

  12. Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation, metastasis, and invasion in triple-negative breast cancer.

    PubMed

    Cheng, Shi-Yann; Yang, Yao-Chih; Ting, Kuan-Lun; Wen, Su-Ying; Viswanadha, Vijaya Padma; Huang, Chih-Yang; Kuo, Wei-Wen

    2017-04-01

    The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis-promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA-MB-231, a triple-negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over-expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390-1398, 2017.

  13. Decreased hematocrit-to-viscosity ratio and increased lactate dehydrogenase level in patients with sickle cell anemia and recurrent leg ulcers.

    PubMed

    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+)--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.

  14. 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.

  15. Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation.

    PubMed

    Oh, Seok-Jeong; Kim, Hwa; Liu, Yingqiu; Han, Hyo-Kyung; Kwon, Kyenghee; Chang, Kyung-Hwa; Park, Kwangsik; Kim, Younghun; Shim, Kyuhwan; An, Seong Soo A; Lee, Moo-Yeol

    2014-03-21

    A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.

  16. Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.

    PubMed

    Holmberg, N; Ryde, U; Bülow, L

    1999-10-01

    L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.

  17. Serum lactate dehydrogenase predicts prognosis and correlates with systemic inflammatory response in patients with advanced pancreatic cancer after gemcitabine-based chemotherapy

    PubMed Central

    Yu, Shu-Lin; Xu, Li-Tao; Qi, Qi; Geng, Ya-Wen; Chen, Hao; Meng, Zhi-Qiang; Wang, Peng; Chen, Zhen

    2017-01-01

    Serum lactate dehydrogenase (LDH) concentrations correlate with tumor progression and poor outcome. We evaluated the predictive value of serum LDH level for overall survival (OS) of patients with advanced pancreatic cancer after gemcitabine-based chemotherapy. We retrospectively enrolled 364 patients with locally advanced or metastatic pancreatic adenocarcinoma who were then allocated to training (n = 139) and validation cohorts (n = 225). We evaluated the association between serum LDH levels and OS as well as with markers of systemic inflammation, including neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR) and lymphocyte/monocyte ratio (LMR). Kaplan–Meier analyses revealed that low serum LDH levels in the training cohort significantly correlated with longer OS. Multivariate analysis identified the serum LDH levels as an independent prognostic predictor of OS (p = 0.005). Serum LDH levels correlated positively with NLR and PLR and correlated negatively with LMR. Similar results were obtained for the validation cohort, except that multivariate analysis identified the serum LDH level as a significant prognostic predictor and only a statistical trend for OS (p = 0.059). We conclude that serum LDH levels were associated with the systemic inflammatory response and served as a significant prognostic predictor of OS. Serum LDH levels predicted OS in patients with advanced pancreatic cancer after gemcitabine-based palliative chemotherapy. PMID:28345594

  18. 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.

  19. 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.

  20. [Glutamate dehydrogenase activity of Bradyrhizobium japonicum in the presence of phytoregulators].

    PubMed

    Leonova, N O; Tytova, L V; Tantsiurenko, O V; Antypchuk, A F

    2006-01-01

    Influence of plant growth regulators ivin and emistim C, and flavonoids daidzein and quercetin on the glutamate dehydrogenase activity of soybean nodule bacteria, with contrasting symbiotic properties, were studied. It was shown that all used phytoregulators stimulated glutamate dehydrogenase activity of Bradyrhizobium japonicum 71t (the strain with highly efficient symbiotic properties) 1.2-4.9 times. Bradyrhizobium japonicum 21110 (the strain with inefficient symbiotic properties) diminished the enzyme activity in the presence of all phythoregulators except for ivin.

  1. 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.

  2. Adrenaline increases skeletal muscle glycogenolysis, pyruvate dehydrogenase activation and carbohydrate oxidation during moderate exercise in humans

    PubMed Central

    Watt, Matthew J; Howlett, Kirsten F; Febbraio, Mark A; Spriet, Lawrence L; Hargreaves, Mark

    2001-01-01

    To evaluate the role of adrenaline in regulating carbohydrate metabolism during moderate exercise, 10 moderately trained men completed two 20 min exercise bouts at 58 ± 2 % peak pulmonary oxygen uptake (V̇O2,peak). On one occasion saline was infused (CON), and on the other adrenaline was infused intravenously for 5 min prior to and throughout exercise (ADR). Glucose kinetics were measured by a primed, continuous infusion of 6,6-[2H]glucose and muscle samples were obtained prior to and at 1 and 20 min of exercise. The infusion of adrenaline elevated (P < 0.01) plasma adrenaline concentrations at rest (pre-infusion, 0.28 ± 0.09; post-infusion, 1.70 ± 0.45 nmol l−1; means ±s.e.m.) and this effect was maintained throughout exercise. Total carbohydrate oxidation increased by 18 % and this effect was due to greater skeletal muscle glycogenolysis (P < 0.05) and pyruvate dehydrogenase (PDH) activation (P < 0.05, treatment effect). Glucose rate of appearance was not different between trials, but the infusion of adrenaline decreased (P < 0.05, treatment effect) skeletal muscle glucose uptake in ADR. During exercise muscle glucose 6-phosphate (G-6-P) (P = 0.055, treatment effect) and lactate (P < 0.05) were elevated in ADR compared with CON and no changes were observed for pyruvate, creatine, phosphocreatine, ATP and the calculated free concentrations of ADP and AMP. The data demonstrate that elevated plasma adrenaline levels during moderate exercise in untrained men increase skeletal muscle glycogen breakdown and PDH activation, which results in greater carbohydrate oxidation. The greater muscle glycogenolysis appears to be due to increased glycogen phosphorylase transformation whilst the increased PDH activity cannot be readily explained. Finally, the decreased glucose uptake observed during exercise in ADR is likely to be due to the increased intracellular G-6-P and a subsequent decrease in glucose phosphorylation. PMID:11433007

  3. 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

  4. 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

  5. Detection of amount and activity of living algae in fresh water by dehydrogenase activity (DHA).

    PubMed

    Xie, Jun; Hu, Wenrong; Pei, Haiyan; Dun, Mina; Qi, Feng

    2008-11-01

    A study was performed to determine the amount and activity of living algae in fresh water by measuring the dehydrogenase activity (DHA) of algae in order to provide a method to assess the effect of algicide treatment. The conditions of measurement were researched with respect to incubating temperature and duration, and selection of extractants. The comparison between this method and an alternative method, chlorophyll a, shows that this method is simple and easy to practice, and can determine the effect of algicide treatment.

  6. Three overlapping lct genes involved in L-lactate utilization by Escherichia coli.

    PubMed Central

    Dong, J M; Taylor, J S; Latour, D J; Iuchi, S; Lin, E C

    1993-01-01

    In Escherichia coli, the lct locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the lct locus contained three overlapping genes in the clockwise order of lctD (encoding a flavin mononucleotide-dependent dehydrogenase), lctR (encoding a putative regulator), and lctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A phi (lctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal. Images PMID:8407843

  7. The BRAF inhibitor vemurafenib activates mitochondrial metabolism and inhibits hyperpolarized pyruvate-lactate exchange in BRAF mutant human melanoma cells

    PubMed Central

    Delgado-Goni, Teresa; Falck Miniotis, Maria; Wantuch, Slawomir; Parkes, Harold G.; Marais, Richard; Workman, Paul; Leach, Martin O.; Beloueche-Babari, Mounia

    2016-01-01

    Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity and potential as non-invasive imaging response biomarkers.1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of HKII, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase and monocarboxylate transporter (MCT) 1 and 4 in BRAF mutant but not BRAFWT cells and, interestingly, decreased BRAF mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as non-invasive imaging of response. PMID:27765851

  8. Fluoride-containing bioactive glasses inhibit pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human osteoblasts.

    PubMed

    Bergandi, Loredana; Aina, Valentina; Garetto, Stefano; Malavasi, Gianluca; Aldieri, Elisabetta; Laurenti, Enzo; Matera, Lina; Morterra, Claudio; Ghigo, Dario

    2010-02-12

    Bioactive glasses such as Hench's 45S5 (Bioglass) have applications to tissue engineering as well as bone repair, and the insertion of fluoride in their composition has been proposed to enhance their bioactivity. In view of a potential clinical application, we investigated whether fluoride-containing glasses exert toxic effects on human MG-63 osteoblasts, and whether and how fluoride, which is released in the cell culture medium, might play a role in such cytotoxicity. A 24h incubation with 50 microg/ml (12.5 microg/cm(2)) of fluoride-containing bioactive glasses termed HCaCaF(2) (F content: 5, 10 and 15 mol.%) caused the release of lactate dehydrogenase in the extracellular medium (index of cytotoxicity), the accumulation of intracellular malonyldialdehyde (index of lipoperoxidation), and the increase of glutathione consumption. Furthermore, fluoride-containing glasses inhibited the pentose phosphate oxidative pathway and the glucose 6-phosphate dehydrogenase activity. These effects are ascribable to the fluoride content/release of glass powders, since they were mimicked by NaF solutions and were prevented by dimethyl sulfoxide and tempol (two radical scavengers), by superoxide dismutase (a superoxide scavenger), and by glutathione (the most important intracellular antioxidant molecule), but not by apocynin (an inhibitor of NADPH oxidase). The presence of fluoride-containing glasses and NaF caused also the generation of reactive oxygen species, which was prevented by superoxide dismutase and catalase. The data suggest that fluoride released from glasses is the cause of MG-63 cell oxidative damage and is independent of NADPH oxidase activation. Our data provide a new mechanism to explain F(-) ions toxicity: fluoride could trigger, at least in part, an oxidative stress via inhibition of the pentose phosphate oxidative pathway and, in particular, through the oxidative inhibition of glucose 6-phosphate dehydrogenase.

  9. 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

  10. Testis-Specific Lactate Dehydrogenase (LDH-C4) in Skeletal Muscle Enhances a Pika’s Sprint-Running Capacity in Hypoxic Environment

    PubMed Central

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

    2015-01-01

    LDH-C4 is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, ldh-c was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living 3000–5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testis and sperm but also in somatic tissues of plateau pika. In this study, the effects of N-propyl oxamate and N-isopropyl oxamate on LDH isozyme kinetics were compared to screens for a selective inhibitor of LDH-C4. To reveal the role and physiological mechanism of LDH-C4 in skeletal muscle of plateau pika, we investigated the effect of N-isopropyl oxamate on the pika exercise tolerance as well as the physiological mechanism. Our results show that Ki of N-propyl oxamate and N-isopropyl oxamate for LDH-A4, LDH-B4, and LDH-C4 were 0.094 mmol/L and 0.462 mmol/L, 0.119 mmol/L and 0.248 mmol/L, and 0.015 mmol/L and 0.013 mmol/L, respectively. N-isopropyl oxamate is a powerful selective inhibitor of plateau pika LDH-C4. In our exercise tolerance experiment, groups treated with inhibitors had significantly lower swimming times than the uninhibited control group. The inhibition rates of LDH, LD, and ATP were 37.12%, 66.27%, and 32.42%, respectively. Our results suggested that ldh-c is expressed in the skeletal muscle of plateau pika, and at least 32.42% of ATP in the skeletal muscle is catalyzed by LDH-C4 by anaerobic glycolysis. This suggests that pika has reduced dependence on oxygen and enhanced adaptation to hypoxic environment due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle. LDH-C4 in plateau pika plays the crucial role in anaerobic glycolysis and generates ATP rapidly since this is the role of LDH-A4 in most species on plain land, which provide evidence that the native humans and animals in Qinghai

  11. 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.

  12. 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

  13. Control of glycolytic flux in Zymomonas mobilis by glucose 6-phosphate dehydrogenase activity

    SciTech Connect

    Snoep, J.L. |; Arfman, N.; Yomano, L.P.; Ingram, L.O.; Westerhoff, H.V.; Conway, T.

    1996-07-20

    Alycolytic genes in Zymomonas mobilis are highly expressed and constitute half of the cytoplasmic protein. The first four genes (glf, zwf, edd, glk) in this pathway form an operon encoding a glucose permease, glucose 6-phosphate dehydrogenase (G6-P dehydrogenase), 6-phosphogluconate dehydratase, and glucokinase, respectively. Each gene was overexpressed from a tac promoter to investigate the control of glycolysis during the early stages of batch fermentation when flux (qCO{sub 2}) is highest. Almost half of flux control appears to reside with G6-P dehydrogenase (C{sub G6-P dehydrogenase}{sup J} = 0.4). Although Z. mobilis exhibits one of the highest rates of glycolysis known, recombinants with elevated G6-P dehydrogenase had a 10% to 13% higher glycolytic flux than the native organism. A small increase in flux was also observed for recombinants expressing glf. Results obtained did not allow a critical evaluation of glucokinase and this enzyme may also represent an important control point. 6-Phosphogluconate dehydratase appears to be saturating at native levels. With constructs containing the full operon, growth rate and flux were both reduced, complicating interpretations. However, results obtained were also consistent with G6-P dehydrogenase as a primary site of control. Flux was 17% higher in operon constructs which exhibited a 17% increase in G6-P dehydrogenase specific activity, relative to the average of other operon constructs which contain a frameshift mutation in zwf.

  14. Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotide) in chemotherapy-naive patients with advanced melanoma and low-normal serum lactate dehydrogenase: 'The AGENDA trial'.

    PubMed

    Bedikian, Agop Y; Garbe, Claus; Conry, Robert; Lebbe, Celeste; Grob, Jean J

    2014-06-01

    In a previous large randomized, open-label study, retrospective subset analysis revealed that the addition of the Bcl-2 antisense oligonucleotide oblimersen to dacarbazine (Dac) significantly improved overall survival, progression-free survival, and the response rate in chemotherapy-naive patients with advanced melanoma and normal baseline serum lactate dehydrogenase (LDH) levels. To confirm and expand on this observation, we conducted a prospective double-blind, placebo-controlled study to determine whether oblimersen augmented the efficacy of Dac in advanced melanoma patients with low-normal baseline LDH levels. A total of 314 chemotherapy-naive patients were randomly assigned to receive Dac (1000 mg/m(2)) preceded by a 5-day continuous intravenous infusion of either oblimersen sodium (7 mg/kg/day) or placebo every 21 days for less than eight cycles. Co-primary efficacy endpoints were overall survival and progression-free survival. Response and progression of the disease were assessed by independent blinded review of computed tomography scan images. No difference in overall nor progression-free survival was observed between the Dac-oblimersen and Dac-placebo groups. Although the overall (17.2 vs. 12.1%) and durable (10.8 vs. 7.6%) response rates numerically favored Dac-oblimersen over Dac-placebo, they did not differ significantly (P=0.19 and 0.32, respectively). The incidence of hematologic adverse events, particularly thrombocytopenia and neutropenia, was higher in the Dac-oblimersen group than in the Dac-placebo group. Withdrawals from the study because of treatment-related adverse events were low (i.e. <2.5%) in both groups. The addition of oblimersen to Dac did not significantly improve overall survival nor progression-free survival in patients with advanced melanoma and low-normal levels of LDH at baseline.

  15. Pretreatment Serum Lactate Dehydrogenase Level as an Independent Prognostic Factor of Nasopharyngeal Carcinoma in the Intensity-Modulated Radiation Therapy Era

    PubMed Central

    Chen, Zhuhong; Guo, Qiaojuan; Lu, Tianzhu; Lin, Shaojun; Zong, Jingfeng; Zhan, Shenghua; Xu, Luying; Pan, Jianji

    2017-01-01

    Background The aims of this study were to analyze the prognostic value of baseline lactate dehydrogenase (LDH) among nasopharyngeal carcinoma (NPC) patients treated with intensity-modulated radiation therapy (IMRT), and to evaluate the potential application of LDH in monitoring treatment efficacy dynamically. Material/Methods From June 2005 to December 2010, 1188 patients with non-metastatic NPC who underwent IMRT with or without chemotherapy were reviewed. Univariate and multivariate analyses were performed to evaluate the predictive value of baseline LDH. Wilcoxon signed-rank test was used to analyze the difference between baseline and post-radiotherapy LDH, and to compare post-radiotherapy LDH with the LDH in cases of distant failure. Results Patients with elevated LDH had significant inferior survival rates, in terms of overall survival (70.0% vs. 83.2%, p=0.010), disease-specific survival (71.1% vs. 85.7%, p=0.002), and distant metastasis-free survival (71.1% vs. 83.4%, p=0.009), but not correlated with locoregional relapse-free survival (p=0.275) or progression-free survival (p=0.104). Subgroup analysis demonstrated that this predictive effect was more significant with advanced stage. Sixty-five post-radiotherapy LDH levels were available from the 90 patients with high LDH at initial diagnosis, and these levels fell in 65 patients, with 62 cases (95.4%) falling within the normal range. Of the 208 patients who experienced distant metastasis, 87 had an available LDH level at that time. Among them, 69 cases (79.3%) had an increased level compared with the post-radiotherapy LDH level. Conclusions Pretreatment LDH is a simple, cost-effective biomarker that could predict survival rates and might be used in individualized treatment. It is also a potential biomarker that might reflect tumor burden and be used to monitor therapy efficacy. PMID:28120819

  16. 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.

  17. Effects of Sesame (Sesamum indicum L.) Supplementation on Creatine Kinase, Lactate Dehydrogenase, Oxidative Stress Markers, and Aerobic Capacity in Semi-Professional Soccer Players

    PubMed Central

    Barbosa, Carlos V. da Silva; Silva, Alexandre S.; de Oliveira, Caio V. C.; Massa, Nayara M. L.; de Sousa, Yasmim R. F.; da Costa, Whyara K. A.; Silva, Ayice C.; Delatorre, Plínio; Carvalho, Rhayane; Braga, Valdir de Andrade; Magnani, Marciane

    2017-01-01

    Nutritional intervention with antioxidants rich foods has been considered a strategy to minimize the effects of overtraining in athletes. This experimental, randomized, and placebo-controlled study evaluated the effects of consumption of sesame (Sesamum indicum L.) on muscle damage markers, oxidative stress, systemic inflammation, and aerobic performance in male semi-professional soccer players. Twenty athletes were randomly assigned to groups that received 40 g (two tablespoons) per day of sesame or a placebo during 28 days of regular training (exposed to routine training that includes loads of heavy training in the final half of the season). Before and after intervention, creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), C-reactive protein (hs-CRP), and aerobic capacity were evaluated. Before intervention, a physiologic imbalance was noted in both groups related to CK and LDH levels. Sesame intake caused a reduction of CK (19%, p < 0.05), LDH (37%, p < 0.05), MDA (55%, p < 0.05) and hs-CRP (53%, p < 0.05) and increased SOD (14%, p < 0.05), vitamin A (25%, p < 0.05), and vitamin E (65%, p < 0.05) in the experimental group. These phenomena were accompanied by increased aerobic capacity (17%, p < 0.05). The placebo group showed an increase in CK (5%, p < 0.05) and no significant change in LDH, SOD or vitamin A. MDA levels decreased (21%, p < 0.05) and vitamin E increased (14%, p < 0.05) in the placebo group, but to a much lesser extent than in the experimental group. These results show that sesame consumption may reduce muscle damage and oxidative stress while improving the aerobic capacity in soccer players.

  18. Lactate dehydrogenase is a prognostic indicator in patients with hepatocellular carcinoma treated by sorafenib: results from the real life practice in HBV endemic area

    PubMed Central

    Li, Mu-xing; Zhao, Hong; Bi, Xin-yu; Li, Zhi-yu; Yao, Xue-song; Li, Huai; Huang, Zhen; Han, Yue; Zhou, Jian-guo; Zhao, Jian-jun; Zhang, Ye-fan; Zhao, Dong-bin; Cai, Jian-qiang

    2016-01-01

    Purpose Lactate dehydrogenase (LDH), which was an indirect marker of hypoxia, was a potentially prognostic factor in several malignancies. There is a lack of evidence about the prognostic value of serum LDH level in patients with hepatocellular carcinoma (HCC) receiving sorafenib treatment from hepatitis B virus endemic areas. Materials and Methods A total of 119 HBV-related HCC patients treated by sorafenib from a Chinese center were included into the study. They were categorized into 2 groups according to the cut-off value of pre-treatment LDH, which was determined by the time dependent receiver operating characteristics (ROC) curve for the overall survival. The prognostic value of LDH was evaluated. The relationships between LDH and other clinicopathological factors were also assessed. Results The cut-off value was 221 U/L. With a median follow up of 15 (range, 3-73) months, 91 patients reached the endpoint. Multivariate analysis proved that pre-treatment serum LDH level was an independent prognostic factor for both overall survival (OS) and progression-free survival (PFS). For patients whose pre-treatment LDH ≥ 221 U/L, increased LDH value after 3 months of sorafenib treatment predicted inferior OS and PFS. And patients with elevated pre-treatment LDH level predisposed to be featured with lower serum albumin, presence of macroscopic vascular invasion, advanced Child-Pugh class, advanced T category, higher AFP, and higher serum total bilirubin. Conclusions Serum LDH level was a potentially prognostic factor in HCC patients treated by sorafenib in HBV endemic area. More relevant studies with reasonable study design are needed to further strengthen its prognostic value. PMID:27880930

  19. 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.

  20. 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.

  1. Plasticity of hypothalamic dopamine neurons during lactation results in dissociation of electrical activity and release.

    PubMed

    Romanò, Nicola; Yip, Siew H; Hodson, David J; Guillou, Anne; Parnaudeau, Sébastien; Kirk, Siobhan; Tronche, François; Bonnefont, Xavier; Le Tissier, Paul; Bunn, Stephen J; Grattan, Dave R; Mollard, Patrice; Martin, Agnès O

    2013-03-06

    Tuberoinfundibular dopamine (TIDA) neurons are the central regulators of prolactin (PRL) secretion. Their extensive functional plasticity allows a change from low PRL secretion in the non-pregnant state to the condition of hyperprolactinemia that characterizes lactation. To allow this rise in PRL, TIDA neurons are thought to become unresponsive to PRL at lactation and functionally silenced. Here we show that, contrary to expectations, the electrical properties of the system were not modified during lactation and that the neurons remained electrically responsive to a PRL stimulus, with PRL inducing an acute increase in their firing rate during lactation that was identical to that seen in non-pregnant mice. Furthermore, we show a long-term organization of TIDA neuron electrical activity with an harmonization of their firing rates, which remains intact during lactation. However, PRL-induced secretion of dopamine (DA) at the median eminence was strongly blunted during lactation, at least in part attributable to lack of phosphorylation of tyrosine hydroxylase, the key enzyme involved in DA synthesis. We therefore conclude that lactation, rather than involving electrical silencing of TIDA neurons, represents a condition of decoupling between electrical activity at the cell body and DA secretion at the median eminence.

  2. Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

    PubMed

    Hesberg, Christine; Hänsch, Robert; Mendel, Ralf R; Bittner, Florian

    2004-04-02

    Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

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

    PubMed Central

    Kristan, Katja; Deluca, Dominga; Adamski, Jerzy; Stojan, Jure; Rižner, Tea Lanišnik

    2005-01-01

    Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor. PMID:16359545

  4. [The role of hepatic and erythrocyte aldehyde dehydrogenase in the development of burn toxemia in rats].

    PubMed

    Solov'eva, A G

    2009-01-01

    The study was designed to examine catalytic properties of non-specific aldehyde dehydrogenase from rat liver and erythrocyte as the main markers of endogenous intoxication after burn. Enzymatic activity was assayed from changes in the rate of NADH synthesis during acetaldehyde oxidation. Burn was shown to decrease it both in the liver and in erythrocytes which resulted in the accumulation of toxic aldehydes and the development of intoxication. Simultaneous fall in alcohol dehydrogenase and lactate dehydrogenase activities is supposed to contribute to the decrease of aldehyde dehydrogenase activity as a result of thermal injury.

  5. INFLUENCE OF MODERATE TEMPERATURE ON GROWTH AND MALIC DEHYDROGENASE ACTIVITY OF A MARINE PSYCHROPHILE.

    PubMed

    MORITA, R Y; BURTON, S D

    1963-11-01

    Morita, Richard Y. (Oregon State University, Corvallis), and Sheril D. Burton. Influence of moderate temperature on growth and malic dehydrogenase activity of a marine psychrophile. J. Bacteriol. 86:1025-1029. 1963.-The maximal and optimal growth temperatures for a marine psychrophilic vibrio (PS 207) were determined to be 30 and 24.5 C, respectively. Malic dehydrogenase was found to be functioning in whole cells at about 1/20 of its observed maximum. Incubation of the cells, prior to or during the assay, at temperatures above the maximal growth temperature permitted the malic dehydrogenase to operate nearer its maximum, but this also inactivated the intracellular enzyme. The heating of whole cells gave an apparent effect of increasing malic dehydrogenase activity. Lysis of the cells permitted the enzyme to function at its full potential but rendered the enzyme more sensitive to heat denaturation. Lysis of the cells also caused the enzyme to lose approximately one-half of its malic dehydrogenase activity with each 10 C drop in temperature, whereas whole cells only lose approximately 1/5 of their enzyme activity at low temperatures with each 10 C drop.

  6. Metabolic flux control at the pyruvate node in an anaerobic Escherichia coli strain with an active pyruvate dehydrogenase.

    PubMed

    Wang, Qingzhao; Ou, Mark S; Kim, Y; Ingram, L O; Shanmugam, K T

    2010-04-01

    During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y(ATP)) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y(ATP) suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

  7. Diagnostic test performance of somatic cell count, lactate dehydrogenase, and N-acetyl-β-D-glucosaminidase for detecting dairy cows with intramammary infection.

    PubMed

    Nyman, A-K; Emanuelson, U; Waller, K Persson

    2016-02-01

    The main objective of this study was to investigate the diagnostic test performance of somatic cell count (SCC), lactate dehydrogenase (LDH), and N-acetyl-β-D-glucosaminidase (NAGase), analyzed in composite test milking samples, for detecting dairy cows with or without intramammary infection (IMI). A second objective was to investigate whether an adjustment of these udder health indicators according to their associations with different influential factors (i.e., parity, days in milk, and season) improved their test performance. Moreover, we wanted to investigate whether test performance of SCC improved if SCC results from previous adjacent test milkings were included in the model. Such test milking data were not available for LDH or NAGase. In this cross-sectional study, quarter milk samples for bacteriological examination were taken from almost 1,000 cows from 25 dairy herds during 3 consecutive days: the day before test milking, the day of test milking, and the day after test milking. From each cow, a composite test milking sample was analyzed for milk composition, SCC, LDH, and NAGase. Among the cows sampled, 485 were IMI negative and 256 were IMI positive in one or more udder quarters according to the definitions used. The remaining cows had inconclusive IMI status. To assess the test performance of SCC, LDH, and NAGase to identify IMI-negative and IMI-positive cows, univariable generalized estimating equation models were used with the udder health indicator of interest as outcome and IMI status as explanatory variable. From these models, receiver-operator characteristic curves were created and the area under cure (AUC) was calculated. From each model, a cut-off was chosen for calculations of the sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and accuracy (ACC) for each udder health indicator. The AUC was similar for the adjusted SCC (0.84), nonadjusted SCC (0.83) and geometric mean SCC (0.80-0.81), but

  8. Toward "homolactic" fermentation of glucose and xylose by engineered Saccharomyces cerevisiae harboring a kinetically efficient l-lactate dehydrogenase within pdc1-pdc5 deletion background.

    PubMed

    Novy, Vera; Brunner, Bernd; Müller, Gerdt; Nidetzky, Bernd

    2017-01-01

    l-Lactic acid is an important platform chemical and its production from the lignocellulosic sugars glucose and xylose is, therefore, of high interest. Tolerance to low pH and a generally high robustness make Saccharomyces cerevisiae a promising host for l-lactic acid fermentation but strain development for effective utilization of both sugars is an unsolved problem. The herein used S. cerevisiae strain IBB10B05 incorporates a NADH-dependent pathway for oxidoreductive xylose assimilation within CEN.PK113-7D background and was additionally evolved for accelerated xylose-to-ethanol fermentation. Selecting the Plasmodium falciparum l-lactate dehydrogenase (pfLDH) for its high kinetic efficiency, strain IBB14LA1 was derived from IBB10B05 by placing the pfldh gene at the pdc1 locus under control of the pdc1 promotor. Strain IBB14LA1_5 additionally had the pdc5 gene disrupted. With both strains, continued l-lactic acid formation from glucose or xylose, each at 50 g/L, necessitated stabilization of pH. Using calcium carbonate (11 g/L), anaerobic shaken bottle fermentations at pH ≥ 5 resulted in l-lactic acid yields (YLA ) of 0.67 g/g glucose and 0.80 g/g xylose for strain IBB14LA1_5. Only little xylitol was formed (≤0.08 g/g) and no ethanol. In pH stabilized aerobic conversions of glucose, strain IBB14LA1_5 further showed excellent l-lactic acid productivities (1.8 g/L/h) without losses in YLA (0.69 g/g glucose). In strain IBB14LA1, the YLA was lower (≤0.18 g/g glucose; ≤0.27 g/g xylose) due to ethanol as well as xylitol formation. Therefore, this study shows that a S. cerevisiae strain originally optimized for xylose-to-ethanol fermentation was useful to implement l-lactic acid production from glucose and xylose; and with the metabolic engineering strategy applied, advance toward homolactic fermentation of both sugars was made. Biotechnol. Bioeng. 2017;114: 163-171. © 2016 Wiley Periodicals, Inc.

  9. In vivo measurement of aldehyde dehydrogenase-2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1-(13) C]pyruvate.

    PubMed

    Josan, Sonal; Xu, Tao; Yen, Yi-Fen; Hurd, Ralph; Ferreira, Julio; Chen, Che-Hong; Mochly-Rosen, Daria; Pfefferbaum, Adolf; Mayer, Dirk; Spielman, Daniel

    2013-06-01

    To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized (13) C-labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10 000-fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T1 relaxation times, are generally poor targets for such (13) C-labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1-(13) C]pyruvate and its conversion to [1-(13) C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in (13) C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R(2) = 0.76).

  10. Non-thermal atmospheric pressure plasma activates lactate in Ringer's solution for anti-tumor effects.

    PubMed

    Tanaka, Hiromasa; Nakamura, Kae; Mizuno, Masaaki; Ishikawa, Kenji; Takeda, Keigo; Kajiyama, Hiroaki; Utsumi, Fumi; Kikkawa, Fumitaka; Hori, Masaru

    2016-11-08

    Non-thermal atmospheric pressure plasma is a novel approach for wound healing, blood coagulation, and cancer therapy. A recent discovery in the field of plasma medicine is that non-thermal atmospheric pressure plasma not only directly but also indirectly affects cells via plasma-treated liquids. This discovery has led to the use of non-thermal atmospheric pressure plasma as a novel chemotherapy. We refer to these plasma-treated liquids as plasma-activated liquids. We chose Ringer's solutions to produce plasma-activated liquids for clinical applications. In vitro and in vivo experiments demonstrated that plasma-activated Ringer's lactate solution has anti-tumor effects, but of the four components in Ringer's lactate solution, only lactate exhibited anti-tumor effects through activation by non-thermal plasma. Nuclear magnetic resonance analyses indicate that plasma irradiation generates acetyl and pyruvic acid-like groups in Ringer's lactate solution. Overall, these results suggest that plasma-activated Ringer's lactate solution is promising for chemotherapy.

  11. Lactate Activates HIF-1 in Oxidative but Not in Warburg-Phenotype Human Tumor Cells

    PubMed Central

    De Saedeleer, Christophe J.; Copetti, Tamara; Porporato, Paolo E.; Verrax, Julien

    2012-01-01

    Cancer can be envisioned as a metabolic disease driven by pressure selection and intercellular cooperativeness. Together with anaerobic glycolysis, the Warburg effect, formally corresponding to uncoupling glycolysis from oxidative phosphorylation, directly participates in cancer aggressiveness, supporting both tumor progression and dissemination. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key contributor to glycolysis. It stimulates the expression of glycolytic transporters and enzymes supporting high rate of glycolysis. In this study, we addressed the reverse possibility of a metabolic control of HIF-1 in tumor cells. We report that lactate, the end-product of glycolysis, inhibits prolylhydroxylase 2 activity and activates HIF-1 in normoxic oxidative tumor cells but not in Warburg-phenotype tumor cells which also expressed lower basal levels of HIF-1α. These data were confirmed using genotypically matched oxidative and mitochondria-depleted glycolytic tumor cells as well as several different wild-type human tumor cell lines of either metabolic phenotype. Lactate activates HIF-1 and triggers tumor angiogenesis and tumor growth in vivo, an activity that we found to be under the specific upstream control of the lactate transporter monocarboxylate transporter 1 (MCT1) expressed in tumor cells. Because MCT1 also gates lactate-fueled tumor cell respiration and mediates pro-angiogenic lactate signaling in endothelial cells, MCT1 inhibition is confirmed as an attractive anticancer strategy in which a single drug may target multiple tumor-promoting pathways. PMID:23082126

  12. [Lipid peroxidation processes and activity of brain succinate dehydrogenase in experimental craniocerebral trauma].

    PubMed

    Demchuk, M L; Medvedev, A E; Promyslov, M Sh; Gorkin, V Z

    1993-01-01

    A statistically significant decrease in the activity of succinate dehydrogenase (SDH) was found in the rabbit brain after craniocerebral injury. The decrease in the activity of brain SDH was not shown to result from "competitive inhibition" by malonate accumulated after activation of lipid peroxidation. The activity of brain SDH was normalized by directed modification of the function of the central nervous system via administration of phenamine (amphetamine) into the injured animals.

  13. INT-dehydrogenase activity test for assessing anaerobic biodegradability of organic compounds.

    PubMed

    Hongwei, Yang; Zhanpeng, Jiang; Shaoqi, Shi; Tang, W Z

    2002-11-01

    This study assessed anaerobic biodegradability of organic compounds from microorganism activity. Dehydrogenase activity can be a good parameter characterizing the microorganism activity. A modified method of 2-(p-iodophenyl-3-(p-nitrophenyl)-5-pheny tetrazolium chloride-dehydrogenase activity determination was proposed in anaerobic biodegradability assessment. Cubic spline curves were adopted to link the data points. This curve was integrated twice to calculate areas. The microorganism activity index in anaerobic biodegradability assessment was calculated by standardizing the integral. According to the results of the activity index, 14 kinds of organic compounds were classified into readily, partially, and poorly biodegradable under anaerobic conditions, respectively. As a result, some conclusions for anaerobic biodegradability of organic compounds were reached, based on the activity index value.

  14. 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

  15. Green tea extract intake during lactation modified cardiac macrophage infiltration and AMP-activated protein kinase phosphorylation in weanling rats from undernourished mother during gestation and lactation.

    PubMed

    Matsumoto, E; Kataoka, S; Mukai, Y; Sato, M; Sato, S

    2017-04-01

    Maternal dietary restriction is often associated with cardiovascular disease in offspring. The aim of this study was to investigate the effect of green tea extract (GTE) intake during lactation on macrophage infiltration, and activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and serine-threonine kinase Akt (Akt) in the hearts of weanlings exposed to maternal dietary protein restriction. Pregnant Wistar rats were fed control (C) or low-protein diets (LP) throughout gestation. Following delivery, the dams received a control or a GTE-containing control diet during lactation: control diet during gestation and lactation (CC), low-protein diet during gestation and lactation (LPC), low-protein diet during gestation and 0.12% GTE-containing low-protein diet during lactation (LPL), and low-protein diet during gestation and 0.24% GTE-containing low-protein diet during lactation (LPH). The female offspring were sacrificed at day 22. Biochemical parameters in the plasma, macrophage infiltration, degree of fibrosis and expression levels of AMPK and Akt were examined. The plasma insulin level increased in LPH compared with LPC. Percentage of the fibrotic areas and the number of macrophages in LPC were higher than those in CC. Conversely, the fibrotic areas and the macrophage number in LPH were smaller (21 and 56%, respectively) than those in LPC. The levels of phosphorylated AMPK in LPL and LPH, and Akt in LPH were greater than those in LPC. In conclusion, maternal protein restriction may induce macrophage infiltration and the decrease of insulin levels. However, GTE intake during lactation may suppress macrophage infiltration and restore insulin secretion function via upregulation of AMPK and insulin signaling in weanlings.

  16. 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

  17. Evidences of basal lactate production in the main white adipose tissue sites of rats. Effects of sex and a cafeteria diet.

    PubMed

    Arriarán, Sofía; Agnelli, Silvia; Sabater, David; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2015-01-01

    Female and male adult Wistar rats were fed standard chow or a simplified cafeteria diet for one month. Then, the rats were killed and the white adipose tissue (WAT) in four sites: perigonadal, retroperitoneal, mesenteric and subcutaneous (inguinal) were sampled and frozen. The complete WAT weight in each site was measured. Gene expression analysis of key lipid and glucose metabolism enzymes were analyzed, as well as tissue and plasma lactate and the activity of lactate dehydrogenase. Lactate gradients between WAT and plasma were estimated. The influence of sex and diet (and indirectly WAT mass) on lactate levels and their relationships with lactate dehydrogenase activity and gene expressions were also measured. A main conclusion is the high production of lactate by WAT, practically irrespective of site, diet or sex. Lactate production is a direct correlate of lactate dehydrogenase activity in the tissue. Furthermore, lactate dehydrogenase activity is again directly correlated with the expression of the genes Ldha and Ldhb for this enzyme. In sum, the ability to produce lactate by WAT is not directly dependent of WAT metabolic state. We postulate that, in WAT, a main function of the lactate dehydrogenase path may be that of converting excess available glucose to 3C fragments, as a way to limit tissue self-utilization as substrate, to help control glycaemia and/or providing short chain substrates for use as energy source elsewhere. More information must be gathered before a conclusive role of WAT in the control of glycaemia, and the full existence of a renewed glucose-lactate-fatty acid cycle is definitely established.

  18. Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone.

    PubMed

    Mezey, E; Potter, J J; Diehl, A M

    1986-01-15

    Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.

  19. Geldanamycin Prevents Hemorrhage-Induced ATP Loss by Overexpressing Inducible HSP70 and Activating Pyruvate Dehydrogenase

    DTIC Science & Technology

    2006-03-24

    levels were determined using the ATP Bioluminescence Assay Kit HS II (Roche; Mannheim, Germany). Luminescence was measured with a TD-20/20...Geldanamycin prevents hemorrhage-induced ATP loss by overexpressing inducible HSP70 and activating pyruvate dehydrogenase Juliann G. Kiang,1,2,3...Geldanamycin prevents hemorrhage-induced ATP loss by overexpressing inducible HSP70 and activating pyruvate dehy- drogenase. Am J Physiol Gastrointest

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

    PubMed

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

    2014-10-20

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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. Pattern of γ-glutamyl transferase activity in cow milk throughout lactation and relationships with metabolic conditions and milk composition.

    PubMed

    Calamari, L; Gobbi, L; Russo, F; Cappelli, F Piccioli

    2015-08-01

    The main objective of this experiment was to study the γ-glutamyl transferase (GGT) activity in milk during lactation and its relationship with metabolic status of dairy cows, milk yield, milk composition, and cheesemaking properties. The study was performed in a tied stall barn and involved 20 lactations from 12 healthy multiparous Italian Friesian dairy cows. During lactation starting at d 10, milk samples were collected weekly and analyzed for composition, somatic cells count, titratable acidity, and milk coagulation properties. The GGT activity was measured in defatted samples. Blood samples were collected weekly to assess biochemical indicators related to energy, protein, and mineral metabolism, markers of inflammation and some enzyme activities. The lactations of each cow were retrospectively categorized into 2 groups according to their milk GGT activity value through lactation. A median value of GGT activity in the milk of all lactations was calculated (3,045 U/L), and 10 lactations with lower GGT activity were classified as low while 10 lactations with greater GGT activity were classified as high. The average value of milk GGT activity during lactation was 3,863 and 3,024 U/L for high and low, respectively. The GGT activity decreased in early lactation and reached minimum values in the second month (3,289 and 2,355 U/L for high and low, respectively). Thereafter GGT activity increased progressively, reaching values in late lactation of 4,511 and 3,540 U/L in high and low, respectively. On average, milk yield was 40.81 and 42.76 kg/d in high and low, respectively, and a negative partial correlation with milk GGT activity was observed. A greater milk protein concentration was observed in high (3.39%) compared with low (3.18%), and a positive partial correlation with milk GGT activity was observed. Greater titratable acidity in high than that in low (3.75 vs. 3.45 degrees Soxhlet-Henkel/50 mL, respectively) was also observed. Plasma glucose was greater in

  6. Effects of Active Recovery on Lactate Concentration, Heart Rate and RPE in Climbing

    PubMed Central

    Draper, Nick; Bird, Ellis L.; Coleman, Ian; Hodgson, Chris

    2006-01-01

    The performance advantage of active rather than passive recovery during subsequent trials for repeated high intensity short-term exercise is well documented. Research findings suggest that shorter periods of active recovery, than traditionally employed, can be prescribed and still retain performance benefits over passive recoveries in successive exercise trials. The aim of this study was to examine the benefits of a short duration active recovery for repeat climbing trials. Ten recreational climbers volunteered for the study. In this randomly assigned crossover study each climber completed five two-minute climbing trails before a two minute active or passive recovery. This was followed by a one and a half minute passive refocusing period for all climbers before the subsequent climbing trial. Heart rate was monitored continuously, RPE immediately post climbing and fingertip capillary blood samples collected during each refocusing phase. There was a non-significant difference between active and passive recoveries for heart rate during climbing. After the active phase climbers had higher heart rates than when following the passive recovery protocol, however, by the end of the refocusing phase the active recovery protocol led to lower heart rates than for the entirely passive recovery. There was a significant difference between active and passive recovery conditions in lactate concentration (F(1,9) = 18.79, p = 0.002) and RPE (F(1,9) = 6.51, p = 0.031). Lactate concentration and RPE were lower across all five climbing trials for the active recovery protocol. After active recovery climbers started the next trial with a lower arterial lactate concentration than for a passive recovery and indicated lower RPE scores at the end of each climb. The refocusing period following active recovery allowed climbers heart rates to return to a lower level at the start of the next climb than for the passive recovery condition. Key Points The three and half minute recovery strategy

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

    PubMed

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

    2012-06-01

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

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

    PubMed

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

    2007-02-01

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

  9. Histochemical modification of the active site of succinate dehydrogenase with N-acetylimidazole.

    PubMed

    Nakae, Y; Shono, M

    1986-04-01

    The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.

  10. Differential contribution of hypothalamic MAPK activity to anxiety-like behaviour in virgin and lactating rats.

    PubMed

    Jurek, Benjamin; Slattery, David A; Maloumby, Rodrigue; Hillerer, Katharina; Koszinowski, Sophie; Neumann, Inga D; van den Burg, Erwin H

    2012-01-01

    The c-Raf - MEK1/2 - ERK1/2 mitogen-activated protein kinase (MAPK) intracellular signalling cascade in neurons plays important roles in the control of a variety of behaviours, including social behaviours and anxiety. These roles partially overlap with those described for oxytocin (OXT), and it has been shown that OXT activates the MAPK pathway in the hypothalamus (of male), and hippocampus (of female) rats. Here, by combining behavioural (light/dark box) and biochemical analyses (western blotting), we tested two hypotheses: (i) that OXT is anxiolytic within the hypothalamus of females, and (ii) that this effect, as well as that of lactation-associated anxiolysis, depends on the recruitment of the MAPK pathway. We found that, when injected bilaterally into the hypothalamic paraventricular nucleus (PVN), OXT decreased anxiety-like behaviour in virgins, and that this effect depended on phosphorylation of MEK1/2. MAPK pathway activation in lactation was evident by high phosphorylated (p) MEK1/2 levels, and nuclear translocation of ERK1. The high pMEK1/2 levels were necessary for the anxiolytic phenotype typically observed during lactation. Interestingly, exogenous OXT in lactating rats reduced pMEK1/2 levels without a concomitant effect on anxiety, indicating that OXT receptor activation can lead to recruitment of additional intracellular pathways to modulate MEK activity. Still other pathways could include MEK, but without subsequent activation of ERK, as we did not observe any increase in OXT-induced ERK phosphorylation. Together the results demonstrate that the MAPK pathway, especially MEK1/2, is critically involved in the regulation of anxiety-like behaviour in female rats.

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

    PubMed

    Jyot, Gagan; Mandal, Kousik; Singh, Balwinder

    2015-05-01

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

  12. Serotonin Regulates Calcium Homeostasis in Lactation by Epigenetic Activation of Hedgehog Signaling

    PubMed Central

    Laporta, Jimena; Keil, Kimberly P.; Weaver, Samantha R.; Cronick, Callyssa M.; Prichard, Austin P.; Crenshaw, Thomas D.; Heyne, Galen W.; Vezina, Chad M.; Lipinski, Robert J.

    2014-01-01

    Calcium homeostasis during lactation is critical for maternal and neonatal health. We previously showed that nonneuronal/peripheral serotonin [5-hydroxytryptamine (5-HT)] causes the lactating mammary gland to synthesize and secrete PTHrP in an acute fashion. Here, using a mouse model, we found that genetic inactivation of tryptophan hydroxylase 1 (Tph1), which catalyzes the rate-limiting step in peripheral 5-HT synthesis, reduced circulating and mammary PTHrP expression, osteoclast activity, and maternal circulating calcium concentrations during the transition from pregnancy to lactation. Tph1 inactivation also reduced sonic hedgehog signaling in the mammary gland during lactation. Each of these deficiencies was rescued by daily injections of 5-hydroxy-L-tryptophan (an immediate precursor of 5-HT) to Tph1-deficient dams. We used immortalized mouse embryonic fibroblasts to demonstrate that 5-HT induces PTHrP through a sonic hedgehog-dependent signal transduction mechanism. We also found that 5-HT altered DNA methylation of the Shh gene locus, leading to transcriptional initiation at an alternate start site and formation of a variant transcript in mouse embryonic fibroblasts in vitro and in mammary tissue in vivo. These results support a new paradigm of 5-HT-mediated Shh regulation involving DNA methylation remodeling and promoter switching. In addition to having immediate implications for lactation biology, identification and characterization of a novel functional regulatory relationship between nonneuronal 5-HT, hedgehog signaling, and PTHrP offers new avenues for the study of these important factors in development and disease. PMID:25192038

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

    PubMed

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

    2014-05-23

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

  14. 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.

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

    PubMed

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

    1989-01-01

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

  16. Metabolic control analysis of L-lactate synthesis pathway in Rhizopus oryzae As 3.2686.

    PubMed

    Ke, Wei; Chang, Shu; Chen, Xiaoju; Luo, Shuizhong; Jiang, Shaotong; Yang, Peizhou; Wu, Xuefeng; Zheng, Zhi

    2015-11-01

    The relationship between the metabolic flux and the activities of the pyruvate branching enzymes of Rhizopus oryzae As 3.2686 during L-lactate fermentation was investigated using the perturbation method of aeration. The control coefficients for five enzymes, pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH), were calculated. Our results indicated significant correlations between PDH and PC, PDC and LDH, PDC and ADH, LDH and ADH, and PDC and PC. It also appeared that PDH, PC, and LDH strongly controlled the L-lactate flux; PDH and ADH strongly controlled the ethanol flux; while PDH and PC strongly controlled the acetyl coenzyme A flux and the oxaloacetate flux. Further, the flux control coefficient curves indicated that the control of the system gradually transferred from PDC to PC during the steady state. Therefore, PC was the key rate-limiting enzyme that controls the flux distribution.

  17. 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

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

    PubMed

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

    1985-01-01

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

  19. 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.

  20. In vitro antibacterial activity of chitosan and chitosan oligosaccharide lactate against important gram negative warmwater fish pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aim: The antibacterial activities of chitosan (CS) and its derivative chitosan oligosaccharide lactate (COL) were evaluated against Aeromonas hydrophila, Edwardsiella ictaluri and Flavobacterium columnare, three highly pathogenic bacteria of warmwater finfish. Methods and Results: The kinetics of ce...

  1. CONVERSION OF LACTATE-C14 TO PROPIONATE BY THE RUMEN MICROFLORA12

    PubMed Central

    Baldwin, R. L.; Wood, W. A.; Emery, R. S.

    1962-01-01

    Baldwin, R. L. (Michigan State University, East Lansing), W. A. Wood, and R. S. Emery. Conversion of lactate-C14 to propionate by the rumen microflora. J. Bacteriol. 83:907–913. 1962.—Rumen microflora enriched on five different diets calculated to present increasing carbohydrate or lactate availability were used to determine the contribution of the randomizing (succinate) and nonrandomizing (acrylate) routes to propionate with lactate-2-C14 and -3-C14 as substrates. Propionate was labeled as though 70 to 90% was formed via the nonrandomizing route. This percentage was highest on diets containing high levels of carbohydrate or lactate or both. Evidence for the presence of succinic dehydrogenase, acetokinase, phosphotransacetylase, and coenzyme A transphorase was obtained with cell-free extracts. Propionate-2-C14 and lactate-2-C14 were converted by extracts to the activated derivatives of acrylate, lactate, propionate, and acetate. PMID:13864343

  2. 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

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

    PubMed

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

    1982-02-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2011-01-01

    The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al13 concentration increase. Our study also found that the effects of Al(III) and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules. PMID:22163924

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

    PubMed

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

    1985-10-01

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

  7. [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.

  8. [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.

  9. Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity.

    PubMed Central

    Bonam, D; Lehman, L; Roberts, G P; Ludden, P W

    1989-01-01

    Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase. Images PMID:2498285

  10. 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

  11. Glutathion peroxidase and glucose-6-phosphate dehydrogenase activities in bovine blood and liver.

    PubMed

    Abd Ellah, Mahmoud Rushdi; Niishimori, Kazuhiro; Goryo, Masanobu; Okada, Keiji; Yasuda, Jun

    2004-10-01

    A total of 46 cattle, including 25 as control, 16 with glycogen degeneration and 5 with severe fatty degeneration were studied. Whole blood and liver tissue specimens were used to measure glutathione peroxidase (GSH-Px) and Glucose-6-Phosphate Dehydrogenase (G6PD) activities. The present study determined the value of these parameters in diagnosing glycogen and fatty degeneration in cattle from the point of the status of antioxidation and lipid peroxidation. The results showed a significant decrease in hepatic GSH-Px activity and a significant increase in hepatic G6PD activity in cases of fatty degeneration. On the other hand, there were no significant changes in erythrocytic and hepatic GSH-Px and G6PD activities in cases of glycogen degeneration. The results indicated lipoperoxidation process in the liver tissues increased in cases of fatty degeneration. Therefore, supplying animals suffering from fatty liver with sufficient quantities of nutrient antioxidants may be valuable when treatment is considered.

  12. Brain regional development of the activity of alpha-ketoglutarate dehydrogenase complex in the rat.

    PubMed

    Buerstatte, C R; Behar, K L; Novotny, E J; Lai, J C

    2000-12-29

    This study was initiated to test the hypothesis that the development of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity, like that of pyruvate dehydrogenase complex, is one of the late developers of tricarboxylic acid (TCA) cycle enzymes. The postnatal development of KGDHC in rat brain exhibits four distinct region-specific patterns. The age-dependent increases in olfactory bulb (OB) and hypothalamus (HYP) form one pattern: low in postnatal days (P) 2 and 4, KGDHC activity rose linearly to attain adult level at P30. The increases in mid-brain (MB) and striatum (ST) constitute a second pattern: being <40% of adult level at P2 and P4, KGDHC activity rose steeply between P10 and P17 and attained adult level by P30. The increases in cerebellum (CB), cerebral cortex (CC), and hippocampus (HIP) form a third pattern: being 25-30% of adult level at P2 and P4, KGDHC activity doubled between P10 and P17 and rose to adult level by P30. KGDHC activity development is unique in pons and medulla (PM): being >60% of the adult level at P2, it rose rapidly to adult level by P10. Thus, KGDHC activity develops earlier in phylogenetically older regions (PM) than in phylogenetically younger regions (CB, CC, HIP). Being lowest in activity among all TCA cycle enzymes, KGDHC activity in any region at any age will exert a limit on the maximum TCA cycle flux therein. The results may have functional and pathophysiological implications in control of brain glucose oxidative metabolism, energy metabolism, and neurotransmitter syntheses.

  13. 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.

  14. E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity

    PubMed Central

    Lacroix, Matthieu; Rodier, Geneviève; Houles, Thibault; Delpech, Hélène; Seyran, Berfin; Gayte, Laurie; Casas, Francois; Pessemesse, Laurence; Heuillet, Maud; Bellvert, Floriant; Portais, Jean-Charles; Berthet, Charlene; Brivet, Michele; Boutron, Audrey; Le Cam, Laurent; Sardet, Claude

    2016-01-01

    The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC. PMID:27621446

  15. SIRT3 DEACETYLATES AND INCREASES PYRUVATE DEHYDROGENASE ACTIVITY IN CANCER CELLS

    PubMed Central

    Wagner, Brett A.; Song, Ha Yong; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R.; Gius, David

    2015-01-01

    Pyruvate dehydrogenase E1 alpha (PDHE1α or PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex (PDC) that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321) and a PDHA1 mutant, mimicking a deacetylated lysine (PDHA1K321R) increases in PDH activity, as compared to the K321 acetylation mimic (PDHA1K321Q) or wild-type PDHA1. Finally, PDHA1K321Q exhibited a more transformed in vitro cellular phenotype as compared to PDHA1K321R. These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyl-lysine suggesting that the acetylome, as well as the kinome, links glycolysis to respiration. PMID:25152236

  16. SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells.

    PubMed

    Ozden, Ozkan; Park, Seong-Hoon; Wagner, Brett A; Yong Song, Ha; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R; Gius, David

    2014-11-01

    Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration.

  17. E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity.

    PubMed

    Lacroix, Matthieu; Rodier, Geneviève; Kirsh, Olivier; Houles, Thibault; Delpech, Hélène; Seyran, Berfin; Gayte, Laurie; Casas, Francois; Pessemesse, Laurence; Heuillet, Maud; Bellvert, Floriant; Portais, Jean-Charles; Berthet, Charlene; Bernex, Florence; Brivet, Michele; Boutron, Audrey; Le Cam, Laurent; Sardet, Claude

    2016-09-27

    The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

  18. Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species.

    PubMed Central

    Lernmark, U.; Gardestrom, P.

    1994-01-01

    Protoplasts from barley (Hordeum vulgare), pea (Pisum sativum), wheat (Triticum aestivum), and spinach (Spinacia oleracea) leaves were fractionated into chloroplast- and mitochondrion-enriched fractions. Pyruvate dehydrogenase complex capacities in mitochondria (mtPDC) and chloroplasts (cpPDC) were measured in appropriate fractions under conditions optimal for each isozyme. The total cellular capacity of PDC was similar in barley and pea but about 50% lower in wheat and spinach. In pea a distribution of 87% mtPDC and 13% cpPDC was found on a cellular basis. In barley, wheat, and spinach the subcellular distribution was the opposite, with about 15% mtPDC and 85% cpPDC. cpPDC activity was constant at about 0.1 nmol cell-1 h-1 in cells from different regions along the developing barley leaf and showed no correlation with developmental patterns of photosynthetic parameters, such as increasing Chl and NADP-glyceraldehyde-3-phosphate dehydrogenase activity. Similarly, the capacity of the mitochondrial isoform did not change during barley leaf development and had a developmental pattern similar to that of citrate synthase and fumarase. Differences in subcellular distribution of PDCs in barley and pea are proposed to be due to differences in regulation, not to changes in isozyme proportions during leaf development or to species-specific differences in phosphorylation state of mtPDC after organelle separation. PMID:12232437

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

    PubMed

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

    2016-06-01

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

  20. 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.

  1. Lactation Consultant

    MedlinePlus

    ... human lactation. Job description Lactation consultants educate women, families, health professionals, and the community about breast feeding and human lactation; facilitate the development of policies which protect, promote, and support breastfeeding; ...

  2. 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).

  3. 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.

  4. Influence of fermentation conditions on specific activity of the enzymes alcohol and aldehyde dehydrogenase from yeasts.

    PubMed

    Mauricio, J C; Ortega, J M

    1993-01-01

    The effects of anaerobic, semi-aerobic and short aeration fermentation conditions and the addition of ergosterol and oleic acid to musts on the specific activity of alcohol and aldehyde dehydrogenase (ADH and ALDH) from two yeast species, Saccharomyces cerevisiae and Torulaspora delbrueckii, were studied. ADH I biosynthesis only occurred during the first few hours of fermentation. ADH II from S. cerevisiae and ALDH-NADP+ from the two yeast species behaved as constitutive enzymes under all fermentation conditions. ADH II from T. delbrueckii was only synthesized in small amounts, and its activity was always lower than in S. cerevisiae, where it was responsible for the termination of alcoholic fermentation during the steady growth phase.

  5. Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines.

    PubMed

    Michishita, M; Akiyoshi, R; Suemizu, H; Nakagawa, T; Sasaki, N; Takemitsu, H; Arai, T; Takahashi, K

    2012-08-01

    Increasing evidence suggests that diverse solid tumours arise from a small population of cells known as cancer stem cells or tumour-initiating cells. Cancer stem cells in several solid tumours are enriched for aldehyde dehydrogenase (ALDH) activity. High levels of ALDH activity (ALDH(high)) were detected in four cell lines derived from canine mammary carcinomas. ALDH(high) cells were enriched in a CD44(+)CD24(-) population having self-renewal capacity. Xenotransplantation into immunodeficient mice demonstrated that 1×10(4) ALDH(high) cells were sufficient for tumour formation in all injected mice, whereas 1×10(4) ALDH(low) cells failed to initiate any tumours. ALDH(high)-derived tumours contained both ALDH(+) and ALDH(-) cells, indicating that these cells had cancer stem cell-like properties.

  6. Mixed lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy.

    PubMed

    Oishi, Yoshimi; Tsukamoto, Hayato; Yokokawa, Takumi; Hirotsu, Keisuke; Shimazu, Mariko; Uchida, Kenji; Tomi, Hironori; Higashida, Kazuhiko; Iwanaka, Nobumasa; Hashimoto, Takeshi

    2015-03-15

    We examined whether a mixed lactate and caffeine compound (LC) could effectively elicit proliferation and differentiation of satellite cells or activate anabolic signals in skeletal muscles. We cultured C2C12 cells with either lactate or LC for 6 h. We found that lactate significantly increased myogenin and follistatin protein levels and phosphorylation of P70S6K while decreasing the levels of myostatin relative to the control. LC significantly increased protein levels of Pax7, MyoD, and Ki67 in addition to myogenin, relative to control. LC also significantly increased follistatin expression relative to control and stimulated phosphorylation of mTOR and P70S6K. In an in vivo study, male F344/DuCrlCrlj rats were assigned to control (Sed, n = 10), exercise (Ex, n = 12), and LC supplementation (LCEx, n = 13) groups. LC was orally administered daily. The LCEx and Ex groups were exercised on a treadmill, running for 30 min at low intensity every other day for 4 wk. The LCEx group experienced a significant increase in the mass of the gastrocnemius (GA) and tibialis anterior (TA) relative to both the Sed and Ex groups. Furthermore, the LCEx group showed a significant increase in the total DNA content of TA compared with the Sed group. The LCEx group experienced a significant increase in myogenin and follistatin expression of GA relative to the Ex group. These results suggest that administration of LC can effectively increase muscle mass concomitant with elevated numbers of myonuclei, even with low-intensity exercise training, via activated satellite cells and anabolic signals.

  7. 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

  8. 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.

  9. Deletion of hexose-6-phosphate dehydrogenase activates the unfolded protein response pathway and induces skeletal myopathy.

    PubMed

    Lavery, Gareth G; Walker, Elizabeth A; Turan, Nil; Rogoff, Daniela; Ryder, Jeffery W; Shelton, John M; Richardson, James A; Falciani, Francesco; White, Perrin C; Stewart, Paul M; Parker, Keith L; McMillan, Daniel R

    2008-03-28

    Hexose-6-phosphate dehydrogenase (H6PD) is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver H6PD is required for the 11-oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1, which converts inactive 11-oxo-glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in type II (fast) fibers, which have increased glycogen content. Here, we show that H6PD null mice develop a severe skeletal myopathy characterized by switching of type II to type I (slow) fibers. Running wheel activity and electrically stimulated force generation in isolated skeletal muscle are both markedly reduced. Affected muscles have normal sarcomeric structure at the electron microscopy level but contain large intrafibrillar membranous vacuoles and abnormal triads indicative of defects in structure and function of the sarcoplasmic reticulum (SR). SR proteins involved in calcium metabolism, including the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), calreticulin, and calsequestrin, show dysregulated expression. Microarray analysis and real-time PCR demonstrate overexpression of genes encoding proteins in the unfolded protein response pathway. We propose that the absence of H6PD induces a progressive myopathy by altering the SR redox state, thereby impairing protein folding and activating the unfolded protein response pathway. These studies thus define a novel metabolic pathway that links ER stress to skeletal muscle integrity and function.

  10. 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.

  11. Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells. A methodological study.

    PubMed

    Hansen, T L; Andersen, H

    1983-01-01

    Through a methodological evaluation, reliable histochemical and biochemical methods for succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells were developed. The histochemical method includes a cleaning of the cultured cells in 1 mM malonate in 0.9% NaCl, air-drying and fixation in acetone (5 min at -20 degrees C), coating of cells with CoQ10 (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37 degrees C in 50 mM succinate and 0.5 mg/ml Nitro BT in 200 mM phosphate buffer, pH 7.6 PMS as an intermediate electron carrier was found inferior to exogenous CoQ10. Both types of cells exhibit equal activity. In the biochemical method homogenizing was performed in 50 mM Tris-HCl buffer, pH 7.5, and 200 mM sucrose. The standard incubation was 2.0 mM INT and 10 mM succinate in 10 mM Tris-HCl buffer, pH 7.5 for 1 h at 37 degrees C. The apparent Km values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I0.5 for malonate was 0.46 mM. Activity in amniotic fluid cells was 18.1 pkat/mg protein and in human skin fibroblasts 20.3 pkat/mg protein. Specificity of the methods was tested by use of a Chinese hamster fibroblast strain B9 known to be succinate dehydrogenase deficient in addition to various control experiments. Congruent results were obtained with the two methods.

  12. 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

  13. Non-thermal atmospheric pressure plasma activates lactate in Ringer’s solution for anti-tumor effects

    PubMed Central

    Tanaka, Hiromasa; Nakamura, Kae; Mizuno, Masaaki; Ishikawa, Kenji; Takeda, Keigo; Kajiyama, Hiroaki; Utsumi, Fumi; Kikkawa, Fumitaka; Hori, Masaru

    2016-01-01

    Non-thermal atmospheric pressure plasma is a novel approach for wound healing, blood coagulation, and cancer therapy. A recent discovery in the field of plasma medicine is that non-thermal atmospheric pressure plasma not only directly but also indirectly affects cells via plasma-treated liquids. This discovery has led to the use of non-thermal atmospheric pressure plasma as a novel chemotherapy. We refer to these plasma-treated liquids as plasma-activated liquids. We chose Ringer’s solutions to produce plasma-activated liquids for clinical applications. In vitro and in vivo experiments demonstrated that plasma-activated Ringer’s lactate solution has anti-tumor effects, but of the four components in Ringer’s lactate solution, only lactate exhibited anti-tumor effects through activation by non-thermal plasma. Nuclear magnetic resonance analyses indicate that plasma irradiation generates acetyl and pyruvic acid-like groups in Ringer’s lactate solution. Overall, these results suggest that plasma-activated Ringer’s lactate solution is promising for chemotherapy. PMID:27824103

  14. Non-thermal atmospheric pressure plasma activates lactate in Ringer’s solution for anti-tumor effects

    NASA Astrophysics Data System (ADS)

    Tanaka, Hiromasa; Nakamura, Kae; Mizuno, Masaaki; Ishikawa, Kenji; Takeda, Keigo; Kajiyama, Hiroaki; Utsumi, Fumi; Kikkawa, Fumitaka; Hori, Masaru

    2016-11-01

    Non-thermal atmospheric pressure plasma is a novel approach for wound healing, blood coagulation, and cancer therapy. A recent discovery in the field of plasma medicine is that non-thermal atmospheric pressure plasma not only directly but also indirectly affects cells via plasma-treated liquids. This discovery has led to the use of non-thermal atmospheric pressure plasma as a novel chemotherapy. We refer to these plasma-treated liquids as plasma-activated liquids. We chose Ringer’s solutions to produce plasma-activated liquids for clinical applications. In vitro and in vivo experiments demonstrated that plasma-activated Ringer’s lactate solution has anti-tumor effects, but of the four components in Ringer’s lactate solution, only lactate exhibited anti-tumor effects through activation by non-thermal plasma. Nuclear magnetic resonance analyses indicate that plasma irradiation generates acetyl and pyruvic acid-like groups in Ringer’s lactate solution. Overall, these results suggest that plasma-activated Ringer’s lactate solution is promising for chemotherapy.

  15. 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.

  16. Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria.

    PubMed

    Tsai, C S; Senior, D J

    1990-04-01

    Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.

  17. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

    DOE PAGES

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; ...

    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

  18. Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease.

    PubMed

    Goldstein, David S; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Kopin, Irwin J; Sharabi, Yehonatan

    2015-04-01

    Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the 'catecholaldehyde hypothesis,' buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 min), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD(+) was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. We report that rotenone, a mitochondrial complex I inhibitor that produces an animal model of Parkinson's disease, increases intracellular levels of the toxic dopamine metabolite 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), via decreased DOPAL metabolism by aldehyde dehydrogenase (ALDH) and decreased vesicular sequestration of cytoplasmic dopamine by the vesicular monoamine transporter (VMAT). The results provide a novel

  19. Nutritional status affects branched-chain oxoacid dehydrogenase activity during exercise in humans.

    PubMed

    Jackman, M L; Gibala, M J; Hultman, E; Graham, T E

    1997-02-01

    We examined the effect of glycogen availability and branched-chain amino acid (BCAA) supplementation on branched-chain oxoacid dehydrogenase (BCOAD) activity during exercise. Six subjects cycled at approximately 75% of their maximal oxygen uptake to exhaustion on three occasions under different preexercise conditions: 1) low muscle glycogen (LOW), 2) low muscle glycogen plus BCAA supplementation (LOW+BCAA), and 3) high muscle glycogen (CON). The LOW trial was performed first, followed by the other two conditions in random order, and biopsies for all trials were obtained at rest, after 15 min of exercise (15 min), and at the point of exhaustion during the LOW trial (49 min). BCOAD activity was not different among the three conditions at rest; however, at 15 min BCOAD activity was higher (P < or = 0.05) for the LOW (31 +/- 5%) and LOW+BCAA (43 +/- 11%) conditions compared with CON (12 +/- 1%). BCOAD activity at 49 min was not different from respective values at 15 min for any condition. These data indicate that BCOAD is rapidly activated during submaximal exercise under conditions associated with low carbohydrate availability. However, there was no relationship between BCOAD activity and glycogen concentration or net glycogenolysis, which suggests that factors other than glycogen availability are important for BCOAD regulation during exercise in humans.

  20. 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

  1. A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase.

    PubMed

    Futer, Olga; Sintchak, Michael D; Caron, Paul R; Nimmesgern, Elmar; DeCenzo, Maureen T; Livingston, David J; Raybuck, Scott A

    2002-01-31

    The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.

  2. 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).

  3. Activation of AMP-activated protein kinase, inhibition of pyruvate dehydrogenase activity, and redistribution of substrate partitioning mediate the acute insulin-sensitizing effects of troglitazone in skeletal muscle cells.

    PubMed

    Fediuc, S; Pimenta, A S; Gaidhu, M P; Ceddia, R B

    2008-05-01

    The aim of this study was to investigate the acute effects of troglitazone on several pathways of glucose and fatty acid (FA) partitioning and the molecular mechanisms involved in these processes in skeletal muscle. Exposure of L6 myotubes to troglitazone for 1 h significantly increased phosphorylation of AMPK and ACC, which was followed by approximately 30% and approximately 60% increases in palmitate oxidation and carnitine palmitoyl transferase-1 (CPT-1) activity, respectively. Troglitazone inhibited basal ( approximately 25%) and insulin-stimulated ( approximately 35%) palmitate uptake but significantly increased basal and insulin-stimulated glucose uptake by approximately 2.2- and 2.7-fold, respectively. Pharmacological inhibition of AMPK completely prevented the effects of troglitazone on palmitate oxidation and glucose uptake. Interestingly, even though troglitazone exerted an insulin sensitizing effect, it reduced basal and insulin-stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to approximately 30%, approximately 60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin-stimulated phosphorylation of Akt(Thr308), Akt(Ser473), and GSK3alpha/beta. Troglitazone also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal ( approximately 3.5-fold) and insulin-stimulated ( approximately 5.5-fold) rates of lactate production by muscle cells. In summary, we provide novel evidence that troglitazone exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism toward lactate production in muscle cells. These effects seem to be at least partially dependent on AMPK activation and may account for potential acute PPAR-gamma-independent anti-diabetic effects of thiazolidinediones in skeletal

  4. 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.

  5. 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.

  6. 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

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

    PubMed Central

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

    2012-01-01

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

  8. 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.

  9. Daily physical activity and blood lactate indices of aerobic fitness in children.

    PubMed Central

    Welsman, J R; Armstrong, N

    1992-01-01

    This study examined the relationship between daily physical activity and aerobic fitness in 11-16-year-olds. Habitual physical activity was assessed in 28 boys (mean(s.d.) age 13.6(1.3) years) and 45 girls (mean(s.d) age 13.7(1.3) years) from minute-by-minute heart rate monitoring during 3 school days. Aerobic fitness was assessed by determining the percentage peak VO2 at blood lactate reference values of 2.5 and 4.0 mmol l-1 during incremental treadmill running. The 4.0 mmol l-1 level occurred at a mean(s.d.) value of 89(7)% peak VO2 in both boys and girls and mean(s.d.) values at the 2.5 mmol l-1 level were 82(9)% peak VO2 in girls. Mean(s.d.) percentage time with heart rates at or above 140 beats min-1 was 6(3)% in boys and 5(3)% in girls. Corresponding values for percentage time at or above 160 beats min-1 were 3(2) for boys and 2(1) for girls. The number of 10- and 20-min periods of activity with the heart rate sustained above the 140 and 160 beats min-1 thresholds were also totalled over the 3 days. No significant relationships were identified between percentage peak VO2 at the 2.5 or 4.0 mmol l-1 blood lactate reference levels and either percentage time or number of 10- or 20-min periods above 140 or 160 beats min-1 (P > 0.05). These results support the hypothesis that daily physical activity levels in 11-16-year-old children do not stress aerobic metabolism sufficiently to influence aerobic fitness. PMID:1490213

  10. 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.

  11. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.

    PubMed

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

    2017-02-08

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

  12. Reference values of blood parameters in beef cattle of different ages and stages of lactation.

    PubMed Central

    Doornenbal, H; Tong, A K; Murray, N L

    1988-01-01

    Reference (normal) values for 12 blood serum components were determined for 48 Shorthorn cows (2-10 years old) and their 48 calves, 357 crossbred cows (12-14 years old), 36 feedlot bulls and 36 feedlot steers. In addition, hemoglobin, hematocrit, triiodothyronine, thyroxine and cortisol levels were determined for the crossbred cows, and feedlot bulls and steers. Reference values were tabulated according to sex, age and stage of lactation. Serum concentrations of urea, total protein and bilirubin, and serum activity of aspartate aminotransferase and lactate dehydrogenase increased with age (P less than 0.05), while calcium, phosphorus and alkaline phosphatase decreased with age (P less than 0.05) from birth to the age of ten years. The Shorthorn cows had the highest levels of glucose at parturition (P less than 0.05) with decreasing levels during lactation. Creatinine concentration decreased during lactation and increased during postweaning. Both lactate dehydrogenase and aspartate aminotransferase levels increased (P less than 0.05) during lactation. Urea and uric acid were present at higher concentrations in lactating than nonlactating cows (P less than 0.05). The values reported, based on a wide age range and large number of cattle, could serve as clinical guides and a basis for further research. PMID:3349406

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

    PubMed

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

    2017-01-01

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

  14. Short-term hypothermia activates hepatic mitochondrial sn-glycerol-3-phosphate dehydrogenase and thermogenic systems.

    PubMed

    Bobyleva, V; Pazienza, L; Muscatello, U; Kneer, N; Lardy, H

    2000-08-15

    The contribution of the sn-glycerol-3-phosphate (G-3-P) shuttle in the control of energy metabolism is well established. It is also known that its activity may be modulated by hormones involved in thermogenesis, such as thyroid hormones or dehydroepiandrosterone and its metabolites, that act by inducing de novo synthesis of mitochondrial G-3-P dehydrogenase (mGPDH). However, little is known as to the factors that may influence the activity without enzyme induction. In the present study we investigated the possible role of the G-3-P shuttle in the thermogenic response to different hypothermic stresses. It was found that a decrease of body temperature causes the liver rapidly to enhance mGPDH activity and G-3-P-dependent respiration. The enhancement, which does not result from de novo synthesis of enzymes, has the potential of increasing heat production both by decreased ATP synthesis during the oxidation of G-3-P and by activation of the glycolytic pathway.

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

    PubMed

    Venkata Mohan, S; Lenin Babu, M

    2011-09-01

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

  16. NADH-dependent decavanadate reductase, an alternative activity of NADP-specific isocitrate dehydrogenase protein.

    PubMed

    Rao, A V; Ramasarma, T

    2000-05-01

    The well known NADP-specific isocitrate dehydrogenase (IDH) obtained from pig heart was found to oxidize NADH with accompanying consumption of oxygen (NADH:O(2)=1:1) in presence of polyvanadate. This activity of the soluble IDH-protein has the following features common with the previously described membrane-enzymes: heat-sensitive, active only with NADH but not NADPH, increased rates in acidic pH, dependence on concentrations of the enzyme, NADH, decavanadate and metavanadate (the two constituents of polyvanadate), and sensitivity to SOD and EDTA. Utilizing NADH as the electron source the IDH protein was able to reduce decavanadate but not metavanadate. This reduced form of vanadyl (V(IV)) was similar in its eight-band electron spin resonance spectrum to vanadyl sulfate but had a 20-fold higher absorbance at its 700 nm peak. This decavanadate reductase activity of the protein was sensitive to heat and was not inhibited by SOD and EDTA. The IDH protein has the additional enzymic activity of NADH-dependent decavanadate reductase and is an example of "one protein--many functions".

  17. 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.

  18. First Description of Reduced Pyruvate Dehydrogenase Enzyme Activity Following Subarachnoid Hemorrhage (SAH)

    PubMed Central

    Lilla, Nadine; Füllgraf, Hannah; Stetter, Christian; Köhler, Stefan; Ernestus, Ralf-Ingo; Westermaier, Thomas

    2017-01-01

    Object: Several previous studies reported metabolic derangements and an accumulation of metabolic products in the early phase of experimental subarachnoid hemorrhage (SAH), which may contribute to secondary brain damage. This may be a result of deranged oxygen utilization due to enzymatic dysfunction in aerobic glucose metabolism. This study was performed to investigate, if pyruvate dehydrogenase enzyme (PDH) is affected in its activity giving further hints for a derangement of oxidative metabolism. Methods: Eighteen male Sprague-Dawley rats were randomly assigned to one of two experimental groups (n = 9): (1) SAH induced by the endovascular filament model and (2) sham-operated controls. Mean arterial blood pressure (MABP), intracranial pressure (ICP), and local cerebral blood flow (LCBF; laser-Doppler flowmetry) were continuously monitored from 30 min before until 3 h after SAH. Thereafter, the animals were sacrificed and PDH activity was measured by ELISA. Results: PDH activity was significantly reduced in animals subjected to SAH compared to controls. Conclusion: The results of this study demonstrate for the first time a reduction of PDH activity following SAH, independent of supply of substrates and may be an independent factor contributing to a derangement of oxidative metabolism, failure of oxygen utilization, and secondary brain damage. PMID:28261039

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

    PubMed

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

    1999-02-01

    Patients with the nephrotic syndrome (NS) exhibit abnormal renal sodium retention which cannot completely explained by a secondary hyperaldosteronism due to reduced renal perfusion. As an alternative mechanism to explain this phenomenon we postulate a cortisol-mediated mineralocorticoid effect as a consequence of a reduced activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). A down-regulation of 11beta-HSD, i.e. of the shuttle of active to inactive glucocorticosteroids, has been shown to cause mineralocorticoid effects. Therefore we investigated the activity of 11beta-HSD by measuring the urinary ratio of (tetrahydrocortisol + 5alpha-tetrahydrocortisol)/tetrahydrocortisone [(THF+5alpha-THF)/THE] by gas-chromatography in 29 NS patients with biopsy-proven glomerulonephritis and 29 healthy control subjects. The ratio of (THF+5alpha-THF)/THE was higher in NS patients (median 1.49, range 0.45-4.07) than in the control subjects (0.98, 0.60-1.36; p<0.01). This ratio was increased as a consequence of a decreased urinary excretion rate of the cortisone metabolite, THE. The present data indicate that a reduced activity of 11beta-HSD is a new mechanism contributing to the exaggerated sodium retention in patients with the NS.

  20. 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

  1. 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.

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

    PubMed Central

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

    2017-01-01

    Reduction of NAD+ by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity. PMID:28361886

  4. Regulation of Human Mitochondrial Aldehyde Dehydrogenase (ALDH-2) Activity by Electrophiles in Vitro*

    PubMed Central

    Oelze, Matthias; Knorr, Maike; Schell, Richard; Kamuf, Jens; Pautz, Andrea; Art, Julia; Wenzel, Philip; Münzel, Thomas; Kleinert, Hartmut; Daiber, Andreas

    2011-01-01

    Recently, mitochondrial aldehyde dehydrogenase (ALDH-2) was reported to reduce ischemic damage in an experimental myocardial infarction model. ALDH-2 activity is redox-sensitive. Therefore, we here compared effects of various electrophiles (organic nitrates, reactive fatty acid metabolites, or oxidants) on the activity of ALDH-2 with special emphasis on organic nitrate-induced inactivation of the enzyme, the biochemical correlate of nitrate tolerance. Recombinant human ALDH-2 was overexpressed in Escherichia coli; activity was determined with an HPLC-based assay, and reactive oxygen and nitrogen species formation was determined by chemiluminescence, fluorescence, protein tyrosine nitration, and diaminonaphthalene nitrosation. The organic nitrate glyceryl trinitrate caused a severe concentration-dependent decrease in enzyme activity, whereas incubation with pentaerythritol tetranitrate had only minor effects. 4-Hydroxynonenal, an oxidized prostaglandin J2, and 9- or 10-nitrooleate caused a significant inhibition of ALDH-2 activity, which was improved in the presence of Mg2+ and Ca2+. Hydrogen peroxide and NO generation caused only minor inhibition of ALDH-2 activity, whereas peroxynitrite generation or bolus additions lead to severe impairment of the enzymatic activity, which was prevented by the thioredoxin/thioredoxin reductase (Trx/TrxR) system. In the presence of glyceryl trinitrate and to a lesser extent pentaerythritol tetranitrate, ALDH-2 may be switched to a peroxynitrite synthase. Electrophiles of different nature potently regulate the enzymatic activity of ALDH-2 and thereby may influence the resistance to ischemic damage in response to myocardial infarction. The Trx/TrxR system may play an important role in this process because it not only prevents inhibition of ALDH-2 but is also inhibited by the ALDH-2 substrate 4-hydroxynonenal. PMID:21252222

  5. Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling

    PubMed Central

    Gomes, Katia M.S.; Campos, Juliane C.; Bechara, Luiz R.G.; Queliconi, Bruno; Lima, Vanessa M.; Disatnik, Marie-Helene; Magno, Paulo; Chen, Che-Hong; Brum, Patricia C.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

    2014-01-01

    Aims We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. Methods and results We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca2+-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. Conclusions Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients. PMID:24817685

  6. Aldehyde dehydrogenase activity in Lactococcus chungangensis: Application in cream cheese to reduce aldehyde in alcohol metabolism.

    PubMed

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

    2016-03-01

    Previous studies have shown that the metabolic capability of colonic microflora may be at least as high as that of the liver or higher than that of the whole human body. Aldehyde dehydrogenase (ALDH) is an enzyme produced by these bacteria that can metabolize acetaldehyde, produce from ethanol to acetate. Lactococcus species, which is commonly used as a starter in dairy products, was recently found to possess the ALDH gene, and the activity of this enzyme was determined. In this study, the ALDH activity of Lactococcus chungangensis CAU 28(T) and 11 other type strains in the genus Lactococcus was studied. Only 5 species, 3 of dairy origin (Lactococcus lactis ssp. lactis KCTC 3769(T), Lactococcus lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T)) and 2 of nondairy origin (Lactococcus fujiensis NJ317(T) and L. chungangensis CAU 28(T)), showed ALDH activity and possessed a gene encoding ALDH. All of these strains were capable of making cream cheese. Among the strains, L. chungangensis produced cream cheese that contained the highest level of ALDH and was found to reduce the level of acetaldehyde in the serum of mice. These results predict a promising role for L. chungangensis CAU28(T) to be used in cheese that can be developed as functional food.

  7. Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex.

    PubMed

    Lib, Margarita; Rodriguez-Mari, Adriana; Marusich, Michael F; Capaldi, Roderick A

    2003-03-01

    Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.

  8. 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

  9. Conformational and activity changes during guanidine denaturation of D-glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Xie, G F; Tsou, C L

    1987-01-05

    Changes in intrinsic protein fluorescence of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been compared with inactivation of the enzyme during denaturation in guanidine solutions. The holoenzyme is completely inactivated at guanidine concentrations less than 0.5 M and this is accompanied by a red shift of the emission maximum at 335 nm and a marked decrease in intensity of the intrinsic fluorescence. At 0.5 M guanidine, the inactivation is a slow process, with a first-order rate constant of 2.4 X 10(-3) s-1. A further red shift in the emission maximum and a decrease in intensity occur at guanidine concentrations higher than 1.5 M. The emission peak at 410 nm of the fluorescent NAD derivative introduced at the active site of this enzyme (Tsou, C.L. et al. (1983) Biochem. Soc. Trans. 11, 425-429) shows both a red shift and a marked decrease in intensity at the same guanidine concentration required to bring about the inactivation and the initial changes in the intrinsic fluorescence of the holoenzyme. It appears that treatment by low guanidine concentrations leads to both complete inactivation and perturbation of the active site conformation and that a tryptophan residue is situated at or near the active site.

  10. Myricetin is a novel inhibitor of human inosine 5'-monophosphate dehydrogenase with anti-leukemia activity.

    PubMed

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang; Lu, Weiqiang; Huang, Jin

    2016-09-02

    Human inosine 5'-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC50 values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity.

  11. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.

  12. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed an invitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana a2b2-hetero tetrameric pyruvate dehydrogenase (E1) plus A.thaliana E1-kinase (AtPDK). Upon addition of MgATP...

  13. Global view of cognate kinase activation by the human pyruvate dehydrogenase complex.

    PubMed

    Guevara, Elena L; Yang, Luying; Birkaya, Barbara; Zhou, Jieyu; Nemeria, Natalia S; Patel, Mulchand S; Jordan, Frank

    2017-02-23

    The human pyruvate dehydrogenase complex (PDC) comprises four multidomain components, E1, E3, E2 and an E3-binding protein (E3BP), the latter two forming the core as E2·E3BP sub-complex. Pyruvate flux through PDC is regulated via phosphorylation (inactivation) at E1 by four PDC kinases (PDKs), and reactivation by two PDC phosphatases. Up-regulation of PDK isoform gene expression is reported in several forms of cancer, while PDKs may be further activated by PDC by binding to the E2·E3BP core. Hence, the PDK: E2·E3BP interaction provides new therapeutic targets. We carried out both functional kinetic and thermodynamic studies to demonstrate significant differences in the activation of PDK isoforms by binding to the E2·E3BP core: (i) PDK2 needs no activation by E2·E3BP for efficient functioning, while PDK4 was the least effective of the four isoforms, and could not be activated by E2·E3BP. Hence, development of inhibitors to the interaction of PDK2 and PDK4 with E2·E3BP is not promising; (ii) Design of inhibitors to interfere with interaction of E2·E3BP with PDK1 and PDK3 is promising. PDK3 needs E2·E3BP core for activation, an activation best achieved by synergistic combination of E2-derived catalytic domain and tridomain.

  14. Global view of cognate kinase activation by the human pyruvate dehydrogenase complex

    PubMed Central

    Guevara, Elena L.; Yang, Luying; Birkaya, Barbara; Zhou, Jieyu; Nemeria, Natalia S.; Patel, Mulchand S.; Jordan, Frank

    2017-01-01

    The human pyruvate dehydrogenase complex (PDC) comprises four multidomain components, E1, E3, E2 and an E3-binding protein (E3BP), the latter two forming the core as E2·E3BP sub-complex. Pyruvate flux through PDC is regulated via phosphorylation (inactivation) at E1 by four PDC kinases (PDKs), and reactivation by two PDC phosphatases. Up-regulation of PDK isoform gene expression is reported in several forms of cancer, while PDKs may be further activated by PDC by binding to the E2·E3BP core. Hence, the PDK: E2·E3BP interaction provides new therapeutic targets. We carried out both functional kinetic and thermodynamic studies to demonstrate significant differences in the activation of PDK isoforms by binding to the E2·E3BP core: (i) PDK2 needs no activation by E2·E3BP for efficient functioning, while PDK4 was the least effective of the four isoforms, and could not be activated by E2·E3BP. Hence, development of inhibitors to the interaction of PDK2 and PDK4 with E2·E3BP is not promising; (ii) Design of inhibitors to interfere with interaction of E2·E3BP with PDK1 and PDK3 is promising. PDK3 needs E2·E3BP core for activation, an activation best achieved by synergistic combination of E2-derived catalytic domain and tridomain. PMID:28230160

  15. 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

  16. Lactate Metabolism is Associated with Mammalian Mitochondria

    PubMed Central

    Chen, Ying-Jr; Mahieu, Nathaniel G.; Huang, Xiaojing; Singh, Manmilan; Crawford, Peter A; Johnson, Stephen L.; Gross, Richard W.; Schaefer, Jacob

    2016-01-01

    It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. Within the fermenting cell itself, however, it is generally assumed that lactate is produced to replenish NAD+ and then is secreted. Here we explored the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. With high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells. PMID:27618187

  17. Comparison of Blood Lactate Elimination in Individuals With Paraplegia and Able-Bodied Individuals During Active Recovery From Exhaustive Exercise

    PubMed Central

    Leicht, Christof; Perret, Claudio

    2008-01-01

    Background/Objective: The aim of the present study was to compare blood lactate elimination between individuals with paraplegia (P) and able-bodied (AB) individuals after strenuous arm exercise. Methods: Eight P and 8 AB men (matched for age, height, and weight) participated in this study. Average weekly arm-training volume for P participants (eg, hand bike, wheelchair basketball) and AB participants (eg, swimming, rowing, cross-country skiing) was 4.1 ± 1.6 vs 2.8 ± 0.8 h. A maximal-arm-cranking intensity-graded exercise test to volitional exhaustion was performed by all test participants. Immediately after the exercise test, the participants performed arm cranking for another 30 minutes at a workload of one third of the maximally achieved power output. During this active recovery, mixed-capillary blood samples were taken for lactate analysis. Results: The lactate accumulation constant was significantly higher for P individuals, whereas the lactate elimination constant showed no significant difference between the two groups. Conclusions: Individuals with paraplegia seem to have no disadvantages in lactate elimination after exhaustive arm exercise compared with able-bodied individuals. PMID:18533413

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

    PubMed Central

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

    2014-01-01

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

  19. 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

  20. 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

  1. 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.

  2. Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities.

    PubMed

    Dong, Yaoyao; Mao, Baiping; Li, Linxi; Guan, Hongguo; Su, Ying; Li, Xiaoheng; Lian, Qingquan; Huang, Ping; Ge, Ren-Shan

    2016-03-01

    Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects.

  3. Rotenone Decreases Intracellular Aldehyde Dehydrogenase Activity: Implications for the Pathogenesis of Parkinson Disease

    PubMed Central

    Goldstein, David S.; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Kopin, Irwin J.; Sharabi, Yehonatan

    2015-01-01

    Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the “catecholaldehyde hypothesis,” buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 minutes), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose-dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD+ was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. PMID:25645689

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

    PubMed

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

    1999-09-01

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

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

    PubMed

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

    2016-04-01

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

  6. Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

    SciTech Connect

    Brodie, A.E.; Reed, D.J. )

    1990-01-01

    The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

  7. Effect of malonate and p-chlorophenoxy acetic acid on hepatic succinic dehydrogenase activity of ageing lizards.

    PubMed

    Jena, B S; Patnaik, B K

    1990-01-01

    The degree of inhibition of hepatic succinic dehydrogenase activity by malonate, a competitive inhibitor, did not differ between young and middle-aged lizards. On the other hand, the same parameter increased significantly between middle-aged and old lizards. The percent inhibition of enzyme activity by p-chlorophenoxy acetic acid was also age-dependent, being higher in middle-aged and old than in young lizards.

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

    PubMed

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

    2005-01-15

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

  9. 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

  10. Effect of chronologic age on induction of cystathionine synthase, uroporphyrinogen I synthase, and glucose-6-phosphate dehydrogenase activities in lymphocytes.

    PubMed Central

    Gartler, S M; Hornung, S K; Motulsky, A G

    1981-01-01

    The activities of cystathionine synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8], and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) have been measured in phytohemagglutinin-stimulated lymphocytes of young and old human subjects. A significant decrease in activity with age was observed for cystathionine synthase and uroporphyrinogen I synthase but not for glucose-6-phosphate dehydrogenase. These changes could not be related to declining phytohemagglutinin response with aging. Age-related decreases in activity of some enzymes may be relevant for an understanding of the biology of aging. False assignment of heterozygosity, and even homozygosity, for certain genetic disorders, such as homocystinuria, may result when low enzyme levels are detected in the lymphocytes of older people. PMID:6940198

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

    PubMed Central

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

    2016-01-01

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