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Sample records for lactate dehydrogenase reaction

  1. 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.

  2. Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4.

    PubMed

    O'Flaherty, C; Breininger, E; Beorlegui, N; Beconi, M T

    2005-10-30

    After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process. PMID:16112812

  3. 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 ...

  4. 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...

  5. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

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

  6. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...dehydrogenase in serum. Lactate 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 infarction, and tumors of the...

  7. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...dehydrogenase in serum. Lactate 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 infarction, and tumors of the...

  8. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...dehydrogenase in serum. Lactate 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 infarction, and tumors of the...

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...activity) in serum. Measurements of lactate dehydrogenase isoenzymes are used in the diagnosis and treatment of liver diseases, such as viral hepatitis, and myocardial infarction. (b) Classification. Class...

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...activity) in serum. Measurements of lactate dehydrogenase isoenzymes are used in the diagnosis and treatment of liver diseases, such as viral hepatitis, and myocardial infarction. (b) Classification. Class...

  11. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1440 Lactate dehydrogenase...

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

    PubMed

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

    2015-03-01

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

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...biological activity) in serum. Measurements of lactate dehydrogenase isoenzymes are used in the diagnosis and treatment of liver diseases, such as viral hepatitis, and myocardial infarction. (b) Classification. Class...

  14. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate dehydrogenase isoenzymes test system. (a)...

  15. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  16. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

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

    SciTech Connect

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

    1989-01-01

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

  18. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  19. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  20. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  1. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  2. 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 ?moles 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

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

    PubMed

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

    2015-09-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD(+) as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn(174) was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases. PMID:26150461

  4. Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase.

    PubMed

    Judge, J L; Owens, K M; Pollock, S J; Woeller, C F; Thatcher, T H; Williams, J P; Phipps, R P; Sime, P J; Kottmann, R M

    2015-10-15

    Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-? (TGF-?) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-? through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-?. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-? to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis. PMID:26254422

  5. 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. PMID:26126931

  6. Plasma lactate dehydrogenase and creatine kinase after anaerobic exercise.

    PubMed

    Pilis, W; Langfort, J; Pilsniak, A; Pyzik, M; Btasiak, M

    1988-04-01

    Eight untrained men performed 15-s and 60-s high-intensity exercise on a bicycle ergometer. Activities of the creatine kinase (CK) and lactate dehydrogenase (LDH) were measured in blood 3 min and 2, 6, and 24 h after cessation of exercise. The results indicate that anaerobic exercise induces a transient increase in plasma LDH activity and a more prolonged elevation in plasma CK activity. A negative correlation was found between CK activity measured before and 3 min after exercise and mean power, and total external work performed in both test types. A similar correlation was ascertained between pre- and post-exercise CK activity and maximal power output measured in the 60-s test. After the 15-s exercise test, only post-exercise plasma CK activity was negatively correlated with the maximal power output. PMID:3384513

  7. Hereditary deficiency of lactate dehydrogenase H-subunit.

    PubMed

    Wakabayashi, H; Tsuchiya, M; Yoshino, K; Kaku, K; Shigei, H

    1996-07-01

    We report herein the fifth family of hereditary deficiency of lactate dehydrogenase (LDH) H-subunit with an autosomal recessive inheritance including two cases of complete deficiency. Their LDH activities were low both in the serum and in the red blood cells (RBC). Electrophoretic analysis revealed that the patients with the complete deficiency had only the LDH5 isozyme. The complete deficiency was associated with marked elevation of fructose-1, 6-diphosphate (FDP) and dihydroxyacetonephosphate (DHAP) and a less marked rise in glyceraldehyde-3-phosphate (GA3P) among glycolytic intermediates in the RBC. Furthermore, hemolysis was observed in the present cases, but this finding was not included in the other reports. PMID:8842761

  8. Placental enzyme polymorphism among Maharashtrians: alkaline phosphatase and lactate dehydrogenase.

    PubMed

    Mukherjee, B N; Das, S K; Malhotra, K C; Kate, S L; Mutalik, G S; Sainani, G S; Bhidya, S

    1978-09-01

    The distribution of placental alkaline phosphatase and lactate dehydrogenase types in 635 placentas from various endogamous groups of Maharashtra have been studied by starch gel electrophoresis. In the case of alkaline phosphatase, 6 common phenotypes and 6 rare phenotypes (F2I1, S1S2, S2S3, I1S2, F1S2, F1I2) are encountered. The highest frequency of Pls1 allele (0.7394) and lowest frequency of Pli1 allele (0.0246) have been found in the Nava-Budha. 6 cases of Cal-1 and 5 cases of Cal-2 types of LDH variants have been observed in the total samples, and Muslims possess the highest frequency of Cal-1 types (3.64%). Population groups are compared with respect to Pl alleles. PMID:727701

  9. D- and L-lactate dehydrogenases during invertebrate evolution

    PubMed Central

    2008-01-01

    Background The L-lactate and D-lactate dehydrogenases, which are involved in the reduction of pyruvate to L(-)-lactate and D(+)-lactate, belong to evolutionarily unrelated enzyme families. The genes encoding L-LDH have been used as a model for gene duplication due to the multiple paralogs found in eubacteria, archaebacteria, and eukaryotes. Phylogenetic studies have suggested that several gene duplication events led to the main isozymes of this gene family in chordates, but little is known about the evolution of L-Ldh in invertebrates. While most invertebrates preferentially oxidize L-lactic acid, several species of mollusks, a few arthropods and polychaetes were found to have exclusively D-LDH enzymatic activity. Therefore, it has been suggested that L-LDH and D-LDH are mutually exclusive. However, recent characterization of putative mammalian D-LDH with significant similarity to yeast proteins showing D-LDH activity suggests that at least mammals have the two naturally occurring forms of LDH specific to L- and D-lactate. This study describes the phylogenetic relationships of invertebrate L-LDH and D-LDH with special emphasis on crustaceans, and discusses gene duplication events during the evolution of L-Ldh. Results Our phylogenetic analyses of L-LDH in vertebrates are consistent with the general view that the main isozymes (LDH-A, LDH-B and LDH-C) evolved through a series of gene duplications after the vertebrates diverged from tunicates. We report several gene duplication events in the crustacean, Daphnia pulex, and the leech, Helobdella robusta. Several amino acid sequences with strong similarity to putative mammalian D-LDH and to yeast DLD1 with D-LDH activity were found in both vertebrates and invertebrates. Conclusion The presence of both L-Ldh and D-Ldh genes in several chordates and invertebrates suggests that the two enzymatic forms are not necessarily mutually exclusive. Although, the evolution of L-Ldh has been punctuated by multiple events of gene duplication in both vertebrates and invertebrates, a shared evolutionary history of this gene in the two groups is apparent. Moreover, the high degree of sequence similarity among D-LDH amino acid sequences suggests that they share a common evolutionary history. PMID:18828920

  10. Nuclear Lactate Dehydrogenase: Bridging Central Metabolism to Epigenetic Modifications in Mammalian Cellular Systems

    E-print Network

    Appanna, Vasu

    1 Nuclear Lactate Dehydrogenase: Bridging Central Metabolism to Epigenetic Modifications to link central metabolism to epigenetic modifications is indeed a novel role for this enzyme, and further and Disease........................................................................23 1.3.1.Diabetes

  11. The D-Lactate Dehydrogenase from Sporolactobacillus inulinus Also Possessing Reversible Deamination Activity

    PubMed Central

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

    2015-01-01

    Hydroxyacid dehydrogenases are responsible for the conversion of 2-keto acids to 2-hydroxyacids and have a wide range of biotechnological applications. In this study, a D-lactate dehydrogenase (D-LDH) from a Sporolactobacillus inulinus strain was experimentally verified to have both the D-LDH and glutamate dehydrogenase (GDH) activities (reversible deamination). The catalytic mechanism was demonstrated by identification of key residues from the crystal structure analysis and site-directed mutagenesis. The Arg234 and Gly79 residues of this enzyme play a significant role in both D-LDH and GDH activities. His295 and Phe298 in DLDH744 were identified to be key residues for lactate dehydrogenase (LDH) activity only whereas Tyr101 is a unique residue that is critical for GDH activity. Characterization of the biochemical properties contributes to understanding of the catalytic mechanism of this novel D-lactate dehydrogenase enzyme. PMID:26398356

  12. Kinetic activation of yeast mitochondrial D-lactate dehydrogenase by carboxylic acids.

    PubMed

    Mourier, Arnaud; Vallortigara, Julie; Yoboue, Edgar D; Rigoulet, Michel; Devin, Anne

    2008-10-01

    Aerobically grown yeast cells express mitochondrial lactate dehydrogenases that localize to the mitochondrial inner membrane. The D-lactate dehydrogenase is a zinc-flavoprotein with high acceptor specificity for cytochrome c, that catalyzes the oxidation of D-lactate into pyruvate. In this paper, we show that mitochondrial respiratory rate in phosphorylating or non-phosphorylating conditions with D-lactate as substrate is stimulated by carboxylic acids. This stimulation does not affect the yield of oxidative phosphorylation. Furthermore, this stimulation lies at the level of the D-lactate dehydrogenase. It is non-competitive, hyperbolic and its dimension is directly related to the number of carboxylic groups on the activator. The physiological meaning of such a regulation is discussed. PMID:18640090

  13. 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.

  14. Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes

    SciTech Connect

    Xue, Q.; Yeung, E.S. Iowa State Univ., Ames, IA )

    1994-04-01

    Trace amounts of enzymes within single human erythrocytes can be quantified by a combination of on-column reaction and capillary electrophoresis. A detection limit of 1.3 x 10[sup [minus]21] mol of LDH was achieved with laser-induced fluorescence by monitoring the product of the enzyme-catalyzed reaction between lactate and NAD[sup +]. Single erythrocyte analysis clearly isolates the major forms of LDH. The variation of total LDH activity in a population of cells from a single individual is large, but the relative activities of the isoenzymes LDH-1 and LDH-2 are fairly constant. This can be explained by the distribution of cell age in the population. A lower enzyme activity is indicative of senescence. The efficient separation of different LDH forms and the high detection sensitivity opens up the possibility of multiple-enzyme assays with a single mammalian cell. 41 refs., 5 figs.

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

    PubMed

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

    2014-12-01

    The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis. PMID:25389710

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

    PubMed

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

    2002-05-01

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

  17. 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 ?-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. PMID:26556099

  18. 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

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

    PubMed Central

    2015-01-01

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

  20. Development of an enzymatic assay system of d-lactate using d-lactate dehydrogenase and a UV-LED fluorescent spectrometer.

    PubMed

    Chen, Chien-Ming; Chen, Shih-Ming; Chien, Po-Jen; Yu, Han-Yin

    2015-12-10

    In this study, we aimed to develop a new enzymatic assay system of d-lactate with good precision, accuracy, and sensitivity for the determination of d-lactate concentrations in rat serum. d-Lactate dehydrogenase (d-LDH) was utilized to catalyze d-lactate and NAD(+) to pyruvate and NADH, respectively. The generated NADH was excited by using a 340-nm UV-light-emitting diode (LED), and the fluorescence at 491nm was detected to determine the concentration of d-lactate in rat serum. The optics, consisting of the sample cuvette, were set on three-dimensional stages to receive the most intensive fluorescence signal into the spectrometer. The optimal conditions of the d-LDH reaction were pH 8.5 and 25°C for 90min. The results showed that the new d-lactate assay system had good linearity (R(2)=0.9964) in the calibration range from 5 to 150?M. Intra-day and inter-day accuracies were in the range of 103.96-109.09% and 102.84-104.59%, respectively, and the intra-day and inter-day precision was 4.28-6.82% and 4.04-12.40%, respectively. Finally, serum d-lactate concentrations determined by the proposed enzymatic assay system were compared with those obtained by a conventional HPLC method. The newly developed d-lactate assay system could detect 10-15 samples in 90min, whereas the HPLC method could detect only one sample over the same time period. PMID:26265307

  1. The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase*

    PubMed Central

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

    2014-01-01

    For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax 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 ? 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 His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, 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 ?, 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. PMID:25258319

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

    PubMed Central

    Dave, Khyati K.; Punekar, Narayan S.

    2015-01-01

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

  3. 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.

  4. 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...

  5. 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...

  6. Incorporating expression data in metabolic modeling: a case study of lactate dehydrogenase

    E-print Network

    Joshua Downer; Joel R. Sevinsky; Natalie G. Ahn; Katheryn A. Resing; M. D. Betterton

    2005-11-12

    Integrating biological information from different sources to understand cellular processes is an important problem in systems biology. We use data from mRNA expression arrays and chemical kinetics to formulate a metabolic model relevant to K562 erythroleukemia cells. MAP kinase pathway activation alters the expression of metabolic enzymes in K562 cells. Our array data show changes in expression of lactate dehydrogenase (LDH) isoforms after treatment with phorbol 12-myristate 13-acetate (PMA), which activates MAP kinase signaling. We model the change in lactate production which occurs when the MAP kinase pathway is activated, using a non-equilibrium, chemical-kinetic model of homolactic fermentation. In particular, we examine the role of LDH isoforms, which catalyze the conversion of pyruvate to lactate. Changes in the isoform ratio are not the primary determinant of the production of lactate. Rather, the total concentration of LDH controls the lactate concentration.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. 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...

  9. 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...

  10. LACTIC ACID PRODUCTION BY SACCHAROMYCES CEREVISIAE EXPRESSING A RHIZOPUS ORYZAE LACTATE DEHYDROGENASE GENE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work demonstrates the first example of a fungal LDH expressed in yeast. A L(+)-lactate dehydrogenase gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adhl promoter and terminator, then placed in a 2 micron contai...

  11. A radiotracer probe to study metal interaction with human lactate dehydrogenase isoenzymes

    SciTech Connect

    Menon, M.P.; Wright, C.E. )

    1989-12-01

    The electrophilic Ag+ ion was found to destroy completely the enzymatic activity of lactate dehydrogenase isoenzyme LDH-1 while other transition metal ions reduced its activity in varying degrees. A radiotracer probe involving 110mAg-labeled silver ion was used to understand the mechanism of denaturation of LDH and also to determine the number of active sites, if any, for substrate binding with the enzyme. Purified LDH-l was reacted with 110mAg-labeled silver ion and the mixture was passed through the sephadex G-75-120 gel to separate the 110mAg-LDH complex that might be formed during the reaction. The resulting elution curve revealed that a stable complex was formed. From the total radioactivity of 110mAg bound LDH, the specific activity of labeled Ag+ and the amount of LDH used the ratio of the number of moles of Ag+ reacted with 1 mol of LDH was computed. This was found to be approximately 4.0, indicating that there are four binding sites in LDD, probably one on each subunit. Kinetic studies of LDH catalysis of L-P reaction in the presence and absence of Ag+ ion suggest that silver ion is involved in competitive inhibition and that the interaction conforms to the lock-and-key model. The inhibition of catalysis by other metals is presumably of a noncompetitive type.

  12. 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.

  13. Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase.

    PubMed

    Markwalter, Christine F; Davis, Keersten M; Wright, David W

    2016-01-15

    We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/?l). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4-6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody. PMID:26475567

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

    PubMed

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

    2013-10-01

    DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson-Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics. PMID:24043813

  15. [Repression and derepression of lactate dehydrogenase loci during mouse development].

    PubMed

    Kolombet, L V; Gapienko, E F

    1977-05-01

    The ultramicroelectrophoretic method was applied to the study of the lactic dehydrogenase (LDH) spectrum alterations during the developmental phases of mice: ovulated ova--zygote--blastocyte--embryo--oocyte--ovulated ova. Only H-subunits were found in the embryo cells before the 5th day of development. After this M-subunits appeared indicating derepression of LDH-A locus. On the 8th day of the embryonal development deres pression of the LDH-B locus was observed to disappear during the oogenesis, being the result of progressive repression of locus LDH-A. Dictiotena of meios prophase is characterised by active H-subunit synthesis and a gradual decrease of the M-subunit synthesis. During the whole dictiotena phase the LDH-spectrum of the follicular cells was of the M-type. PMID:884268

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

    PubMed Central

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

    2015-01-01

    ABSTRACT NAD-independent l-lactate dehydrogenases (l-iLDHs) play important roles in l-lactate utilization of different organisms. All of the previously reported l-iLDHs were flavoproteins that catalyze the oxidation of l-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of l-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive l-iLDH activity was detected. The expressed l-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified l-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified l-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing l-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the l-lactate oxidation. LldABC has narrow substrate specificity, and only l-lactate and dl-2-hydrobutyrate were rapidly oxidized. Mg2+ could activate l-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the l-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the l-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized l-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor. IMPORTANCE Providing new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent l-lactate dehydrogenase (l-iLDH) encoded by the gene cluster lldABC is indispensable for the l-lactate metabolism in Pseudomonas stutzeri A1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containing l-iLDH in other microbes, LldABC in P. stutzeri A1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor. PMID:25917905

  17. Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target

    PubMed Central

    Vudriko, Patrick; Masatani, Tatsunori; Cao, Shinuo; Terkawi, Mohamad Alla; Kamyingkird, Ketsarin; Mousa, Ahmed A; Adjou Moumouni, Paul F; Nishikawa, Yoshifumi; Xuan, Xuenan

    2014-01-01

    Babesia microti is an emerging zoonotic protozoan organism that causes “malaria-like” symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD+) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The Km values of NAD+ and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 ?M, while at 2.5 ?M, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection. PMID:25125971

  18. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    SciTech Connect

    Ma, L.

    2000-09-12

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

  19. Binding ligands and cofactor to L-lactate dehydrogenase from human skeletal and heart muscles.

    PubMed

    ?widerek, Katarzyna; Paneth, Piotr

    2011-05-19

    Binding affinities of cofactor and ligands to the active site of two different isoforms of lactate dehydrogenase (LDH), heart and skeletal muscles (H4 and M4, respectively), can be used for medical and biological applications. Herein, a hybrid QM/MM computational approach based on free energy perturbation methods has been carried out to estimate binding affinities and binding isotope effects (BIEs) for NADH/NAD(+) and oxamate, pyruvate, L-lactate, and D-lactate ligands to the M4 and H4 isoforms of L-LDH. Here, we show that determining how cofactor and ligands interact with the active site of LDH isoforms advanced the still open discussion on the intracellular lactate shuttle hypothesis. In our discussion we deny the key concept of this hypothesis showing, based on interaction energy values, that there is no evidence that the M4 type of LDH in the skeletal muscles cells served as a catalyst of the conversion of lactate to pyruvate. Additionally, theoretical determination of BIEs for H4 and M4 types of LDH shows that there is a way of using the BIEs as a tool capable to distinguish these isoforms, and for this purpose D-lactate labeled with deuterium in positions 11 or 7, 8, 9 ([11-2H]-BIE and [7,8,9-2H3]-BIE) or L-lactate labeled only in position 11 ([11-2H]-BIE) could be used. We propose the BIEs as a useful tool which can be applied in order to experimentally determine the types of LDH. PMID:21526780

  20. 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. PMID:26008622

  1. Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

    PubMed Central

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

    2014-01-01

    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. PMID:25245810

  2. 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.

  3. Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase, a Biomarker for Malaria, Using the Specific DNA Aptamer

    PubMed Central

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

    2014-01-01

    A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum. PMID:24992632

  4. Suppression of lactate dehydrogenase A compromises tumor progression by downregulation of the Warburg effect in glioblastoma.

    PubMed

    Li, Juan; Zhu, Shuchai; Tong, Jing; Hao, Hui; Yang, Jie; Liu, Zhikun; Wang, Yuxiang

    2016-01-20

    Reprogrammed glucose metabolism is an emerging hallmark of cancer cells, which show a unique metabolic phenotype known as the Warburg effect. Lactate dehydrogenase A (LDHA), a key enzyme in the glycolytic process, executes the final step by conversion of lactate into pyruvate. However, little is known about the roles of LDHA in human glioblastoma (GBM). In this study, we aimed to determine the effects of LDHA and elucidate related underlying mechanisms. Data derived from Oncomine database showed that LDHA is commonly upregulated in GBM tissues in comparison with corresponding normal controls. Silencing of LDHA expression resulted in reduced glycolysis, decreased cell growth, increased cell apoptosis, and attenuated invasive ability. In the presence of 2-deoxyglucose, a glycolysis inhibitor, the oncogenic activities of LDHA were completely blocked. These findings provide evidence of the cellular functions of LDHA in the progression of GBM and suggest that LDHA might act as a potential therapeutic target for GBM treatment. PMID:26694942

  5. Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli

    SciTech Connect

    Contag, P.R.; Williams, M.G.; Rogers, P. )

    1990-12-01

    A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids. pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grown anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pPC58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent M{sub r} of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.

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

    SciTech Connect

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

    1988-12-01

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

  7. 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. PMID:21625872

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

    SciTech Connect

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

    2012-08-01

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

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

    PubMed

    Grabowski, Micha?; Banecki, Bogdan; Kadzi?ski, Leszek; Jakóbkiewicz-Banecka, Joanna; Ka?mierkiewicz, Rajmund; Gabig-Cimi?ska, Magdalena; W?grzyn, Grzegorz; W?grzyn, 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. PMID:26253470

  10. Lactate Dehydrogenase A is a potential prognostic marker in clear cell renal cell carcinoma

    PubMed Central

    2014-01-01

    Background Over 90% of cancer-related deaths in clear cell renal cell carcinoma (RCC) are caused by tumor relapse and metastasis. Thus, there is an urgent need for new molecular markers that can potentiate the efficacy of the current clinical-based models of prognosis assessment. The objective of this study is to evaluate the potential significance of lactate dehydrogenase A (LDHA), assessed by immunohistochemical staining, as a prognostic marker in clear cell renal cell carcinoma in relation to clinicopathological features and clinical outcome. Methods We assessed the expression of LDHA at the protein level, by immunohistochemistry, and correlated its expression with multiple clinicopathological features including tumor size, clinical stage, histological grade, disease-free and overall survival in 385 patients with primary clear cell renal cell carcinoma. We also correlated the LDHA expression with overall survival, at mRNA level, in an independent data set of 170 clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases. Cox proportional hazards models adjusted for the potential clinicopathological factors were used to test for associations between the LDHA expression and both disease-free survival and overall survival. Results There is statistically significant positive correlation between LDHA level of expression and tumor size, clinical stage and histological grade. Moreover, LDHA expression shows significantly inverse correlation with both disease-free survival and overall survival in patients with clear cell renal cell carcinoma. Our results are validated by examining LDHA expression, at the mRNA level, in the independent data set of clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases which also shows that higher lactate dehydrogenase A expression is associated with significantly shorter overall survival. Conclusion Our results indicate that LDHA up-regulation can be a predictor of poor prognosis in clear cell renal cell carcinoma. Thus, it represents a potential prognostic biomarker that can boost the accuracy of other prognostic models in patients with clear cell renal cell carcinoma. PMID:24885701

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

    PubMed

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

    2015-12-15

    The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution. PMID:26201980

  12. Positive selection on D-lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus.

    PubMed

    Zhang, Jifeng; Gong, Guangyu; Wang, Xiao; Zhang, Hao; Tian, Weidong

    2015-08-01

    Lactobacillus delbrueckii has been widely used for yogurt fermentation. It has genes encoding both D- and L-type lactate dehydrogenases (LDHs) that catalyse the production of L(+) or D(-) stereoisomer of lactic acid. D-lactic acid is the primary lactate product by L. delbrueckii, yet it cannot be metabolised by human intestine. Since it has been domesticated for long time, an interesting question arises regarding to whether the selection pressure has affected the evolution of both L-LDH and D-LDH genes in the genome. To answer this question, in this study the authors first investigated the evolution of these two genes by constructing phylogenetic trees. They found that D-LDH-based phylogenetic tree could better represent the phylogenetic relationship in the acidophilus complex than L-LDH-based tree. They next investigated the evolutions of LDH genes of L. delbrueckii at amino acid level, and found that D-LDH gene in L. delbrueckii is positively selected, possibly a consequence of long-term domestication. They further identified four amino acids that are under positive selection. One of them, V261, is located at the centre of three catalytic active sites, indicating likely functional effects on the enzyme activity. The selection from the domestication process thus provides direction for future engineering of D-LDH. PMID:26243834

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

    PubMed Central

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

    2015-01-01

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

  14. 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

  15. 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

  16. Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide-tetrazolium coupling reactions for quantitative l-lactate analysis.

    PubMed

    Kan, Shu-Chen; Chang, Wei-Feng; Lan, Min-Chi; Lin, Chia-Chi; Lai, Wei-Shiang; Shieh, Chwen-Jen; Hsiung, Kuang-Pin; Liu, Yung-Chuan

    2015-02-15

    In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 ?l, 2(-6)U/?l) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 ?l, 12 mM), l-lactate dehydrogenase (1 ?l, 0.25U/?l), and NAD(+) (2?l, 1.5×10(-5)M) were added into the mobile phase (100 ?l) composed of 0.1% (w/w) Tween 20 in 10mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications. PMID:25454507

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

    SciTech Connect

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition, evaporation rate can be controlled or adjusted in this method during the crystallization process to favor either nucleation or growing processes for optimizing crystallization process. The protein crystals gotten by this method were experimentally proven to possess high x-ray diffraction qualities. Finally, we crystallized human lactate dehydrogenase 1 (H4) complexed with NADH and determined its structure by x-ray crystallography. The structure of LDH/NADH displays a significantly different structural feature, compared with LDH/NADH/inhibitor ternary complex structure, that subunits in LDH/NADH complex show open conformation or two conformations on the active site while the subunits in LDH/NADH/inhibitor are all in close conformation. Multiple LDH/NADH crystals were obtained and used for x-ray diffraction experiments. Difference in subunit conformation was observed among the structures independently solved from multiple individual LDH/NADH crystals. Structural differences observed among crystals suggest the existence of multiple conformers in solution.

  18. 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.

  19. Gene expression variation in duplicate lactate dehydrogenase genes: do ecological species show distinct responses?

    PubMed

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

    2014-01-01

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

  20. Lactate dehydrogenase as a biomarker for early renal damage in patients with sickle cell disease.

    PubMed

    Alzahri, Mohammad S; Mousa, Shaker A; Almomen, Abdulkareem M; Hasanato, Rana M; Polimeni, John M; Racz, Michael J

    2015-01-01

    Among many complications of sickle cell disease, renal failure is the main contributor to early mortality. It is present in up to 21% of patients with sickle cell disease. Although screening for microalbuminuria and proteinuria is the current acceptable practice to detect and follow renal damage in patients with sickle cell disease, there is a crucial need for other, more sensitive biomarkers. This becomes especially true knowing that those biomarkers start to appear only after more than 60% of the kidney function is lost. The primary purpose of this study is to determine whether lactate dehydrogenase (LDH) correlates with other, direct and indirect bio-markers of renal insufficiency in patients with sickle cell disease and, therefore, could be used as a biomarker for early renal damage in patients with sickle cell disease. Fifty-five patients with an established diagnosis of sickle cell disease were recruited to in the study. Blood samples were taken and 24-h urine collection samples were collected. Using Statcrunch, a data analysis tool available on the web, we studied the correlation between LDH and other biomarkers of kidney function as well as the distribution and relationship between the variables. Regression analysis showed a significant negative correlation between serum LDH and creatinine clearance, R (correlation coefficient) = -0.44, P = 0.0008. This correlation was more significant at younger age. This study shows that in sickle cell patients LDH correlates with creatinine clearance and, therefore, LDH could serve as a biomarker to predict renal insufficiency in those patients. PMID:26586054

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

    PubMed Central

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

    2014-01-01

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

  2. Low intensity microwave radiation as modulator of the L-lactate dehydrogenase activity.

    PubMed

    Vojisavljevic, Vuk; Pirogova, Elena; Cosic, Irena

    2011-07-01

    In this study, we investigated experimentally the possibility of modulating protein activity by low intensity microwaves by measuring alternations of L: -Lactate Dehydrogenase enzyme (LDH) activity. The LDH enzyme solutions were irradiated by microwaves of the selected frequencies and powers using the Transverse Electro-Magnetic (TEM) cell. The kinetics of the irradiated LDH was measured by continuous monitoring of nicotine adenine dinucleotide, reduced (NADH) absorbance at 340 nm. A comparative analysis of changes in the activity of the irradiated LDH enzyme versus the non-radiated enzyme was performed for the selected frequencies and powers. It was found that LDH activity can be selectively increased only by irradiation at the particular frequencies of 500 MHz [electric field: 0.02 V/m (1.2 × 10?? W/m²)-2.1 V/m (1.2 × 10?² W/m²)] and 900 MHz [electric field: 0.021-0.21 V/m (1.2 × 10?? W/m²)]. Based on results obtained it was concluded that LDH enzyme activity can be modulated by specific frequencies of low power microwave radiation. This finding can serve to support the hypothesis that low intensity microwaves can induce non-thermal effects in bio-molecules. PMID:21308416

  3. Quantification of lactate-dehydrogenase and cell viability in postmortem human dental pulp.

    PubMed

    Caviedes-Bucheli, Javier; Avendaño, Nuvia; Gutierrez, Rhina; Hernández, Sandra; Moreno, Gloria Cristina; Romero, María Consuelo; Muñoz, Hugo Roberto

    2006-03-01

    Understanding pulp repair and regeneration requires being familiar with this tissue's behavior under extreme conditions, such as postmortem state where an abrupt interruption of tissue blood supply occurs. The purpose of this study was to quantify cell viability and the amount of lactate-dehydrogenase (LDH) expressed in human pulp tissue 6, 12, and 24 hours postmortem to establish how long dental pulp remains viable after death. Pulp samples were obtained from 14 unidentified corpses of people who had received lethal injuries in car accidents or from gunshot wounds; they had at least three caries- and restoration-free incisors. Half of each sample was used for determining cell viability at three different time intervals. The rest of each sample was used for quantifying LDH expression at the same time intervals. Another 14 pulp samples were obtained from live patients' healthy premolars where extraction was indicated for orthodontic reasons to assess normal LDH value in pulp tissue. The results showed cell viability decreasing from 89 to 68 to 41% measured 6, 12, and 24 hours postmortem, respectively. LDH expression in healthy pulps was 246 U/mg pulp weight. Expression increased after death from 249 U/mg at 6 hours to 337 U/mg at 12 hours. LDH expression decreased to 131 U/mg 24 hours postmortem. These findings are valuable in understanding dental pulp survival capability under extreme conditions that may have important clinical significance in terms of repair and regeneration. PMID:16500222

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

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

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

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

    PubMed

    Südi, J

    1974-04-01

    The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k(obs.)=120s(-1), k(-2)=1200s(-1). PMID:4377095

  6. Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria*

    PubMed Central

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

    2013-01-01

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the 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. PMID:23720742

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

    PubMed

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

    2014-08-01

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

  8. Antimalarial Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by Docking Studies

    PubMed Central

    Penna-Coutinho, Julia; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; França, Tanos Celmar Costa; Krettli, Antoniana Ursine

    2011-01-01

    The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ?90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use. PMID:21779323

  9. 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 ?M). 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 ?M). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships. PMID:23628333

  10. 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:26587373

  11. 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. PMID:12545384

  12. 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

  13. 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-?ldhA increased 1.5 fold. Metabolic flux analysis showed both IAM1183-?ldhA and IAM1183-?adh exhibited significant changes in flux, including enhanced flux towards the hydrogen generation. The lactate production of IAM1183-?ldhA significantly decreased by 91.42%, while the alcohol yield of IAM1183-?adh decreased to 30%. The mutant IAM1183-?ldhA with better hydrogen-producing performance was selected for further investigation in a 5-L fermentor. The hydrogen production of IAM1183-?ldhA 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. PMID:26188552

  14. Creatine kinase and lactate dehydrogenase isoenzymes in serum of patients suffering burns, blunt trauma, or myocardial infarction.

    PubMed

    Shahangian, S; Ash, K O; Wahlstrom, N O; Warden, G D; Saffle, J R; Taylor, A; Green, L S

    1984-08-01

    Medical records of 53 burn and trauma patients were reviewed to assess the possibility of myocardial damage. Except for electrophoretically detectable creatine kinase MB isoenzyme, none showed evidence of myocardial injury. Lactate dehydrogenase isoenzyme tests, electrocardiograms, myocardial pyrophosphate scans, clinical course, and results of (two) autopsies were all negative for myocardial necrosis or ischemia. Types of patient, number, mean peak value (U/L) for serum creatine kinase, and ranges of percentage MB isoenzyme were as follows. Burns from direct electrical contact: 28, 16 600, 0-29; electrical flash or other thermal burns: 10, 4340, 0-22; blunt trauma (mostly from automobile accidents): 15, 3430, 0-18; myocardial infarction: 57, 1520, 4-46. Evidently creatine kinase MB isoenzyme is nonspecific in burn and trauma patients and should not be the only test result used to assess myocardial involvement. PMID:6744581

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

    SciTech Connect

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

    2012-01-01

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

  16. Highly stereoselective biosynthesis of (R)-?-hydroxy carboxylic acids through rationally re-designed mutation of d-lactate dehydrogenase

    PubMed Central

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

    2013-01-01

    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 ?-keto carboxylic acids such as phenylpyruvic acid (PPA), ?-ketobutyric acid, ?-ketovaleric acid, ?-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)-?-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 ?-hydroxy carboxylic acids. PMID:24292439

  17. Production of l-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases

    PubMed Central

    2013-01-01

    Background Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding l-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. Results Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. Conclusions We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional ethanol pathway, at neutral and low pH, generated a comprehensive picture of lactic acid production in this yeast. The findings are applicable in generation other lactic acid producing yeast, thus providing a significant contribution to the field of biotechnical production of lactic acid. PMID:23706009

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

    PubMed

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

    2015-08-14

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

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

    PubMed Central

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

    2015-01-01

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

  20. The Cost of Capturing Prey: Measuring Largemouth Bass Foraging Activity using Glycolytic Enzymes (Lactate Dehydrogenase)

    E-print Network

    The Cost of Capturing Prey: Measuring Largemouth Bass Foraging Activity using Glycolytic Enzymes THE COST OF CAPTURING PREY: MEASURING LARGEMOUTH BASS FORAGING ACTIVITY USING GLYCOLYTIC ENZYMES (LACTATE #12;11 The Cost of Capturing Prey: Measuring Largemouth Bass Foraging Activity using Glycolytic

  1. Mitochondrial Lactate Dehydrogenase Is Involved in Oxidative-Energy Metabolism in Human Astrocytoma

    E-print Network

    Appanna, Vasu

    product of anaerobic energy production and its fate in cerebral metabolism has not been precisely@laurentian.ca Introduction Since lactate is produced during anaerobic energy metabolism, this monocarboxylic acid has been postulated that this monocarboxylic acid may be supporting oxidative respiration in the neurons. This may

  2. 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 period when the use of artificial teats and dummies (pacifiers) may condition the infant to different oral actions that are inappropriate for breast-feeding. Comparisons of breast milk and cow's milk fail to describe the many important differences between them, e.g., the structural and qualitative differences in proteins and fats, and the bioavailability of minerals. The protection against infection and allergies conferred on the infant, which is impossible to attain through any other feeding regimen, is one of breast milk's most outstanding qualities. The maximum birth-spacing effect of lactation is achieved when an infant is fully, or nearly fully, breast-fed and the mother consequently remains amenorrhoeic. PMID:20604468

  3. 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.

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

    PubMed

    Zhang, Haili; Guo, Fengguang; Zhu, Guan

    2015-11-01

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

  5. 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. PMID:19472341

  6. Biochemical, biophysical, and molecular genetic studies on the membrane-bound D-lactate dehydrogenase from Escherichia coli

    SciTech Connect

    Rule, G.S.

    1986-01-01

    The membrane-bound D-lactate dehydrogenase (D-LDH) from E. coli has been used as a model system in order to study structure-function relationships in membrane proteins. This enzyme is activated by various lipids and detergents in vitro, and appears to provide energy for the active transport of amino acids and sugars in E. coli membrane vesicles. In order to obtain sufficient amounts of enzyme for /sup 19/F nuclear magnetic resonance (NMR) and circular dichroism (CD) experiments, the cloned dld gene was used to construct a plasmid in which the expression of D-LDH is induced by a temperature shift. Upon temperature induction, the presence of this plasmid results in levels of D-LDH in the cell which are 300-fold higher than wild type levels. The cloned dld gene was also sequenced and the primary structure of D-LDH, as deduced from the DNA sequence, was verified by determination of the amino-terminal sequence and the amino acid composition of D-LDH. The dld gene codes for a protein which does not contain a signal sequence and is 571 residues long.

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

    PubMed Central

    Zhu, Guan

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. 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.

  10. 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

  11. The effect of foetal bovine serum supplementation upon the lactate dehydrogenase cytotoxicity assay: Important considerations for in vitro toxicity analysis.

    PubMed

    Thomas, Martin G; Marwood, Roxanne M; Parsons, Anna E; Parsons, Richard B

    2015-12-25

    The lactate dehydrogenase (LDH) assay is a commonly-used tool for assessing toxicity in vitro. However, anecdotal reports suggest that foetal bovine serum (FBS) may contain LDH at concentrations significant enough to interfere with the assay and thus reduce its sensitivity. A series of experiments were performed to determine whether addition of FBS to culture medium significantly elevated culture media LDH content, and whether replacement of FBS with heat inactivated foetal bovine serum (HI-FBS) reduced LDH content and interfered with cell response to cytotoxic challenge. The addition of FBS at 5, 10 and 15% final concentrations increased culture medium LDH content in a dose-dependent manner. The substitution of HI-FBS for FBS reduced culture medium LDH content and increased the dynamic range of the assay. Cell viability of the SH-SY5Y human neuroblastoma and N27 rat mesencephalic neurone cell lines were significantly reduced as measured using the MTT reduction assay, whilst HI-FBS only affected toxicity response in a cell- and toxin-specific manner, although these effects were small. Hence, for cell lines with a high FBS requirement, the use of HI-FBS or alternative toxicity assays can be considered, or the use of alternative formulations, such as chemically-defined serum-free media, be adopted. PMID:26498060

  12. Identification of N-acylhydrazone derivatives as novel lactate dehydrogenase A inhibitors.

    PubMed

    Rupiani, Sebastiano; Buonfiglio, Rosa; Manerba, Marcella; Di Ianni, Lorenza; Vettraino, Marina; Giacomini, Elisa; Masetti, Matteo; Falchi, Federico; Di Stefano, Giuseppina; Roberti, Marinella; Recanatini, Maurizio

    2015-08-28

    Glycolysis is drastically increased in tumors and it is the main route to energy production with a minor use of oxidative phosphorylation. Among the key enzymes in the glycolytic process, LDH is emerging as one of the most interesting targets for the development of new inhibitors. In this context, in the present work, we carried out a virtual screening procedure followed by chemical modifications of the identified structures according to a "hit-to-lead" process. The effects of the new molecules were preliminary probed against purified human LDH-A. The compounds active at low micromolar level were additionally characterized for their activity on some cellular metabolic processes by using Raji human cell line. Within the series, 1 was considered the best candidate, and a more detailed characterization of its biological properties was performed. In Raji cells exposed to compound 1 we evidenced the occurrence of effects usually observed in cancer cells after LDH-A inhibition: reduced lactate production and NAD/NADH ratio, apoptosis. The flow cytometry analysis of treated cells also showed cell cycle changes compatible with effects exerted at the glycolytic level. Finally, in agreement with the data obtained with other inhibitors or by silencing LDH-A expression, compound 1 was found to increase Raji cells response to some commonly used chemotherapeutic agents. Taken together, all these finding are in support of the LDH-A inhibiting activity of compound 1. PMID:26114812

  13. Structure and reaction pathways of methyl lactate on Pd(1 1 1)

    NASA Astrophysics Data System (ADS)

    Burkholder, Luke; Tysoe, Wilfred T.

    2009-09-01

    The adsorption and reaction of methyl lactate (CH 3CH(OH)COOCH 3) is studied in ultrahigh vacuum on a Pd(1 1 1) surface using temperature-programmed desorption (TPD) and reflection-absorption infrared spectroscopy (RAIRS). Methyl lactate reacts at relatively low temperatures (˜220 K) by O-H bond scission. This intermediate can either react with hydrogen to reform methyl lactate at ˜280-300 K or undergo ?-hydride elimination to form flat-lying methyl pyruvate. This decomposes to form acetyl and methoxy carbonyl species as found previously following methyl pyruvate adsorption on Pd(1 1 1). These species predominantly react to form carbon monoxide, methane and hydrogen.

  14. Expression of lactate dehydrogenase A and B genes in different tissues of rats adapted to chronic hypobaric hypoxia.

    PubMed

    Rossignol, Fabrice; Solares, Magali; Balanza, Elfride; Coudert, Jean; Clottes, Eric

    2003-05-01

    Lactate dehydrogenase (LDH) is a tetramer made up of two different subunits A and B. In cellular models, severe hypoxia increases LDH A gene expression whereas LDH B gene does not exhibit any regulation. The aim of our work was to characterise LDH expression in different tissues of rats bred at high altitude. For this purpose, we chose a Sprague-Dawley rat strain adapted to chronic hypoxia in La Paz (3700 m), Bolivia. Two normoxic control groups were bred at low altitude in Clermont-Ferrand (350 m), France, one group was ad libitum with free access to food and water as was the hypoxic one, and the second normoxic group was nourished with the food intakes measured for the animals from La Paz. We measured total LDH specific activity, isoform distribution and LDH A and B mRNA amounts in three skeletal muscles (soleus, extensor digitorum longus (EDL), plantaris), heart and brain. Our study demonstrates that, unlike what has been shown in cellular models under severe hypoxia, LDH A gene is not systematically up-regulated in tissues of rats living at high altitude. Furthermore, chronic hypoxia limits LDH B gene transcription or its mRNA stability in both soleus and EDL. These regulations occur at various molecular levels like gene transcription, mRNA stabilisation or translation and protein stability, depending on the tissue studied, and are partly attributed to caloric restriction provoked by high altitude. These data provide insight into LDH gene expression underlying the diverse and complex tissue-specific response to chronic hypoxia. PMID:12682909

  15. 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

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

    SciTech Connect

    Liao, Ya-Tang; Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan; Genomics Research Center, Academia Sinica, Taiwan ; Chen, Chien-Jen; Genomics Research Center, Academia Sinica, Taiwan ; Li, Wan-Fen; Hsu, Ling-I; Tsai, Li-Yu; Huang, Yeou-Lih; Sun, Chien-Wen; Chen, Wei J.; Wang, Shu-Li; Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan

    2012-08-01

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

  17. 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

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

    PubMed Central

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

    1997-01-01

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

  19. Lactate dehydrogenase test

    MedlinePLUS

    ... value range is 105 - 333 IU/L (international units per liter). Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or test different samples. Talk to your doctor about the ...

  20. Changes in milk yield, lactate dehydrogenase, milking frequency, and interquarter yield ratio persist for up to 8 weeks after antibiotic treatment of mastitis.

    PubMed

    Fogsgaard, K K; Løvendahl, P; Bennedsgaard, T W; Østergaard, S

    2015-11-01

    Within the dairy industry, the appearance of milk and withdrawal time due to antibiotic residuals in the milk are used to determine recovery status after cases of treated mastitis. However, both milk production and dairy cow behavior have been shown to be affected after the normalization of milk appearance, indicating that animals may not have fully recovered. The aim of the present study was to describe the changes in milk yield, lactate dehydrogenase activity, milking frequency, and interquarter yield ratio (defined as the coefficient of variation between the active quarters) after cases of naturally occurring mastitis with special focus on the recovery period after antibiotic treatment. A second aim was to examine whether these changes were affected by the pathogens present at the time of mastitis diagnosis. This retrospective study was based on a cohort data set including 1,032 lactations from 795 dairy cows kept on 2 Danish farms and milked by an automatic milking system. A total of 174 treated mastitis cases were compared with nontreated control cows from 5wk before treatment and until 8wk after. Treated mastitis resulted in reduced milk yield, elevated lactate dehydrogenase activity, lower milking frequency, and elevated interquarter yield ratio. Within these measures, deviations from baseline levels and from the control cows were found as early as 1 to 3wk before the antibiotic treatment and peaked around the days of treatment. In some cases, the mastitic cows returned to premastitis levels, whereas in others they remained affected throughout the rest of the observation period. To correctly estimate the effects of treated mastitis and the recovery status of cows, it is important to take the individual cow into account and not only compare with herd levels, as this might mask the true degree of the changes. The effects on each outcome variable depended on the involved pathogen and differences were found between primiparous cows and older animals. However, in general, the changes in milk production, lactate dehydrogenase activity, and interquarter yield ratio showed parallels, suggesting that the recovery period continued for weeks after antibiotic treatment. These results call for further investigation into management of mastitic dairy cows to optimize recovery, limit milk loss, and ensure animal welfare during the period after mastitis. PMID:26364092

  1. 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±6.43?mol(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. PMID:24412354

  2. Stereoselective titanium-mediated aldol reactions of a chiral lactate-derived ethyl ketone with ketones.

    PubMed

    Alcoberro, Sandra; Gómez-Palomino, Alejandro; Solà, Ricard; Romea, Pedro; Urpí, Fèlix; Font-Bardia, Mercè

    2014-01-17

    Aldol reactions of titanium enolates of lactate-derived ethyl ketone 1 with other ketones proceed in a very efficient and stereocontrolled manner provided that a further equivalent of TiCl4 is added to the reacting mixture. The scope of these reactions encompasses simple ketones such as acetone or cyclohexanone as well as other ketones that contain potential chelating groups such as pyruvate esters or ?- and ?-hydroxy ketones. PMID:24372372

  3. 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 parasitaemias. OptiMAL-IT® performed best overall and would perform best in an area of high malaria prevalence among screened fever cases. However, heat stability was unacceptable and the number of steps to perform this test is a significant drawback in the field. A reliable, heat-stable, highly sensitive RDT, capable of diagnosing all Plasmodium species has yet to be identified. PMID:19860920

  4. Reanalysis of mGWAS results and in vitro validation show that lactate dehydrogenase interacts with branched-chain amino acid metabolism.

    PubMed

    Heemskerk, Mattijs M; van Harmelen, Vanessa Ja; van Dijk, Ko Willems; van Klinken, Jan Bert

    2016-01-01

    The assignment of causative genes to noncoding variants identified in genome-wide association studies (GWASs) is challenging. We show how combination of knowledge from gene and pathway databases and chromatin interaction data leads to reinterpretation of published quantitative trait loci for blood metabolites. We describe a previously unidentified link between the rs2403254 locus, which is associated with the ratio of 3-methyl-2-oxobutanoate and alpha-hydroxyisovalerate levels, and the distal LDHA gene. We confirmed that lactate dehydrogenase can catalyze the conversion between these metabolites in vitro, suggesting that it has a role in branched-chain amino acid metabolism. Examining datasets from the ENCODE project we found evidence that the locus and LDHA promoter physically interact, showing that LDHA expression is likely under control of distal regulatory elements. Importantly, this discovery demonstrates that bioinformatic workflows for data integration can have a vital role in the interpretation of GWAS results. PMID:26014429

  5. 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. PMID:25217009

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

    PubMed Central

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

    2014-01-01

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

  7. (4B-3H) NADH-H2O exchange reaction of the mitochondrial NADH dehydrogenase

    SciTech Connect

    Chen, S.; Guillory, R.J.

    1985-06-14

    The purified mitochondrial NADH dehydrogenase enzyme has been shown to catalyze a rapid (4B-/sup 3/H) NADH-H/sub 2/O exchange reaction. When the enzyme is subjected to a single freeze-thaw cycle there is a complete loss of NADH dehydrogenation without a measurable decrease in the (4B-/sup 3/H) NADH-H/sub 2/O exchange. Complete loss of the (4B-/sup 3/H) NADH-H/sub 2/O exchange follows brief exposure to ultraviolet photoirradiation. The differential sensitivity of the water exchange reaction and the dehydrogenase activity suggests a direct involvement of the enzymes flavin cofactor in the catalysis of the (4B-/sup 3/H) NADH-H/sub 2/O exchange. Arylazido-beta-alanyl NAD+ (A3'-0-(3-(N-4-azido-2-nitrophenyl)amino) propionyl)NAD+) is shown to be a potent photodependent inhibitor of the (4B-3H) NADH-H/sub 2/O exchange activity following photoirradiation with visible light. This is consistent with the observed photodependent inhibition of the dehydrogenase activity by this photoprobe.

  8. 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.

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

    PubMed

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

    2011-03-24

    Highly invasive tumor cells are characterized by a metabolic switch, known as the Warburg effect, from "normal" oxidative phosphorylation to increased glycolysis even under sufficiently oxygenated conditions. This dependence on glycolysis also confers a growth advantage to cells present in hypoxic regions of the tumor. One of the key enzymes involved in glycolysis, the muscle isoform of lactate dehydrogenase (LDH-A), is overexpressed by metastatic cancer cells and is linked to the vitality of tumors in hypoxia. This enzyme may be considered as a potential target for new anticancer agents, since its inhibition cuts cancer energetic and anabolic supply, thus reducing the metastatic and invasive potential of cancer cells. We have discovered new and efficient N-hydroxyindole-based inhibitors of LDH-A, which are isoform-selective (over LDH-B) and competitive with both the substrate (pyruvate) and the cofactor (NADH). The antiproliferative activity of these compounds was confirmed on a series of cancer cell lines, and they proved to be particularly effective under hypoxic conditions. Moreover, NMR experiments showed that these compounds are able to reduce the glucose-to-lactate conversion inside the cell. PMID:21332213

  10. Dependence of Substrate Irradiation Reaction Rate Stimulation on Lactic Dehydrogenase Source

    E-print Network

    George Bass; James Chenevey

    2013-04-16

    Stimulation of LDH initial reaction rates by timed pre-irradiation of crystalline sodium pyruvate and lithium lactate is reported for enzymes isolated from rabbit muscle, pig heart, human erythrocytes and chicken heart. The phenomenon investigated is referred to as the Comorosan effect. For the mammalian source enzymes, the pyruvate irradiation stimulations occurred at irradiation times of 5 and 35 sec. and the lactate irradiation times at 15 and 45 seconds. In contrast, for the chicken heart enzyme, the pyruvate irradiation stimulations occurred at 15 and 35 sec., while those for lactate occurred at 5 and 20 sec. Thus, a shift in stimulatory irradiation times is found on going from the mammalian enzymes to the avian enzyme. A similar shift between mammalian and yeast enzymes has been established by Comorosan and co-workers. For the chicken heart LDH, the separation between successive irradiation times is different for the forward and reverse reactions. This is the first reported incidence of the separation not being the same.

  11. Indoleamine 2,3?dioxygenase downregulates T?cell receptor complex ??chain and c?Myc, and reduces proliferation, lactate dehydrogenase levels and mitochondrial glutaminase in human T?cells.

    PubMed

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Tsogka, Konstantina; Sounidaki, Maria; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2016-01-01

    Indoleamine 2,3?dioxygenase (IDO), through L?tryptophan depletion, activates general control non?derepressible (GCN) 2 kinase and suppresses T?cell proliferation, in addition to suppressing aerobic glycolysis and glutaminolysis, which are required for these rapidly proliferating cells. A number of, however not all of these alterations, are partially mediated through IDO?induced p53 upregulation. In two?way mixed lymphocyte reactions (MLRs), IDO reduced cellular proliferation. In MLR?derived T?cells, IDO induced the expression levels of p53 and p21, however concurrently reduced the levels of ??chain, c?Myc, lactate dehydrogenase A (LDH?A) and glutaminase (GLS)2. However, p53 had no effect on the expression of the above proteins. These results were recapitulated in T?cells activated with anti?CD2, anti?CD3 and anti?CD28 by direct activation of the GCN2 kinase with tryptophanol. In conclusion, IDO, through GCN2 kinase activation, downregulates the levels of TCR?complex ??chain and c?Myc, resulting in the suppression of T?cell proliferation and a reduction in the levels of LDH?A and GLS2, which are key enzymes involved in aerobic glycolysis and glutaminolysis, respectively. PMID:26647830

  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. PMID:25988244

  13. Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.

    PubMed

    Moxley, Michael A; Becker, Donald F

    2012-01-10

    The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and ?(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters. PMID:22148640

  14. The effects of lactate on nitrosylmyoglobin formation from nitrite and metmyoglobin in a cured meat system.

    PubMed

    McClure, Brooke N; Sebranek, Joseph G; Kim, Yuan H; Sullivan, Gary A

    2011-12-01

    Two experiments were conducted to determine the effects of lactate on nitrite during meat curing. In the first experiment, using a model system, eight reaction components including nitrite and lactate, were used to assess the effect of each component on metmyoglobin reducing activity by excluding one component at a time. Excluding lactate, nicotinamide adenine dinucleotide (NAD), l-lactate dehydrogenase (LDH) or phenazine methosulfate (PMS) resulted in no reducing activity. A second experiment, utilising a meat mixture, investigated the effects of lactate (0%, 2%, 4% or 6%), nitrite (0 or 156ppm), and packaging (oxygen-permeable or vacuum) on residual nitrite, meat colour and pH. Addition of lactate reduced residual nitrite in the meat mixtures. Both experiments support the hypothesis that lactate generates NADH which then reduces metmyoglobin to deoxymyoglobin. The resulting greater concentration of reduced myoglobin subsequently reacted with nitrite to produce more nitric oxide, reducing nitrite concentration and accelerating curing reactions. PMID:25212339

  15. Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase.

    PubMed

    Wang, Vincent C-C; Islam, Shams T A; Can, Mehmet; Ragsdale, Stephen W; Armstrong, Fraser A

    2015-10-29

    Protein film electrochemistry has been used to investigate reactions of highly active nickel-containing carbon monoxide dehydrogenases (CODHs). When attached to a pyrolytic graphite electrode, these enzymes behave as reversible electrocatalysts, displaying CO2 reduction or CO oxidation at minimal overpotential. The O2 sensitivity of CODH is suppressed by adding cyanide, a reversible inhibitor of CO oxidation, or by raising the electrode potential. Reduction of N2O, isoelectronic with CO2, is catalyzed by CODH, but the reaction is sluggish, despite a large overpotential, and results in inactivation. Production of H2 and formate under highly reducing conditions is consistent with calculations predicting that a nickel-hydrido species might be formed, but the very low rates suggest that such a species is not on the main catalytic pathway. PMID:26176986

  16. 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.

  17. Duck lens epsilon-crystallin and lactate dehydrogenase B4 are identical: a single-copy gene product with two distinct functions.

    PubMed Central

    Hendriks, W; Mulders, J W; Bibby, M A; Slingsby, C; Bloemendal, H; de Jong, W W

    1988-01-01

    To investigate whether or not duck lens epsilon-crystallin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck epsilon-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens. Images PMID:3174623

  18. Immune evasion mediated by tumor-derived lactate dehydrogenase induction of NKG2D ligands on myeloid cells in glioblastoma patients.

    PubMed

    Crane, Courtney A; Austgen, Kathryn; Haberthur, Kristen; Hofmann, Carly; Moyes, Kara White; Avanesyan, Lia; Fong, Lawrence; Campbell, Michael J; Cooper, Stewart; Oakes, Scott A; Parsa, Andrew T; Lanier, Lewis L

    2014-09-01

    Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses. PMID:25136121

  19. The oxidative half-reaction of xanthine dehydrogenase with NAD; reaction kinetics and steady-state mechanism.

    PubMed

    Harris, C M; Massey, V

    1997-11-01

    The reaction between reduced xanthine dehydrogenase (XDH) from bovine milk and NAD has been studied in detail. An understanding of this reaction is necessary for a complete description of XDH turnover with its presumed natural electron acceptor and to address the preference of XDH for NAD over oxygen as a substrate. The reaction between pre-reduced XDH and NAD was studied by stopped-flow spectrophotometry. The reaction was found to involve two rounds of oxidation with 2 eq of NAD. The first round goes to completion, and the second round reaches a slightly disfavored equilibrium. Rapid binding of NAD with an apparent Kd of 25 +/- 2 microM is followed by NAD reduction at a rate constant of 130 +/- 13 s-1. NADH dissociation at a rate constant of 42 +/- 12 s-1 completes a round of oxidation. These steps have been successfully tested and modeled to repeat themselves in the second round of oxidation. The association rate constant for NAD binding was estimated to be much greater than any rate constant measured in the oxidation by molecular oxygen, thus explaining how NAD competes with oxygen for reducing equivalents. Rate constants for NAD reduction and NADH dissociation are respectively 21- and 7-fold greater than kcat, indicating that the reductive half-reaction of the enzyme by xanthine is mostly rate-limiting in xanthine/NAD turnover. A steady-state mechanism for XDH is discussed. PMID:9353290

  20. Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

    SciTech Connect

    Malik, Radhika; Viola, Ronald E.

    2010-10-28

    The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

  1. 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. PMID:24861375

  2. 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.

  3. Biocatalytic carbon capture via reversible reaction cycle catalyzed by isocitrate dehydrogenase.

    PubMed

    Xia, Shunxiang; Frigo-Vaz, Benjamin; Zhao, Xueyan; Kim, Jungbae; Wang, Ping

    2014-09-12

    The practice of carbon capture and storage (CCS) requires efficient capture and separation of carbon dioxide from its gaseous mixtures such as flue gas, followed by releasing it as a pure gas which can be subsequently compressed and injected into underground storage sites. This has been mostly achieved via reversible thermochemical reactions which are generally energy-intensive. The current work examines a biocatalytic approach for carbon capture using an NADP(H)-dependent isocitrate dehydrogenase (ICDH) which catalyzes reversibly carboxylation and decarboxylation reactions. Different from chemical carbon capture processes that rely on thermal energy to realize purification of carbon dioxide, the biocatalytic strategy utilizes pH to leverage the reaction equilibrium, thereby realizing energy-efficient carbon capture under ambient conditions. Results showed that over 25 mol of carbon dioxide could be captured and purified from its gas mixture for each gram of ICDH applied for each carboxylation/decarboxylation reaction cycle by varying pH between 6 and 9. This work demonstrates the promising potentials of pH-sensitive biocatalysis as a green-chemistry route for carbon capture. PMID:25152403

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

    PubMed

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

    2014-10-01

    Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D-form of PLA is known to be more active than the L-isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating D-lactate dehydrogenase (D-ldh) were used to produce D-PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (?50 mmol l(-1)), growing cells produced a maximum yield of 35 mmol l(-1) of D-PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0-7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. D-ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l(-1) and 5645 s(-1), respectively. The conditions required for the efficient production of D-PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield. Significance and impact of the study: This is the first study on the production of D-phenyllactic acid, which is a natural antimicrobial compound, from phenylpyruvate using Leuconostoc mesenteroides cells. The strain, ATCC 8293, that was used in the study, possesses high stereoselectivity and delivers a high yield. Therefore, it might be a promising candidate for use in large-scale production facilities and in fermented foods. PMID:24888766

  5. Pretreatment Serum Lactate Dehydrogenase and N Classification Predict Long-Term Survival and Distant Metastasis in Patients With Nasopharyngeal Carcinoma Who Have A Positive Family History of Cancer

    PubMed Central

    Zhang, Wenna; Chen, Yupei; Zhou, Guanqun; Liu, Xu; Chen, Lei; Tang, Linglong; Mao, Yanping; Sun, Ying; Ma, Jun

    2015-01-01

    Abstract The purpose of the present study was to evaluate prognostic factors in patients with nasopharyngeal carcinoma (NPC) from the endemic area of southern China who have a positive family history (FH) of cancer. Retrospective analysis of 600 patients with nondisseminated NPC and a positive FH was conducted. The prognostic value of different factors for overall survival (OS), distant metastasis-free survival (DMFS), disease-free survival (DFS), and local relapse-free survival (LRFS) were assessed using Cox regression models. The 3-year OS, DMFS, DFS, and LRFS rates were 93.8%, 91.3%, 86.3%, and 93.8%, respectively. The FH tumor type was NPC for 226/600 (37.7%) patients and other cancers for 374/600 (62.3%) patients. The 3-year OS and DMFS rates for patients with an FH of NPC were 91.2% and 89.8%, respectively. Thirty of 600 (5.0%) patients had elevated pretreatment serum lactate dehydrogenase (LDH >245.0?IU/L). In multivariate analysis, N classification (HR 4.56, 95% CI 2.13–9.74, P?

  6. A study of salivary lactate dehydrogenase isoenzyme levels in patients with oral leukoplakia and squamous cell carcinoma by gel electrophoresis method

    PubMed Central

    Joshi, Priya Shirish; Golgire, Someshwar

    2014-01-01

    Context: The enzyme lactate dehydrogenase (LDH), which is found in almost all the cells of body tissues, can be separated into five fractions and the isoenzyme pattern is believed to vary according to the metabolic requirement of each tissue. LDH concentration in saliva, as an expression of cellular necrosis, could be considered to be a specific indicator for oral lesions that affect the integrity of the oral mucosa. Aim: The present study was designed to evaluate salivary LDH isoenzyme pattern in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and to correlate between LDH isoenzyme levels and histopathologic grading in selected cases of OL and OSCC. Materials and Methods: Clinically diagnosed 30 cases each of OL and OSCC were selected for the study and 30 healthy individuals of comparable age served as control. Unstimulated whole saliva was aseptically collected and was processed immediately for LDH isoenzymes measurement by agarose gel electrophoresis. Biopsy specimen obtained was processed and stained by hematoxylin and eosin. Sections of OL and OSCC cases were scrutinized histopathologically and appropriately graded for epithelial dysplasia and differentiation of carcinoma respectively. Statistical Analysis Used: Two sample t test for testing the significance of difference between two group means was used. Results and Conclusion: The present salivary analysis for LDH isoenzyme reveals an overall increased salivary LDH isoenzyme level in OL and OSCC cases and a significant correlation between levels of salivary LDH isoenzymes and histopathologic grades of dysplasia in OL and OSCC. Salivary analysis of LDH will definitely provide the clinician and/or the patient himself with an efficient, non invasive and friendly new tool for diagnosis and monitoring of oral precancer and cancer. PMID:25364177

  7. 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.

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

    PubMed Central

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

    2013-01-01

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

  9. Evidence for hysteretic substrate channeling in the proline dehydrogenase and ?1-pyrroline-5-carboxylate dehydrogenase coupled reaction of proline utilization A (PutA).

    PubMed

    Moxley, Michael A; Sanyal, Nikhilesh; Krishnan, Navasona; Tanner, John J; Becker, Donald F

    2014-02-01

    PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and ?(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to ?(1)-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-?-semialdehyde (the hydrolyzed form of ?(1)-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD(+)-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed. PMID:24352662

  10. Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and ?1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)*

    PubMed Central

    Moxley, Michael A.; Sanyal, Nikhilesh; Krishnan, Navasona; Tanner, John J.; Becker, Donald F.

    2014-01-01

    PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and ?1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to ?1-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-?-semialdehyde (the hydrolyzed form of ?1-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD+-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed. PMID:24352662

  11. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...crystalline mass. It is prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous sulfate with...

  12. Age-dependent poliomyelitis of mice: expression of endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons.

    PubMed Central

    Contag, C H; Plagemann, P G

    1989-01-01

    The widespread presence of endogenous retroviruses in the genomes of animals and humans has suggested that these viruses may be involved in both normal and abnormal developmental processes. Previous studies have indicated the involvement of endogenous ecotropic murine leukemia virus (MuLV) in the development of age-dependent poliomyelitis caused by infection of old C58 or AKR mice by lactate dehydrogenase-elevating virus (LDV). The only genetic components which segregate with susceptibility to LDV-induced paralytic disease are multiple proviral copies of ecotropic MuLV and the permissive allele, at the Fv-1 locus, for N-tropic, ecotropic virus replication (Fv-1n/n). Using in situ hybridization and Northern (RNA) blot hybridization, we have correlated the expression of the endogenous MuLV, both temporally and spatially, with LDV infection of anterior horn motor neurons and the development of paralysis. Our data indicate that treatment of 6- to 7-month-old C58/M mice with cyclophosphamide, which renders these mice susceptible to LDV-induced paralytic disease, results in transient increases in ecotropic MuLV RNA levels in motor neurons throughout the spinal cord. Peripheral inoculation of C58/M mice with LDV, at the time of elevated MuLV RNA levels, results in a rapid spread of LDV to some spinal cord motor neurons. LDV infections then spread slowly but progressively throughout the spinal cord, involving an increasing number of motor neurons. LDV replication is cytocidal and results in neuron destruction and paralysis of the infected animals 2 to 3 weeks postinfection. The slow replication of LDV in the spinal cord contrasts sharply with the rapid replication of LDV in macrophages, the normal host cells for LDV, during the acute phase of infection. The data indicate that the interaction between the endogenous MuLV with the generally nonpathogenic murine togavirus LDV occurs at the level of the motor neuron. We discuss potential mechanisms for the novel dual-virus etiology of age-dependent poliomyelitis of mice. Images PMID:2550670

  13. Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates.

    PubMed

    Graf, Michael M H; Sucharitakul, Jeerus; Bren, Urban; Chu, Dinh Binh; Koellensperger, Gunda; Hann, Stephan; Furtmüller, Paul G; Obinger, Christian; Peterbauer, Clemens K; Oostenbrink, Chris; Chaiyen, Pimchai; Haltrich, Dietmar

    2015-11-01

    Monomeric Agaricus meleagris pyranose dehydrogenase (AmPDH) belongs to the glucose-methanol-choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. AmPDH can double oxidize various mono- and oligosaccharides at different positions (C1 to C4). To study the structure/function relationship of selected active-site residues of AmPDH pertaining to substrate (carbohydrate) turnover in more detail, several active-site variants were generated, heterologously expressed in Pichia pastoris, and characterized by biochemical, biophysical and computational means. The crystal structure of AmPDH shows two active-site histidines, both of which could take on the role as the catalytic base in the reductive half-reaction. Steady-state kinetics revealed that His512 is the only catalytic base because H512A showed a reduction in (kcat /KM )glucose by a factor of 10(5) , whereas this catalytic efficiency was reduced by two or three orders of magnitude for His556 variants (H556A, H556N). This was further corroborated by transient-state kinetics, where a comparable decrease in the reductive rate constant was observed for H556A, whereas the rate constant for the oxidative half-reaction (using benzoquinone as substrate) was increased for H556A compared to recombinant wild-type AmPDH. Steady-state kinetics furthermore indicated that Gln392, Tyr510, Val511 and His556 are important for the catalytic efficiency of PDH. Molecular dynamics (MD) simulations and free energy calculations were used to predict d-glucose oxidation sites, which were validated by GC-MS measurements. These simulations also suggest that van der Waals interactions are the main driving force for substrate recognition and binding. PMID:26284701

  14. Application of artificial neural networks and DFT-based parameters for prediction of reaction kinetics of ethylbenzene dehydrogenase.

    PubMed

    Szaleniec, Maciej; Witko, Ma?gorzata; Tadeusiewicz, Ryszard; Goclon, Jakub

    2006-03-01

    Artificial neural networks (ANNs) are used for classification and prediction of enzymatic activity of ethylbenzene dehydrogenase from EbN1 Azoarcus sp. bacterium. Ethylbenzene dehydrogenase (EBDH) catalyzes stereo-specific oxidation of ethylbenzene and its derivates to alcohols, which find its application as building blocks in pharmaceutical industry. ANN systems are trained based on theoretical variables derived from Density Functional Theory (DFT) modeling, topological descriptors, and kinetic parameters measured with developed spectrophotometric assay. Obtained models exhibit high degree of accuracy (100% of correct classifications, correlation between predicted and experimental values of reaction rates on the 0.97 level). The applicability of ANNs is demonstrated as useful tool for the prediction of biochemical enzyme activity of new substrates basing only on quantum chemical calculations and simple structural characteristics. Multi Linear Regression and Molecular Field Analysis (MFA) are used in order to compare robustness of ANN and both classical and 3D-quantitative structure-activity relationship (QSAR) approaches. PMID:16779618

  15. Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

    PubMed

    Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

    2015-07-01

    Klebsiella oxytoca KMS005 (?adhE?ackA-pta?ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. PMID:25895450

  16. Structural features of aluminium(III) complexes with bioligands in glutamate dehydrogenase reaction system--a review.

    PubMed

    Yang, Xiaodi; Zhang, Qianqian; Li, Laifa; Shen, Renfang

    2007-09-01

    Aluminium(III) complexes are essential for understanding the toxicity, bioavailability and transport mechanisms of aluminium in environmental and biological systems. Since elucidation of the exact structures of these weakly coordinated systems is very difficult, the structures of Al(III) complexes in glutamate dehydrogenase reactions system were investigated recently from the following four aspects: (1) Constitutional studies: The keto-enol tautomerism of the complexes between aluminium(III) ion and alpha-ketoglutarate ligands in acidic aqueous solutions was studied. It is clearly demonstrated that Al(III) can promote the keto-enol tautomerization of alpha-ketoglutarate. (2) Configurational studies: Compared with L-Glu, the complex stability of D-Glu-Al is stronger, especially for the tridentate species. The result was further supported by computational results in the molecular mechanics model with the UFF forcefield. It is implied that Al(III) complexation may favor the racemization from L- to D-amino acids. (3) Conformational studies: At biologically relevant pH and concentrations of Al(III) and NADH, Al(III) was found to increase the percentage of folded forms of NADH, which results in reducing the activity of the coenzyme NADH in the hollow-dehydrogenase reactions system. However, the conformations of NAD(+) and Al-NAD(+) are dependent upon the solvents and other ligands in the complexes. (4) Biological effects: The effects of Al(III) on the activity of the glutamate dehydrogenase-catalyzed reactions were studied by monitoring the differential-pulse polarography reduction current of NAD(+). At the physiologically relevant pH values (pH 6.5 and 7.5), the activity of the GDH enzyme was strongly dependent on the concentration of the Al(III) in the assayed mixture solutions. PMID:17643493

  17. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... Specific Substances Affirmed as GRAS § 184.1311 Ferrous lactate. (a) Ferrous lactate (iron (II) lactate... lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of...

  18. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... Specific Substances Affirmed as GRAS § 184.1311 Ferrous lactate. (a) Ferrous lactate (iron (II) lactate... lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of...

  19. Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

    PubMed

    Tramontana, S; Bionaz, M; Sharma, A; Graugnard, D E; Cutler, E A; Ajmone-Marsan, P; Hurley, W L; Loor, J J

    2008-08-01

    High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species. PMID:18650282

  20. The Levels of Serum C-Reactive Protein, Beta 2 Microglobulin, Ferritin, Lactate Dehydrogenase and Some Specific Proteins in Patients with Non-Hodgkin’s Lymphoma Before and After Treatment

    PubMed Central

    Yildirim, Rahsan; Gundogdu, Mehmet; Erdem, Fuat; Kiki, lhami; Bilici, Mehmet

    2009-01-01

    Objective: The aim of this study was to measure serum C reactive protein, ?2 microglobulin, ferritin, lactate dehydrogenase, complement 3, complement 4, immunoglobulin A, immunoglobulin M, immunoglobulin G and transferrin levels in patients with Non-Hodgkin Lymphoma before and after treatment, and to determine whether any differences occur with treatment, investigate relationship between these parameters and systemic symptoms, and to determine whether they could be used as tumor markers. Materials and Methods: The parameters listed above were studied before and after treatment in sera of 27 patients with the diagnosis of Non-Hodgkin Lymphoma who admitted to our department. Of the patients, 10 (37%) were females and 17 (63%) were males. Mean age was 57.7 ± 16.5 (19–82) years. The subjects were newly diagnosed and treatment. Results: Post-treatment serum ferritin and CRP levels were found to be significantly decreased in patients with NHL compared to pre-treatment levels (p=0.009 and p=0.015, respectively). In addition, ferritin levels measured before treatment were significantly lower in subjects with B symptoms than those without B symptoms (p=0.02). IgA levels of patients with B symptom were significantly increased compared to those without B symptoms following treatment (p=0.03). Conclusions: We are in the opinion that serum ferritin and CRP parameters may be used as tumor markers and may be indicators in the efficacy evaluation of treatment in Non-Hodgkin’s Lymphoma. PMID:25610096

  1. Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction.

    PubMed

    Williamson, G; Engel, P C

    1984-03-01

    The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate. PMID:6712628

  2. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... to 155, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51... reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous sulfate with...

  3. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... (1996), pp. 154 to 155, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR... prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous...

  4. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW.... It is prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of...

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

    PubMed Central

    Clementi, Marisa A; Deis, Ricardo P; Telleria, Carlos M

    2004-01-01

    Background In the rat, the maintenance of gestation is dependent on progesterone production from the corpora lutea (CL), which are under the control of pituitary, decidual and placental hormones. The luteal metabolism of progesterone during gestation has been amply studied. However, the regulation of progesterone synthesis and degradation during pseudopregnancy (PSP), in which the CL are mainly under the control of pituitary prolactin (PRL), is not well known. The objectives of this investigation were: i) to study the luteal metabolism of progesterone during PSP by measuring the activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3betaHSD), involved in progesterone biosynthesis, and that of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD), involved in progesterone catabolism; and ii) to determine the role of decidualization on progesterone metabolism in PSP. Methods PSP was induced mechanically at 10:00 h on the estrus of 4-day cycling Wistar rats, and the stimulus for decidualization was provided by scratching the uterus on day 4 of PSP. 3betaHSD and 20alphaHSD activities were measured in the CL isolated from ovaries of PSP rats using a spectrophotometric method. Serum concentrations of progesterone, PRL, androstenedione, and estradiol were measured by radioimmunoassay (RIA). Results The PSP stage induced mechanically in cycling rats lasted 11.3 ± 0.09 days (n = 14). Serum progesterone concentration was high until day 10 of PSP, and declined thereafter. Serum PRL concentration was high on the first days of PSP but decreased significantly from days 6 to 9, having minimal values on days 10 and 11. Luteal 3betaHSD activities were elevated until day 6 of PSP, after which they progressively declined, reaching minimal values at the end of PSP. Luteal 20alphaHSD activities were very low until day 9, but abruptly increased at the end of PSP. When the deciduoma was induced by scratching the uterus of pseudopregnant animals on day 4 (PSP+D), PSP was extended to 18 ± 2.2 days (n = 8). In PSP + D rats, serum progesterone and PRL levels, and luteal 3betaHSD activities were higher than in pseudopregnant rats on day 11. Decidualization also prevented the increase in luteal 20alphaHSD activities observed on day 11 of PSP. Administration of the dopaminergic agonist CB154 in PSP + D rats on day 10 of PSP induced a decline in both serum PRL and progesterone on day 11 of PSP, values that were not different from that of pseudopregnant controls. Conclusions We have established that during the final period of PSP a decline in progesterone biosynthesis occurs before the increase in progesterone catabolism. We have also shown that decidualization in pseudopregnant rats extends the life of the CL by prolonging the production of pituitary PRL, and by maintaining high 3betaHSD and low 20alphaHSD activities within the CL leading to sustained production of progesterone. PMID:15140254

  6. Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production

    PubMed Central

    Wang, Yujiao; Lv, Min; Zhang, Yingxin; Xiao, Xieyue; Jiang, Tianyi; Zhang, Wen; Hu, Chunhui; Gao, Chao; Ma, Cuiqing; Xu, Ping

    2014-01-01

    As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. PMID:25373400

  7. D-lactate metabolism in starved Octopus ocellatus.

    PubMed

    Fujisawa, Tomomi; Akagi, Shinsuke; Kawase, Michi; Yamamoto, Masamichi; Ohmori, Shinji

    2005-06-01

    The concentrations of D- and L-lactate, methylglyoxal and pyruvate were measured in tissues of normal and starved Octopus ocellatus. D-Lactate was always more abundant than L-lactate in the tissues. D-Lactate, pyruvate and methylglyoxal were present in 320, 94 and 43 times higher concentrations in tentacle of O. ocellatus of control group than those in normal rat skeletal muscle. The D-lactate concentration in the tentacle of O. ocellatus was 17-fold higher than that in Octopus vulgars. The activities of enzymes involved with D-lactate metabolism such as pyruvate kinase, octopine dehydrogenase, glyoxalase I and II and lactate dehydrogenase were measured in those tissues. The activities of glyoxalase I and II, and D-lactate dehydrogenase were increased in mantle and tentacle of starved octopus, while the levels of D-lactate and related metabolites were lowered in these tissues. The experimental results presented in this report and up to the present indicate that D-lactate is actively used for energy production in the tentacle and mantle of the starved animals. In octopus, especially starved octopus D-lactate was actively produced from methylglyoxal, which is formed via aminoacetone from threonine and glycine. PMID:15880764

  8. Towards catalyst compartimentation in combined chemo- and biocatalytic processes: immobilization of alcohol dehydrogenases for the diastereoselective reduction of a ?-hydroxy ketone obtained from an organocatalytic aldol reaction.

    PubMed

    Rulli, Giuseppe; Heidlindemann, Marcel; Berkessel, Albrecht; Hummel, Werner; Gröger, Harald

    2013-11-01

    The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with "free" enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor. PMID:24036136

  9. Lactate metabolism in the fetal rabbit lung

    SciTech Connect

    Engle, M.J.; Brown, D.J.; Dooley, M.

    1986-05-01

    Lactate is frequently overlooked as a potential substrate for the fetal lung, even though it is present in the fetal circulation in concentrations as high as 8 mM. These high concentrations, coupled with the relatively low levels of glucose in the fetal blood, may indicate that lactate can substitute for glucose in pulmonary energy generation and phospholipid synthesis. A series of experiments was therefore undertaken in order to investigate the role of lactate in perinatal pulmonary development. Explants from 30 day gestation fetal rabbit lungs were incubated in Krebs-Ringer bicarbonate buffer supplemented with 3 mM (U-/sup 14/C)-glucose and varying levels of lactate. In the absence of medium lactate, fetal rabbit lung explants were capable of producing lactate at a rate of approximately 200 etamoles/mg protein/hour. The addition of lactate to the bathing medium immediately reduced net lactate production and above 4 mM, fetal rabbit lung explants became net utilizers of lactate. Media lactate concentrations of 2.5 mM, 5 mM and 10 mM also decreased glucose incorporation into total tissue disaturated phosphatidylcholine by approximately 20%, 35%, and 45%, respectively. Glucose incorporation into surfactant phosphatidylcholine was also reduced by approximately 50%, when lactate was present in the incubation medium at a concentration of 5 mM. Additional experiments also revealed that fetal lung lactate dehydrogenase activity was almost twice that found in the adult rabbit lung. These data indicate that lactate may be an important carbon source for the developing lung and could be a significant component in the manufacture of surfactant phosphatidylcholine during late gestation.

  10. Hydride Transfer in Liver Alcohol Dehydrogenase: Quantum Dynamics, Kinetic Isotope Effects, and Role of Enzyme Motion

    E-print Network

    Hammes-Schiffer, Sharon

    Hydride Transfer in Liver Alcohol Dehydrogenase: Quantum Dynamics, Kinetic Isotope Effects dynamics of the hydride transfer reaction catalyzed by liver alcohol dehydrogenase (LADH) are studied and the coenzyme. I. Introduction Liver alcohol dehydrogenase (LADH) catalyzes the reversible oxidation of alcohols

  11. Genetics Home Reference: Pyruvate dehydrogenase deficiency

    MedlinePLUS

    ... conversion is essential to begin the series of chemical reactions that produce energy for cells. The pyruvate dehydrogenase ... E3, each of which performs part of the chemical reaction that converts pyruvate to acetyl-CoA. In addition, ...

  12. Glycolysis and the significance of lactate in traumatic brain injury

    PubMed Central

    Carpenter, Keri L. H.; Jalloh, Ibrahim; Hutchinson, Peter J.

    2015-01-01

    In traumatic brain injury (TBI) patients, elevation of the brain extracellular lactate concentration and the lactate/pyruvate ratio are well-recognized, and are associated statistically with unfavorable clinical outcome. Brain extracellular lactate was conventionally regarded as a waste product of glucose, when glucose is metabolized via glycolysis (Embden-Meyerhof-Parnas pathway) to pyruvate, followed by conversion to lactate by the action of lactate dehydrogenase, and export of lactate into the extracellular fluid. In TBI, glycolytic lactate is ascribed to hypoxia or mitochondrial dysfunction, although the precise nature of the latter is incompletely understood. Seemingly in contrast to lactate's association with unfavorable outcome is a growing body of evidence that lactate can be beneficial. The idea that the brain can utilize lactate by feeding into the tricarboxylic acid (TCA) cycle of neurons, first published two decades ago, has become known as the astrocyte-neuron lactate shuttle hypothesis. Direct evidence of brain utilization of lactate was first obtained 5 years ago in a cerebral microdialysis study in TBI patients, where administration of 13C-labeled lactate via the microdialysis catheter and simultaneous collection of the emerging microdialysates, with 13C NMR analysis, revealed 13C labeling in glutamine consistent with lactate utilization via the TCA cycle. This suggests that where neurons are too damaged to utilize the lactate produced from glucose by astrocytes, i.e., uncoupling of neuronal and glial metabolism, high extracellular levels of lactate would accumulate, explaining the association between high lactate and poor outcome. Recently, an intravenous exogenous lactate supplementation study in TBI patients revealed evidence for a beneficial effect judged by surrogate endpoints. Here we review the current state of knowledge about glycolysis and lactate in TBI, how it can be measured in patients, and whether it can be modulated to achieve better clinical outcome. PMID:25904838

  13. 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.

  14. 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 ?26-28 was highly disrupted on D-lactate, whereas the growth of mutant ?32-33 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

  15. Heavy atom isotope effects on enzymatic reactions

    NASA Astrophysics Data System (ADS)

    Paneth, Piotr

    1994-05-01

    The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

  16. Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction

    PubMed Central

    Zhang, Jinrui; Pierick, Angela ten; van Rossum, Harmen M.; Maleki Seifar, Reza; Ras, Cor; Daran, Jean-Marc; Heijnen, Joseph J.; Aljoscha Wahl, S.

    2015-01-01

    Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: . The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D?=?0.1?h?1) conditions, to be 22.0?±?2.6 and 15.6?±?0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05?±?0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway. PMID:26243542

  17. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-30

    Shewanella oneidensis MR-1 is a facultative anaerobe growing by coupling organic matter oxidation to reduction of wide range of electron acceptors. Here we quantitatively assessed lactate and pyruvate metabolism of these bacteria under three distinct conditions: electron acceptor limited growth on lactate with O2 and fumarate, and pyruvate fermentation, which does not sustain growth but allows cells to survive for prolonged period. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of all ATP needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute much to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, and TCA cycle did not contribute significantly to substrate oxidation. Pyruvate dehydrogenase reaction was not involved in lactate metabolism under O2 limitation, however was important for anaerobic growth probably supplying reducing equivalents for biosynthesis. Unexpectedly, obtained results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination between substrate-level phosphorylation and a respiratory process, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). Based on involved enzymes localization we hypothesize that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  18. [High-efficiency L-lactate production from glycerol by metabolically engineered Escherichia coli].

    PubMed

    Tian, Kangming; Shi, Guiyang; Lu, Fuping; Singh, Suren; Wang, Zhengxiang

    2013-09-01

    High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence (P(ldhA)) of the D-lactate dehydrogenase (LdhA) from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, P(ldhA)-BcoaLDH. Then, the cassette was chromosomally integrated into an ldhA mutant strain, Escherichia coli CICIM B0013-080C, by replacing lldD encoding an FMN-dependent L-lactate dehydrogenase. An L-lactate higher-producer strain, designated as E. coli B0013-090B, possessing genotype of lldD::P(ldhA)-BcoaLDH, deltaack-pta deltapps deltapflB deltadld deltapoxB deltaadhE deltafrdA and deltaldhA, was generated. Under the optimal condition, 132.4 g/L L-lactate was accumulated by B0013-090B with the lactate productivity of 4.90 g/Lh and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%. PMID:24409690

  19. 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

  20. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant malate dehydrogenase. However, when expressed in a strain of E. coli unable to ferment glucose, the mutant enzyme restored growth and produced lactic acid as the sole fermentation product.

  1. A comparative proteomic analysis of Bacillus coagulans in response to lactate stress during the production of L-lactic acid.

    PubMed

    Wang, Xiuwen; Qin, Jiayang; Wang, Landong; Xu, Ping

    2014-12-01

    The growth rate and maximum biomass of Bacillus coagulans 2-6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2-6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571 ± 117, 1,281 ± 231 and 904 ± 127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress. PMID:25214213

  2. Production of optically pure d-lactate from glycerol by engineered Klebsiella pneumoniae strain.

    PubMed

    Feng, Xinjun; Ding, Yamei; Xian, Mo; Xu, Xin; Zhang, Rubing; Zhao, Guang

    2014-11-01

    In this study, glycerol was used to produce optically pure d-lactate by engineered Klebsiella pneumoniae strain. In the recombinant strain, d-lactate dehydrogenase LdhA was overexpressed, and two genes, dhaT and yqhD for biosynthesis of main byproduct 1,3-propanediol, were knocked out. To further improve d-lactate production, the culture condition was optimized and the results demonstrated that aeration rate played an important role in d-lactate production. In microaerobic fed-batch fermentation, the engineered strain accumulated 142.1g/L optically pure d-lactate with a yield of 0.82g/g glycerol, which represented the highest d-lactate production from glycerol so far. This study showed that K. pneumoniae strain has high efficiency to convert glycerol into d-lactate and high potentiality in utilization of crude glycerol from biodiesel industry. PMID:25270041

  3. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  4. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  5. Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export

    PubMed Central

    van Maris, Antonius J. A.; Winkler, Aaron A.; Porro, Danilo; van Dijken, Johannes P.; Pronk, Jack T.

    2004-01-01

    Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export. PMID:15128549

  6. BACTERIAL EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pyruvate dehydrogenase complex (PDC) is a very large multi-component structure that catalyzes decarboxylation of pyruvate, yielding CO2, NADH, and acetyl-CoA as products. The decarboxylation reaction is catalyzed by pyruvate dehydrogenase (E1). The PDC occupies a key position in intermediary met...

  7. 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. PMID:26288952

  8. Lactate Racemization as a Rescue Pathway for Supplying d-Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

    PubMed Central

    Goffin, Philippe; Deghorain, Marie; Mainardi, Jean-Luc; Tytgat, Isabelle; Champomier-Vergès, Marie-Christine; Kleerebezem, Michiel; Hols, Pascal

    2005-01-01

    Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon inactivation or inhibition of the LdhD enzyme. PMID:16166538

  9. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver...myocardial infarction, and tumors of the lung or kidneys. (b) Classification. Class II (special...

  10. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver...myocardial infarction, and tumors of the lung or kidneys. (b) Classification. Class II (special...

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

    ERIC Educational Resources Information Center

    Meany, J. E.

    2007-01-01

    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…

  12. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

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

  13. Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.

    PubMed

    Park, Jae Mo; Josan, Sonal; Mayer, Dirk; Hurd, Ralph E; Chung, Youngran; Bendahan, David; Spielman, Daniel M; Jue, Thomas

    2015-10-01

    The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 3 s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusion of dichloroacetate stimulates pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation from [1-(13)C]lactate increases sharply and acetyl-l-carnitine, acetoacetate and glutamate levels also rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study thus introduces an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products. PMID:26347554

  14. The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

    PubMed Central

    Holert, Johannes; Jagmann, Nina

    2013-01-01

    In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7?,12?-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7?,12?,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD+ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage. PMID:23708132

  15. The essential function of genes for a hydratase and an aldehyde dehydrogenase for growth of Pseudomonas sp. strain Chol1 with the steroid compound cholate indicates an aldolytic reaction step for deacetylation of the side chain.

    PubMed

    Holert, Johannes; Jagmann, Nina; Philipp, Bodo

    2013-08-01

    In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7?,12?-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7?,12?,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD(+) as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage. PMID:23708132

  16. Lactate does not activate NF-?B 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 ?-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-?B (NF-?B) 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-?B activity in endothelial cells promotes angiogenesis, NF-?B 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-?B 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-?B 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 of lactate: similar to glycolysis that aliments OXPHOS with pyruvate produced by pyruvate kinase and NADH produced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), oxidative lactate metabolism aliments OXPHOS in oxidative tumor cells with pyruvate and NADH produced by LDH1. PMID:26528183

  17. 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. PMID:25611453

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

    PubMed

    Yao, Shuo; Mikkelsen, Marie Just

    2010-01-01

    Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (AdhB), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ?adhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major aldehyde dehydrogenase in the cell and functions predominantly in the acetyl-CoA reduction to acetaldehyde in the ethanol formation pathway. Finally, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield. PMID:20924198

  19. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Paj?cka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and ?-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from ?-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids. GLIA 2015;63:2313-2326. PMID:26221781

  20. Modeling Extended Lactations of Holsteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modeling extended lactations for the US Holsteins is useful as a majority (>55%) of the cows in the present population produce lactations longer than 305 d. In this study nine empirical and mechanistic models were compared on their suitability for modeling 305-d and 999-d lactations of US Holsteins...

  1. Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.

    PubMed

    Liu, Hongbo; Yang, Xiaolan; Liu, Lin; Dang, Jizheng; Xie, Yanling; Zhang, Yi; Pu, Jun; Long, Gaobo; Li, Yuanli; Yuan, Yonghua; Liao, Juan; Liao, Fei

    2013-02-19

    Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (?(A)) much larger than that of Product B (?(B)) formed by Enzyme B action on Substrate B; ?(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (?(0)); (b) absorbance at ?(A) and ?(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with ?-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. ?-Glutamyltransferase releases p-nitroaniline from ?-glutamyl-p-nitroaniline with ?(0) at 344 nm and ?(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing ?(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of ?-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and ?-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with ?(0) at 405 nm and ?(A) at 458 nm, ?-D-galactosidase releases 4-nitrophenol from ?-D-(4-nitrophenyl)-galactoside with ?(B) at 405 nm. No interference from substrates/products made SDESA of ?-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising. PMID:23305208

  2. Postpartum Exercise and Lactation.

    PubMed

    Bane, Susan M

    2015-12-01

    Many women who are breastfeeding also want to participate in exercise, but have concerns about the safety of their newborn. The following chapter reviews issues related to postpartum exercise and lactation. The goals of the chapter are to help clinicians understand the benefits of exercise, examine the impact of postpartum exercise on breastfeeding, and provide practical recommendations for exercise during the postpartum period in women who are breastfeeding. PMID:26398298

  3. Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A {sup 13}C-NMR study

    SciTech Connect

    Renault, Sophie; Faiz, Hassan; Gadet, Rudy; Ferrier, Bernard; Martin, Guy; Baverel, Gabriel; Conjard-Duplany, Agnes

    2010-01-01

    As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-{sup 13}C]-, or L-[2-{sup 13}C]-, or L-[3-{sup 13}C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

  4. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  5. Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals previously uncharacterized machinery for lactate utilization

    SciTech Connect

    Pinchuk, Grigoriy E.; Rodionov, Dmitry A.; Yang, Chen; Li, Xiaoqing; Osterman, Andrei L.; Dervyn, Etienne; Geydebrekht, Oleg V.; Reed, Samantha B.; Romine, Margaret F.; Collart, Frank R.; Scott, J.; Fredrickson, Jim K.; Beliaev, Alex S.

    2009-02-24

    The ability to utilize lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D- or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. Using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D- and L-lactate dehydrogenase enzymes. The predicted D-LDH gene (dldD, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by three genes with previously unknown functions (lldEGF, SO1520-19-18). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dldD and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is the first described example of a multi-subunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.

  6. MODELING EXTENDED LACTATIONS IN HOLSTEINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to develop an equation for predicting average yield of cows still in milk from 1 to 999 days. Test day yields (kg/d) of 903,529 lactations of 305,202 Holstein cows calved between 1997 and 2003 were used. Average daily yield (Y) for each 30-d interval of lactation wa...

  7. Systematic Engineering of Escherichia coli for d-Lactate Production from Crude Glycerol.

    PubMed

    Wang, Zei Wen; Saini, Mukesh; Lin, Li-Jen; Chiang, Chung-Jen; Chao, Yun-Peng

    2015-11-01

    Crude glycerol resulting from biodiesel production is an abundant and renewable resource. However, the impurities in crude glycerol usually make microbial fermentation problematic. This issue was addressed by systematic engineering of Escherichia coli for the production of d-lactate from crude glycerol. First, mgsA and the synthetic pathways of undesired products were eliminated in E. coli, rendering the strain capable of homofermentative production of optically pure d-lactate. To direct carbon flux toward d-lactate, the resulting strain was endowed with an enhanced expression of glpD-glpK in the glycerol catabolism and of a heterologous gene encoding d-lactate dehydrogenase. Moreover, the strain was evolved to improve its utilization of cruder glycerol and subsequently equipped with the FocA channel to export intracellular d-lactate. Finally, the fed-batch fermentation with two-phase culturing was carried out with a bioreactor. As a result, the engineered strain enabled production of 105 g/L d-lactate (99.9% optical purity) from 121 g/L crude glycerol at 40 h. The result indicates the feasibility of our approach to engineering E. coli for the crude glycerol-based fermentation. PMID:26477354

  8. Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of l-Leucine to Isocaproate by Clostridium difficile

    PubMed Central

    Kim, Jihoe; Darley, Daniel; Selmer, Thorsten; Buckel, Wolfgang

    2006-01-01

    The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO2 and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (Km = 68 ?M, k cat = 31 s?1) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate. PMID:16957230

  9. Lactate and lactate clearance in acute cardiac care patients

    PubMed Central

    Lazzeri, Chiara; Picariello, Claudio; Dini, Carlotta Sorini; Gensini, Gian Franco; Valente, Serafina

    2012-01-01

    Hyperlactataemia is commonly used as a diagnostic and prognostic tool in intensive care settings. Recent studies documented that serial lactate measurements over time (or lactate clearance), may be clinically more reliable than lactate absolute value for risk stratification in different pathological conditions. While the negative prognostic role of hyperlactataemia in several critical ill diseases (such as sepsis and trauma) is well established, data in patients with acute cardiac conditions (i.e. acute coronary syndromes) are scarce and controversial. The present paper provides an overview of the current available evidence on the clinical role of lactic acid levels and lactate clearance in acute cardiac settings (acute coronary syndromes, cardiogenic shock, cardiac surgery), focusing on its prognostic role. PMID:24062898

  10. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  11. Substrate specificity of sheep liver sorbitol dehydrogenase.

    PubMed Central

    Lindstad, R I; Köll, P; McKinley-McKee, J S

    1998-01-01

    The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

  12. 21 CFR 73.165 - Ferrous lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 1 2011-04-01 2011-04-01 false Ferrous lactate. 73.165 Section 73.165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Foods § 73.165 Ferrous lactate. (a) Identity. The color additive ferrous lactate is the ferrous lactate defined...

  13. L-lactate production from biodiesel-derived crude glycerol by metabolically engineered Enterococcus faecalis: cytotoxic evaluation of biodiesel waste and development of a glycerol-inducible gene expression system.

    PubMed

    Doi, Yuki

    2015-03-01

    Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD(+) ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD600]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (?pfl mutant) expressing the ldhL1LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h(-1) (1.6 g liter(-1) h(-1)). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste. PMID:25576618

  14. l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

    PubMed Central

    2015-01-01

    Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD+ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD600]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (?pfl mutant) expressing the ldhL1LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h?1 (1.6 g liter?1 h?1). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste. PMID:25576618

  15. Towards an effective biosensor for monitoring AD leachate: a knockout E. coli mutant that cannot catabolise lactate.

    PubMed

    Sweeney, Joseph; Murphy, Cormac D; McDonnell, Kevin

    2015-12-01

    Development of a biosensor for the convenient measurement of acetate and propionate concentrations in a two-phase anaerobic digestor (AD) requires a bacterium that will be unresponsive to the other organic acids present in the leachate, of which lactate is the most abundant. Successive gene knockouts of E.coli W3110 D-lactate dehydrogenase (dld), L-lactate dehydrogenase (lldD), glycolate oxidase (glcD) and a suspected L-lactate dehdrogenase (ykgF) were performed. The resulting quadruple mutant (IMD Wldgy) was incapable of growth on D- and L-lactate, whereas the wild type grew readily on these substrates. Furthermore, the O2 consumption rates of acetate-grown IMD Wldgy cell suspensions supplied with either acetate (0.1 mM) or a synthetic leachate including acetate (0.1 mM) and DL-lactate (1 mM) were identical (2.79 and 2.70 mg l(-1) min(-1), respectively). This was in marked contrast to similar experiments with the wild type which gave initial O2 consumption rates of 2.00, 2.36 and 2.97 mg l(-1) min(-1) when cell suspensions were supplied with acetate (0.1 mM), acetate (0.1 mM) plus D-lactate (1 mM) or acetate (0.1 mM) plus L-lactate (1 mM), respectively. The knockout strain provides a platform for the design of a biosensor that can accessibly monitor acetate and propionate concentrations in AD leachate via O2-uptake measurements. PMID:26272093

  16. Lactate Utilization Is Regulated by the FadR-Type Regulator LldR in Pseudomonas aeruginosa

    PubMed Central

    Gao, Chao; Hu, Chunhui; Zheng, Zhaojuan; Jiang, Tianyi; Dou, Peipei; Zhang, Wen; Che, Bin; Wang, Yujiao; Lv, Min

    2012-01-01

    NAD-independent l-lactate dehydrogenase (l-iLDH) and NAD-independent d-lactate dehydrogenase (d-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an l-iLDH), and lldE (encoding a d-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses d-iLDH and l-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldRM, lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. l-Lactate and d-lactate interfered with the DNA-binding activity of LldR. Thus, l-iLDH and d-iLDH were expressed when the operon was induced in the presence of l-lactate or d-lactate. PMID:22408166

  17. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of ?,?-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  18. Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase.

    PubMed Central

    McAlister-Henn, L; Thompson, L M

    1987-01-01

    The mitochondrial tricarboxylic acid cycle enzyme malate dehydrogenase was purified from Saccharomyces cerevisiae, and an antibody to the purified enzyme was obtained in rabbits. Immunoscreening of a yeast genomic DNA library cloned into a lambda gt11 expression vector with anti-malate dehydrogenase immunoglobulin G resulted in identification of a lambda recombinant encoding an immunoreactive beta-galactosidase fusion protein. The yeast DNA portion of the coding region for the fusion protein translates into an amino acid sequence which is very similar to carboxy-terminal sequences of malate dehydrogenases from other organisms. In s. cerevisiae transformed with a multicopy plasmid carrying the complete malate dehydrogenase gene, the specific activity and immunoreactivity of the mitochondrial isozyme are increased by eightfold. Expression of both the chromosomal and plasmid-borne genes is repressed by growth on glucose. Disruption of the chromosomal malate dehydrogenase gene in haploid S. cerevisiae produces mutants unable to grow on acetate and impaired in growth on glycerol plus lactate as carbon sources. Images PMID:3312168

  19. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    MedlinePLUS

    ... Rare Diseases Information Center (GARD) Print friendly version Glucose-6-phosphate dehydrogenase deficiency Table of Contents Overview ... of the National Institutes of Health. Overview Listen Glucose 6 phosphate dehydrogenase (G6PD) deficiency is a hereditary ...

  20. Nutritional aspects of human lactation*

    PubMed Central

    Thomson, A. M.; Black, A. E.

    1975-01-01

    This paper reviews the literature on the incidence and duration of breast-feeding in various countries, the volume and composition of breast milk, the health and nutrition of breast-fed babies as judged by growth and morbidity, maternal nutritional requirements during lactation, and the effect of prolonged lactation on maternal health. It appears that lactation can be as well sustained by impoverished as by affluent mothers, and that even in communities where malnutrition is common the average growth of infants is satisfactory up to the age of about 3 months on a diet of breast milk alone. Breast milk appears to have specific anti-infective properties, but prolonged breast-feeding will not prevent infections among older infants reared in a poor environment. The authors believe that breast-feeding is the best form of nutrition for the young infant and deplore its decline in modern industrial societies. The recommendations of various FAO/WHO Expert Groups on nutritional intakes during lactation are summarized. The need for an increased daily energy intake of 4.2 MJ (1 000 kcal) is questioned, and an increase of 2.5 MJ (600 kcal) is suggested. Data on the effect of prolonged lactation on the health of the mother are scanty; body weight appears to be maintained even among poorly nourished mothers. The authors stress the need for well-planned and technically adequate studies of the material and psychological factors involved in breast feeding. PMID:816479

  1. Boosting D-lactate production in engineered cyanobacteria using sterilized anaerobic digestion effluents.

    PubMed

    Hollinshead, Whitney D; Varman, Arul M; You, Le; Hembree, Zachary; Tang, Yinjie J

    2014-10-01

    Anaerobic digestion (AD) is an environmentally friendly approach to waste treatment, which can generate N and P-rich effluents that can be used as nutrient sources for microalgal cultivations. Modifications of AD processes to inhibit methanogenesis leads to the accumulation of acetic acid, a carbon source that can promote microalgal biosynthesis. This study tested different AD effluents from municipal wastes on their effect on D-lactate production by an engineered Synechocystis sp. PCC 6803 (carrying a novel lactate dehydrogenase). The results indicate that: (1) AD effluents can be supplemented into the modified BG-11 culture medium (up to 1:4 volume ratio) to reduce N and P cost; (2) acetate-rich AD effluents enhance D-lactate synthesis by ? 40% (1.2g/L of D-lactate in 20 days); and (3) neutral or acidic medium had a deleterious effect on lactate secretion and biomass growth by the engineered strain. This study demonstrates the advantages and guidelines in employing wastewater for photomixotrophic biosynthesis using engineered microalgae. PMID:25084044

  2. FDH: an Aldehyde Dehydrogenase Fusion Enzyme in Folate Metabolism

    PubMed Central

    Krupenko, Sergey A.

    2009-01-01

    FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO2 in a NADP+-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1–310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400–902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to class 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311–399), which is a structural and functional homolog of carrier proteins possessing a 4?-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO2. PMID:18848533

  3. Exogenous lactate supply affects lactate kinetics of rainbow trout, not swimming performance

    PubMed Central

    Omlin, Teye; Langevin, Karolanne

    2014-01-01

    Intense swimming causes circulatory lactate accumulation in rainbow trout because lactate disposal (Rd) is not stimulated as strongly as lactate appearance (Ra). This mismatch suggests that maximal Rd is limited by tissue capacity to metabolize lactate. This study uses exogenous lactate to investigate what constrains maximal Rd and minimal Ra. Our goals were to determine how exogenous lactate affects: 1) Ra and Rd of lactate under baseline conditions or during graded swimming, and 2) exercise performance (critical swimming speed, Ucrit) and energetics (cost of transport, COT). Results show that exogenous lactate allows swimming trout to boost maximal Rd lactate by 40% and reach impressive rates of 56 ?mol·kg?1·min?1. This shows that the metabolic capacity of tissues for lactate disposal is not responsible for setting the highest Rd normally observed after intense swimming. Baseline endogenous Ra (resting in normoxic water) is not significantly reduced by exogenous lactate supply. Therefore, trout have an obligatory need to produce lactate, either as a fuel for oxidative tissues and/or from organs relying on glycolysis. Exogenous lactate does not affect Ucrit or COT, probably because it acts as a substitute for glucose and lipids rather than extra fuel. We conclude that the observed 40% increase in Rd lactate is made possible by accelerating lactate entry into oxidative tissues via monocarboxylate transporters (MCTs). This observation together with the weak expression of MCTs and the phenomenon of white muscle lactate retention show that lactate metabolism of rainbow trout is significantly constrained by transmembrane transport. PMID:25121611

  4. Antipsychotics in pregnancy and lactation.

    PubMed

    Babu, Girish N; Desai, Geetha; Chandra, Prabha S

    2015-07-01

    Research on psychotropic medications during pregnancy and lactation is limited as often involves complex ethical issues. Information on safety of psychotropic drugs during these critical phases is either inconclusive or undetermined. Many women with severe mental illness have unplanned pregnancies and require antipsychotic medication during pregnancy and lactation. Multiple issues have to be considered while choosing safe treatments for pregnant and lactating women and the best approach is to individualize the treatment. Medication should be guided primarily by its safety data and by the psychiatric history of the patient. Important issues to be kept in mind include pre-pregnancy counseling for all women, including planning pregnancies; folate supplementation, discussion with patient and family regarding options, and active liaison with obstetricians, ultrasonologists and pediatricians. Whenever possible, non-pharmacological approaches should be used in addition. PMID:26330648

  5. Antipsychotics in pregnancy and lactation

    PubMed Central

    Babu, Girish N.; Desai, Geetha; Chandra, Prabha S.

    2015-01-01

    Research on psychotropic medications during pregnancy and lactation is limited as often involves complex ethical issues. Information on safety of psychotropic drugs during these critical phases is either inconclusive or undetermined. Many women with severe mental illness have unplanned pregnancies and require antipsychotic medication during pregnancy and lactation. Multiple issues have to be considered while choosing safe treatments for pregnant and lactating women and the best approach is to individualize the treatment. Medication should be guided primarily by its safety data and by the psychiatric history of the patient. Important issues to be kept in mind include pre-pregnancy counseling for all women, including planning pregnancies; folate supplementation, discussion with patient and family regarding options, and active liaison with obstetricians, ultrasonologists and pediatricians. Whenever possible, non-pharmacological approaches should be used in addition. PMID:26330648

  6. L-lactate utilization by dairy goats

    SciTech Connect

    Rodriguez, N.R.

    1984-01-01

    Three Toggenberg goats were used to investigate utilization of L-lactate as substrate for lipogenesis and gluconeogenesis. Objectives were: (1) to determine the extent lactate could be used for body and milk fat synthesis; (2) to estimate contribution of lactate to glucose synthesis; (3) to assess differences in these measurements during early lactation, mid-lactation and the dry period; and (4) to observe differences in labeling of glycerol and free fatty acid (FFA) fractions in body and milk fat 7 days post-infusion of isotopes. Goats were fed in metabolism crates a 70% concentrate ration in hourly increments to meet individual requirements. After a pulse dose, U-/sup 14/C-lactate (34 uCi/hr) and 6-/sup 3/H-Glucose (100 uCi/hr) was infused via jugular cannula for 8 hours. Blood an milk were sampled hourly beginning 3 and 3.5 hours, respectively, after the pulse dose. Body fat was biopsied after the infusion (Day 0) and one week post-infusion (Day 7). Plasma glucose and lactate concentrations were greater in early 70.4 and 7.7 mg/dl, respectively) compared to mid-lactation (50.8 and 5.9 gm/dl). Mid-lactation and dry period values were similar. Glucose turnover differed for early and mid-lactation and the dry period (141, 86, and 70 mmol/hr, respectively). Percentage of glucose derived from lactate tended to decrease through lactation into the dry period (28% vs 10%). Plasma lactate turnover was greater during lactation as opposed to the dry period (124 and 35 mmol/hr). During early lactation a greater proportion of lactate was incorporated into glucose than during either mid-lactation or the dry period.

  7. The Occurrence of Glycolate Dehydrogenase and Glycolate Oxidase in Green Plants

    PubMed Central

    Frederick, Sue Ellen; Gruber, Peter J.; Tolbert, N. E.

    1973-01-01

    Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O2 uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance. Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate. PMID:16658555

  8. Lactate dehydrogenase regulation of the metmyoglobin reducing system to improve color stability of bovine muscles through lactate enhancement 

    E-print Network

    Kim, Yuan Hwan

    2009-05-15

    in highoxygen modified atmosphere package, irradiated, stored in the dark at 1°C for 14 days. Instrumental color, TRA, lipid oxidation, and NADH were measured. LD remained the most red, whereas PM was most discolored. LD had a significantly higher level of LDH-1...

  9. Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

    PubMed Central

    Neuberger, M S; Patterson, R A; Hartley, B S

    1979-01-01

    An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization. Images Fig. 1. Fig. 2. PMID:393250

  10. 21 CFR 582.5311 - Ferrous lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5311 Ferrous lactate. (a) Product. Ferrous lactate. (b) Conditions of use. This...

  11. 21 CFR 582.5311 - Ferrous lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5311 Ferrous lactate. (a) Product. Ferrous lactate. (b) Conditions of use. This...

  12. 21 CFR 582.5311 - Ferrous lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5311 Ferrous lactate. (a) Product. Ferrous lactate. (b) Conditions of use....

  13. Cell-cell and intracellular lactate shuttles.

    PubMed

    Brooks, George A

    2009-12-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. 'Cell-cell' and 'intracellular lactate shuttle' concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell-cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other. PMID:19805739

  14. Cell–cell and intracellular lactate shuttles

    PubMed Central

    Brooks, George A

    2009-01-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. ‘Cell–cell’ and ‘intracellular lactate shuttle’ concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell–cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other. PMID:19805739

  15. The origin and evolution of lactation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of mammary glands are the defining morphological feature of mammals, and a successful lactation is crucial to mammalian reproductive strategies. Among mammalian species, the nature of lactation and the composition of milk vary greatly. The evolution of lactation and its diversity amon...

  16. Imaging Pregnant and Lactating Patients.

    PubMed

    Tirada, Nikki; Dreizin, David; Khati, Nadia J; Akin, Esma A; Zeman, Robert K

    2015-10-01

    As use of imaging in the evaluation of pregnant and lactating patients continues to increase, misperceptions of radiation and safety risks have proliferated, which has led to often unwarranted concerns among patients and clinicians. When radiologic examinations are appropriately used, the benefits derived from the information gained usually outweigh the risks. This review describes appropriateness and safety issues, estimated doses for imaging examinations that use iodizing radiation (ie, radiography, computed tomography, nuclear scintigraphy, and fluoroscopically guided interventional radiology), radiation risks to the mother and conceptus during various stages of pregnancy, and use of iodinated or gadolinium-based contrast agents and radiotracers in pregnant and lactating women. Maternal radiation risk must be weighed with the potential consequences of missing a life-threatening diagnosis such as pulmonary embolus. Fetal risks (ie, spontaneous abortion, teratogenesis, or carcinogenesis) vary with gestational age and imaging modality and should be considered in the context of the potential benefit of medically necessary diagnostic imaging. When feasible and medically indicated, modalities that do not use ionizing radiation (eg, magnetic resonance imaging) are preferred in pregnant and lactating patients. Radiologists should strive to minimize risks of radiation to the mother and fetus, counsel patients effectively, and promote a realistic understanding of risks related to imaging during pregnancy and lactation. (©)RSNA, 2015. PMID:26466183

  17. BREASTFEEDING SUPPORT Campus Lactation Rooms

    E-print Network

    Militzer, Burkhard

    BREASTFEEDING SUPPORT On Campus Campus Lactation Rooms Equipped with hospital-grade, electric-mail Health*Matters. Product descriptions are available on the Health*Matters website. Breastfeeding Your Baby breastfeeding basics and problem solving. The last hour focuses on planning for return-to work or school

  18. PHYLOGENY AND EVOLUTION OF ALDEHYDE DEHYDROGENASE-HOMOLOGOUS FOLATE ENZYMES

    PubMed Central

    Strickland, Kyle C.; Holmes, Roger S.; Oleinik, Natalia V.; Krupenko, Natalia I.; Krupenko, Sergey A.

    2011-01-01

    Folate coenzymes function as one-carbon group carriers in intracellular metabolic pathways. Folate-dependent reactions are compartmentalized within the cell and are catalyzed by two distinct groups of enzymes, cytosolic and mitochondrial. Some folate enzymes are present in both compartments and are likely the products of gene duplications. A well-characterized cytosolic folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), contains a domain with significant sequence similarity to aldehyde dehydrogenases. This domain enables FDH to catalyze the NADP+-dependent conversion of short-chain aldehydes to corresponding acids in vitro. The aldehyde dehydrogenase-like reaction is the final step in the overall FDH mechanism, by which a tetrahydrofolate-bound formyl group is oxidized to CO2 in an NADP+-dependent fashion. We have recently cloned and characterized another folate enzyme containing an ALDH domain, a mitochondrial FDH. Here the biological roles of the two enzymes, a comparison of the respective genes, and some potential evolutionary implications are discussed. The phylogenic analysis suggests that the vertebrate ALDH1L2 gene arose from a duplication event of the ALDH1L1 gene prior to the emergence of osseous fish >500 millions years ago. PMID:21215736

  19. Conformational changes and catalysis by alcohol dehydrogenase.

    PubMed

    Plapp, Bryce V

    2010-01-01

    As shown by X-ray crystallography, horse liver alcohol dehydrogenase undergoes a global conformational change upon binding of NAD(+) or NADH, involving a rotation of the catalytic domain relative to the coenzyme binding domain and the closing up of the active site to produce a catalytically efficient enzyme. The conformational change requires a complete coenzyme and is affected by various chemical or mutational substitutions that can increase the catalytic turnover by altering the kinetics of the isomerization and rate of dissociation of coenzymes. The binding of NAD(+) is kinetically limited by a unimolecular isomerization (corresponding to the conformational change) that is controlled by deprotonation of the catalytic zinc-water to produce a negatively-charged zinc-hydroxide, which can attract the positively-charged nicotinamide ring. The deprotonation is facilitated by His-51 acting through a hydrogen-bonded network to relay the proton to solvent. Binding of NADH also involves a conformational change, but the rate is very fast. After the enzyme binds NAD(+) and closes up, the substrate displaces the hydroxide bound to the catalytic zinc; this exchange may involve a double displacement reaction where the carboxylate group of a glutamate residue first displaces the hydroxide (inverting the tetrahedral coordination of the zinc), and then the exogenous ligand displaces the glutamate. The resulting enzyme-NAD(+)-alcoholate complex is poised for hydrogen transfer, and small conformational fluctuations may bring the reactants together so that the hydride ion is transferred by quantum mechanical tunneling. In the process, the nicotinamide ring may become puckered, as seen in structures of complexes of the enzyme with NADH. The conformational changes of alcohol dehydrogenase demonstrate the importance of protein dynamics in catalysis. PMID:19583966

  20. Formate dehydrogenase of Clostridium pasteurianum.

    PubMed Central

    Liu, C L; Mortenson, L E

    1984-01-01

    Formate dehydrogenase was purified to electrophoretic homogeneity from N2-fixing cells of Clostridium pasteurianum W5. The purified enzyme has a minimal Mr of 117,000 with two nonidentical subunits with molecular weights of 76,000 and 34,000, respectively. It contains 2 mol of molybdenum, 24 mol of nonheme iron, and 28 mol of acid-labile sulfide per mol of enzyme; no other metal ions were detected. Analysis of its iron-sulfur centers by ligand exchange techniques showed that 20 iron atoms of formate dehydrogenase can be extruded as Fe4S4 centers. Fluorescence analysis of its isolated molybdenum centers suggests it is a molybdopterin. The clostridial formate dehydrogenase has a pH optimum between 8.3 and 8.5 and a temperature optimum of 52 degrees C. The Km for formate is 1.72 mM with a Vmax of 551 mumol of methyl viologen reduced per min per mg of protein. Sodium azide competes competitively with formate (K1 = 3.57 microM), whereas the inactivation by cyanide follows pseudo-first-order kinetics with K = 5 X 10(2) M-1 s-1. PMID:6547435

  1. Effects of decreased lactate accumulation after dichloroacetate administration on exercise training–induced mitochondrial adaptations in mouse skeletal muscle

    PubMed Central

    Hoshino, Daisuke; Tamura, Yuki; Masuda, Hiroyuki; Matsunaga, Yutaka; Hatta, Hideo

    2015-01-01

    Recent studies suggested that lactate accumulation can be a signal for mitochondrial biogenesis in skeletal muscle. We investigated whether reductions in lactate concentrations in response to dichloroacetate (DCA), an activator of pyruvate dehydrogenase, attenuate mitochondrial adaptations after exercise training in mice. We first confirmed that DCA administration (200 mg/kg BW by i.p. injection) 10 min before exercise decreased muscle and blood lactate concentrations after high-intensity interval exercise (10 bouts of 1 min treadmill running at 40 m/min with a 1 min rest). At the same time, exercise-induced signal cascades did not change by pre-exercise DCA administration. These results suggested that DCA administration affected only lactate concentrations after exercise. We next examined the effects of acute DCA administration on mRNA expressions involved with mitochondrial biogenesis after same high-intensity interval exercise and the effects of chronic DCA administration on mitochondrial adaptations after high-intensity interval training (increasing intensity from 38 to 43 m/min by the end of training period). Acute DCA administration did not change most of the exercise-induced mRNA upregulation. These data suggest that lactate reductions by DCA administration did not affect transcriptional activation after high-intensity interval exercise. However, chronic DCA administration attenuated, in part, mitochondrial adaptations such as training-induced increasing rates of citrate synthase (P = 0.06), ?-hydroxyacyl CoA dehydrogenase activity (P < 0.05), cytochrome c oxidase IV (P < 0.05) and a fatty acid transporter, fatty acid translocase/CD36 (P < 0.05), proteins after exercise training. These results suggest that lactate accumulation during high-intensity interval exercise may be associated with mitochondrial adaptations after chronic exercise training. PMID:26416973

  2. Effects of decreased lactate accumulation after dichloroacetate administration on exercise training-induced mitochondrial adaptations in mouse skeletal muscle.

    PubMed

    Hoshino, Daisuke; Tamura, Yuki; Masuda, Hiroyuki; Matsunaga, Yutaka; Hatta, Hideo

    2015-09-01

    Recent studies suggested that lactate accumulation can be a signal for mitochondrial biogenesis in skeletal muscle. We investigated whether reductions in lactate concentrations in response to dichloroacetate (DCA), an activator of pyruvate dehydrogenase, attenuate mitochondrial adaptations after exercise training in mice. We first confirmed that DCA administration (200 mg/kg BW by i.p. injection) 10 min before exercise decreased muscle and blood lactate concentrations after high-intensity interval exercise (10 bouts of 1 min treadmill running at 40 m/min with a 1 min rest). At the same time, exercise-induced signal cascades did not change by pre-exercise DCA administration. These results suggested that DCA administration affected only lactate concentrations after exercise. We next examined the effects of acute DCA administration on mRNA expressions involved with mitochondrial biogenesis after same high-intensity interval exercise and the effects of chronic DCA administration on mitochondrial adaptations after high-intensity interval training (increasing intensity from 38 to 43 m/min by the end of training period). Acute DCA administration did not change most of the exercise-induced mRNA upregulation. These data suggest that lactate reductions by DCA administration did not affect transcriptional activation after high-intensity interval exercise. However, chronic DCA administration attenuated, in part, mitochondrial adaptations such as training-induced increasing rates of citrate synthase (P = 0.06), ?-hydroxyacyl CoA dehydrogenase activity (P < 0.05), cytochrome c oxidase IV (P < 0.05) and a fatty acid transporter, fatty acid translocase/CD36 (P < 0.05), proteins after exercise training. These results suggest that lactate accumulation during high-intensity interval exercise may be associated with mitochondrial adaptations after chronic exercise training. PMID:26416973

  3. Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

    SciTech Connect

    Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

    2011-12-01

    Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of wide range of electron acceptors. Here, we quantitatively assessed lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor limited growth on lactate with O2; lactate with fumarate; and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the TCA cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under O2 limitation but was required for anaerobic growth likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

  4. EFFECTS OF EXERCISE TRAINING ON BRAIN BIOENERGETICS: LACTATE AS A KEY MEDIATOR

    E-print Network

    E, Lezi

    2013-12-31

    Carbonylcyanide p-trifluoromethoxyphenylhydrazone FOXO1 Forkhead box protein O1 FOXO3a Forkhead box O3 G6Pase Glucose 6-phosphatase GAPDH Glyceraldehyde-3-phosphate dehydrogenase GSK3? Glycogen synthase kinase 3 beta HBSS Hank’s Balanced Salt Solution HDAC...1 Histone deacetylase 1 HIF-1? Hypoxia-inducible factor alpha HOMA-IR Homeostasis model assessment of insulin resistance IL-1? Interleukin-1 beta LAC Lactate LSD Fisher’s Least Significant Difference post-hoc test MCT Monocarboxylate...

  5. Maternal flaxseed diet during lactation changes adrenal function in adult male rat offspring.

    PubMed

    Figueiredo, Mariana Sarto; da Conceição, Ellen Paula Santos; de Oliveira, Elaine; Lisboa, Patricia Cristina; de Moura, Egberto Gaspar

    2015-10-14

    Flaxseed (Linum usitatissimum L.) has been a focus of interest in the field of functional foods because of its potential health benefits. However, we hypothesised that maternal flaxseed intake during lactation could induce several metabolic dysfunctions in adult offspring. In the present study, we aimed to characterise the adrenal function of adult offspring whose dams were supplemented with whole flaxseed during lactation. At birth, lactating Wistar rats were divided into two groups: rats from dams fed the flaxseed diet (FLAX) with 25% of flaxseed and controls dams. Pups received standard diet after weaning and male offspring were killed at age 180 days old to collect blood and tissues. We evaluated body weight and food intake during development, corticosteronaemia, adrenal catecholamine content, hepatic cholesterol, TAG and glycogen contents, and the protein expression of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), 11-?-hydroxysteroid dehydrogenase type 1 (11?-HSD1) and adrenaline ?2 receptor at postnatal day 180 (PN180). After weaning, pups from the FLAX group had a higher body weight (+10 %) and food intake (+10%). At PN180, the FLAX offspring exhibited higher serum corticosterone (+48%) and lower adrenal catecholamine ( - 23%) contents, lower glycogen ( - 30%), higher cholesterol (4-fold increase) and TAG (3-fold-increase) contents in the liver, and higher 11?-HSD1 (+62%) protein expression. Although the protein expression of hypothalamic CRH was unaffected, the FLAX offspring had lower protein expression of pituitary ACTH ( - 34%). Therefore, induction of hypercorticosteronaemia by dietary flaxseed during lactation may be due to an increased hepatic activation of 11?-HSD1 and suppression of ACTH. The changes in the liver fat content of the FLAX group are suggestive of steatosis, in which hypercorticosteronaemia may play an important role. Thus, it is recommended that lactating women restrict the intake of flaxseed during lactation. PMID:26337632

  6. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

    PubMed Central

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of ?-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different ?-, ?-, and ?-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each ?-subunit together with common ?- and ?-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  7. Dichloroacetate increases glucose use and decreases lactate in developing rat brain

    SciTech Connect

    Miller, A.L.; Hatch, J.P.; Prihoda, T.J. )

    1990-12-01

    Dichloroacetate (DCA) activates pyruvate dehydrogenase (PDH) by inhibiting PDH kinase. Neutralized DCA (100 mg/kg) or saline was intravenously administered to 20 to 25-day-old rats (50-75g). Fifteen minutes later a mixture of {sup 6-14}C glucose and {sup 3}H fluorodeoxyglucose (FDG) was administered intravenously and the animals were sacrificed by microwave irradiation (2450 MHz, 8.0 kW, 0.6-0.8 sec) after 2 or 5 min. Brain regional rates of glucose use and metabolite levels were determined. DCA-treated rats had increased rates of glucose use in all regions studied (cortex, thalamus, striatum, and brain stem), with an average increase of 41%. Lactate levels were lower in all regions, by an average of 35%. There were no significant changes in levels of ATP, creatine phosphate, or glycogen in any brain region. Blood levels of lactate did not differ significantly between the DCA- and the saline-treated groups. Blood glucose levels were higher in the DCA group. In rats sacrificed by freeze-blowing, DCA treatment caused lower brain levels of both lactate and pyruvate. These results cannot be explained by any systemic effect of DCA. Rather, it appears that in the immature rat, DCA treatment results in activation of brain PDH, increased metabolism of brain pyruvate and lactate, and a resulting increase in brain glycolytic rate.

  8. Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression.

    PubMed

    Chaube, Balkrishna; Malvi, Parmanand; Singh, Shivendra Vikram; Mohammad, Naoshad; Meena, Avtar Singh; Bhat, Manoj Kumar

    2015-11-10

    Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance. Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment. In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression. We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels. In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis. Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo. Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice. Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis. Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation. Inhibition of these two augments synthetic lethality in melanoma. PMID:26484566

  9. Inhibition of Amyloid-? (A?) Peptide-Binding Alcohol Dehydrogenase-A? Interaction Reduces A? Accumulation and Improves Mitochondrial Function in a Mouse Model of Alzheimer's Disease

    E-print Network

    Yao, Jun; Du, Heng; Yan, Shiqiang; Fang, Fang; Wang, Chaodong; Lue, Lih-Fen; Guo, Lan; Chen, Doris; Stern, David M.; Moore, Frank J. Gunn; Chen, John Xi; Arancio, Ottavio; Yan, Shirley ShiDu

    2011-02-09

    dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal. Biochemistry 37:15835–15841. Lin MT, Beal MF (2006) Alzheimer’s APP mangles mitochondria. Nat Med 12:1241–1243. Lissy NA, Davis PK, Irwin M, Kaelin WG, Dowdy SF (2000) A...

  10. The Crystal Structure of a Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight Into the Reaction Mechansim and Shows A Novel Binding Mode of the 2'-Phosphate of NADP+ and A Novel Cation Binding Site

    SciTech Connect

    Gonzalez-Segura, L.; Rudino-Pinera, E; Munoz-Clares, R; Horjales, E

    2009-01-01

    In the human pathogen Pseudomonas aeruginosa, the NAD(P)+-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors-abundant at infection sites-and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP+ and one of the even fewer that require K+ ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP+ and K+ ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the 'oxyanion hole.' The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2?-phosphate of the NADP+, thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K+ binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The othersingle bondnot described before for any ALDH but most likely present in most of themsingle bondis located in between the dimeric unit, helping structure a region involved in coenzyme binding and catalysis. This may explain the effects of K+ ions on the activity and stability of PaBADH.

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

    MedlinePLUS

    ... PubMed Recent literature OMIM Genetic disorder catalog Conditions > Glucose-6-phosphate dehydrogenase deficiency On this page: Description ... names Glossary definitions Reviewed May 2006 What is glucose-6-phosphate dehydrogenase deficiency? Glucose-6-phosphate dehydrogenase ...

  12. Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

    PubMed Central

    Arrabal, Sergio; Lucena, Miguel Angel; Canduela, Miren Josune; Ramos-Uriarte, Almudena; Rivera, Patricia; Serrano, Antonia; Pavón, Francisco Javier; Decara, Juan; Vargas, Antonio; Baixeras, Elena; Martín-Rufián, Mercedes; Márquez, Javier; Fernández-Llébrez, Pedro; De Roos, Baukje; Grandes, Pedro; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of ?-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD. PMID:26671069

  13. Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

    PubMed Central

    Köpke, Michael; Gerth, Monica L.; Maddock, Danielle J.; Mueller, Alexander P.; Liew, FungMin

    2014-01-01

    Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 ?M h?1 optical density unit?1), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

  14. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  15. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  16. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  17. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and....1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid with sodium hydroxide. (b)...

  18. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  19. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  20. The effect of exercise on lactate metabolism

    PubMed Central

    Hubbard, Judith L.

    1973-01-01

    1. An I.V. injection of 5 ?c [U-14C]sodium L(+)-lactate was given to four subjects at rest and again 10 min after beginning a 40-50 min period of heavy exercise at an estimated 62-72% of their maximum aerobic power (V?O2 max.). Both blood lactate concentration and V?O2 remained relatively constant after the first few minutes of exercise. 2. In all subjects both at rest and during exercise blood lactate and total radioactivity were measured at frequent intervals after injection of [14C]lactate. Timed expired gas collections were made and the quantity of 14CO2 present in each collection measured. In two subjects the specific activity of lactate and of glucose isolated from blood was also measured. 3. It was found that during 30 min of exercise 35-68% of the administered [14C]lactate was recovered as 14CO2 in the expired gas, whereas at rest only 3-7% was recovered in the same period. 4. After injection of [14C]lactate the blood 14C concentration and the specific activity of the blood lactate declined very rapidly. This decline was more rapid during exercise than at rest. 5. In the two subjects in whom it was measured the specific activity of blood glucose was lower during exercise than at rest. 6. These results show that both at rest and during heavy exercise, lactate is removed from the blood and metabolized, and that during exercise this metabolism is much more rapid. 7. In the light of these findings the sustained blood lactate concentration observed in these experiments is regarded as representing a dynamic equilibrium between the production and metabolism of lactate during exercise. The results give no support to the hypothesis that lactate is produced only during the first few minutes of submaximal work. PMID:4715350

  1. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... carbonate or calcium hydroxide. (b) The ingredient meets the specifications of the Food Chemicals Codex, 3d... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O, where x is any integer up to...

  2. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient meets the specifications of... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... Specific Substances Affirmed as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O,...

  3. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient meets the specifications of... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... Specific Substances Affirmed as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O,...

  4. Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

  5. Genetics Home Reference: Succinic semialdehyde dehydrogenase deficiency

    MedlinePLUS

    ... anxiety. Less frequently, affected individuals may have increased aggression, hallucinations, obsessive-compulsive disorder (OCD), and self-injurious ... semialdehyde dehydrogenase deficiency? Mutations in the ALDH5A1 gene cause succinic semialdehyde dehydrogenase deficiency. The ALDH5A1 gene provides ...

  6. MECHANISTIC ANALYSIS OF PYRUVATE DEHYDROGENASE KINASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex. The PDK is a member of the ATPase/kinase superfamily. Member proteins of this family are characterized by four signature sequences in the catalytic domain (N-, D-, F-, and G...

  7. Pyruvate Dehydrogenase Kinases: Therapeutic Targets for Diabetes and Cancers

    PubMed Central

    2015-01-01

    Impaired glucose homeostasis is one of the risk factors for causing metabolic diseases including obesity, type 2 diabetes, and cancers. In glucose metabolism, pyruvate dehydrogenase complex (PDC) mediates a major regulatory step, an irreversible reaction of oxidative decarboxylation of pyruvate to acetyl-CoA. Tight control of PDC is critical because it plays a key role in glucose disposal. PDC activity is tightly regulated using phosphorylation by pyruvate dehydrogenase kinases (PDK1 to 4) and pyruvate dehydrogenase phosphatases (PDP1 and 2). PDKs and PDPs exhibit unique tissue expression patterns, kinetic properties, and sensitivities to regulatory molecules. During the last decades, the up-regulation of PDKs has been observed in the tissues of patients and mammals with metabolic diseases, which suggests that the inhibition of these kinases may have beneficial effects for treating metabolic diseases. This review summarizes the recent advances in the role of specific PDK isoenzymes on the induction of metabolic diseases and describes the effects of PDK inhibition on the prevention of metabolic diseases using pharmacological inhibitors. Based on these reports, PDK isoenzymes are strong therapeutic targets for preventing and treating metabolic diseases. PMID:26124988

  8. Glutamate Dehydrogenase from Pumpkin Cotyledons

    PubMed Central

    Chou, Kuo-Howere; Splittstoesser, Walter E.

    1972-01-01

    Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH4+ or ?-ketoglutarate. The soluble enzyme was more sensitive to NH4+ inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found. PMID:16657999

  9. Application of Hyperpolarized [1-13C]Lactate for the In Vivo Investigation of Cardiac Metabolism

    PubMed Central

    Mayer, Dirk; Yen, Yi-Fen; Josan, Sonal; Park, Jae Mo; Pfefferbaum, Adolf; Hurd, Ralph E.; Spielman, Daniel M.

    2012-01-01

    In addition to cancer imaging, 13C-MRS of hyperpolarized pyruvate also has demonstrated utility for the investigation of cardiac metabolism and ischemic heart disease. Although no adverse effects have yet been reported for doses commonly used in vivo, high substrate concentrations lead to supraphysiological pyruvate levels that can affect the underlying metabolism and have to be taken into account when interpreting the results. With lactate serving as an important energy source for the heart and with physiological lactate levels one to two orders of magnitude higher than for pyruvate, hyperpolarized lactate could potentially be used as an alternative to pyruvate for probing cardiac metabolism. In this study, hyperpolarized [1-13C]lactate was used to acquire time-resolved spectra from the healthy rat heart in vivo and to measure dichloroacetate (DCA)-modulated changes in flux through pyruvate dehydrogenase (PDH). Both the primary oxidation of lactate to pyruvate and the subsequent conversion of pyruvate to alanine and bicarbonate could reliably be detected. As DCA stimulates the activity of PDH through inhibition of PDH kinase, a more than 2.5-fold increase in bicarbonate-to-substrate ratio was found after administration of DCA similar to the effect when using [1-13C]pyruvate as the substrate. PMID:22278751

  10. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  11. The expression of lactate dehydrogenase in Zea mays seedlings under hypoxic and anoxic conditions 

    E-print Network

    MacAlpine, David Michael

    1995-01-01

    The ability of root cells to survive flooded (low 02) conditions may depend, in part, on energy produced anaerobically during fermentation to ethanol or lactic acid. The Davies-Roberts hypothesis predicts only a transient formation of lactic acid...

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

    SciTech Connect

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

    1991-03-11

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

  13. Interdemic variation in haematocrit and lactate dehydrogenase in the African cyprinid Barbus

    E-print Network

    Fussman, Gregor

    in these traits correlates with the environmental dissolved oxygen concentrations. A related objective. neumayeri vary according to the dissolved oxygen concentrations measured in the field. When compared cyprinid Barbus neumayeri from Rwembaita Swamp (low-oxygen) and Njuguta River (high-oxygen) in the Kibale

  14. Relationship between polymorphisms in lactate dehydrogenase B gene and milk characteristics in beef cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450s are a group of heme-containing monooxygenases necessary for the oxidative metabolism of foreign biological substances. Our goal was to determine the frequency of single nucleotide polymorphism (SNP) 994 in the CYP3A28 sequence of three breed types of cattle. The distribution of geno...

  15. Lactate Dehydrogenase Undergoes a Substantial Structural Change to Bind its Substrate

    E-print Network

    Callender, Robert

    in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed

  16. 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…

  17. SAXS fingerprints of aldehyde dehydrogenase oligomers

    PubMed Central

    Tanner, John J.

    2015-01-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren–Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), ?-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513–5522; Luo et al., J. Mol. Biol. 425 (2013) 3106–3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  18. Identification of a dehydrogenase acting on D-2-hydroxyglutarate.

    PubMed

    Achouri, Younes; Noël, Gaëtane; Vertommen, Didier; Rider, Mark H; Veiga-Da-Cunha, Maria; Van Schaftingen, Emile

    2004-07-01

    Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into a-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme. PMID:15070399

  19. Computational Studies of the Mechanism for Proton and Hydride Transfer in Liver Alcohol Dehydrogenase

    E-print Network

    Hammes-Schiffer, Sharon

    studies directed at elucidating the mechanism of the oxidation of benzyl alcohol by liver alcoholComputational Studies of the Mechanism for Proton and Hydride Transfer in Liver Alcohol dehydrogenase (LADH). This enzyme reaction involves a hydride transfer from the alcohol substrate

  20. NDRG3-mediated lactate signaling in hypoxia

    PubMed Central

    Park, Kyung Chan; Lee, Dong Chul; Yeom, Young Il

    2015-01-01

    Hypoxia is associated with many pathological conditions as well as the normal physiology of metazoans. We identified a lactate-dependent signaling pathway in hypoxia, mediated by the oxygen- and lactate-regulated protein NDRG family member 3 (NDRG3). Oxygen negatively regulates NDRG3 expression at the protein level via the PHD2/VHL system, whereas lactate, produced in excess under prolonged hypoxia, blocks its proteasomal degradation by binding to NDRG3. We also found that the stabilized NDRG3 protein promotes angiogenesis and cell growth under hypoxia by activating the Raf-ERK pathway. Inhibiting cellular lactate production abolishes NDRG3-mediated hypoxia responses. The NDRG3-Raf-ERK axis therefore provides the genetic basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases in addition to advancing our understanding of the normal physiology of hypoxia responses. [BMB Reports 2015; 48(6): 301-302] PMID:25936780

  1. Regulation of xanthine dehydrogenase in chick liver

    PubMed Central

    Corte, E. Della; Stirpe, F.

    1967-01-01

    1. It has been confirmed that the xanthine-dehydrogenase activity of chick liver is enhanced by starvation and by administration of inosine; the effects of these treatments are not additive. 2. Inosine has no effect when given to chicks depleted of the enzyme by feeding a low-protein diet. 3. Actinomycin D prevents the effect of inosine, but itself enhances the activity of xanthine dehydrogenase. 4. The xanthine-dehydrogenase activity is unchanged after addition of orotic acid to the diet, and is stimulated by injection of inorganic iron. PMID:6029610

  2. [The effect of lactation on the parasitism of rats by Strongyloides ratti (author's transl)].

    PubMed

    Bailenger, J; Cabannes, A

    1976-01-01

    In lactating females of many animal species infested by Nematoda, the self-cure is, if not suppressed, at least very distinctly delayed. It does not appear that an immunological deficiency is the cause of this. We show that this phenomenon also exists in lactating female rats with Strongyloides ratti parasites. In fact, for Strongyloides ratti, the maintenance of the worms is not the only notable modification determined by lactation; much more important is the decrease in the intensity of the parasitism. This aspect is not mentioned by writers who have only studied the different parasitic states in their final phase. Parallel to these alterations in the parasitism, the evolution of the corticosteronemy differs, from two points of view, from that described in infested virgin rats: --Suppression of the hypercorticosteronemy which normally appears 48 hours after infestation; --Attenuation of the hypocorticosteronemy which usually sets in from the tenth day of infestation. This opposition of lactation to the variations in the corticosteronemy induced by the worms is explained by the effect of lactation on the secretion of gluco-cortico-steroids, described under the term of "buffer effect of lactation". The decrease in the intensity of the parasitism may be explained by the fact that lactation, by preventing the hypercorticosteronemy normally caused by larval migration, permits the intervention of aspecific defences. As for the prolongation of the parasitism, it would seem to result on one hand, from a reduced solicitation of the means of defence owing to a smaller number of worms and, on another hand, from the slowing down of the hypocorticosteronemy through the buffer effect of lactation with all the consequences flowing from this at the level of the specific and aspecific defence reactions. PMID:1020890

  3. Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Tsuge, Yota; Yamamoto, Shogo; Kato, Naoto; Suda, Masako; Vertès, Alain A; Yukawa, Hideaki; Inui, Masayuki

    2015-06-01

    We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale. PMID:25820644

  4. Simultaneous measurements of lactate turnover rate and umbilical lactate uptake in the fetal lamb.

    PubMed Central

    Sparks, J W; Hay, W W; Bonds, D; Meschia, G; Battaglia, F C

    1982-01-01

    Lactic acid represents a major exogenous nutrient for the developing fetal lamb in utero. Our study was undertaken (a) to quantitate the net consumption of lactate by the fetus, (b) to quantitate the net lactate production and metabolism by the placenta, and (c) to compare the net fetal lactate consumption with fetal lactate use, measured simultaneously with radioactive tracers. 14 pregnant sheep were prepared with catheters in the maternal femoral artery and uterine vein and in the fetal aorta and umbilical vein. By simultaneous application of the Fick principle to the uterine and umbilical circulations, placental glucose consumption and placental lactate production were rapid, averaging 39.8 +/- 5.1 and 11.8 +/- 0.7 mg.min-1. Net lactate umbilical uptake averaged 1.95 +/- 0.16 mg-1.kg.min-1. During infusion of L-[14C(U)]lactate, fetal lactate turnover was much more rapid, averaging 6.5 +/- 0.8 mg.kg-1.min-1, and lactate utilization within the anatomic fetus was 5.9 +/- 0.7 mg.kg-1.min-1. During infusion of tracer glucose, endogenous fetal lactate production from glucose and nonglucose substrates averaged 3.0 and 1.5 mg.kg-1.min-1, respectively. The present studies have quantitated under well oxygenated, steady-state conditions, the rapid placental metabolism and production of lactate, the net fetal consumption of lactate, and the rapid endogenous fetal lactate production from glucose and nonglucose substrates. PMID:7085882

  5. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of ?-NAD(+) with a redox potential of -324mV and a 341nm ?max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of ?-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with ?-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with ?-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. PMID:25280628

  6. Etiology and therapeutic approach to elevated lactate

    PubMed Central

    Andersen, Lars W.; Mackenhauer, Julie; Roberts, Jonathan C.; Berg, Katherine M.; Cocchi, Michael N.; Donnino, Michael W.

    2014-01-01

    Lactate levels are commonly evaluated in acutely ill patients. Although most commonly used in the context of evaluating shock, lactate can be elevated for many reasons. While tissue hypoperfusion is probably the most common cause of elevation, many other etiologies or contributing factors exist. Clinicians need to be aware of the many potential causes of lactate elevation as the clinical and prognostic importance of an elevated lactate varies widely by disease state. Moreover, specific therapy may need to be tailored to the underlying cause of elevation. The current review is based on a comprehensive PubMed search and contains an overview of the pathophysiology of lactate elevation followed by an in-depth look at the varied etiologies, including medication-related causes. The strengths and weaknesses of lactate as a diagnostic/prognostic tool and its potential use as a clinical endpoint of resuscitation will be discussed. The review ends with some general recommendations on management of patients with elevated lactate. PMID:24079682

  7. Investigation of single nucleotide polymorphisms based on the intronic sequences of the propylene alcohol dehydrogenase gene in Chinese tobacco genotypes

    PubMed Central

    Wei, Ji-Cheng; Qiu, En-Jian; Guo, Hui-Yan; Hao, Ai-Ping; Chen, Rong-Ping

    2014-01-01

    A pair of primers was designed to amplify the propylene alcohol dehydrogenase gene sequence based on the cDNA sequence of the tobacco allyl-alcohol dehydrogenase gene. All introns were sequenced using traditional polymerase chain reaction (PCR) methods and T-A cloning. The sequences from common tobacco (Nicotiana tabaccum L.) and rustica tobacco (Nicotiana rustica L.) were analysed between the third intron and the fourth intron of the propylene alcohol dehydrogenase gene. The results showed that the alcohol dehydrogenase gene is a low-copy nuclear gene. The intron sequences have a combination of single nucleotide polymorphisms and length polymorphisms between common tobacco and rustica tobacco, which are suitable to identify the different germplasms. Furthermore, there are some single nucleotide polymorphism sites in the target sequence within common tobacco that can be used to distinguish intraspecific varieties.

  8. L-lactate dehydrogenation in flavocytochrome b2: a first principles molecular dynamics study.

    PubMed

    Tabacchi, Gloria; Zucchini, Daniela; Caprini, Gianluca; Gamba, Aldo; Lederer, Florence; Vanoni, Maria A; Fois, Ettore

    2009-04-01

    First principles molecular dynamics studies on active-site models of flavocytochrome b2 (L-lactate : cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidation reaction, a still-debated issue. In the calculated enzyme-substrate model complex, the L-lactate alpha-OH hydrogen is hydrogen bonded to the active-site base H373 Nepsilon, whereas the Halpha is directed towards flavin N5, suggesting that the reaction is initiated by alpha-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidation led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to flavin mononucleotide, without intermediates, but with alpha-OH proton abstraction preceding Halpha transfer and a calculated free energy barrier (12.1 kcal mol(-1)) consistent with that determined experimentally (13.5 kcal mol(-1)). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a number of flavoenzymes. Namely, they highlighted the role of: (a) the flavin mononucleotide-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin reduction; (b) an active site water molecule belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction. PMID:19348008

  9. Cotton fabric-based electrochemical device for lactate measurement in saliva.

    PubMed

    Malon, Radha S P; Chua, K Y; Wicaksono, Dedy H B; Córcoles, Emma P

    2014-06-21

    Lactate measurement is vital in clinical diagnostics especially among trauma and sepsis patients. In recent years, it has been shown that saliva samples are an excellent applicable alternative for non-invasive measurement of lactate. In this study, we describe a method for the determination of lactate concentration in saliva samples by using a simple and low-cost cotton fabric-based electrochemical device (FED). The device was fabricated using template method for patterning the electrodes and wax-patterning technique for creating the sample placement/reaction zone. Lactate oxidase (LOx) enzyme was immobilised at the reaction zone using a simple entrapment method. The LOx enzymatic reaction product, hydrogen peroxide (H2O2) was measured using chronoamperometric measurements at the optimal detection potential (-0.2 V vs. Ag/AgCl), in which the device exhibited a linear working range between 0.1 to 5 mM, sensitivity (slope) of 0.3169 ?A mM(-1) and detection limit of 0.3 mM. The low detection limit and wide linear range were suitable to measure salivary lactate (SL) concentration, thus saliva samples obtained under fasting conditions and after meals were evaluated using the FED. The measured SL varied among subjects and increased after meals randomly. The proposed device provides a suitable analytical alternative for rapid and non-invasive determination of lactate in saliva samples. The device can also be adapted to a variety of other assays that requires simplicity, low-cost, portability and flexibility. PMID:24776756

  10. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    SciTech Connect

    Hedstrom,L.; Gan, L.

    2006-01-01

    Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

  11. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    PubMed Central

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  12. The phytoene dehydrogenase gene of Phycomyces: regulation of its expression by blue light and vitamin A.

    PubMed

    Ruiz-Hidalgo, M J; Benito, E P; Sandmann, G; Eslava, A P

    1997-02-27

    By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene. PMID:9079885

  13. Isocitrate dehydrogenase mutations in gliomas.

    PubMed

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

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate ?-ketoglutarate (?KG), but recurrent mutations at Arg(132) of IDH1 and Arg(172) of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of ?KG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits ?KG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy. PMID:26188014

  14. Characterization of a Pseudomonas putida Allylic Alcohol Dehydrogenase Induced by Growth on 2-Methyl-3-Buten-2-ol

    PubMed Central

    Malone, Vincent F.; Chastain, Amy J.; Ohlsson, John T.; Poneleit, Loelle S.; Nemecek-Marshall, Michele; Fall, Ray

    1999-01-01

    We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB. PMID:10347052

  15. Thiol-catalyzed formation of lactate and glycerate from glyceraldehyde. [significance in molecular evolution

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    1983-01-01

    The rate of lactate formation from glyceraldehyde, catalyzed by N-acetyl-cysteine at ambient temperature in aqueous sodium phosphate (pH 7.0), is more rapid at higher sodium phosphate concentrations and remains essentially the same in the presence and absence of oxygen. The dramatic increase in the rate of glycerate formation that is brought about by this thiol, N-acetylcysteine, is accompanied by commensurate decreases in the rates of glycolate and formate production. It is suggested that the thiol-dependent formation of lactate and glycerate occurs by way of their respective thioesters. Attention is given to the significance of these reactions in the context of molecular evolution.

  16. [Effect of low-frequency vibration on the activity of dehydrogenase in neurones of anterior vestibular nucleus in rats].

    PubMed

    Nasibullin, B A; Rozanov, V A; Ianovsky?, M B

    1993-01-01

    To obtain the characteristics of the main changes in oxidative metabolism in the neurons of the nucleus vestibularis anterior (NVA) under the influence of low frequency vibration in rat brain the activities of some dehydrogenases (NADN-DH, NADPH-DH, succinate-DH, malate-DH, beta-oxybutyrate-DH, alpha-glycerolphosphate-DH, lactate-DH, glutamate-DH and 6-phosphogluconate-DH) were measured using histochemical methods. The sizes of subpopulations of neurons differing in enzyme activities were estimated. PMID:8335122

  17. Lactate shuttling and lactate use as fuel after traumatic brain injury: metabolic considerations

    PubMed Central

    Dienel, Gerald A

    2014-01-01

    Lactate is proposed to be generated by astrocytes during glutamatergic neurotransmission and shuttled to neurons as ‘preferred' oxidative fuel. However, a large body of evidence demonstrates that metabolic changes during activation of living brain disprove essential components of the astrocyte–neuron lactate shuttle model. For example, some glutamate is oxidized to generate ATP after its uptake into astrocytes and neuronal glucose phosphorylation rises during activation and provides pyruvate for oxidation. Extension of the notion that lactate is a preferential fuel into the traumatic brain injury (TBI) field has important clinical implications, and the concept must, therefore, be carefully evaluated before implementation into patient care. Microdialysis studies in TBI patients demonstrate that lactate and pyruvate levels and lactate/pyruvate ratios, along with other data, have important diagnostic value to distinguish between ischemia and mitochondrial dysfunction. Results show that lactate release from human brain to blood predominates over its uptake after TBI, and strong evidence for lactate metabolism is lacking; mitochondrial dysfunction may inhibit lactate oxidation. Claims that exogenous lactate infusion is energetically beneficial for TBI patients are not based on metabolic assays and data are incorrectly interpreted. PMID:25204393

  18. LactMed: Drugs and Lactation Database

    MedlinePLUS

    TOXNET Home > LactMed LactMed A TOXNET DATABASE Drugs and Lactation Database (LactMed) SEARCH LACTMED BROWSE LACTMED ADVANCED SEARCH Search Search Term Records with Include Synonyms and CAS Numbers in Search ...

  19. Regulation of bone mineral loss during lactation

    NASA Technical Reports Server (NTRS)

    Brommage, R.; Deluca, H. F.

    1985-01-01

    The effects of varyng dietary calcium and phosphorous levels, vitamin D deficiency, oophorectomy, adrenalectomy, and simultaneous pregnancy on bone mineral loss during lactation in rats are studied. The experimental procedures and evaluations are described. The femur ash weight of lactating and nonlactating rats are calculated. The data reveals that a decrease in dietary calcium of 0.02 percent results in an increased loss of bone mineral, an increase in calcium to 1.4 percent does not lessen bone mineral loss, and bone mineral loss in vitamin D deficient rats is independent of calcium levels. It is observed that changes in dietary phosphorous level, oophorectomy, adrenalectomy, and simultaneous pragnancy do not reduce bone mineral loss during lactation. The analysis of various hormones to determine the mechanism that triggers bone mineral loss during lactation is presented.

  20. Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

    PubMed

    Rodrigues, A F; Guerreiro, M R; Formas-Oliveira, A S; Fernandes, P; Blechert, A-K; Genzel, Y; Alves, P M; Hu, W S; Coroadinha, A S

    2016-01-01

    Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrolment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype. Biotechnol. Bioeng. 2016;113: 150-162. © 2015 Wiley Periodicals, Inc. PMID:26134455

  1. Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy

    NASA Astrophysics Data System (ADS)

    Li, Chao; Tao, Fei; Ni, Jun; Wang, Yu; Yao, Feng; Xu, Ping

    2015-05-01

    It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO2. However, cofactor bias of cyanobacteria is different from bacteria that prefer NADH, which hampers cyanobacterial strain engineering. In this study, the key enzyme D-lactate dehydrogenase (LdhD) from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH. Then, the engineered enzyme was introduced into Synechococcus elongatus PCC7942 to construct an efficient light-driven system that produces D-lactic acid from CO2. Mutation of LdhD drove a fundamental shift in cofactor preference towards NADPH, and increased D-lactate productivity by over 3.6-fold. We further demonstrated that introduction of a lactic acid transporter and bubbling CO2-enriched air also enhanced D-lactate productivity. Using this combinational strategy, increased D-lactate concentration and productivity were achieved. The present strategy may also be used to engineer cyanobacteria for producing other useful chemicals.

  2. Enhancing the light-driven production of d-lactate by engineering cyanobacterium using a combinational strategy

    PubMed Central

    Li, Chao; Tao, Fei; Ni, Jun; Wang, Yu; Yao, Feng; Xu, Ping

    2015-01-01

    It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO2. However, cofactor bias of cyanobacteria is different from bacteria that prefer NADH, which hampers cyanobacterial strain engineering. In this study, the key enzyme d-lactate dehydrogenase (LdhD) from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH. Then, the engineered enzyme was introduced into Synechococcus elongatus PCC7942 to construct an efficient light-driven system that produces d-lactic acid from CO2. Mutation of LdhD drove a fundamental shift in cofactor preference towards NADPH, and increased d-lactate productivity by over 3.6-fold. We further demonstrated that introduction of a lactic acid transporter and bubbling CO2-enriched air also enhanced d-lactate productivity. Using this combinational strategy, increased d-lactate concentration and productivity were achieved. The present strategy may also be used to engineer cyanobacteria for producing other useful chemicals. PMID:25940225

  3. Lactation: historical patterns and potential for manipulation.

    PubMed

    Blackburn, D G

    1993-10-01

    The advent of biotechnology has made data on undomesticated mammals relevant to dairy science. Such data indicate the potential of lactation for modification, reveal genetic material available for use through bioengineering, help distinguish adaptive features from historical artifacts, and clarify limits on lactational diversity that date from early evolution. Evolutionary analysis indicates that a complex degree of lactation preceded divergence of the extant mammalian lineages during the Mesozoic Era. Although aspects of monotreme lactation appear to be ancestral for extant mammals, the marsupials and eutherians exhibit divergent specializations. Evidence is consistent with the idea that protolacteal glands evolved by combining features of skin gland populations into a new functional complex. Secretions of these ancestral glands may have had antimicrobial properties that protected the eggs or hatchlings and organic components that supplemented offspring nutrition. Following development of highly nutritious milks, evolution produced diversity in milk composition and function, milk output, length of lactation, mammary gland anatomy, and contributions of lactation to offspring nutrition. Certain marsupials are specialized in terms of functional independence and temporal plasticity of mammary tissues. Mammalian diversity indicates that artificial selection and physiological manipulation of domestic artiodactyls has only modestly exploited the potential of mammary glands as a nutritional source for humans. PMID:8227641

  4. Detection and characterization of sorbitol dehydrogenase from apple callus tissue.

    PubMed

    Negm, F B; Loescher, W H

    1979-07-01

    Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity. PMID:16660917

  5. Continuous and simultaneous electrochemical measurements of glucose, lactate, and ascorbate in rat brain following brain ischemia.

    PubMed

    Lin, Yuqing; Yu, Ping; Hao, Jie; Wang, Yuexiang; Ohsaka, Takeo; Mao, Lanqun

    2014-04-15

    Developing new tools and technologies to enable recording the dynamic changes of multiple neurochemicals is the essence of better understanding of the molecular basis of brain functions. This study demonstrates a microfluidic chip-based online electrochemical system (OECS) for in vivo continuous and simultaneous monitoring of glucose, lactate, and ascorbate in rat brain. To fabricate the microfluidic chip-based detecting system, a microfluidic chip with patterned channel is developed into an electrochemical flow cell by incorporating the chip with three surface-modified indium-tin oxide (ITO) electrodes as working electrodes, a Ag/AgCl wire as reference electrode, and a stainless steel tube as counter electrode. Selective detection of ascorbate is achieved by the use of single-walled carbon nanotubes (SWNTs) to largely facilitate the electrochemical oxidation of ascorbate, while a dehydrogenase-based biosensing mechanism with methylene green (MG) adsorbed onto SWNTs as an electrocatalyst for the oxidation of dihydronicotiamide adenine dinucleotide (NADH) is employed for biosensing of glucose and lactate. To avoid the crosstalk among three sensors, the sensor alignment is carefully designed with the SWNT-modified electrode in the upstream channel and paralleled glucose and lactate biosensors in the downstream channels. With the microfluidic chip-based electrochemical flow cell as the detector, an OECS is successfully established by directly integrating the microfluidic chip-based electrochemical flow cell with in vivo microdialysis. The OECS exhibits a good linear response toward glucose, lactate, and ascorbate with less crosstalk. This property, along with the high stability and selectivity, enables the OECS for continuously monitoring three species in rat brain following brain ischemia. PMID:24621127

  6. Blunted HPA axis response in lactating, vasopressin-deficient Brattleboro rats.

    PubMed

    Fodor, Anna; Pintér, Ottó; Domokos, Agnes; Langnaese, Kristina; Barna, István; Engelmann, Mario; Zelena, Dóra

    2013-11-01

    Adaptation to stress is a basic phenomenon in mammalian life that is mandatorily associated with the activity of the hypothalamic-pituitary-adrenal (HPA) axis. An increased resting activity of the HPA axis can be measured during pregnancy and lactation, suggesting that these reproductive states lead to chronic load in females. In this study, we examined the consequences of the congenital lack of vasopressin on the activity of the HPA axis during lactation using vasopressin-deficient Brattleboro rats. Virgin and lactating, homozygous vasopressin-deficient rats were compared with control, heterozygous rats. In control dams compared with virgins, physiological changes similar to those observed in a chronic stress state (thymus involution, adrenal gland hyperplasia, elevation of proopiomelanocortin mRNA levels in the adenohypophysis, and resting plasma corticosterone levels) were observed. In vasopressin-deficient dams, adrenal gland hyperplasia and resting corticosterone level elevations were not observed. Corticotropin-releasing hormone (Crh) mRNA levels in the hypothalamic paraventricular nucleus were elevated in only the control dams, while oxytocin (OT) mRNA levels were higher in vasopressin-deficient virgins and lactation induced a further increase in both the genotypes. Suckling-induced ACTH and corticosterone level elevations were blunted in vasopressin-deficient dams. Anaphylactoid reaction (i.v. egg white) and insulin-induced hypoglycemia stimulated the HPA axis, which were blunted in lactating rats compared with the virgins and in vasopressin-deficient rats compared with the controls without interaction of the two factors. Vasopressin seems to contribute to the physiological changes observed during lactation mimicking a chronic stress state, but its role in acute HPA axis regulation during lactation seems to be similar to that observed in virgins. If vasopressin is congenitally absent, OT, but not the CRH, compensates for the missing vasopressin; however, the functional restitution remains incomplete. PMID:23943883

  7. The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G.

    2015-01-01

    ABSTRACT Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. IMPORTANCE Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth. PMID:25666131

  8. Proline dehydrogenase (oxidase) in cancer.

    PubMed

    Liu, Wei; Phang, James M

    2012-01-01

    Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the proline degradative pathway, plays a special role in tumorigenesis and tumor development. Proline metabolism catalyzed by PRODH/POX is closely linked with the tricarboxylic acid (TCA) cycle and urea cycle. The proline cycle formed by the interconversion of proline and ?(1) -pyrroline-5-carboxylate (P5C) between mitochondria and cytosol interlocks with pentose phosphate pathway. Importantly, by catalyzing proline to P5C, PRODH/POX donates electrons into the electron transport chain to generate ROS or ATP. In earlier studies, we found that PRODH/POX functions as a tumor suppressor to initiate apoptosis, inhibit tumor growth, and block the cell cycle, all by ROS signaling. It also suppresses hypoxia inducible factor signaling by increasing ?-ketoglutarate. During tumor progression, PRODH/POX is under the control of various tumor-associated factors, such as tumor suppressor p53, inflammatory factor peroxisome proliferator-activated receptor gamma (PPAR?), onco-miRNA miR-23b*, and oncogenic transcription factor c-MYC. Recent studies revealed the two-sided features of PRODH/POX-mediated regulation. Under metabolic stress such as oxygen and glucose deprivation, PRODH/POX can be induced to serve as a tumor survival factor through ATP production or ROS-induced autophagy. The paradoxical roles of PRODH/POX can be understood considering the temporal and spatial context of the tumor. Further studies will provide additional insights into this protein and on its metabolic effects in tumors, which may lead to new therapeutic strategies. PMID:22886911

  9. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  10. PRODUCTIVE LIFE INCLUDING ALL LACTATIONS AND LONGER LACTATIONS WITH DIMINISHING CREDITS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alternative measures of productive life (PL) were compared and life expectancy factors were updated to replace estimates from 1993. Alternatives were proposed with extra credits for lactations longer than 10 mo and beyond 84 mo of age, and for each calf produced so that an extremely long lactation w...

  11. Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

    E-print Network

    Grant, Gregory

    Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase EXTREME of D-3-phosphoglyc- erate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 Å bacteria such as Mycobacterium, Bacillus subtilis, Corynebacterium, plants such as Arabidopsis, and higher

  12. Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol.

    PubMed

    Yamada-Onodera, Keiko; Nakajima, Akira; Tani, Yoshiki

    2006-12-01

    Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols. PMID:17270720

  13. Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum

    SciTech Connect

    Ragsdale, S.W.

    1992-01-01

    The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H[sub 4]folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe[sup 3-4]S[sub 4

  14. Pyruvate Dehydrogenase | High Resolution Electron Microscopy

    Cancer.gov

    This image shows the inner and outer shells of the large protein pyruvate dehydrogenase, which is important for a cell’s metabolism. This protein turns pyruvate, which comes from sugars like glucose, into a molecule called acetyl-coA. This transformation helps energize the cell.

  15. Pharmacologic inhibition of lactate production prevents myofibroblast differentiation.

    PubMed

    Kottmann, Robert Matthew; Trawick, Emma; Judge, Jennifer L; Wahl, Lindsay A; Epa, Amali P; Owens, Kristina M; Thatcher, Thomas H; Phipps, Richard P; Sime, Patricia J

    2015-12-01

    Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. Transforming growth factor-? (TGF-?) has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-?. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-?-induced myofibroblast differentiation. Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-? and or gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Gossypol inhibited TGF-?-induced expression of the myofibroblast marker ?-smooth muscle actin (?-SMA) in a dose-dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3, and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose-dependent manner. Furthermore, gossypol inhibited TGF-? bioactivity in a dose-dependent manner. Concurrent treatment with an LDH siRNA increased the ability of gossypol to inhibit TGF-?-induced myofibroblast differentiation. Gossypol inhibits TGF-?-induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid, and inhibition of TGF-? bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis. PMID:26408551

  16. Lactate production in McArdle's disease.

    PubMed Central

    Baksi, A. K.; Buxton, P. H.; Cochrane, P.; Hughes, R. R.

    1977-01-01

    A case of McArdle's disease in a man is described in detail and a less complete study of his family is reported. This patient showed the classical features of McArdle's disease and the diagnosis was confirmed by muscle biopsy. Unlike other reported cases of this disorder, this case showed a normal rise in blood lactate levels on ischaemic exercise. This apparently paradoxical finding is discussed. It is suggested that a normal rise in the level of blood lactate on ischaemic exercise should not exclude myophosphorylase deficiency. Images Fig. 1 Fig. 2 Fig. 3 PMID:266169

  17. Breast diseases during pregnancy and lactation

    PubMed Central

    Yu, Ji Hoon; Kim, Min Jeong; Cho, Hyonil; Liu, Hyun Ju; Han, Sei-Jun

    2013-01-01

    Breast is a typical female sexual physiologic organ that is influenced by steroid hormone from menarche until menopause. Therefore various diseases can be developed by continuous action of estrogen and progesterone. Breast diseases are mainly categorized as benign and malignant. It is very important to distinguish the malignancy from breast diseases. However, it is very difficult to diagnose malignancy in pregnant and lactating women even though the same breast diseases took place. Therefore, we will review breast diseases such as breast carcinoma during pregnancy and lactation. PMID:24327995

  18. Diet for a Healthy Lactating Woman.

    PubMed

    Kolasa, Kathryn M; Firnhaber, Gina; Haven, Kelley

    2015-12-01

    The nutrient and caloric requirements for lactation are set by the Institute of Medicine. The dietary pattern to meet those needs is found in the Dietary Guidelines for Americans. Only deficiency states for selected nutrients and/or prolonged inadequate caloric intake appear to affect the volume and quality of breast milk. Other dietary concerns of lactating women include "dieting" to return to prepregnancy weight; low maternal intake of selected nutrients due to health conditions or food choices; need for supplementation of calcium, vitamin D, and fatty acids; and use of non-nutritive sweeteners, caffeine, herbal supplements, and alcohol. PMID:26398295

  19. Enzymatic reaction paths as determined by transition path sampling

    NASA Astrophysics Data System (ADS)

    Masterson, Jean Emily

    Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems, we observed changes in the reaction mechanism and altered contributions of the mutated residues to the enzymatic reaction coordinate, but we did not detect a substantial change in the time of barrier crossing. These results confirm the importance of maintaining the dynamics and structural scaffolding of the hhLDH PV in order to facilitate facile barrier passage. We also utilized TPS to investigate the possible role of fast protein dynamics in the enzymatic reaction coordinate of human dihydrofolate reductase (hsDHFR). We found that sub-picosecond dynamics of hsDHFR do contribute to the reaction coordinate, whereas this is not the case in the E. coli version of the enzyme. This result indicates a shift in the DHFR family to a more dynamic version of catalysis. The second inquiry we addressed in this thesis regarding enzymatic barrier passage concerns the variability of paths through reactive phase space for a given enzymatic reaction. We further investigated the hhLDH-catalyzed reaction using a high-perturbation TPS algorithm. Though we saw that alternate reaction paths were possible, the dominant reaction path we observed corresponded to that previously elucidated in prior hhLDH TPS studies. Since the additional reaction paths we observed were likely high-energy, these results indicate that only the dominant reaction path contributes significantly to the overall reaction rate. In conclusion, we show that the enzymes hhLDH and hsDHFR exhibit paths through reactive phase space where fast protein motions are involved in the enzymatic reaction coordinate and exhibit a non-negligible contribution to chemical barrier crossing.

  20. Novel Membrane Based Process for Producing Lactate Esters

    SciTech Connect

    1999-02-01

    Lactate Esters from Renewable Carbohydrate Feedstocks can Replace Petroleum-Derived Solvents. Lactate esters are versatile solvents that are biodegradable, nontoxic, and applicable to a wide range of industrial and consumer uses.

  1. LactMed: New NLM Database on Drugs and Lactation

    MedlinePLUS

    ... Current Issue Past Issues Research News From NIH LactMed: New NLM Database on Drugs and Lactation Past ... milk, infant levels in blood, potential effects in breast-feeding infants and on lactation itself. The American Academy ...

  2. Evaluation on the responses of succinate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase and glucose-6-phosphate dehydrogenase to acid shock generated acid tolerance in Escherichia coli

    PubMed Central

    Jain, Pradeep Kumar; Jain, Vivek; Singh, Ashish Kumar; Chauhan, Ankur; Sinha, Sarika

    2013-01-01

    Background: Escherichia coli have an optimum pH range of 6-7 for growth and survival that's why, called neutrophiles. The ?pH across the cytoplasmic membrane is linked to cellular bioenergetics and metabolism of the body which is the major supplier of the proton motive force, so homeostasis of cellular pH is essential. When challenged by low pH, protons enter the cytoplasm; as a result, mechanisms are required to alleviate the effects of lowered cytoplasmic pH. Materials and Methods: The activities of Succinate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase and glucose-6-phosphate dehydrogenase in acid shocked cells of E. coli DH5 ? and E. coli W3110 subjected to pH 3, 4, and 5 by two types of acidification, like external (using 0.1 N HCl), external along with the monensin (1 ?M) and cytoplasmic acidification using the sodium benzoate as an acid permeant (20 mM) which is coupled to the electron transport chain by the reducing power, as yet another system possessed by E. coli as an armor against harsh acidic environments. Result: Results showed that an exposure to acidic environment (pH 3, 4 and 5) for a short period of time increased the activities of these dehydrogenases in all types of acidification except cytoplasmic acidification, which shows that higher recycling of reducing power results in pumping out of protons from the cytoplasm through the electron transport chain complexes, thereby restoring the cytoplasmic pH of the bacteria in the range of 7.4-7.8. Conclusion: Study indicates that acid shocked E. coli for a period of 2 h can survive for a sustained period. PMID:24223390

  3. Targeting Tumor Metabolism for Cancer Treatment: Is Pyruvate Dehydrogenase Kinases (PDKs) a Viable Anticancer Target?

    PubMed Central

    Zhang, Wen; Zhang, Shao-Lin; Hu, Xiaohui; Tam, Kin Yip

    2015-01-01

    Cancer remains a lethal threat to global lives. Development of novel anticancer therapeutics is still a challenge to scientists in the field of biomedicine. In cancer cells, the metabolic features are significantly different from those of normal ones, which are hallmarks of several malignancies. Recent studies brought atypical cellular metabolism, such as aerobic glycolysis or the Warburg effect, into the scientific limelight. Targeting these altered metabolic pathways in cancer cells presents a promising therapeutic strategy. Pyruvate dehydrogenase kinases (PDKs), key enzymes in the pathway of glucose metabolism, could inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylating it and preserving the substrates pyruvate, lactate and alanine for gluconeogenesis. Overexpression of PDKs could block the oxidative decarboxylation of pyruvate to satisfy high oxygen demand in cancer cells, while inhibition of PDKs could upregulate the activity of PDC and rectify the balance between the demand and supply of oxygen, which could lead to cancer cell death. Thus, inhibitors targeting PDKs represent a promising strategy for cancer treatment by acting on glycolytic tumors while showing minimal side effects on the oxidative healthy organs. This review considers the role of PDKs as regulator of PDC that catalyzes the oxidative decarboxylation of pyruvate in mitochondrion. It is concluded that PDKs are solid therapeutic targets. Inhibition of PDKs could be an attractive therapeutic approach for the development of anti-cancer drugs. PMID:26681918

  4. Structurally Integrated Photoluminescence-Based Lactate Sensor Using Organic Light Emitting Devices (OLEDs) as the Light Source

    SciTech Connect

    Chengliang Qian

    2006-08-09

    Multianalyte bio(chemical) sensors are extensively researched for monitoring analytes in complex systems, such as blood serum. As a step towards developing such multianalyte sensors, we studied a novel, structurally integrated, organic light emitting device (OLED)-based sensing platform for detection of lactate. Lactate biosensors have attracted numerous research efforts, due to their wide applications in clinical diagnosis, athletic training and food industry. The OLED-based sensor is based on monitoring the oxidation reaction of lactate, which is catalyzed by the lactate oxidase (LOX) enzyme. The sensing component is based on an oxygen-sensitive dye, Platinum octaethyl porphyrin (PtOEP), whose photoluminescence (PL) lifetime {tau} decreases as the oxygen level increases. The PtOEP dye was embedded in a thin film polystyrene (PS) matrix; the LOX was dissolved in solution or immobilized in a sol-gel matrix. {tau} was measured as a function of the lactate concentration; as the lactate concentration increases, {tau} increases due to increased oxygen consumption. The sensors performance is discussed in terms of the detection sensitivity, dynamic range, and response time. A response time of {approx}32 sec was achieved when the LOX was dissolved in solution and kept in a closed cell. Steps towards development of a multianalyte sensor array using an array of individually addressable OLED pixels were also presented.

  5. Americium(iii) and europium(iii) complex formation with lactate at elevated temperatures studied by spectroscopy and quantum chemical calculations.

    PubMed

    Barkleit, Astrid; Kretzschmar, Jerome; Tsushima, Satoru; Acker, Margret

    2014-08-01

    Thermodynamic parameters for the complex formation of Am(iii) and Eu(iii) with lactate were determined with UV-vis and time-resolved laser-induced fluorescence spectroscopy (TRLFS) in a temperature range between 25 and 70 °C. The reaction enthalpy decreased with increasing ionic strength. ATR FT-IR and NMR spectroscopy in combination with density functional theory (DFT) calculations revealed structural details of the Eu(iii) lactate 1?:?1 complex: a chelating coordination mode of the lactate with a monodentate binding carboxylate group and the hydroxyl group being deprotonated. PMID:24828353

  6. LACTATION PERSISTENCY: INSIGHTS FROM MAMMARY CELL PROLIFERATION STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A persistent lactation is dependent upon maintaining number and activity of milk secreting cells with advancing lactation. When dairy cows are milked twice daily, the increase in milk yield from parturition to peak lactation is due to increased secretory activity per cell, rather than to accretion ...

  7. Microbial production of lactate-containing polyesters

    PubMed Central

    Yang, Jung Eun; Choi, So Young; Shin, Jae Ho; Park, Si Jae; Lee, Sang Yup

    2013-01-01

    Due to our increasing concerns on environmental problems and limited fossil resources, biobased production of chemicals and materials through biorefinery has been attracting much attention. Optimization of the metabolic performance of microorganisms, the key biocatalysts for the efficient production of the desired target bioproducts, has been achieved by metabolic engineering. Metabolic engineering allowed more efficient production of polyhydroxyalkanoates, a family of microbial polyesters. More recently, non-natural polyesters containing lactate as a monomer have also been produced by one-step fermentation of engineered bacteria. Systems metabolic engineering integrating traditional metabolic engineering with systems biology, synthetic biology, protein/enzyme engineering through directed evolution and structural design, and evolutionary engineering, enabled microorganisms to efficiently produce natural and non-natural products. Here, we review the strategies for the metabolic engineering of microorganisms for the in vivo biosynthesis of lactate-containing polyesters and for the optimization of whole cell metabolism to efficiently produce lactate-containing polyesters. Also, major problems to be solved to further enhance the production of lactate-containing polyesters are discussed. PMID:23718266

  8. HEXONEOGENESIS IN THE HUMAN BREAST DURING LACTATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lactose is the major osmotic agent in milk. Therefore, lactose synthesis indirectly regulates milk volume. The aim of this study was to determine the source of glucose and galactose in lactose. Six healthy lactating women were studied twice, during a 24 h fast and during ingestion of a mixed macr...

  9. 21 CFR 184.1639 - Potassium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium lactate. 184.1639 Section 184.1639 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS...

  10. Biomonitoring Polybrominated Diphenyl Ethers in Lactating Women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Breast milk is a valuable biological specimen for biomonitoring lipid-soluble polybrominated diphenyl ethers (PBDEs). The goal of this project was to determine the levels of PBDEs in breast milk of lactating women from the Seacoast region of New Hampshire and to examine potential relationships betw...

  11. 21 CFR 73.165 - Ferrous lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 1 2013-04-01 2013-04-01 false Ferrous lactate. 73.165 Section 73.165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies...

  12. 21 CFR 73.165 - Ferrous lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 1 2012-04-01 2012-04-01 false Ferrous lactate. 73.165 Section 73.165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies...

  13. 21 CFR 73.165 - Ferrous lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 1 2014-04-01 2014-04-01 false Ferrous lactate. 73.165 Section 73.165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies...

  14. 21 CFR 73.165 - Ferrous lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Ferrous lactate. 73.165 Section 73.165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies...

  15. Fermentative biohydrogen production from lactate and acetate.

    PubMed

    Wu, Chao-Wei; Whang, Liang-Ming; Cheng, Hai-Hsuan; Chan, Kan-Chi

    2012-06-01

    In this study, a continuous-flow stirred tank reactor (CSTR) fed with lactate and acetate was operated to enrich hydrogen-producing bacteria. By varying the influent substrate concentrations and hydraulic retention times (HRT), the volumetric loading rate (VLR) of 55.64 kg-COD/m(3)/day seemed to be optimum for this enriched culture for fermentative hydrogen production from lactate and acetate. The results of batch experiments confirmed that the enriched culture tended to fulfill the e(-) equiv requirement for cell growth at a lower VLR condition (21.77 kg-COD/m(3)/day), while it could largely distribute the e(-) equiv for hydrogen production at a higher VLR condition. However, a maximum lactate/acetate concentration allowed for enriching this culture existed, especially at a lower HRT condition in which wash-out can be an issue for this enriched culture. Finally, the results of cloning and sequencing indicated that Clostridium tyrobutyricum was considered the major hydrogen-producing bacteria in the CSTR fed with lactate and acetate. PMID:22318084

  16. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS...

  17. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium lactate. 184.1207 Section 184.1207 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS §...

  18. Effect of nutrition on pregnancy and lactation*

    PubMed Central

    Gopalan, C.

    1962-01-01

    Pregnancy and lactation constitute states of considerable physiological stress which impose increased nutritional demands. If these demands are not adequately met, it may be expected that not only the nutritional status of the subject will be affected, but also the course of pregnancy and lactation. While a great deal of work with experimental animals has been carried out to elucidate the role of nutrition in pregnancy and lactation, the question arises how far these experimental results are applicable to human subjects. The unfortunate nutritional situation prevalent in certain under-developed countries affords opportunities for the study of the effects of nutritional deficiencies on the course of pregnancy and lactation in the human subject. In this paper, the available literature on the effect of maternal nutrition on the course of pregnancy and the condition of the infant at birth is reviewed, as is the effect of the state of maternal nutrition on the output and chemical composition of milk in nursing mothers. The review reveals many important gaps in our knowledge and highlights the need for further work on this important problem. PMID:13900365

  19. Photo-production of lactate from glyoxylate: how minerals can facilitate energy storage in a prebiotic world

    E-print Network

    Photo-production of lactate from glyoxylate: how minerals can facilitate energy storage fixation and energy storage at the time life originated (Scheme 1A).2­5 Moreover, products of the r) of semiconductors can facilitate rapid reactions promoted by radical formation and energy storage.7 The example

  20. Exposure to Mother's Pregnancy and Lactation in Infancy is Associated with Sexual Attraction to Pregnancy and Lactation

    E-print Network

    Enquist, Magnus

    Exposure to Mother's Pregnancy and Lactation in Infancy is Associated with Sexual Attraction to Pregnancy and Lactation in Adulthoodjsm_2065 140..147 Magnus Enquist, PhD,* Hanna Aronsson, B.Sc.,* Stefano that pregnancy or lactation may become sexually attractive in adulthood following an exposure to pregnant

  1. Hormonal and behavioral responses to stress in lactating and non-lactating female common marmosets (Callithrix jacchus)

    E-print Network

    Saltzman, Wendy

    Hormonal and behavioral responses to stress in lactating and non-lactating female common marmosets hormone Cortisol Lactation Maternal care Parental care Stress In several mammalian species, hypothalamic preventing stress-induced disruptions of maternal care. Experimental elevations of HPA axis hormones have

  2. A novel OFET-based biosensor for the selective and sensitive detection of lactate levels.

    PubMed

    Minami, Tsuyoshi; Sato, Tsubasa; Minamiki, Tsukuru; Fukuda, Kenjiro; Kumaki, Daisuke; Tokito, Shizuo

    2015-12-15

    Biosensors based on organic field effect transistors (OFETs) are one of the more promising device applications in organic electronics. However, OFET-based biosensors are still in their early stages of development compared to other electrochemical biosensors. This study is the first to report on an extended-gate type organic field effect transistor (OFET) for lactate detection in aqueous media. Here, the extended-gate electrode of the OFET was modified with layers of a lactate oxidase and a horseradish peroxidase osmium-redox polymer on a flexible plastic film substrate for an enzymatic redox reaction of lactate. The device exhibited both high selectivity and sensitivity. The limit of detection (LOD) and the limit of quantification (LOQ) were estimated to be 66 nM and 220 nM, respectively, which are the sufficient detection limit for practical sensor applications. The obtained results confirm that extended-gate type OFET devices are applicable to enzyme-based biosensors for detecting lactate levels. PMID:26101795

  3. Inhibition kinetics of catabolic dehydrogenases by elevated moieties of ATP and ADP--implication for a new regulation mechanism in Lactococcus lactis.

    PubMed

    Cao, Rong; Zeidan, Ahmad A; Rådström, Peter; van Niel, Ed W J

    2010-04-01

    ATP and ADP inhibit, in varying degrees, several dehydrogenases of the central carbon metabolism of Lactococcus lactis ATCC 19435 in vitro, i.e. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH). Here we demonstrate mixed inhibition for GAPDH and competitive inhibition for LDH and ADH by adenine nucleotides in single inhibition studies. The nonlinear negative co-operativity was best modelled with Hill-type kinetics, showing greater flexibility than the usual parabolic inhibition equation. Because these natural inhibitors are present simultaneously in the cytoplasm, multiple inhibition kinetics was determined for each dehydrogenase. For ADH and LDH, the inhibitor combinations ATP plus NAD and ADP plus NAD are indifferent to each other. Model discrimination suggested that the weak allosteric inhibition of GAPDH had no relevance when multiple inhibitors are present. Interestingly, with ADH and GAPDH the combination of ATP and ADP exhibits lower dissociation constants than with either inhibitor alone. Moreover, the concerted inhibition of ADH and GAPDH, but not of LDH, shows synergy between the two nucleotides. Similar kinetics, but without synergies, were found for horse liver and yeast ADHs, indicating that dehydrogenases can be modulated by these nucleotides in a nonlinear manner in many organisms. The action of an elevated pool of ATP and ADP may effectively inactivate lactococcal ADH, but not GAPDH and LDH, providing leverage for the observed metabolic shift to homolactic acid formation in lactococcal resting cells on maltose. Therefore, we interpret these results as a regulation mechanism contributing to readjusting the flux of ATP production in L. lactis. PMID:20193044

  4. The energetics of lactation in cooperatively breeding meerkats Suricata suricatta.

    PubMed

    Scantlebury, M; Russell, A F; McIlrath, G M; Speakman, J R; Clutton-Brock, T H

    2002-10-22

    Species may become obligate cooperative breeders when parents are unable to raise their offspring unassisted. We measured the daily energy expenditure of mothers, helpers and offspring during peak lactation in cooperatively breeding meerkats Suricata suricatta using the doubly labelled water technique. Lactating mothers expended more energy per day than allo-lactating subordinate females, non-lactating females or suckling offspring. Metabolizable energy intakes of lactating mothers were calculated from isotope-based estimates of offspring milk energy intake, and were not significantly different from the previously suggested maximal limit for mammals. Allo-lactating females were the only category of animals that lost weight during the period of study, probably because they spent more time babysitting than non-lactating females. Daily energy expenditure (DEE) of lactating mothers increased with litter size but decreased with the number of helpers. Calculations show that for every 10 helpers, even in the absence of allo-lactators, mothers are able to reduce their DEE during peak lactation by an amount equivalent to the energy cost of one pup. These results indicate that helpers have beneficial energetic consequences for lactating mothers in an obligate cooperatively breeding mammal. PMID:12396490

  5. Proline dehydrogenase contributes to pathogen defense in Arabidopsis.

    PubMed

    Cecchini, Nicolás Miguel; Monteoliva, Mariela Inés; Alvarez, María Elena

    2011-04-01

    L-proline (Pro) catabolism is activated in plants recovering from abiotic stresses associated with water deprivation. In this catabolic pathway, Pro is converted to glutamate by two reactions catalyzed by proline dehydrogenase (ProDH) and ?(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH), with ?(1)-pyrroline-5-carboxylate (P5C) as the intermediate. Alternatively, under certain conditions, the P5C derived from Pro is converted back to Pro by P5C reductase, thus stimulating the Pro-P5C cycle, which may generate reactive oxygen species (ROS) as a consequence of the ProDH activity. We previously observed that Pro biosynthesis is altered in Arabidopsis (Arabidopsis thaliana) tissues that induce the hypersensitive response (HR) in response to Pseudomonas syringae. In this work, we characterized the Pro catabolic pathway and ProDH activity in this model. Induction of ProDH expression was found to be dependent on salicylic acid, and an increase in ProDH activity was detected in cells destined to die. To evaluate the role of ProDH in the HR, ProDH-silenced plants were generated. These plants displayed reduced ROS and cell death levels as well as enhanced susceptibility in response to avirulent pathogens. Interestingly, the early activation of ProDH was accompanied by an increase in P5C reductase but not in P5CDH transcripts, with few changes occurring in the Pro and P5C levels. Therefore, our results suggest that in wild-type plants, ProDH is a defense component contributing to HR and disease resistance, which apparently potentiates the accumulation of ROS. The participation of the Pro-P5C cycle in the latter response is discussed. PMID:21311034

  6. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    SciTech Connect

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  7. Kinetic characterization of branched chain ketoacid dehydrogenase.

    PubMed

    Boyer, B; Odessey, R

    1991-02-15

    Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition. PMID:1990967

  8. Malate dehydrogenase-2 inhibitor LW6 promotes metabolic adaptations and reduces proliferation and apoptosis in activated human T-cells

    PubMed Central

    ELEFTHERIADIS, THEODOROS; PISSAS, GEORGIOS; ANTONIADI, GEORGIA; LIAKOPOULOS, VASSILIOS; STEFANIDIS, IOANNIS

    2015-01-01

    Activated T cells rely on aerobic glycolysis and glutaminolysis in order to proliferate and differentiate into effector cells. Therefore, intervention in these metabolic pathways inhibits proliferation. The aim of the present study was to evaluate the effects of Krebs' cycle inhibition at the level of malate dehydrogenase-2 (MDH2) in human activated T cells using the MDH2 inhibitor LW6. Activated T cells from healthy volunteers were cultured in the presence or absence of LW6 and cytotoxicity, cell proliferation and the expression levels of hypoxia-inducible factor (HIF)-1?, c-Myc, p53, cleaved caspase-3 and certain enzymes involved in glucose metabolism and glutaminolysis were evaluated. The results revealed that LW6 was not toxic and decreased apoptosis and the levels of the pro-apoptotic tumor suppressor p53. In addition, LW6 inhibited T-cell proliferation and decreased the levels of c-Myc, HIF-1?, glucose transporter-1, hexokinase-II, lactate dehydrogenase-A and phosphorylated pyruvate dehydrogenase. By contrast, LW6 increased the levels of pyruvate dehydrogenase. These alterations may lead to decreased production of pyruvate, which preferentially enters into the Krebs' cycle. Furthermore, LW6 decreased the levels of glutaminase-2, while increasing those of glutaminase-1, which may preserve glutaminolysis, and possibly pyruvate-malate cycling, potentially protecting the cells from energy collapse. In summary, the inhibition of MDH2 in activated T cells abrogates proliferation without adversely affecting cell survival. Adaptations of cellular glucose and glutamine metabolism may prevent energy collapse. PMID:26640580

  9. On line continuous monitoring of blood lactate in men by a wearable device based upon an enzymatic amperometric lactate sensor.

    PubMed

    Meyerhoff, C; Bischof, F; Mennel, F J; Sternberg, F; Bican, J; Pfeiffer, E F

    1993-01-01

    A wearable device for the continuous measurement of lactate in the blood was constructed by the combination of continuous blood sampling employing a double lumen catheter with an amperometric lactate sensor. In vitro, the lactate sensor turned out to have a linear concentration range between 0 and 15 mmol/l. The response time of the sensor itself amounted to 100 sec, whereas the lag time for blood sampling amounted to 2.2 min. In vivo, the lactate sensor was successfully used for the detection of changes of the blood lactate concentration following strenuous exercise in 7 healthy volunteers, in two cases up to 22 h. In conclusion, the technique of continuous blood sampling by the use of a double lumen catheter in combination with the amperometric lactate sensor is feasible and simplifies frequent blood lactate estimations. PMID:8311937

  10. CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE

    PubMed Central

    Fahimi, H. Dariush; Amarasingham, Chandra Raj

    1964-01-01

    The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum. PMID:14195619

  11. Immunolocalization of mannitol dehydrogenase in celery plants and cells.

    PubMed Central

    Zamski, E; Yamamoto, Y T; Williamson, J D; Conkling, M A; Pharr, D M

    1996-01-01

    Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants. PMID:8938403

  12. Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase

    PubMed Central

    Bandaria, Jigar N.; Dutta, Samrat; Nydegger, Michael W.; Rock, William; Kohen, Amnon; Cheatum, Christopher M.

    2010-01-01

    The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzyme-catalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using two-dimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (). We observe that the frequency–frequency time correlation functions (FFCF) for the ternary complexes with NAD+ and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window. PMID:20876138

  13. Intramolecular Hydrogen Bonding in Methyl Lactate.

    PubMed

    Schrøder, Sidsel D; Wallberg, Jens H; Kroll, Jay A; Maroun, Zeina; Vaida, Veronica; Kjaergaard, Henrik G

    2015-09-17

    The intramolecular hydrogen bonding in methyl lactate was studied with Fourier transform infrared (FTIR) spectroscopy, intracavity laser photoacoustic spectroscopy, and cavity ring-down spectroscopy. Vapor phase spectra were recorded in the ?vOH = 1-4 OH-stretching regions, and the observed OH-stretching transitions were compared with theoretical results. Transition frequencies and oscillator strengths were obtained using a one-dimensional anharmonic oscillator local mode model with potential energy and dipole moment surfaces calculated at the CCSD(T)-F12a/VDZ-F12 level. The three most abundant conformers of methyl lactate all appear to possess an intramolecular hydrogen bond, with the hydroxyl group forming a hydrogen bond with either the carbonyl or ester oxygen. The intramolecular hydrogen bonds were investigated theoretically by analyses based on electron density topology, natural bond orbital analysis, and visualization of the electrostatic potential energy. PMID:26296230

  14. Extended lactation in dairy cows: effects of milking frequency, calving season and nutrition on lactation persistency and milk quality.

    PubMed

    Sorensen, Annette; Muir, D Donald; Knight, Christopher H

    2008-02-01

    Twelve spring-calving and twelve winter-calving cows were managed for extended lactation cycles of 18-months duration, with the former group then completing a second extended lactation. Half of the cows were fed according to standard management practice for the herd; the other half received supplementary concentrate from week 9 of lactation onwards. Commencing at the same time, half of the udder of each cow was subjected to increased milking frequency (thrice daily rather than twice daily). Lactation persistency (and hence total milk yield) was significantly increased by frequent milking. Winter calving cows and supplemented cows also exhibited better persistency, but this was only evident up until the point of re-breeding, at around lactation week 33. Milk composition was measured in the spring-calving cows in both their first and second extended lactations. Composition altered during the course of the lactation, protein and fat percentages increasing and lactose percentage decreasing, irrespective of treatment. The quality of the milk for processing into cheese, fermented products, heat-treated products and cream liqueurs was assessed by calculation of casein number (casein protein as a proportion of total protein). Processing quality declined across the course of lactation in those groups that showed poor persistency but not in those that maintained a persistent lactation. Milk hygienic quality (somatic cell counts) showed parallel changes. Body condition score increased during the course of lactation but was not affected by supplementation; none of the cows became excessively fat. All cows remained healthy throughout the extended lactations and the majority (33/36) re-bred successfully. By demonstrating that lactation persistency is plastic and can be improved by simple management interventions, the results lend support to the economic arguments in favour of extended lactation cycles. The likely welfare benefits of extended lactation are also discussed. PMID:18226299

  15. Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes.

    PubMed

    Antonenkov, V D

    1989-07-15

    Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed. PMID:2753047

  16. Use of a Novel Escherichia coli-Leuconostoc Shuttle Vector for Metabolic Engineering of Leuconostoc citreum To Overproduce d-Lactate

    PubMed Central

    Chae, Han Seung; Lee, Seung Hwan; Lee, Ju-Hoon; Park, Si Jae

    2013-01-01

    Determination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC13302 revealed that it contains 20,721 bp, a G+C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stable Escherichia coli-Leuconostoc shuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced into Leuconostoc, Lactococcus lactis, and Pediococcus. This shuttle vector was used to engineer Leuconostoc citreum 95 to overproduce d-lactate. The L. citreum 95 strain engineered using plasmid pMBLT02, which overexpresses d-lactate dehydrogenase, exhibited enhanced production of optically pure d-lactate (61 g/liter, which is 6 times greater than the amount produced by the control strain) when cultured in a reactor supplemented with 140 g/liter glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of a d-lactate overproduction system in other Leuconostoc strains and Lactococcus lactis. PMID:23241984

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

    PubMed

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

    2015-12-01

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

  18. Characterization of Lactate Sensors Based on Lactate Oxidase and Palladium Benzoporphyrin Immobilized in Hydrogels

    PubMed Central

    Andrus, Liam P.; Unruh, Rachel; Wisniewski, Natalie A.; McShane, Michael J.

    2015-01-01

    An optical biosensor for lactate detection is described. By encapsulating enzyme-phosphor sensing molecules within permeable hydrogel materials, lactate-sensitive emission lifetimes were achieved. The relative amount of monomer was varied to compare three homo- and co-polymer materials: poly(2-hydroxyethyl methacrylate) (pHEMA) and two copolymers of pHEMA and poly(acrylamide) (pAam). Diffusion analysis demonstrated the ability to control lactate transport by varying the hydrogel composition, while having a minimal effect on oxygen diffusion. Sensors displayed the desired dose-variable response to lactate challenges, highlighting the tunable, diffusion-controlled nature of the sensing platform. Short-term repeated exposure tests revealed enhanced stability for sensors comprising hydrogels with acrylamide additives; after an initial “break-in” period, signal retention was 100% for 15 repeated cycles. Finally, because this study describes the modification of a previously developed glucose sensor for lactate analysis, it demonstrates the potential for mix-and-match enzyme-phosphor-hydrogel sensing for use in future multi-analyte sensors. PMID:26198251

  19. Comparison of Field Groundwater Biostimulation Experiments Using Polylactate and Lactate Solutions at the Chromium-Contaminated Hanford 100-H Site

    NASA Astrophysics Data System (ADS)

    Hazen, T. C.; Faybishenko, B.; Beller, H. R.; Brodie, E. L.; Sonnenthal, E. L.; Steefel, C.; Larsen, J.; Conrad, M. E.; Bill, M.; Christensen, J. N.; Brown, S. T.; Joyner, D.; Borglin, S. E.; Geller, J. T.; Chakraborty, R.; Nico, P. S.; Long, P. E.; Newcomer, D. R.; Arntzen, E.

    2011-12-01

    The primary contaminant of concern in groundwater at the DOE Hanford 100 Area (Washington State) is hexavalent chromium [Cr(VI)] in Hanford coarse-grained sediments. Three lactate injections were conducted in March, August, and October 2010 at the Hanford 100-H field site to assess the efficacy of in situ Cr(VI) bioreductive immobilization. Each time, 55 gal of lactate solution was injected into the Hanford aquifer. To characterize the biogeochemical regimes before and after electron donor injection, we implemented a comprehensive plan of groundwater sampling for microbial, geochemical, and isotopic analyses. These tests were performed to provide evidence of transformation of toxic and soluble Cr(VI) into less toxic and poorly soluble Cr(III) by bioimmobilization, and to quantify critical and interrelated microbial metabolic and geochemical mechanisms affecting chromium in situ reductive immobilization and the long-term sustainability of chromium bioremediation. The results of lactate injections were compared with data from two groundwater biostimulation tests that were conducted in 2004 and 2008 by injecting Hydrogen Release Compound (HRC°), a slow-release glycerol polylactate, into the Hanford aquifer. In all HRC and lactate injection tests, 13C-labeled lactate was added to the injected solutions to track post-injection carbon pathways. Monitoring showed that despite a very low initial total microbial density (from <104 to 105 cells/mL), both HRC and lactate injections stimulated anaerobic microbial activity, which led to an increase in biomass to >107 cells/mL (including sulfate- and nitrate-reducing bacteria), resulting in a significant decrease in soluble Cr(VI) concentrations to below the MCL. In all tests, lactate was consumed nearly completely within the first week, much faster than HRC. Modeling of biogeochemical and isotope fractionation processes with the reaction-transport code TOUGHREACT captured the biodegradation of lactate, fermentative production of acetate and propionate, the evolution of 13C in bicarbonate, and the rate of sulfate reduction. In contrast to the slow-release HRC injections, no long-term effects of biostimulation and Cr bioreduction were observed in groundwater after the lactate injections. The presentation will address these patterns of the geochemical, ?13C of DIC, and biomass changes in groundwater before and after the polylactate and lactate injections.

  20. Brain lactate metabolism: the discoveries and the controversies

    PubMed Central

    Dienel, Gerald A

    2012-01-01

    Potential roles for lactate in the energetics of brain activation have changed radically during the past three decades, shifting from waste product to supplemental fuel and signaling molecule. Current models for lactate transport and metabolism involving cellular responses to excitatory neurotransmission are highly debated, owing, in part, to discordant results obtained in different experimental systems and conditions. Major conclusions drawn from tabular data summarizing results obtained in many laboratories are as follows: Glutamate-stimulated glycolysis is not an inherent property of all astrocyte cultures. Synaptosomes from the adult brain and many preparations of cultured neurons have high capacities to increase glucose transport, glycolysis, and glucose-supported respiration, and pathway rates are stimulated by glutamate and compounds that enhance metabolic demand. Lactate accumulation in activated tissue is a minor fraction of glucose metabolized and does not reflect pathway fluxes. Brain activation in subjects with low plasma lactate causes outward, brain-to-blood lactate gradients, and lactate is quickly released in substantial amounts. Lactate utilization by the adult brain increases during lactate infusions and strenuous exercise that markedly increase blood lactate levels. Lactate can be an ‘opportunistic', glucose-sparing substrate when present in high amounts, but most evidence supports glucose as the major fuel for normal, activated brain. PMID:22186669

  1. Role and regulation of 11?-hydroxysteroid dehydrogenase in lung inflammation 

    E-print Network

    Yang, Fu

    2010-01-01

    Glucocorticoids are steroid hormones that have potent anti-inflammatory actions. Endogenous glucocorticoid action is modulated by 11?-hydroxysteroid dehydrogenase (11?-HSD) which catalyses the interconversion of ...

  2. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  3. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  4. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  5. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1380 Hydroxybutyric dehydrogenase test system. (a)...

  6. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1500 Malic dehydrogenase test system. (a)...

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

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

    PubMed

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

    2015-11-01

    Colostrum has a different composition compared with milk in established lactation. This difference is in part due to the partially open blood-milk barrier, which, when closed, is designed to prevent the interdiffusion of blood and milk components. In the first days of lactation, ?-lactalbumin (?-LA), a milk protein, is typically present in blood and several blood-derived proteins are also present in milk, such as IgG1, IgG2, serum albumin (SA), and lactate dehydrogenase (LDH). With the exception of IgG1, which is known to be transferred by active transcellular transport, the other proteins are thought to pass paracellularly through the temporarily open barrier. Along with an exchange of blood and milk components, somatic cell count (SCC) is typically high in colostrum. The decline of these proteins and SCC can be used as indicators to determine transcellular or paracellular transport. Two hypotheses were tested. The first hypothesis was that the decline curve for a protein or SCC would be the same as IgG1, indicating transcellular transport, or the decline curve would be different than IgG1, indicating paracellular transport. The second hypothesis was that the decline curves of SCC and all proteins that are thought to have paracellular transport would be the same. Ten Holstein cows were milked at 4 h after parturition, the next 5 consecutive milkings, and the afternoon milking on d 5, 8, 10, and 14 of lactation for a total of 10 milking time points, and sequential jugular blood samples were also taken. Blood and milk samples were analyzed for the concentrations of LDH, SA, IgG1, IgG2, and ?-LA and milk samples were measured for SCC. Protein concentration and SCC curves were generated from all 10 time points and were evaluated using the tau time constant model to determine the rate of decline of the slope of each protein. When examining the first hypothesis, the concentration of IgG1 declined significantly faster in the milk than the proteins IgG2 and LDH, but declined at the same rate as SA. Immunoglobulin G1 also declined significantly faster than SCC and ?-LA in plasma. The second hypothesis showed that IgG2, LDH, and SA in milk were declining at the same rate, but were declining significantly faster than SCC and ?-LA in plasma. These results indicate that only active transcellular transport of IgG1 occurred, with a sharp decline at parturition, compared with IgG2, SA, LDH, ?-LA, and SCC, which are likely following paracellular transport. PMID:26298756

  9. [Characterization of the pathology of lactating cows based on the level of lactation. Principal factors in the variation and typing of pathologic profiles of lactation].

    PubMed

    Landais, E; Coulon, J B; Garel, J P; Hoden, A

    1989-01-01

    The health disturbances investigated were observed during a long-term trial (six years) conducted at an experimental station located at 1,100 m elevation. The study dealt with 487 lactations involving 190 cows of the Montbéliarde and French Friesian breeds, which produced on average 4,200 kg milk per lactation. The disturbances concerned 59% of monitored lactations, with a mean incidence of 2.1 disturbances per lactation. Lameness and mastitis accounted respectively for 52 and 24% of the clinical affections. Pathology was significantly influenced by breed, basic diet (hay or grass silage), concentrate quantities, lactation rank and year. The authors describe a method permitting an independent analysis of the effects of lactation stage and of season on mastitis and lameness frequency, by limiting the biases due to grouping of calvings and to culling. The study of lactations affected by several pathological disturbances shows that the different types of affections recorded are mutually independent but that successive occurrences of the same affection are not. On the basis of these results, the authors have proposed to globally characterize the "pathological profiles" of lactations. PMID:2817732

  10. A 3D-printed device for a smartphone-based chemiluminescence biosensor for lactate in oral fluid and sweat.

    PubMed

    Roda, Aldo; Guardigli, Massimo; Calabria, Donato; Calabretta, Maria Maddalena; Cevenini, Luca; Michelini, Elisa

    2014-12-21

    Increasingly, smartphones are used as portable personal computers, revolutionizing communication styles and entire lifestyles. Using 3D-printing technology we have made a disposable minicartridge that can be easily prototyped to turn any kind of smartphone or tablet into a portable luminometer to detect chemiluminescence derived from enzyme-coupled reactions. As proof-of-principle, lactate oxidase was coupled with horseradish peroxidase for lactate determination in oral fluid and sweat. Lactate can be quantified in less than five minutes with detection limits of 0.5 mmol L(-1) (corresponding to 4.5 mg dL(-1)) and 0.1 mmol L(-1) (corresponding to 0.9 mg dL(-1)) in oral fluid and sweat, respectively. A smartphone-based device shows adequate analytical performance to offer a cost-effective alternative for non-invasive lactate measurement. It could be used to evaluate lactate variation in relation to the anaerobic threshold in endurance sport and for monitoring lactic acidosis in critical-care patients. PMID:25343380

  11. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 ...864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device...

  12. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 ...864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device...

  13. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 ...864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device...

  14. Hepatic metabolic response of Holstein cows in early and mid lactation is altered by nutrient supply and lipopolysaccharide in vitro.

    PubMed

    Garcia, M; Bequette, B J; Moyes, K M

    2015-10-01

    The metabolic response of the liver during periods of inflammation is poorly understood. The objective of this study was to characterize the effects of nutrient supply and lipopolysaccharide (LPS) challenge on hepatic intermediate metabolism of early- and mid-lactation cows by employing gas chromatography-mass spectrometry with stable isotope tracer. Twelve multiparous Holstein-Friesian cows in early (n = 6; 12 ± 4.2 d in milk) and mid (n = 6; 115 ± 13.5 d in milk) lactation were used for this study. Liver biopsies were performed on all cows. Liver slices (40-60 mg) were incubated in a 37°C water bath for 2 h with either control (phosphate buffered saline), pyruvate (PYR; 1mM unlabeled pyruvate and 1mM [(13)C3]pyruvate), pyruvate + propionate (PYR+PRO; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM sodium propionate), or pyruvate + AA (PYR+AA; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM AA solution), and LPS (0.0 or 0.2 ?g/mL) was added to flasks per treatment. Enrichment of isotopomers in metabolic equilibrium with Krebs cycle intermediates was assessed. Pyruvate fluxes and the enzymatic activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) and phosphoenol pyruvate carboxykinase (PEPCK) were calculated. Media were analyzed for concentrations of tumor necrosis factor-? (TNF-?), glucose, and haptoglobin. Data were analyzed as randomized block (stage of lactation) design in a factorial arrangement of nutrient treatments by LPS dose. Challenge with LPS increased the mRNA abundance of TNF-?, haptoglobin, and serum amyloid A 2, and the concentration of TNF-? in media. Challenge with LPS increased mRNA abundance of PC but reduced the enrichment of (13)C1[M1] and (13)C2[M2]alanine and tended to reduce the enzymatic activity of PEPCK. Incubation with PYR+PRO and PYR+AA increased the flux of pyruvate to acetyl CoA. However, only PYR+PRO increased the enzymatic activity of PEPCK and PDH versus PC and decreased the mRNA abundance of PC. Cows in early lactation tended to receive a greater contribution of pyruvate to the oxaloacetate flux via the lower PDH versus PC activity and a higher mRNA abundance of PC than cows in mid lactation. Our results suggest that regardless of stage of lactation and nutrient supplement, hepatic gluconeogenesis was impaired during inflammation. Further research examining how various nutrients support liver function and improve the immunometabolic response of liver during inflammation is warranted. PMID:26233455

  15. Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Lin, Ying; Alguindigue, Susan S; Volkman, Jerome; Nicholas, Kenneth M; West, Ann H; Cook, Paul F

    2007-01-23

    The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M. PMID:17223711

  16. Enhanced Responsiveness to Selective Serotonin Reuptake Inhibitors during Lactation

    PubMed Central

    Jury, Nicholas J.; McCormick, Betsy A.; Horseman, Nelson D.; Benoit, Stephen C.; Gregerson, Karen A.

    2015-01-01

    The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence has cast doubt on the effectiveness of SSRIs, these results support their therapeutic use in the treatment of PPD. PMID:25689282

  17. Clinical correlates of arterial lactate levels in STEMI patients.

    PubMed

    Weil, Max Harry; Tang, Wanchun

    2011-01-01

    Increases in blood lactate reflect decreases in systemic blood flows associated with low blood flow states characteristic of circulatory shock. Accordingly, the report by Vermeulen and colleagues documents the use of the blood lactate measurement as a prognostic indicator in settings of ST elevation myocardial infarction. That lactate value therefore identified high-risk patients as a complication, often with clinical signs of cardiogenic shock of corresponding severities. PMID:21345276

  18. Enhanced responsiveness to selective serotonin reuptake inhibitors during lactation.

    PubMed

    Jury, Nicholas J; McCormick, Betsy A; Horseman, Nelson D; Benoit, Stephen C; Gregerson, Karen A

    2015-01-01

    The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence has cast doubt on the effectiveness of SSRIs, these results support their therapeutic use in the treatment of PPD. PMID:25689282

  19. Hexose-6-Phosphate Dehydrogenase and 11?-Hydroxysteroid Dehydrogenase-1 Tissue Distribution in the Rat

    PubMed Central

    Gomez-Sanchez, Elise P.; Romero, Damian G.; de Rodriguez, Angela F.; Warden, Mary P.; Krozowski, Zygmunt; Gomez-Sanchez, Celso E.

    2008-01-01

    Intracellular concentrations of the glucocorticoids cortisol and corticosterone are modulated by the enzymes 11?-hydroxysteroid dehydrogenase (11?-HSD) 1 and 2. 11?-HSD1 is a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal reductase that converts the inactive glucocorticoids cortisone and 11-dehydrocorticosterone to their active forms, cortisol and corticosterone. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme that generates NADPH from oxidized NADP (NADP+) within the endoplasmic reticulum. In the absence of NADPH or H6PDH to regenerate NADPH, 11?-HSD1 acts as a dehydrogenase and inactivates glucocorticoids, as does 11?-HSD2. A monoclonal antibody against H6PDH was produced to study the possibility that 11?-HSD1 in the absence of H6PDH may be responsible for hydroxysteroid dehydrogenase activity in tissues that do not express significant amounts of 11?-HSD2. H6PDH and 11?-HSD1 expression was surveyed in a variety of rat tissues by real-time RT-PCR, Western blot analysis, and immunohistochemistry. H6PDH was found in a wide variety of tissues, with the greatest concentrations in the liver, kidney, and Leydig cells. Although the brain as a whole did not express significant amounts of H6PDH, some neurons were clearly immunoreactive by immunohistochemistry. H6PDH was amply expressed in most tissues examined in which 11?-HSD1 was also expressed, with the notable exception of the renal interstitial cells, in which dehydrogenase activity by 11?-HSD1 probably moderates activation of the glucocorticoid receptor because rat renal interstitial cells do not have significant amounts of mineralocorticoid receptors. This antibody against the H6PDH should prove useful for further studies of enzyme activity requiring NADPH generation within the endoplasmic reticulum. PMID:18039793

  20. Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

    PubMed Central

    Alp, P R; Newsholme, E A; Zammit, V A

    1976-01-01

    1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium. PMID:8036

  1. Lactate's effect on human neuroblastoma cell bioenergetic fluxes.

    PubMed

    E, Lezi; Swerdlow, Russell H

    2016-01-01

    Lactate, once considered a metabolic dead-end, has been recently proposed to support neuron bioenergetics. To better understand how lactate specifically influences cell energy metabolism, we studied the effects of lactate supplementation on SH-SY5Y human neuroblastoma cell bioenergetic fluxes. Lactate supplementation increased cell respiration, there was no change in respiratory coupling efficiency, and lactate itself appeared to directly support the respiratory flux increase. Conversely, lactate supplementation reduced the glycolysis flux. This apparent pro-aerobic shift in the respiration:glycolysis ratio was accompanied by post-translational modifications and compartmental redistributions of proteins that respond to and modify bioenergetic fluxes, including cAMP-response element binding protein (CREB), p38 mitogen-activated protein kinases (p38 MAPK), AMP-activated protein kinase (AMPK), peroxisome-proliferator activated receptor gamma coactivator 1 ? (PGC-1?), Akt, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FOXO1). mRNA levels for PGC-1?, nuclear respiratory factor 1 (NRF1), and cytochrome c oxidase subunit 1 (COX1) increased. Some effects depended on the direct presence of lactate, while others were durable and evident several hours after lactate was removed. We conclude lactate can be used to manipulate cell bioenergetics. PMID:26592660

  2. Lactate transport and receptor actions in cerebral malaria

    PubMed Central

    Mariga, Shelton T.; Kolko, Miriam; Gjedde, Albert; Bergersen, Linda H.

    2014-01-01

    Cerebral malaria (CM), caused by Plasmodium falciparum infection, is a prevalent neurological disorder in the tropics. Most of the patients are children, typically with intractable seizures and high mortality. Current treatment is unsatisfactory. Understanding the pathogenesis of CM is required in order to identify therapeutic targets. Here, we argue that cerebral energy metabolic defects are probable etiological factors in CM pathogenesis, because malaria parasites consume large amounts of glucose metabolized mostly to lactate. Monocarboxylate transporters (MCTs) mediate facilitated transfer, which serves to equalize lactate concentrations across cell membranes in the direction of the concentration gradient. The equalizing action of MCTs is the basis for lactate’s role as a volume transmitter of metabolic signals in the brain. Lactate binds to the lactate receptor GPR81, recently discovered on brain cells and cerebral blood vessels, causing inhibition of adenylyl cyclase. High levels of lactate delivered by the parasite at the vascular endothelium may damage the blood–brain barrier, disrupt lactate homeostasis in the brain, and imply MCTs and the lactate receptor as novel therapeutic targets in CM. PMID:24904266

  3. Is lactate the new panacea for endothelial dysfunction?

    PubMed

    Nalos, Marek; Tang, Benjamin M; Nanan, Ralph

    2014-01-01

    Fluid resuscitation in the critically ill is a hot topic. The current strategy of rapid and adequate resuscitation in shock followed by conservative fluid administration is often difficult to achieve with standard crystalloid solutions. Research into alternative intravenous fluids tailored to individual patient needs is required. In the previous issue of Critical Care, Somasetia and colleagues compare the effects of hypertonic sodium lactate with the World Health Organization-recommended strategy of Ringer's lactate resuscitation in children with severe Dengue, a viral infection for which causal treatment and vaccination are not available. The results not only suggest unimpaired lactate metabolism during shock in children but document improvement in endothelial barrier function, limited coagulopathy, and avoidance of fluid overload with hypertonic sodium lactate. Their study invites several important questions to be answered. Is hypertonicity or lactate per se important for the beneficial effects? Are the metabolic or anti-inflammatory effects responsible? Is the raised lactate in shock an adaptive response? Should reduction in lactate levels be the goal of resuscitation? These questions may trigger further research into the role of lactate and lactate-based intravenous fluids in resuscitation of the critically ill. PMID:25672811

  4. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Sorbitol dehydrogenase test system. 862.1670 Section 862.1670 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1670 Sorbitol dehydrogenase...

  5. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Malic dehydrogenase test system. 862.1500 Section 862.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1500 Malic dehydrogenase test system....

  6. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological...

  7. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological...

  8. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Malic dehydrogenase test system. 862.1500 Section 862.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1500 Malic dehydrogenase test system....

  9. Immobilization of alcohol dehydrogenase onto metal-chelated cryogels.

    PubMed

    Uygun, Deniz Akta?; Akduman, Begüm; Uygun, Murat; Akgöl, Sinan; Denizli, Adil

    2015-01-01

    In this presented work, poly(HEMA-GMA) cryogel was synthesized and used for the immobilization of alcohol dehydrogenase. For this, synthesized cryogels were functionalized with iminodiacetic acid and chelated with Zn(2+). This metal-chelated cryogels were used for the alcohol dehydrogenase immobilization and their kinetic parameters were compared with free enzyme. Optimum pH was found to be 7.0 for both immobilized and free enzyme preparations, while temperature optima for free and immobilized alcohol dehydrogenase was 25 °C. Kinetic constants such as K(m), V(max), and k(cat) for free and immobilized form of alcohol dehydrogenase were also investigated. k(cat) value of free enzyme was found to be 3743.9 min(-1), while k(cat) for immobilized enzyme was 3165.7 min(-1). Thermal stability of the free and immobilized alcohol dehydrogenase was studied and stability of the immobilized enzyme was found to be higher than free form. Also, operational stability and reusability profile of the immobilized alcohol dehydrogenase were investigated. Finally, storage stability of the free and immobilized alcohol dehydrogenase was studied, and at the end of the 60 days storage, it was demonstrated that, immobilized alcohol dehydrogenase was exhibited high stability than that of free enzyme. PMID:25715869

  10. Induction of Glycerol Phosphate Dehydrogenase Gene Expression During Seizure

    E-print Network

    Kuhl, Dietmar

    Induction of Glycerol Phosphate Dehydrogenase Gene Expression During Seizure and Analgesia Wolfgang display, we found that the gene for NAD -dependent glycerol phosphate dehy- drogenase (GPDH; EC 1 as it is mimicked by exogenously applied analgesic drugs. Key Words: Glycerol phosphate dehydrogenase

  11. Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N-Propargylglycine†‡

    PubMed Central

    White, Tommi A.; Johnson, William H.; Whitman, Christian P.; Tanner, John J.

    2009-01-01

    The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 Å resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the ?-amino group of Lys99 via a 3-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25°, which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this ?,?-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a 3-carbon chain. The presumed non-enzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely. PMID:18426222

  12. Relationship between season, lactation number and incidence of clinical mastitis in different stages of lactation in a Holstein dairy farm

    PubMed Central

    Moosavi, Maede; Mirzaei, Abdolah; Ghavami, Mohsen; Tamadon, Amin

    2014-01-01

    The aim of the present study was to compare the occurrence and duration of clinical mastitis in different seasons, stages of lactation period and parities in a Holstein dairy farm in Iran. A retrospective epidemiological survey from April 2005 to March 2008 was conducted on 884 clinical mastitis cases of 7437 lactations. Data of each case including calendar-date of mastitis onset, days in milk (DIM) of mastitis onset (early: 0-74 DIM; middle: 75-150 DIM, and late ? 150 DIM), duration of mastitis, and parity (1, 2, and ? 3) were recorded. Based on date of mastitis onset, cases were classified into stages of lactation. Moreover, beginning of mastitis was seasonally categorized. Duration of clinical mastitis after treatment in early lactation was less than late lactation in the first-parity cows (p = 0.005). In early lactation period, the first-parity cows suffered clinical mastitis in days earlier than two other parity groups (p < 0.001). Moreover, in late lactation period, the first-parity cows had clinical mastitis in days later than cows in the third and more parities (p = 0.002). Occurrence of clinical mastitis in summer increased in late lactation period but in winter increased in early lactation period (p = 0.001). In addition, occurrence time of clinical mastitis in summer were in days later than in spring (p = 0.02) and winter (p = 0.03) in early lactation period. In conclusion, occurrence of mastitis in winter and spring during early lactation and in summer during late lactation period were more prevalent especially in lower parities. PMID:25568687

  13. Workplace lactation program: a nursing friendly initiative.

    PubMed

    Angeletti, Michelle A

    2008-01-01

    The U.S. is experiencing a nursing shortage that is threatening its quality of healthcare. One contributing factor that has been identified is the level of dissatisfaction that nurses have with their working conditions. Health Services Organizations can use female and family friendly initiatives, such as workplace lactation programs to demonstrate that they are willing to support a female employee's task of balancing familial and profession roles. By meeting the needs of breastfeeding mothers, organizations can have a positive impact on employees' levels of satisfaction, which can positively impact recruitment efforts, productivity and retention. PMID:18998524

  14. Kinetic Analysis of the Amino Terminal End of Active Site Loop of Lactate Deyhdrogenase from Plasmodium Vivax

    PubMed Central

    Mutlu, Özal; Bal?k, Dilek Turgut

    2012-01-01

    Objective: In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH’s. Material and Methods: Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by Ni-NTA agarose matrix and kinetic properties were analysed. Results: Observing increase of Km values of mutant enzymes showed that mutations in this place caused decreasing affinity of enzyme for its substrate. However kcat values were about the same throughout all mutant proteins. Conclusion: Sensitivity of the studied region emphasizes the significance of this site for drug design studies for both Plasmodium and some other Apicomplexans. PMID:25207035

  15. Structures of the hydrolase domain of human 10-formyltetrahydrofolate dehydrogenase and its complex with a substrate analogue.

    PubMed

    Kursula, Petri; Schüler, Herwig; Flodin, Susanne; Nilsson-Ehle, Petra; Ogg, Derek J; Savitsky, Pavel; Nordlund, Pär; Stenmark, Pål

    2006-11-01

    10-Formyltetrahydrofolate dehydrogenase is a ubiquitously expressed enzyme in the human body. It catalyses the formation of tetrahydrofolate and carbon dioxide from 10-formyltetrahydrofolate, thereby playing an important role in the human metabolism of one-carbon units. It is a two-domain protein in which the N-terminal domain hydrolyses 10-formyltetrahydrofolate into formate and tetrahydrofolate. The high-resolution crystal structure of the hydrolase domain from human 10-formyltetrahydrofolate dehydrogenase has been determined in the presence and absence of a substrate analogue. The structures reveal conformational changes of two loops upon ligand binding, while key active-site residues appear to be pre-organized for catalysis prior to substrate binding. Two water molecules in the structures mark the positions of key oxygen moieties in the catalytic reaction and reaction geometries are proposed based on the structural data. PMID:17057331

  16. An enzyme-amplified microtiter plate assay for ethanol: Its application to the detection of peanut ethanol and alcohol dehydrogenase

    SciTech Connect

    Chung, S.Y.; Vercellotti, J.R.; Sanders, T.H.

    1995-12-01

    A calorimetric microliter plate assay for ethanol amplified by aldehyde dehydrogenase (ALDH) was developed. In the assay ethanol from a sample took part in a chain-reaction catalyzed by alcohol dehydrogenase (ADH) and amplified by ALDH in the presence of NAD{sup +}, diaphorase, and p-ibdonitrotetrazolium-violet (INT-violet)(a precursor of red product). The resultant reaction gave a red color, the intensity of which was proportional to the amount of ethanol present. Using the technique, the content of activity from peanuts of differing maturity and curing stages were determined respectively. Data showed that immature peanuts had a higher level of ethanol and a lower ADH activity than mature peanuts, and that the level of ethanol and ADH activity decreased with the curing time. This indicates that peanut maturity and curing have an effect on ethanol. Also, this implies that other peanut volatiles could be affected in the same way as ethanol, a major volatile in peanuts.

  17. Rat lung phospholipid fatty acid composition in prepregnant, pregnant, and lactating rats: relationship to ozone-induced pulmonary toxicity.

    PubMed

    Gunnison, A F; Finkelstein, I

    1997-01-01

    Our laboratory has demonstrated recently that pulmonary inflammation induced by acute ozone exposure is much more severe in late stage pregnant and lactating rats than in postlactating rats or age-matched virgin females. It is currently widely believed that such pulmonary damage results, at least in part, from the reaction of ozone at sites of unsaturation in phospholipid fatty acid (PLFA) molecules located in the epithelial fluid layer lining the lung surfaces and/or the plasma membranes of epithelial cells underlying this fluid layer. The objective of this study was to compare the PLFA composition of lung tissue and surfactant from ozone-sensitive late stage pregnant and lactating rats with comparable tissue from relatively ozone-insensitive age-matched prepregnant (virgin female) rats to explore the possibility that changes in lung PLFA composition during pregnancy and/or lactation contribute to the enhanced sensitivity of these physiologic states to ozone. In addition, the correlation of changes in plasma PLFA composition with those in lung was investigated. There were minor differences in the composition of lung tissue and surfactant PLFAs between prepregnant rats and pregnant rats at day 17 of gestation and only slightly greater differences between prepregnant and lactating rats. Changes from the prepregnant state in the PLFA composition of lung tissue, but not surfactant, correlated with changes in the plasma only in lactating rats and not in pregnant rats. Overall, the double bond index of PLFAs in surfactant and lung tissue was decreased in pregnant and lactating rats compared with prepregnant rats. Thus, the increased sensitivity of pregnant and lactating rats to ozone-induced lung injury cannot be attributed to an increased availability of unsaturated fatty acids. In addition, the arachidonic acid composition of phospholipids did not appear to explain differences between prepregnant rats and pregnant or lactating rats in their inflammatory response to ozone. In conclusion, there is no evidence that the relatively minor changes in lung tissue PLFA composition which occur during pregnancy and lactation predispose rats in these physiologic states to ozone-induced pulmonary toxicity. PMID:9042669

  18. Native and Modified Lactate Dehydrogenase Expression in a Fumaric Acid Producing isolate Rhizopus oryzae 99-880

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is a filamentous fungus that is of broad importance to the industrial, agricultural, and medical community. R. oryzae can be subdivided into two groups based on genetic and phenotypic differences. Type-I isolates accumulate primarily lactic acid when grown in the presence of a ferm...

  19. Identification of a potent inhibitor targeting human lactate dehydrogenase A and its metabolic modulation for cancer cell line.

    PubMed

    Chen, Chun-Ye; Feng, Yan; Chen, Jing-Yu; Deng, Hao

    2016-01-01

    Targeting LDHA represents a promising strategy for the development of new anti-cancer agents. We report herein the identification of a potent compound as a direct LDHA inhibitor. The in vitro enzymatic assay revealed that the VS-2 had good inhibitory potency (IC50=0.25?M) to LDHA. Cytotoxic assay suggested that the VS-2 could inhibit MCF-7 cancer cell growth, with the IC50 value low to 1.54?M. The seahorse XF24 experiment validated that the VS-2 served as a modulator to reprogram MCF-7 cancer cell metabolism from glycolysis to mitochondrial respiration. PMID:26597536

  20. Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

    PubMed Central

    Lampel, K A; Uratani, B; Chaudhry, G R; Ramaley, R F; Rudikoff, S

    1986-01-01

    The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase. Images PMID:3082854

  1. Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

    PubMed Central

    Morawski, B; Casy, G; Illaszewicz, C; Griengl, H; Ribbons, D W

    1997-01-01

    Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies. PMID:9190820

  2. Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

    PubMed

    Morawski, B; Casy, G; Illaszewicz, C; Griengl, H; Ribbons, D W

    1997-06-01

    Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies. PMID:9190820

  3. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  4. d-Glyceraldehyde 3-Phosphate Dehydrogenases of Higher Plants 12

    PubMed Central

    Schulman, Marvin D.; Gibbs, Martin

    1968-01-01

    The d-glyceraldehyde 3-P dehydrogenases of spinach leaf, pea seed, and pea shoot were purified. The NADP and NAD-linked enzymes of either spinach leaves and pea shoots could not be separated. Changes in the ratio of NADP- to NAD-linked activity of the spinach leaf and pea shoot enzymes were observed during both purification and storage of crude extracts. The spinach leaf, pea shoot, and pea seed enzymes differ electrophoretically from each other and from the rabbit muscle enzyme. The pea seed and shoot enzymes contain bound nucleotide cofactor, resist proteolytic attack, have similar Michaelis-Menton kinetic constants and are competitively inhibited by d-sedoheptulose-7-phosphate and d-sedoheptulose 1,7-diphosphate. Charcoal removes the bound nucleotide from the pea seed enzyme but not from the pea shoot enzymes. NADP and NADPH were found to inhibit the reductive but not oxidative reaction catalyzed by the charcoal treated seed enzyme. The function of the pea shoot NADP and NAD-linked enzymes in chloroplast metabolism is discussed in regard to their location and catalytic properties. Although the NADP-linked activity can be assigned a primary, if not exclusive function in photosynthesis, the assignment of a distinct metabolic function to the NAD-linked activity cannot be made at present. PMID:4387009

  5. Immobilization and enzymatic properties of Bacillus megaterium glucose dehydrogenase

    SciTech Connect

    Baron, M.; Fontana, J.D.; Guimaraes, M.F.; Woodward, J.

    1996-12-31

    The enzymatic production of hydrogen gas from renewable sources of energy; e.g., cellulose, starch, lactose, can be obtained by coupling the reactions catalyzed by glucose dehydrogenase (GDH) and hydrogenase. In order to enhance the thermostability of GDH from Bacillus megaterium, the enzyme was immobilized by ionic adsorption using the polycationic polymer DEAE-(dextran)Sephadex. The effect of enzyme concentration on immobilization showed a tendency to increase the activity of the immobilized enzyme with the increase of the amount of added GDH. When the enzyme: support ratio was 15.97 U: 100 mg, the immobilization yield was 84.76%. The enzymatic profiles for the immobilized GDH were a little different when compared to those for free enzyme with respect to the effects of pH and temperature. Concerning the effect of incubation time carried at pH 7.5 and at 40{degrees}C, the maximum production of reduced coenzyme by the immobilized enzyme was reached within 4 h and it was maintained up to 16 h without loss of enzymatic activity. The coupling of the immobilized GDH activity with that for free alkaline cellulose (Novozym. 342) demonstrated the possibility for obtaining reduced coenzyme from the cellulose hydrolysis and the immobilized GDH could be reassayed 10 times maintaining its enzyme activity.

  6. An animal model of human aldehyde dehydrogenase deficiency

    SciTech Connect

    Chang, C.; Mann, J.; Yoshida, A.

    1994-09-01

    The genetic deficiency of ALDH2, a major mitochondrial aldehyde dehydrogenase, is intimately related to alcohol sensitivity and the degree of predisposition to alcoholic diseases in humans. The ultimate biological role of ALDH2 can be exposed by knocking out the ALDH2 gene in an animal model. As the first step for this line of studies, we cloned and characterized the ALDH2 gene from mouse C57/6J strain which is associated with a high alcohol preference. The gene spans 26 kbp and is composed of 13 exons. Embryonic stem cells were transfected with a replacement vector which contains a partially deleted exon3, a positive selection cassette (pPgk Neo), exon 4 with an artificial stop codon, exons 5, 6, 7, and a negative selection cassette (pMCI-Tk). Genomic DNAs prepared from drug resistant clones were analyzed by polymerase chain reaction and by Southern blot analysis to distinguish random integration from homologous recombination. Out of 132 clones examined, 8 had undergone homologous recombination at one of the ALDH2 alleles. The cloned transformed embryonic stem cells with a disrupted ALDH2 allele were injected into blastocysts. Transplantation of the blastocysts into surrogate mother mice yielded chimeric mice. The role of ALDH2 in alcohol preference, alcohol sensitivity and other biological and behavioral characteristics can be elucidated by examining the heterozygous and homozygous mutant strains produced by breeding of chimeric mice.

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

    PubMed

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

    2014-02-01

    Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents. PMID:24064207

  8. Lactate response to different volume patterns of power clean.

    PubMed

    Date, Anand S; Simonson, Shawn R; Ransdell, Lynda B; Gao, Yong

    2013-03-01

    The ability to metabolize or tolerate lactate and produce power simultaneously can be an important determinant of performance. Current training practices for improving lactate use include high-intensity aerobic activities or a combination of aerobic and resistance training. Excessive aerobic training may have undesired physiological adaptations (e.g., muscle loss, change in fiber types). The role of explosive power training in lactate production and use needs further clarification. We hypothesized that high-volume explosive power movements such as Olympic lifts can increase lactate production and overload lactate clearance. Hence, the purpose of this study was to assess lactate accumulation after the completion of 3 different volume patterns of power cleans. Ten male recreational athletes (age 24.22 ± 1.39 years) volunteered. Volume patterns consisted of 3 sets × 3 repetition maximum (3RM) (low volume [LV]), 3 sets × 6 reps at 80-85% of 3RM (midvolume [MV]), and 3 sets × 9 reps at 70-75% of 3RM (high volume [HV]). Rest period was identical at 2 minutes. Blood samples were collected immediately before and after each volume pattern. The HV resulted in the greatest lactate accumulation (7.43 ± 2.94 mmol·L) vs. (5.27 ± 2.48 and 4.03 ± 1.78 mmol·L in MV and LV, respectively). Mean relative increase in lactate was the highest in HV (356.34%). The findings indicate that lactate production in power cleans is largely associated with volume, determined by number of repetitions, load, and rest interval. High-volume explosive training may impose greater metabolic demands than low-volume explosive training and may improve ability to produce power in the presence of lactate. The role of explosive power training in overloading the lactate clearance mechanism should be examined further, especially for athletes of intermittent sport. PMID:22648139

  9. Clinical significance of lactate in acute cardiac patients

    PubMed Central

    Lazzeri, Chiara; Valente, Serafina; Chiostri, Marco; Gensini, Gian Franco

    2015-01-01

    Lactate, as a metabolite of easy and quick assessment, has been studied over time in critically ill patients in order to evaluate its prognostic ability. The present review is focused on the prognostic role of lactate levels in acute cardiac patients (that is with acute coronary syndrome, cardiogenic shock, cardiac arrest, non including post cardiac surgery patients). In patients with ST-elevation myocardial infarction treated with mechanical revascularization, hyperlactatemia identified a subset of patients at higher risk for early death and in-hospital complications, being strictly related mainly to hemodynamic derangement. The prognostic impact of hyperlactatemia on mortality has been documented in patients with cardiogenic shock and in those with cardiac arrest even if there is no cut-off value of lactate to be associated with worse outcome or to guide resuscitation or hemodynamic management. Therapeutic hypothermia seems to affect per se lactate values which have been shown to progressively decrease during hypothermia. The mechanism(s) accounting for lactate levels during hypothemia seem to be multiple ranging from the metabolic effects of reduced temperatures to the hemodynamic effects of hypothermia (i.e., reduced need of vasopressor agents). Serial lactate measurements over time, or lactate clearance, have been reported to be clinically more reliable than lactate absolute value also in acute cardiac patients. Despite differences in study design, timing of lactate measurements and type of acute cardiac conditions (i.e., cardiogenic shock, cardiac arrest, refractory cardiac arrest), available evidence strongly suggests that higher lactate levels can be observed on admission in non-survivors and that higher lactate clearance is associated with better outcome. PMID:26322188

  10. Utilization of Oxalacetate by Acinetobacter calcoaceticus: Evidence for Coupling Between Malic Enzyme and Malic Dehydrogenase

    PubMed Central

    Dolin, M. I.; Juni, E.

    1978-01-01

    Growth of Acinetobacter calcoaceticus strain BD413 in malate-mineral medium resulted in the excretion of large quantities of oxalacetate. Malate was virtually depleted by the time the cell density reached 60% of its final value; most of the remaining growth took place at the expense of oxalacetate. Experiments in which oxalacetate was used as the initial substrate showed that pyruvate was not utilized until most of the oxalacetate disappeared. The generation time for growth on malate or oxalacetate was ?40 min; the generation time for growth on pyruvate was 62 min, which implies that pyruvate transport may be rate limiting. Oxalacetate and pyruvate, however, supported approximately the same growth yield. These observations suggested that the first step in the utilization of oxalacetate as an energy source consisted of an enzymatic decarboxylation of the keto acid to pyruvate and CO2. Three enzyme reactions that carry out this decarboxylation have been detected in extracts of A. calcoaceticus. The first, which functioned maximally at pH 4.8, was attributable to the oxalacetate decarboxylase activity of oxidized diphosphopyridine nucleotide-malic enzyme. The second and third, which functioned in the neutral pH range, resulted from coupling of oxidized diphosphopyridine nucleotide-malic enzyme to reduced diphosphopyridine nucleotide-dependent malic dehydrogenase, and oxidized triphosphopyridine nucleotide-malic enzyme to a reduced triphosphopyridine nucleotide-dependent malic dehydrogenase. The efficiency of these coupled reactions was high enough so that the overall reaction could be physiologically significant. PMID:627536

  11. Crystallization and preliminary X-ray diffraction studies of Staphylococcus aureus homoserine dehydrogenase

    PubMed Central

    Navratna, Vikas; Gopal, Balasubramanian

    2013-01-01

    Staphylococcus aureus is a Gram-positive nosocomial pathogen. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in cell-wall maintenance and essential amino-acid biosynthesis are significant drug targets. Homoserine dehydrogenase (HSD) is an oxidoreductase that is involved in the reversible conversion of l-aspartate semialdehyde to l-homoserine in a dinucleotide cofactor-dependent reduction reaction. HSD is thus a crucial intermediate enzyme linked to the biosynthesis of several essential amino acids such as lysine, methionine, isoleucine and threonine. PMID:24192352

  12. The Influence of Reproductive Experience on Milk Energy Output and Lactation Performance in the Grey Seal

    E-print Network

    Bowen, W. Don

    The Influence of Reproductive Experience on Milk Energy Output and Lactation Performance of lactation performance, whether reproductive experience may have a significant influence on milk energy influence on lactation performance. Although there was no difference between primiparous females in milk

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

    PubMed Central

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

    2015-01-01

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

  14. [The biological bases of human lactation].

    PubMed

    Villalpando, S; De Santiago, S

    1993-12-01

    The mammary gland integrated by secretory acini has two main physiological goals: the lactogenesis and the milk ejection. The first is controlled by estrogens, prolactin and cortisol; the former by oxytocin. Milk is synthesized through five mechanisms namely: I. Cellular synthesis and exocytosis; 2. Cellular synthesis and exocytosis packed in cell membrane fragment; 3. Passive transport through both cell membranes; 4. Active transport via receptors through both cell membranes, and 5. Paracellular transport. The human milk has a high concentration of lactose and relatively low concentrations of fat and protein in comparison to other species. Its composition varies little in relationship to maternal diet, nevertheless, it holds a close correlation, with body composition of the mother. Exclusively breast-fed infants grow in a similar manner to those formula-fed up to 4-6 months; afterwards growth decelerates, to spurt up the need for complementary food. It has been amply demonstrated that human milk protects against acute infections, both bacterial and viral. The current hospital practices of perinatal care should be changed in our country in order to favour the initiation of breast-feeding. Concerned physicians should have more and precise knowledge about the physiology of lactation in order to help mothers to pursue a successful lactation. PMID:8110409

  15. Effect of manual lymph drainage on removal of blood lactate after submaximal exercise.

    PubMed

    Bakar, Yesim; Coknaz, Hakk?; Karl?, Ümid; Semsek, Önder; Ser?n, Erdinc; Pala, Ömer Osman

    2015-11-01

    [Purpose] It has been well-established that exercise-induced muscle damage occurs following intense exercise. Massage is commonly used to manage muscle damage resulting from exercise. However the effect of massage after exercise is still not clear. The purpose of this study was to examine the effect of manual lymph drainage on muscle damage and on the removal of blood lactate following submaximal exercise (SE), as part of a solution to the challenging problem in sports medicine of muscular recovery after exercise. [Subjects and Methods] Eighteen healthy male students, with moderate exercise training, were randomly assigned to either receive manual lymph drainage (MLD) or serve as controls. Both groups were subjected to a graded exercise test, performed on a treadmill ergometer, to determine each subject's individual anaerobic threshold (IAT). Seven days later, all subjects were made to run for 30 minutes on the same treadmill ergometer, at a running speed equivalent to the IAT. One group received MLD treatment, while the control subjects received no treatment. [Results] Following an increase immediately after exercise, lactic acid (LA) and lactate dehydrogenase (LDH) serum levels dropped rapidly and significantly at the end of MLD application and two hours after SE in the subjects receiving MLD. The course of creatine kinase (CK) and myoglobin levels was comparable, and with myoglobin showing a significant difference at 2?h after SE, and CK at 24?h after SE. [Conclusion] Manual lymph drainage after SE correlated with a more rapid fall in LA and of the muscular enzymes of LDH, CK and myoglobin, and may have resulted in an improvement in the regenerative processes elicted by structural damage to the muscle cells. PMID:26696704

  16. Effect of manual lymph drainage on removal of blood lactate after submaximal exercise

    PubMed Central

    Bakar, Yesim; Coknaz, Hakk?; Karl?, Ümid; Semsek, Önder; Ser?n, Erdinc; Pala, Ömer Osman

    2015-01-01

    [Purpose] It has been well-established that exercise-induced muscle damage occurs following intense exercise. Massage is commonly used to manage muscle damage resulting from exercise. However the effect of massage after exercise is still not clear. The purpose of this study was to examine the effect of manual lymph drainage on muscle damage and on the removal of blood lactate following submaximal exercise (SE), as part of a solution to the challenging problem in sports medicine of muscular recovery after exercise. [Subjects and Methods] Eighteen healthy male students, with moderate exercise training, were randomly assigned to either receive manual lymph drainage (MLD) or serve as controls. Both groups were subjected to a graded exercise test, performed on a treadmill ergometer, to determine each subject’s individual anaerobic threshold (IAT). Seven days later, all subjects were made to run for 30 minutes on the same treadmill ergometer, at a running speed equivalent to the IAT. One group received MLD treatment, while the control subjects received no treatment. [Results] Following an increase immediately after exercise, lactic acid (LA) and lactate dehydrogenase (LDH) serum levels dropped rapidly and significantly at the end of MLD application and two hours after SE in the subjects receiving MLD. The course of creatine kinase (CK) and myoglobin levels was comparable, and with myoglobin showing a significant difference at 2?h after SE, and CK at 24?h after SE. [Conclusion] Manual lymph drainage after SE correlated with a more rapid fall in LA and of the muscular enzymes of LDH, CK and myoglobin, and may have resulted in an improvement in the regenerative processes elicted by structural damage to the muscle cells. PMID:26696704

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

    PubMed Central

    Pavlova, Sylvia I.; Jin, Ling; Gasparovich, Stephen R.

    2013-01-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci. PMID:23637459

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

    PubMed

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

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci. PMID:23637459

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

    PubMed Central

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

    2013-01-01

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. PMID:24970175

  20. 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.

  1. METABOLIC ADAPTATION TO FEEDING AND FASTING DURING LACTATION IN HUMANS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of these studies was to determine the metabolic adaptation to fasting and feeding during lactation. Normal lactating (L) and nonlactating (NL) women (n = 6 each) were studied using infusions of [U-13C]glucose and [2-13C]glycerol during: 1) a 24-h fast, and 2) ingestion of Sustacal (protocol ...

  2. VISCERAL TISSUE GROWTH AND PROLIFERATION DURING THE BOVINE LACTATION CYCLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty one multiparous, nonpregnant, lactating dairy cows were used to assess the impact of stage of lactation on visceral tissue mass and small intestinal cell proliferation. Cows were housed in tie stalls with 12 h of light/dark and were milked twice daily at 0700 and 1800 h. Cows had ad libitum...

  3. GENETIC EVALUATION AND BEST PREDICTION OF LACTATION PERSISTENCY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cows with high persistency tend to milk less than expected at the beginning of lactation and more than expected at the end. Best prediction of persistency was calculated as a function of a trait-specific standard lactation curve and the linear regression of a cow’s test day deviations on days in mil...

  4. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

    Monkenbusch, M; Stadler, A; Biehl, R; Ollivier, J; Zamponi, M; Richter, D

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  5. Fast internal dynamics in alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Monkenbusch, M.; Stadler, A.; Biehl, R.; Ollivier, J.; Zamponi, M.; Richter, D.

    2015-08-01

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  6. Aldehyde dehydrogenases and cancer stem cells.

    PubMed

    Xu, Xia; Chai, Shoujie; Wang, Pingli; Zhang, Chenchen; Yang, Yiming; Yang, Ying; Wang, Kai

    2015-12-01

    Aldehyde dehydrogenases (ALDHs), as essential regulators of aldehyde metabolism in the human body, protect organisms from damage induced by active aldehydes. Given their roles in different cancer types, ALDHs have been evaluated as potential prognostic markers of cancer. ALDHs exhibit high activity in cancer stem cells (CSCs) and may serve as markers of CSCs. Moreover, studies indicated that ALDHs and their regulated retinoic acid, reactive oxygen species and reactive aldehydes metabolism were strongly related with various properties of CSCs. Besides, recent research evidences have demonstrated the transcriptional and post-translational regulation of ALDH expression and activation in CSCs. Thus, this review focuses on the function and regulation of ALDHs in CSCs, particularly ALDH1A1 and ALDH1A3. PMID:26319899

  7. Stability of immobilized yeast alcohol dehydrogenase

    SciTech Connect

    Ooshima, H.; Genko, Y.; Harano, Y.

    1981-12-01

    The effects of substrate on stabilities of native (NA) and three kinds of immobilized yeast alcohol dehydrogenase (IMA), namely PGA (the carrier; porous glass), SEA (agarose gel) prepared covalently, and AMA (anion-exchange resin) prepared ionically, were studied. The following results were obtained. 1) The deactivations of NA and IMA free from the substrate or in the presence of ethanol obey the first-order kinetics, whereas, in the presence of butyraldehyde, their deactivation behaviors are explained on the basis of coexistence of two components of YADHs, namely the labile E1 and the comparatively stable E2, with different first-order deactivation constants. (2) A few attempts for stabilization of IMA were carried out from the viewpoint of the effects of crosslinkages among the subunits of YADH for PGA and the multibonding between the carrier and enzyme for SEA. The former is effective for the stabilization, whereas the latter is not. (Refs. 19).

  8. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion. PMID:18819805

  9. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  10. Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Xu, Hengyu; West, Ann H; Cook, Paul F

    2006-10-01

    Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value. PMID:17002315

  11. Sorbitol production from lactose by engineered Lactobacillus casei deficient in sorbitol transport system and mannitol-1-phosphate dehydrogenase.

    PubMed

    De Boeck, Reinout; Sarmiento-Rubiano, Luz Adriana; Nadal, Inmaculada; Monedero, Vicente; Pérez-Martínez, Gaspar; Yebra, María J

    2010-02-01

    Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main L-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTS(Gut)) in BL251. These results showed that the PTS(Gut) did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Deltaldh1 DeltagutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate. PMID:19784641

  12. Gene expression and biochemical analysis of cheese-ripening yeasts: focus on catabolism of L-methionine, lactate, and lactose.

    PubMed

    Cholet, Orianne; Hénaut, Alain; Casaregola, Serge; Bonnarme, Pascal

    2007-04-01

    DNA microarrays of 86 genes from the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Yarrowia lipolytica were developed to determine which genes were expressed in a medium mimicking a cheese-ripening environment. These genes were selected for potential involvement in lactose/lactate catabolism and the biosynthesis of sulfur-flavored compounds. Hybridization conditions to follow specifically the expression of homologous genes belonging to different species were set up. The microarray was first validated on pure cultures of each yeast; no interspecies cross-hybridization was observed. Expression patterns of targeted genes were studied in pure cultures of each yeast, as well as in coculture, and compared to biochemical data. As expected, a high expression of the LAC genes of K. marxianus was observed. This is a yeast that efficiently degrades lactose. Several lactate dehydrogenase-encoding genes were also expressed essentially in D. hansenii and K. marxianus, which are two efficient deacidifying yeasts in cheese ripening. A set of genes possibly involved in l-methionine catabolism was also used on the array. Y. lipolytica, which efficiently assimilates l-methionine, also exhibited a high expression of the Saccharomyces cerevisiae orthologs BAT2 and ARO8, which are involved in the l-methionine degradation pathway. Our data provide the first evidence that the use of a multispecies microarray could be a powerful tool to investigate targeted metabolism and possible metabolic interactions between species within microbial cocultures. PMID:17308183

  13. Neuroendocrine regulation of lactation and milk production.

    PubMed

    Crowley, William R

    2015-01-01

    Prolactin (PRL) released from lactotrophs of the anterior pituitary gland in response to the suckling by the offspring is the major hormonal signal responsible for stimulation of milk synthesis in the mammary glands. PRL secretion is under chronic inhibition exerted by dopamine (DA), which is released from neurons of the arcuate nucleus of the hypothalamus into the hypophyseal portal vasculature. Suckling by the young activates ascending systems that decrease the release of DA from this system, resulting in enhanced responsiveness to one or more PRL-releasing hormones, such as thyrotropin-releasing hormone. The neuropeptide oxytocin (OT), synthesized in magnocellular neurons of the hypothalamic supraoptic, paraventricular, and several accessory nuclei, is responsible for contracting the myoepithelial cells of the mammary gland to produce milk ejection. Electrophysiological recordings demonstrate that shortly before each milk ejection, the entire neurosecretory OT population fires a synchronized burst of action potentials (the milk ejection burst), resulting in release of OT from nerve terminals in the neurohypophysis. Both of these neuroendocrine systems undergo alterations in late gestation that prepare them for the secretory demands of lactation, and that reduce their responsiveness to stimuli other than suckling, especially physical stressors. The demands of milk synthesis and release produce a condition of negative energy balance in the suckled mother, and, in laboratory rodents, are accompanied by a dramatic hyperphagia. The reduction in secretion of the adipocyte hormone, leptin, a hallmark of negative energy balance, may be an important endocrine signal to hypothalamic systems that integrate lactation-associated food intake with neuroendocrine systems. PMID:25589271

  14. Effects of Fusarium mycotoxins in rations with different concentrate proportions on serum haptoglobin and hepatocellular integrity in lactating dairy cows.

    PubMed

    Kinoshita, A; Keese, C; Beineke, A; Meyer, U; Starke, A; Sauerwein, H; Dänicke, S; Rehage, J

    2015-10-01

    It was hypothesized that long-term intake of a diet contaminated with deoxynivalenol (DON) and differing in the proportion of concentrate might affect hepatocellular integrity and function as well as biomarkers of systemic inflammation in lactating dairy cows. In Period 1 (11 weeks), 26 lactating German Holstein cows (13 primiparous and 13 pluriparous, 31 days in milk, 522 kg body weight, on average) were divided into two groups and fed diets (50% concentrate) with (MYC, n = 12; on average 5.3 mg DON/kg DM) or without (CON, n = 14) DON contaminations. In Period 2 (16 weeks), each group was further divided into two groups to test whether elevated concentrate proportion as additional burden might enhance the toxicity of DON. The cows in MYC60 (n = 6; 4.6 mg DON/kg DM) and CON60 (n = 7) received the diet with 60% concentrate, while cows in MYC30 (n = 6; 4.4 mg DON/kg DM) and CON30 (n = 7) received the diet with 30% concentrate. Blood samples were taken in biweekly intervals for activities of aspartate amino transferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase as well as for concentration of total bilirubin and haptoglobin. Biopsies from liver were collected in week 27 for morphological analyses. No DON effect was found for the variables assessed in blood. The diet with 60% concentrate led to higher activities of AST and GLDH in Period 2. No morphological change was found by both light and electron microscopic analyses of liver samples. Results indicated that long-term intake of DON-contaminated diet over 27 weeks led to neither relevant damages of hepatocytes nor systemic inflammatory responses in lactating dairy cows, even if the dietary concentrate proportion was increased to 60%. PMID:25845993

  15. l-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum?

    PubMed Central

    Blombach, Bastian; Schreiner, Mark E.; Holátko, Ji?í; Bartek, Tobias; Oldiges, Marco; Eikmanns, Bernhard J.

    2007-01-01

    Corynebacterium glutamicum was engineered for the production of l-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ?aceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, l-alanine, and l-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum ?aceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and l-alanine towards l-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum ?aceE(pJC4ilvBNCE) produced up to 210 mM l-valine with a volumetric productivity of 10.0 mM h?1 (1.17 g l?1 h?1) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose. PMID:17293513

  16. Influence of Altered NADH Metabolic Pathway on the Respiratory-deficient Mutant of Rhizopus oryzae and its L-lactate Production.

    PubMed

    Shu, Chang; Guo, Chenchen; Luo, Shuizhong; Jiang, Shaotong; Zheng, Zhi

    2015-08-01

    Respiratory-deficient mutants of Rhizopus oryzae (R. oryzae) AS 3.3461 were acquired by ultraviolet (UV) irradiation to investigate changes in intracellular NADH metabolic pathway and its influence on the fermentation characteristics of the strain. Compared with R. oryzae AS 3.3461, the intracellular ATP level of the respiratory-deficient strain UV-1 decreased by 52.7 % and the glucose utilization rate rose by 8.9 %; When incubated for 36 h, the activities of phosphofructokinase (PFK), hexokinase (HK), and pyruvate kinase (PK) in the mutant rose by 74.2, 7.2, and 12.0 %, respectively; when incubated for 48 h, the intracellular NADH/NAD(+) ratio of the mutant rose by 14.6 %; when a mixed carbon source with a glucose/gluconic acid ratio of 1:1 was substituted to culture the mutant, the NADH/NAD(+) ratio decreased by 4.6 %; the ATP content dropped by 27.6 %; the lactate dehydrogenase (LDH) activity rose by 22.7 %; and the lactate yield rose by 11.6 %. These results indicated that changes to the NADH metabolic pathway under a low-energy charge level can effectively increase the glycolytic rate and further improve the yield of L-lactate of R. oryzae. PMID:26047930

  17. Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

    SciTech Connect

    Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T.

    2012-02-27

    The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

  18. Lactate Profile During Greco-Roman Wrestling Matchx

    PubMed Central

    Karnincic, Hrvoje; Tocilj, Zoran; Uljevic, Ognjen; Erceg, Marko

    2009-01-01

    The objective of this study was to determine and compare lactate profile of two groups of Greco-Roman wrestlers with different competences and training experience. Study was conducted on 10 wrestles that were members of Croatian national team and 10 wrestlers that were members of Wrestling club Split. Lactate samples were collected at four intervals during control fights that were held according to international wrestling rules of World wrestling federation FILA. Values of lactate increased as competition progressed, and they were highest at the end of the match for both groups of wrestlers. According to this study there were no significant differences in lactate between two groups at the end of the match, while significant differences were noted during the match. The information about lactate profile presented in this study can be used by coaches and wrestlers to develop condition programs. Key Points There were no significant differences in lactate concentrations at the end of the match between two proficiency levels of wrestlers. More proficient (elite) wrestlers raise lactates gradually through the wrestling match while less proficient (club) wrestlers raise it abruptly at the end of the first bout. Both groups of wrestlers are unable to sustain same level of activity through the match suggesting that they are utilizing too much energy from anaerobic glycolysis. PMID:24474881

  19. Lactate profile during greco-roman wrestling matchx.

    PubMed

    Karnincic, Hrvoje; Tocilj, Zoran; Uljevic, Ognjen; Erceg, Marko

    2009-11-01

    The objective of this study was to determine and compare lactate profile of two groups of Greco-Roman wrestlers with different competences and training experience. Study was conducted on 10 wrestles that were members of Croatian national team and 10 wrestlers that were members of Wrestling club Split. Lactate samples were collected at four intervals during control fights that were held according to international wrestling rules of World wrestling federation FILA. Values of lactate increased as competition progressed, and they were highest at the end of the match for both groups of wrestlers. According to this study there were no significant differences in lactate between two groups at the end of the match, while significant differences were noted during the match. The information about lactate profile presented in this study can be used by coaches and wrestlers to develop condition programs. Key PointsThere were no significant differences in lactate concentrations at the end of the match between two proficiency levels of wrestlers.More proficient (elite) wrestlers raise lactates gradually through the wrestling match while less proficient (club) wrestlers raise it abruptly at the end of the first bout.Both groups of wrestlers are unable to sustain same level of activity through the match suggesting that they are utilizing too much energy from anaerobic glycolysis. PMID:24474881

  20. Targeting lactate transport suppresses in vivo breast tumour growth

    PubMed Central

    Morais-Santos, Filipa; Granja, Sara; Miranda-Gonçalves, Vera; Moreira, António H.J.; Queirós, Sandro; Vilaça, João L.; Schmitt, Fernando C.; Longatto-Filho, Adhemar; Paredes, Joana

    2015-01-01

    Background Most cancers, including breast cancer, have high rates of glucose consumption, associated with lactate production, a process referred as “Warburg effect”. Acidification of the tumour microenvironment by lactate extrusion, performed by lactate transporters (MCTs), is associated with higher cell proliferation, migration, invasion, angiogenesis and increased cell survival. Previously, we have described MCT1 up-regulation in breast carcinoma samples and demonstrated the importance of in vitro MCT inhibition. In this study, we performed siRNA knockdown of MCT1 and MCT4 in basal-like breast cancer cells in both normoxia and hypoxia conditions to validate the potential of lactate transport inhibition in breast cancer treatment. Results The effect of MCT knockdown was evaluated on lactate efflux, proliferation, cell biomass, migration and invasion and induction of tumour xenografts in nude mice. MCT knockdown led to a decrease in in vitro tumour cell aggressiveness, with decreased lactate transport, cell proliferation, migration and invasion and, importantly, to an inhibition of in vivo tumour formation and growth. Conclusions This work supports MCTs as promising targets in cancer therapy, demonstrates the contribution of MCTs to cancer cell aggressiveness and, more importantly, shows, for the first time, the disruption of in vivo breast tumour growth by targeting lactate transport. PMID:26203664

  1. Lactate as a Hemodynamic Marker in the Critically Ill

    PubMed Central

    Fuller, Brian M.; Dellinger, R. Phillip

    2013-01-01

    Purpose of review An early quantitative resuscitation strategy improves outcome in critically ill patients. The hemodynamic endpoints of such a strategy have been a topic of debate in the literature. This review focuses on the use of lactate as a marker for risk stratification, lactate clearance as a hemodynamic endpoint, and its use compared to mixed venous oxygenation as a resuscitation goal Recent findings Lactate clearance is associated with improved outcome across several cohorts of critically ill patients. Lactate levels and central venous oxygen saturations are frequently discordant. Targeting lactate clearance as part of a quantitative resuscitation strategy may be as effective as targeting central venous oxygen saturation. Summary Resuscitation of the critically ill patient should be aimed at the reversal of tissue hypoxia. The use of lactate as a hemodynamic marker and resuscitation endpoint makes physiologic sense and is supported by recent data. The use of lactate clearance versus other traditional endpoints of resuscitation, such as mixed venous oxygen saturation, should be based on the clinical characteristics and response of the individual patient. PMID:22517402

  2. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    SciTech Connect

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari; Nara, Takeshi; Aoki, Takashi; Harada, Shigeharu; Kita, Kiyoshi

    2005-10-01

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (? = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup ?1} and a solvent content of 44%.

  3. [Modeling of Ni-Fe-center of Ni-CO-dehydrogenases by nickel complexes with thiaazaligands].

    PubMed

    Pavlishchuk, V V; Kolotilov, S V; Edison, A V; Prushan, M D; Butcher, R D; Sinn, E

    2001-01-01

    Three new nickel(II) complexes with ligands 1,8-bis(2'-pyridyl)-3,6-dithiaoctane (Pdto) and dithiosemicarbazone of 4,7-dithiadecane-2,9-dione (DtdtzH2) of composition Ni(Pdto)(H2O)2(ClO4)2, Ni(DtdtzH2)(ClO4)2 and Ni(Dtdtz) were prepared, their molecular structures, spectral and redox-properties were studied. The possibilities of chemical reduction of Ni(Pdto)(H2O)2(ClO4)2 to nickel(I) and nickel(0) species and the reaction of nickel(I) complex with CO were shown, which may be described as the modeling of one of the stages of reactions with CO on active Ni-Fe-site of Ni-CO-dehydrogenases. It was found that Ni(DtdtzH2)(ClO4)2 reacted with (Et4N)2[Fe4S4(SBz)4] (BzSH = C6H5 CH2SH) forming adduct. In the row of studied complexes Ni(Pdto) (H2O)2(ClO4)2 may be described as the best structural model of Ni-Fe-site of Ni-CO-dehydrogenases on the redox properties. PMID:12035560

  4. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  5. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...levels of lactic dehydrogenase are found in a variety of conditions, including megaloblastic anemia (decrease in the number of mature red blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood-forming...

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

    MedlinePLUS

    ... pubic hair growth, and excessive body hair growth (hirsutism). Females with 3?-HSD deficiency have difficulty conceiving ... dehydration ; dehydrogenase ; difficulty conceiving ; enzyme ; fertility ; gene ; genitalia ; hirsutism ; hormone ; hyperplasia ; hypospadias ; infertile ; inflammation ; inherited ; kidney ; menstruation ; ...

  7. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  8. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  9. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  10. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  11. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  12. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...serum and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease such as pulmonary infarction (local...

  13. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...serum and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease such as pulmonary infarction (local...

  14. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...serum and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease such as pulmonary infarction (local...

  15. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...Measurements of 6-phosphogluconate dehydrogenase are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  16. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...Measurements of 6-phosphogluconate dehydrogenase are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  17. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...Measurements of 6-phosphogluconate dehydrogenase are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  18. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...Measurements of 6-phosphogluconate dehydrogenase are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  19. Cerebral lactate dynamics across sleep/wake cycles

    PubMed Central

    Rempe, Michael J.; Wisor, Jonathan P.

    2015-01-01

    Cerebral metabolism varies dramatically as a function of sleep state. Brain concentration of lactate, the end product of glucose utilization via glycolysis, varies as a function of sleep state, and like slow wave activity (SWA) in the electroencephalogram (EEG), increases as a function of time spent awake or in rapid eye movement sleep and declines as a function of time spent in slow wave sleep (SWS). We sought to determine whether lactate concentration exhibits homeostatic dynamics akin to those of SWA in SWS. Lactate concentration in the cerebral cortex was measured by indwelling enzymatic biosensors. A set of equations based conceptually on Process S (previously used to quantify the homeostatic dynamics of SWA) was used to predict the sleep/wake state-dependent dynamics of lactate concentration in the cerebral cortex. Additionally, we applied an iterative parameter space-restricting algorithm (the Nelder-Mead method) to reduce computational time to find the optimal values of the free parameters. Compared to an exhaustive search, this algorithm reduced the computation time required by orders of magnitude. We show that state-dependent lactate concentration dynamics can be described by a homeostatic model, but that the optimal time constants for describing lactate dynamics are much smaller than those of SWA. This disconnect between lactate dynamics and SWA dynamics does not support the concept that lactate concentration is a biochemical mediator of sleep homeostasis. However, lactate synthesis in the cerebral cortex may nonetheless be informative with regard to sleep function, since the impact of glycolysis on sleep slow wave regulation is only just now being investigated. PMID:25642184

  20. Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

    SciTech Connect

    Dubois, J.; Chapman, S.K.; Mathews, F.S.; Reid, G.A.; Lederer, F. )

    1990-07-10

    A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.

  1. Inclusion compounds between ?-, ?- and ?-cyclodextrins: iron II lactate: a theoretical and experimental study using diffusion coefficients and molecular mechanics

    NASA Astrophysics Data System (ADS)

    Leite, Rosiley A.; Lino, Antonio C. S.; Takahata, Yuji

    2003-01-01

    The inclusion compounds between iron II lactate and three different cyclodextrins (CDs) were studied by means of experimental and theoretical data. The importance of iron II in the human metabolism effort the necessity of a minimum concentration to the human life. Malnutrition is one great problem in social politics of many countries on the world. The possibility to the development of novel medicines with the iron II species stable look for an increase on the efficiency for this kind of aid. Kinetics measurements confirm the possibility to stop the oxidation reaction. It was the first indication of efficient molecular encapsulation. Diffusion coefficient measurements were carried out by Taylor-Aris diffusion technique. The decrease of diffusion coefficients measured for iron II lactate when alone and forming the inclusion complexes was obtained for all hosts molecules used. Molecular Mechanics calculations were performed to elucidate the perfect arrange of iron II lactate inside CDs cavity. No great differences were obtained to the binding energy for the different hosts. Using the software HyperChem6.03v MM+, AMBER94 and OPLS Forced Fields for iron atom in two chemical environments (a) vacuum and (b) with addition of 250 water molecules (MM+). The solvent treatment was decisive to the order of stability. This order was ?-CD>?-CD>?-CD, the same order of solubility in water. The results contained in this work confirm the possibility to protect iron II lactate against oxidation.

  2. Comparative Transcriptome Analysis Reveals Different Molecular Mechanisms of Bacillus coagulans 2-6 Response to Sodium Lactate and Calcium Lactate during Lactic Acid Production

    PubMed Central

    Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

    2015-01-01

    Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that ‘ATP-binding cassette transporters’ were significantly affected by calcium lactate stress, and ‘amino sugar and nucleotide sugar metabolism’ was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of ‘glycolysis/gluconeogenesis’ genes but positive effect on the expression of ‘citrate cycle (TCA cycle)’ genes. However, calcium lactate stress had positive influence on the expression of ‘glycolysis/gluconeogenesis’ genes and had minor influence on ‘citrate cycle (TCA cycle)’ genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans. PMID:25875592

  3. Comparative transcriptome analysis reveals different molecular mechanisms of Bacillus coagulans 2-6 response to sodium lactate and calcium lactate during lactic acid production.

    PubMed

    Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

    2015-01-01

    Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that 'ATP-binding cassette transporters' were significantly affected by calcium lactate stress, and 'amino sugar and nucleotide sugar metabolism' was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of 'glycolysis/gluconeogenesis' genes but positive effect on the expression of 'citrate cycle (TCA cycle)' genes. However, calcium lactate stress had positive influence on the expression of 'glycolysis/gluconeogenesis' genes and had minor influence on 'citrate cycle (TCA cycle)' genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans. PMID:25875592

  4. Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis

    E-print Network

    Locasale, Jason W.

    Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood [1, 2]. Using a metabolomics approach with ...

  5. Purification and properties of milk xanthine dehydrogenase.

    PubMed

    Hunt, J; Massey, V

    1992-10-25

    Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin. PMID:1328233

  6. Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase

    PubMed Central

    Cerff, R.; Quail, P. H.

    1974-01-01

    Ammonium sulfate chromatography has been employed to separate glyceraldehyde 3-phosphate dehydrogenases (GPD) of Sinapis alba cotyledons of various developmental stages. Cotyledons of dark-grown seedlings possess one major NAD-specific enzyme designated NAD-GPD I. Irradiation with continuous far red light leads to a strong increase in NADP-GPD activity and to the formation of a second NAD activity designated NAD-GPD II. These two activities occur in a constant ratio during cotyledon development, and they are eluted together in ammonium sulfate chromatography. In a later stage of cotyledon development the light-dependent increase in NAD-GPD II is matched by an equivalent decrease in NAD-GPD I. These data suggest that the chloroplast marker enzyme NADP-GPD (EC 1.2.1.13) also has NAD activity and that the light-dependent formation of this bifunctional enzyme is correlated with activity changes of the NAD-GPD of cytoplasmic glycolysis (EC 1.2.1.12). PMID:16658822

  7. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

    Chen, Che-Hong; Ferreira, Julio Cesar Batista; Gross, Eric R.; Mochly-Rosen, Daria

    2014-01-01

    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme. PMID:24382882

  8. [Glucose-6-phosphate dehydrogenase deficiency in Japan].

    PubMed

    Kanno, Hitoshi; Ogura, Hiromi

    2015-07-01

    In the past 10 years, we have diagnosed congenital hemolytic anemia in 294 patients, approximately 33% of whom were found to have glucose-6-phosphate dehydrogenase (G6PD) deficiency. It is becoming more common for Japanese to marry people of other ethnic origins, such that G6PD deficiency is becoming more prevalent in Japan. Japanese G6PD deficiency tends to be diagnosed in the neonatal period due to severe jaundice, while G6PD-deficient patients with foreign ancestors tend to be diagnosed at the onset of an acute hemolytic crisis before the age of six. It is difficult to predict the clinical course of each patient by G6PD activity, reduced glutathione content, or the presence/absence of severe neonatal jaundice. We propose that both neonatal G6PD screening and systematic analyses of G6PD gene mutations may be useful for personalized management of patients with G6PD-deficient hemolytic anemia. PMID:26251139

  9. Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole.

    PubMed

    Goulas, P

    1987-10-15

    Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated. PMID:3665930

  10. Influence of supplemental monensin on gestating and lactating ewes 

    E-print Network

    Peel, Richard Kraig

    1997-01-01

    Twenty-five multiparous Rambouillet ewes were used to determine the effects of feeding monensin during late gestation and early lactation on prepartum and postpartum ewe body weight, milk production, blood glucose levels, and lamb production. Ewes...

  11. Postpartum depression in the absence of lactation: an evolutionary perspective 

    E-print Network

    Peyton, Kelly J

    1996-01-01

    between the hormonal profiles of women in Western and non-Western countries. Specifically, the activity of oxytocin, a neurohormone released during parturition and lactation, is reduced in the postpartum period in mothers who do not breastfeed. Because...

  12. Role of the Maternal Liver in Lactating Mice

    E-print Network

    Camba-Colon, Joanna Irene Rosa

    2009-01-01

    syndrome       The liver acts as a detoxification center liver is important in lactation.   It plays an important role in the  detoxification detoxification center for both.  Nonetheless,  despite the critical role of the liver 

  13. [Maintenance of lactation: a challenge for hospitalized premature infant's mothers].

    PubMed

    de Azevedo, Melissa; Mendes, Eliane Norma Wagner

    2008-03-01

    Preterm birth is a difficult situation for all family members, interfering with the establishment of parental bonding and attachment with the baby. This paper is a qualitative and collective case study, carried out with the purpose of identifying the perception of mothers concerning the maintenance of lactation during the hospital stay of premature infants at Hospital de Clínicas de Porto Alegre (Clinic Hospital of Porto Alegre), Rio Grande do Sul, Brazil. The data were collected from March to April/2006 through interviews and observations and resulted in four categories. This article focuses only on the categories that refer to the mechanisms used by mothers to maintain lactation during hospitalization: beliefs and attitudes related to maintenance of lactation and breast milk expression at the human milk bank. The maintenance of lactation constitutes a complex process to be learnt by mothers involved with their infant's prematurity and hospitalization. PMID:18767363

  14. Lack of Influence of the Menstrual Cycle on Blood Lactate.

    ERIC Educational Resources Information Center

    Lamont, Linda S.

    1986-01-01

    Nine healthy women were tested before, during, and after exercise during the follicular and luteal phases of their menstrual cycles to determine the effect of menstruation on blood lactate levels. Findings are discussed. (Author/MT)

  15. Pten Regulates Development and Lactation in the Mammary Glands of Dairy Cows

    PubMed Central

    Wang, Zhuoran; Hou, Xiaoming; Qu, Bo; Wang, Jie; Gao, Xuejun; Li, Qingzhang

    2014-01-01

    Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of ?-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for ?-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPAR?, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of ?-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased ?-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of ?-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways. PMID:25009983

  16. Pten regulates development and lactation in the mammary glands of dairy cows.

    PubMed

    Wang, Zhuoran; Hou, Xiaoming; Qu, Bo; Wang, Jie; Gao, Xuejun; Li, Qingzhang

    2014-01-01

    Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of ?-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for ?-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPAR?, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of ?-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased ?-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of ?-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways. PMID:25009983

  17. Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases.

    PubMed

    Rodríguez-Zavala, José Salud; Allali-Hassani, Abdellah; Weiner, Henry

    2006-06-01

    Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step. PMID:16731973

  18. Chronic muscle stimulation increases lactate transport in rat skeletal muscle.

    PubMed

    McCullagh, K J; Juel, C; O'Brien, M; Bonen, A

    1996-03-01

    The aim of this study was to examine the effects of chronic low frequency stimulation on the lactate transport across the plasma membrane of the tibialis anterior (TA) muscle of the rat. Stimulating electrodes were implanted on either side of the peroneal nerve in one hindlimb. Chronic stimulation (10 Hz, 50 microsecond bursts, 24h/day) commenced 7 days after surgery, and were continued for 7 days. Animals were then left for 24 h, and thereafter muscles were obtained. Cytochrome C-oxidase activity was increased 1.9-fold in the stimulated TA compared to the control TA (p < 0.05). Lactate transport (zero-trans) was measured in giant sarcolemmal vesicles obtained from the chronically stimulated TA and the control TA. At each of the concentrations used in these studies a significant increase in lactate transport was observed; 2.8-fold increase at 1 mM lactate p < 0.05); 2-fold increases at both 30 mM and 50 mM lactate p < 0.05). These studies have shown that lactate transport capacity is markedly increased in response to chronic muscle contraction. PMID:8709976

  19. Lactation protects against myocardial ischemia-reperfusion injury in rats.

    PubMed

    Shekarforoush, S; Safari, F

    2015-12-01

    Some researchers have reported that lactation is effective in reducing cardiovascular disease risk factors. The purpose of this study was to investigate whether lactation may improve intrinsic tolerance against ischemia reperfusion (IR) injury. The rats were randomly divided into two groups (n = 8 in each group). In the lactation (Lact) group, the surgery was performed on postpartum day 21 (at the end of lactation period) and the results were compared with those of virgin female rats (control group). Cardiac IR injury was induced by means of left anterior descending coronary artery occlusion for 30 min followed by reperfusion for 120 min. Infarct size was measured using the staining agent 2,3,5-triphenyltetrazolium chloride. At the end of the experiment, Mean arterial pressure in the control group was significantly lower than that in the Lact group. Myocardial infarct size was significantly reduced in the Lact group (23 ± 3% vs. 45 ± 8%, p < 0.05 in the control group). Lactation reduced the extent of myocardial injury induced by ischemia and reperfusion. So, lactation may increase cardiac tolerance to ischemic injury. PMID:26690029

  20. Effects of ammonium lactate on 2,3-pentanedione formation from lactic acid

    SciTech Connect

    Tam, M.S.; Jackson, J.E.; Miller, D.J.

    1999-10-01

    Ammonia is often used for pH adjustment during fermentation of glucose to lactic acid. Its presence as ammonium lactate in the catalytic upgrading of lactic acid to 2,3-pentanedione over CsOH/silica reduces diketone yields to nearly zero at high ammonia levels. Removal of ammonia from the feed restores 2,3-pentanedione yield, indicating that the catalyst itself is not poisoned by ammonia. Instead, 2,3-pentanedione continues to form in the presence of ammonia and is consumed in secondary reactions downstream of the catalyst bed. Both base-catalyzed self-condensation of 2,3-pentanedione to duroquinone (and oligomeric species) and direct reaction of ammonia with the diketone are observed.

  1. Maximal lactate steady state in Judo

    PubMed Central

    de Azevedo, Paulo Henrique Silva Marques; Pithon-Curi, Tania; Zagatto, Alessandro Moura; Oliveira, João; Perez, Sérgio

    2014-01-01

    Summary Background: the purpose of this study was to verify the validity of respiratory compensation threshold (RCT) measured during a new single judo specific incremental test (JSIT) for aerobic demand evaluation. Methods: to test the validity of the new test, the JSIT was compared with Maximal Lactate Steady State (MLSS), which is the gold standard procedure for aerobic demand measuring. Eight well-trained male competitive judo players (24.3 ± 7.9 years; height of 169.3 ± 6.7cm; fat mass of 12.7 ± 3.9%) performed a maximal incremental specific test for judo to assess the RCT and performed on 30-minute MLSS test, where both tests were performed mimicking the UchiKomi drills. Results: the intensity at RCT measured on JSIT was not significantly different compared to MLSS (p=0.40). In addition, it was observed high and significant correlation between MLSS and RCT (r=0.90, p=0.002), as well as a high agreement. Conclusions: RCT measured during JSIT is a valid procedure to measure the aerobic demand, respecting the ecological validity of Judo. PMID:25332923

  2. Mood stabilizers in pregnancy and lactation

    PubMed Central

    Grover, Sandeep; Avasthi, Ajit

    2015-01-01

    Management of bipolar during pregnancy and postpartum is very challenging. The treating clinicians have to take into account various factors like current mental state, longitudinal history of the patient, past history of relapse while off medication, response to medication, time of pregnancy at which patient presents to the clinician, etc. The choice of drug should depend on the balance between safety and efficacy profile. Whenever patient is on psychotropic medication, close and intensive monitoring should be done. Among the various mood stabilizers, use of lithium during the second and third trimester appears to be safe. Use of valproate during first trimester is associated with major malformation and long-term sequalae in the form of developmental delay, lower intelligence quotient, and higher risk of development of autism spectrum disorder. Similarly use of carbamazepine in first trimester is associated with higher risk of major congenital malformation and its use in first trimester is contraindicated. Data for lamotrigine (LTG) appears to be more favorable than other antiepileptics. During lactation, use of valproate and LTG is reported to be safe. Use of typical and/atypical antipsychotic is a good option during pregnancy in women with bipolar disorder. PMID:26330649

  3. Mood stabilizers in pregnancy and lactation.

    PubMed

    Grover, Sandeep; Avasthi, Ajit

    2015-07-01

    Management of bipolar during pregnancy and postpartum is very challenging. The treating clinicians have to take into account various factors like current mental state, longitudinal history of the patient, past history of relapse while off medication, response to medication, time of pregnancy at which patient presents to the clinician, etc. The choice of drug should depend on the balance between safety and efficacy profile. Whenever patient is on psychotropic medication, close and intensive monitoring should be done. Among the various mood stabilizers, use of lithium during the second and third trimester appears to be safe. Use of valproate during first trimester is associated with major malformation and long-term sequalae in the form of developmental delay, lower intelligence quotient, and higher risk of development of autism spectrum disorder. Similarly use of carbamazepine in first trimester is associated with higher risk of major congenital malformation and its use in first trimester is contraindicated. Data for lamotrigine (LTG) appears to be more favorable than other antiepileptics. During lactation, use of valproate and LTG is reported to be safe. Use of typical and/atypical antipsychotic is a good option during pregnancy in women with bipolar disorder. PMID:26330649

  4. Foraging decisions in a capital breeder: trade-offs between mass gain and lactation.

    PubMed

    Hamel, Sandra; Côté, Steeve D

    2009-08-01

    The high energetic costs of lactation can lead to fundamental trade-offs in life-history traits, particularly in young females that reproduce before completing body growth. We assessed whether lactating female mountain goats (Oreamnos americanus) used behavioural tactics at fine spatio-temporal scales to increase energy intake to compensate for the costs of lactation. Lactating females increased bite rate and chewing rate compared with non-lactating females, but selected similar foraging sites in terms of plant quality and abundance. At peak lactation, forage intake of lactating females was >40% greater than that of non-lactating females. For females that had reached asymptotic body mass (i.e. > or =6 years old), summer mass gain of lactating females was similar to that of non-lactating females. At 4 and 5 years of age, however, daily mass gain of lactating females was about 20% lower than that of non-lactating females. We conclude that increased foraging may allow fully-grown lactating females to compensate for the energetic costs of lactation, but that there is a major trade-off between mass gain and lactation for younger females. PMID:19488787

  5. Yeast alcohol dehydrogenase structure and catalysis.

    PubMed

    Raj, Savarimuthu Baskar; Ramaswamy, S; Plapp, Bryce V

    2014-09-16

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of "back-to-back" dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  6. Structural Studies of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

  7. A critical review of cellobiose dehydrogenases.

    PubMed

    Henriksson, G; Johansson, G; Pettersson, G

    2000-03-10

    Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion. Monosaccharides, maltose and molecular oxygen are poor substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested. PMID:10725534

  8. The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris.

    PubMed

    Hadj-Saïd, Jessica; Pandelia, Maria-Eirini; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2015-12-01

    Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO?and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe-4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooS(C), respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooS(C) is Ni-loaded, reversibly converts CO and CO?, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooS(C) reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site. PMID:26255854

  9. Yeast Alcohol Dehydrogenase Structure and Catalysis

    PubMed Central

    2015-01-01

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  10. Quinoprotein-catalysed reactions.

    PubMed Central

    Anthony, C

    1996-01-01

    This review is concerned with the structure and function of the quinoprotein enzymes, sometimes called quinoenzymes. These have prosthetic groups containing quinones, the name thus being analogous to the flavoproteins containing flavin prosthetic groups. Pyrrolo-quinoline quinone (PQQ) is non-covalently attached, whereas tryptophan tryptophylquinone (TTQ), topaquinone (TPQ) and lysine tyrosylquinone (LTQ) are derived from amino acid residues in the backbone of the enzymes. The mechanisms of the quinoproteins are reviewed and related to their recently determined three-dimensional structures. As expected, the quinone structures in the prosthetic groups play important roles in the mechanisms. A second common feature is the presence of a catalytic base (aspartate) at the active site which initiates the reactions by abstracting a proton from the substrate, and it is likely to be involved in multiple reactions in the mechanism. A third common feature of these enzymes is that the first part of the reaction produces a reduced prosthetic group; this part of the mechanism is fairly well understood. This is followed by an oxidative phase involving electron transfer reactions which remain poorly understood. In both types of dehydrogenase (containing PQQ and TTQ), electrons must pass from the reduced prosthetic group to redox centres in a second recipient protein (or protein domain), whereas in amine oxidases (containing TPQ or LTQ), electrons must be transferred to molecular oxygen by way of a redox-active copper ion in the protein. PMID:9003352

  11. In Vitro Lactate Metabolism by Ruminal Ingesta1

    PubMed Central

    Satter, L. D.; Esdale, W. J.

    1968-01-01

    Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-14C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range. PMID:5659361

  12. Substrate specificity of human and yeast aldehyde dehydrogenases.

    PubMed

    Wang, Ming-Fang; Han, Chih-Li; Yin, Shih-Jiun

    2009-03-16

    Human aldehyde dehydrogenase (ALDH) family may contribute to metabolism of hydrocarbons, biogenic amines, retinoids, steroids, and lipid peroxidation. We previously reported kinetic properties of human cytosolic ALDH1 and mitochondrial ALDH2 towards oxidation of the straight-chain and branched-chain aliphatic aldehydes with various chain lengths [S.J. Yin, M.F. Wang, C.L. Han, S.L. Wang, Substrate binding pocket structure of human aldehyde dehydrogenases: a substrate specificity approach, Adv. Exp. Med. Biol. 372 (1995) 9-16]. We present here substrate specificities for aromatic and heterocyclic aldehydes with purified human liver ALDH1 and ALDH2, and also with yeast mitochondrial ALDH2 for comparison. Kinetic assay for human ALDHs was performed in 50mM HEPES, pH 7.5 and 25 degrees C, containing 0.5mM NAD(+), 1.7% (v/v) acetonitrile (as a solvent carrier for aldehydes) and varied concentrations of substrate, and for yeast ALDH2 the assay was determined in the same reaction mixture except containing 3mM NAD(+) and addition of 200 mM KCl. With respect to phenylacetaldehyde, 2-phenylpropionaldehyde, benzaldehyde, p-nitrobenzaldehyde, cinnamaldehyde, 2-furaldehyde and indole-3-acetaldehyde, human liver ALDH1 exhibited K(M) ranging from 0.25 to 4.8 microM, V(max) of 0.34-2.4U/mg, and catalytic efficiency, V(max)/K(M), 0.070-3.9U/(mg microM); human ALDH2 exhibited K(M) ranging from less than 0.15-0.74 microM, V(max) of 0.039-0.51 U/mg, and V(max)/K(M), 0.15-1.0U/(mg microM). Human ALDH1 and ALDH2 exhibited substate inhibition constants (K(i)) for phenylacetaldehyde, 95 and 430 microM, respectively. Yeast ALDH2 exhibited K(M) for straight-chain aliphatic aldehydes (C1-C10), 2.3-210 microM, and substrate inhibition constants (C2-C10), 79-2900 microM, with a trend of being smaller K(M) and K(i) for longer chain lengths; and K(M) for cinnamaldehyde, benzaldehyde, and 2-furaldehyde, 5.0, 79, and 1000 microM, respectively. Therefore human ALDH1/ALDH2 and yeast ALDH2 can contribute to detoxification or metabolism of various exogenous/endogenous aliphatic and aromatic aldehydes. The systematic changes in kinetic parameters for oxidation of structurally related aldehydes may reflect subtle functional topographic distinctions of substrate pocket for human and yeast ALDHs. PMID:18983993

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

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; D?uga, Joanna; Socha, Jaros?aw

    2015-01-01

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

  14. Characterization of cDNAs encoding human pyruvate dehydrogenase alpha subunit.

    PubMed Central

    Ho, L; Wexler, I D; Liu, T C; Thekkumkara, T J; Patel, M S

    1989-01-01

    A cDNA clone (1423 base pairs) comprising the entire coding region of the precursor form of the alpha subunit of pyruvate dehydrogenase (E1 alpha) has been isolated from a human liver cDNA library in phage lambda gt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E1 alpha peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E1 alpha cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E1 alpha cDNA resolves existing discrepancies among three previously reported human E1 alpha cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients. Images PMID:2748588

  15. Properties of purified carbon monoxide dehydrogenase from Clostridium thermoaceticum, a nickel, iron-sulfur protein

    SciTech Connect

    Ragsdale, S.W.; Clark, J.E.; Ljungdahl, L.G.; Lundie, L.L.; Drake, H.L.

    1983-02-25

    Carbon monoxide dehydrogenase from Clostridium thermoaceticum has been purified to homogeneity using a strict anaerobic procedure. The enzyme has a molecular weight of about 440,000 and it consists of three each of two different subunits giving the composition ..cap alpha../sub 3/..beta../sub 3/. The molecular weight of the ..cap alpha..-subunit is 78,000 and that of the ..beta..-subunit is 71,000. Pore limit gel electrophoresis gave a molecular weight of 161,000 indicating that the enzyme dissociates to form a dimer with an ..cap alpha beta.. structure. The dimer apparently contains per mol 2 nickel, 1 zinc, 11 iron, and 14 acid-labile sulfur. The anaerobic enzyme has an iron-sulfur type spectrum, which is changed in the presence of the substrate, CO. In the presence of oxygen, which destroys the activity or CO/sub 2/, the spectrum is that of a typical iron-sulfur protein. Under acidic conditions a low molecular weight nickel factor separates from the enzyme. Viologens, methylene blue, ferredoxin, flavodoxin, and rubredoxin serve as electron acceptorrs. The enzyme has a pH optimum around 8.4. Cyanide and methyliodide inhibit the enzyme. CO reverses the cyanide inhibition but promotes the reaction with methyl iodide. The pure enzyme has no hydrogenase or formate dehydrogenase activity.

  16. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-?-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ?3-fold in the presence of NADH and ?4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  17. Cloning and characterization of the ADH5 gene encoding human alcohol dehydrogenase 5, formaldehyde dehydrogenase.

    PubMed

    Hur, M W; Edenberg, H J

    1992-11-16

    Human chi-alcohol dehydrogenase (chi-ADH) is a zinc-containing dimeric enzyme responsible for the oxidation of long-chain alcohols and omega-hydroxyfatty acids. Class-III ADHs, of which chi-ADH is the prototype, are widely produced and well conserved during evolution. This suggests that they fulfill important housekeeping roles in cellular metabolism. Recent evidence suggests that class-III ADH and formaldehyde dehydrogenase (FDH) are the same enzyme. We have isolated and characterized two overlapping genomic clones that cover the entire ADH5 (FDH) gene. ADH5 is composed of nine exons and eight introns. Two major transcription start points were identified by primer extension. The 5' nontranslated region is unusual in that it contains two additional upstream ATG codons, which would encode peptides of 20 and 10 amino acids. Neither of the upstream ATGs is in a good context for translation initiation, whereas the ATG initiating &khgr;-ADH is in a favorable context. The 5' region of ADH5 is a CpG island; it is extremely G+C rich and has many CpG doublets. It does not contain either a TATA box or a CAAT box. This is consistent with ubiquitous expression, and contrasts with the promoters of all previously cloned ADH genes, which are expressed in a tissue-specific manner. The 5' region of ADH5 contains consensus binding sites for the transcriptional regulatory proteins, Sp1, AP2, LF-A1, NF-1, NF-A2, and NF-E1. A 1.5-kb upstream fragment from ADH5 was able to drive the transcription of a cat reporter gene at high levels in monkey kidney cells (CV-1). Several processed pseudogenes were also isolated. PMID:1446828

  18. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    SciTech Connect

    Park, Yun-Hee; Patel, Mulchand S.

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  19. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    SciTech Connect

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C.

    2013-09-18

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases that includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in their active sites that help explain the variations in their respective substrate specificities.

  20. Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Morita, Tomotake; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2010-01-01

    Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant DeltaadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. DeltaadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting DeltaadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on DeltaadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production. PMID:20622460