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Sample records for pyruvate kinase pklr

  1. Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR Variants in a Thai Population.

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    Rebekah van Bruggen

    Full Text Available Pyruvate kinase (PKLR is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41 is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

  2. Alu element insertion in PKLR gene as a novel cause of pyruvate kinase deficiency in Middle Eastern patients.

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    Lesmana, Harry; Dyer, Lisa; Li, Xia; Denton, James; Griffiths, Jenna; Chonat, Satheesh; Seu, Katie G; Heeney, Matthew M; Zhang, Kejian; Hopkin, Robert J; Kalfa, Theodosia A

    2018-03-01

    Pyruvate kinase deficiency (PKD) is the most frequent red blood cell enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Over 250 PKLR-gene mutations have been described, including missense/nonsense, splicing and regulatory mutations, small insertions, small and gross deletions, causing PKD and hemolytic anemia of variable severity. Alu retrotransposons are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. Alu insertions have been associated with a number of human diseases either by disrupting a coding region or a splice signal. Here, we report on two unrelated Middle Eastern patients, both born from consanguineous parents, with transfusion-dependent hemolytic anemia, where sequence analysis revealed a homozygous insertion of AluYb9 within exon 6 of the PKLR gene, causing precipitous decrease of PKLR RNA levels. This Alu element insertion consists a previously unrecognized mechanism underlying pathogenesis of PKD. © 2017 Wiley Periodicals, Inc.

  3. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Jaouani Mouna

    2015-09-26

    Sep 26, 2015 ... to several mutations at the Pyruvate Kinase gene (PKLR) located on chromosome .... Tunisians (Fig. 2) [21]. The screening of whole PKLR gene revealed the presence of ..... newborns: the pitfalls of diagnosis. J Pediatr 2007 ...

  4. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

  5. Novel mutations associated with pyruvate kinase deficiency in Brazil

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    Maria Carolina Costa Melo Svidnicki

    2018-01-01

    Full Text Available Background: Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype–phenotype correlations. Method: Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A. Results: Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. Conclusion: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants. Keywords: Red cell disorder, Pyruvate kinase, Mutation, Hemolytic anemia, PKLR gene

  6. Crystal structure of Cryptosporidium parvum pyruvate kinase.

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    William J Cook

    Full Text Available Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303-320 of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

  7. Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

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    Zita Garate

    2015-12-01

    Full Text Available Pyruvate kinase deficiency (PKD is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs from peripheral blood mononuclear cells (PB-MNCs of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR. Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.

  8. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Phenotypic and molecular genetic analysis of Pyruvate Kinase deficiency in a Tunisian family. Jaouani Mouna, Hamdi Nadia, Chaouch Leila, Kalai Miniar, Mellouli Fethi, Darragi Imen, Boudriga Imen, Chaouachi Dorra, Bejaoui Mohamed, Abbes Salem ...

  9. The Role of Pyruvate Dehydrogenase Kinase in Diabetes and Obesity

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    In-Kyu Lee

    2014-06-01

    Full Text Available The pyruvate dehydrogenase complex (PDC is an emerging target for the treatment of metabolic syndrome. To maintain a steady-state concentration of adenosine triphosphate during the feed-fast cycle, cells require efficient utilization of fatty acid and glucose, which is controlled by the PDC. The PDC converts pyruvate, coenzyme A (CoA, and oxidized nicotinamide adenine dinucleotide (NAD+ into acetyl-CoA, reduced form of nicotinamide adenine dinucleotide (NADH, and carbon dioxide. The activity of the PDC is up- and down-regulated by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. In addition, pyruvate is a key intermediate of glucose oxidation and an important precursor for the synthesis of glucose, glycerol, fatty acids, and nonessential amino acids.

  10. Lack of association between PKLR rs3020781 and NOS1AP rs7538490 and type 2 diabetes, overweight, obesity and related metabolic phenotypes in a Danish large-scale study: case-control studies and analyses of quantitative traits

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    Almind Katrine

    2008-12-01

    Full Text Available Abstract Background Several studies in multiple ethnicities have reported linkage to type 2 diabetes on chromosome 1q21-25. Both PKLR encoding the liver pyruvate kinase and NOS1AP encoding the nitric oxide synthase 1 (neuronal adaptor protein (CAPON are positioned within this chromosomal region and are thus positional candidates for the observed linkage peak. The C-allele of PKLR rs3020781 and the T-allele of NOS1AP rs7538490 are reported to strongly associate with type 2 diabetes in various European-descent populations comprising a total of 2,198 individuals with a combined odds ratio (OR of 1.33 [1.16–1.54] and 1.53 [1.28–1.81], respectively. Our aim was to validate these findings by investigating the impact of the two variants on type 2 diabetes and related quantitative metabolic phenotypes in a large study sample of Danes. Further, we intended to expand the analyses by examining the effect of the variants in relation to overweight and obesity. Methods PKLR rs3020781 and NOS1AP rs7538490 were genotyped, using TaqMan allelic discrimination, in a combined study sample comprising a total of 16,801 and 16,913 individuals, respectively. The participants were ascertained from four different study groups; the population-based Inter99 cohort (nPKLR = 5,962, nNOS1AP = 6,008, a type 2 diabetic patient group (nPKLR = 1,873, nNOS1AP = 1,874 from Steno Diabetes Center, a population-based study sample (nPKLR = 599, nNOS1AP = 596 from Steno Diabetes Center and the ADDITION Denmark screening study cohort (nPKLR = 8,367, nNOS1AP = 8,435. Results In case-control studies we evaluated the potential association between rs3020781 and rs7538490 and type 2 diabetes and obesity. No significant associations were observed for type 2 diabetes (rs3020781: pAF = 0.49, OR = 1.02 [0.96–1.10]; rs7538490: pAF = 0.84, OR = 0.99 [0.93–1.06]. Neither did we show association with overweight or obesity. Additionally, the PKLR and the NOS1AP genotypes were demonstrated not

  11. Pyruvate kinase M2: a potential target for regulating inflammation

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    Jose Carlos eAlves-Filho

    2016-04-01

    Full Text Available Pyruvate kinase (PK is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signalling pathways, affecting both the enzymatic activity of PKM2 as a pyruvate kinase and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for PKM2 as a therapeutic target in inflammatory and metabolic disorders.

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

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    Gopinath eSutendra

    2013-03-01

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

  13. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

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    Fabian V Filipp

    2013-01-01

    Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2) is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor acti...

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

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    Michael I. Koukourakis

    2005-01-01

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

  15. The crystal structure of Toxoplasma gondii pyruvate kinase 1.

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    Rebecca Bakszt

    2010-09-01

    Full Text Available Pyruvate kinase (PK, which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population.We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers.We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  16. The crystal structure of Toxoplasma gondii pyruvate kinase 1.

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    Bakszt, Rebecca; Wernimont, Amy; Allali-Hassani, Abdellah; Mok, Man Wai; Hills, Tanya; Hui, Raymond; Pizarro, Juan C

    2010-09-14

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  17. The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

    Energy Technology Data Exchange (ETDEWEB)

    Bakszt, R.; Wernimont, A; Allali-Hassani, A; Mok, M; Hills, T; Hui, R; Pizarro, J

    2010-01-01

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two {alpha}-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  18. Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity.

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    Netsanet Worku

    Full Text Available Human African Trypanosomiasis (HAT also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties.The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM. The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions.Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross

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

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    Hayden Bell

    2016-06-01

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

  20. Pyruvate Kinase Triggers a Metabolic Feedback Loop that Controls Redox Metabolism in Respiring Cells

    NARCIS (Netherlands)

    Grüning, N.M.; Rinnerthaler, M.; Bluemlein, K.; Mulleder, M.; Wamelink, M.M.C.; Lehrach, H.; Jakobs, C.A.J.M.; Breitenbach, M.; Ralser, M.

    2011-01-01

    In proliferating cells, a transition from aerobic to anaerobic metabolism is known as the Warburg effect, whose reversal inhibits cancer cell proliferation. Studying its regulator pyruvate kinase (PYK) in yeast, we discovered that central metabolism is self-adapting to synchronize redox metabolism

  1. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

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    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

  2. Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

    Science.gov (United States)

    Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

    2012-10-12

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

  3. Erythrocyte pyruvate kinase deficiency in the Ohio Amish: origin and characterization of the mutant enzyme.

    OpenAIRE

    Muir, W A; Beutler, E; Wasson, C

    1984-01-01

    We have identified eight individuals in an Amish population in Geauga County, Ohio, who have a congenital hemolytic anemia and red cell pyruvate kinase (PK) deficiency. The mutant enzyme is a low Km phosphoenolpyruvate (PEP) variant associated with a slower (77.5% of normal) electrophoretic mobility in starch gel. Because of the high consanguinity in this population, we assume the affected individuals are homozygous for the mutant gene. Genealogical records allow us to trace all eight cases b...

  4. Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

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    Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

    1983-07-01

    Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

  5. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    International Nuclear Information System (INIS)

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P.

    2005-01-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

  6. Domain interaction in rabbit muscle pyruvate kinase. II. Small angle neutron scattering and computer simulation.

    Science.gov (United States)

    Consler, T G; Uberbacher, E C; Bunick, G J; Liebman, M N; Lee, J C

    1988-02-25

    The effects of ligands on the structure of rabbit muscle pyruvate kinase were studied by small angle neutron scattering. The radius of gyration, RG, decreases by about 1 A in the presence of the substrate phosphoenolpyruvate, but increases by about the same magnitude in the presence of the allosteric inhibitor phenylalanine. With increasing pH or in the absence of Mg2+ and K+, the RG of pyruvate kinase increases. Hence, there is a 2-A difference in RG between two alternative conformations. Length distribution analysis indicates that, under all experimental conditions which increase the radius of gyration, there is a pronounced increase observed in the probability for interatomic distance between 80 and 110 A. These small angle neutron scattering results indicate a "contraction" and "expansion" of the enzyme when it transforms between its active and inactive forms. Using the alpha-carbon coordinates of crystalline cat muscle pyruvate kinase, a length distribution profile was calculated, and it matches the scattering profile of the inactive form. These observations are expected since the crystals were grown in the absence of divalent cations (Stuart, D. I., Levine, M., Muirhead, H., and Stammers, D. K. (1979) J. Mol. Biol. 134, 109-142). Hence, results from neutron scattering, x-ray crystallographic, and sedimentation studies (Oberfelder, R. W., Lee, L. L.-Y., and Lee, J.C. (1984) Biochemistry 23, 3813-3821) are totally consistent with each other. With the aid of computer modeling, the crystal structure has been manipulated in order to effect changes that are consistent with the conformational change described by the solution scattering data. The structural manipulation involves the rotation of the B domain relative to the A domain, leading to the closure of the cleft between these domains. These manipulations resulted in the generation of new sets of atomic (C-alpha) coordinates, which were utilized in calculations, the result of which compared favorably with the

  7. Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas.

    Science.gov (United States)

    González, R; Carvajal, N; Morán, A

    1984-01-01

    In contrast to the Mg2+-activated enzyme, in the presence of Mn2+ pyruvate kinase exhibits hyperbolic kinetics with respect to the substrate phosphoenolpyruvate and is insensitive to fructose 1,6-biphosphate, phenylalanine and alanine. However, with both metal activated species inhibition by excess ADP is observed. In contrast with Mg2+, which affords significant protection against inactivation caused by 5,5'-dithiobis (2-nitrobenzoic acid), the rate of inactivation by this reagent is increased in the presence of Mn2+. Differences in conformational changes induced by combination of pyruvate kinase with Mg2+ or Mn2+ were indicated by u.v. difference spectra.

  8. Heavy-atom isotope effects on binding of reactants to lactate dehydrogenase and pyruvate kinase

    International Nuclear Information System (INIS)

    Gawlita, E.

    1993-04-01

    18 O and 13 C kinetic isotope effects have been measured on the reaction of pyruvate kinase with phospho-enol-pyruvate and ADP using a remote label technique. The magnitude of both investigated isotope effects showed a dependence on the concentration of ADP. However, while the carbon effect was simply 'washed out' to unity at high ATP concentration, the oxygen effect becomes inverse and reached 0.9928 at the highest used concentration of ADP. Such a result testifies that the assumption of the negligible effect of isotopic substitution on enzyme-substrate associations remains correct only for carbon effects. An equilibrium 18 O isotope effect on association of oxalate with lactate dehydrogenase in the presence of NADHP has been evaluated by both experimental and theoretical means. Experimental methods, which involved equilibrium dialysis and gas chromatographic/mass spectrometric measurement of isotopic ration, yielded an inverse value of 0.9840. Semiempirical methods involved vibrational analysis of oxalate in two different environments. The comparison of calculated values with the experimentally determined isotope effect indicated that the AM 1 Hamiltonian proved superior to its PM 3 counterpart in this modelling. 160 refs, 8 figs, 18 tabs

  9. Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase.

    Science.gov (United States)

    Rodriguez-Contreras, Dayana; Hamilton, Nicklas

    2014-11-21

    Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

    Science.gov (United States)

    Stamm, Irmela; Lottspeich, Friedrich; Plaga, Wulf

    2005-06-01

    Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

  11. Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer

    Directory of Open Access Journals (Sweden)

    Gopinath Prakasam

    2018-02-01

    Full Text Available Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis, i.e., Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through various non-glycolytic moonlight functions, such as gene transcription, protein kinase activity, and redox balance. A large body of data have shown that several distinct posttranslation modifications (PTMs regulate PKM2 in a way that benefits cancer growth, e.g., formation of PKM2 dimers. This review discusses the recent advancements in our understanding of various PTMs and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anticancer strategies.

  12. Data regarding the growth of Lactobacillus acidophilus NCFM on different carbohydrates and recombinant production of elongation factor G and pyruvate kinase

    DEFF Research Database (Denmark)

    Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie

    2017-01-01

    The present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further...

  13. An improved strategy for the crystallization of Leishmania mexicana pyruvate kinase

    International Nuclear Information System (INIS)

    Morgan, Hugh P.; McNae, Iain W.; Hsin, Kun-Yi; Michels, Paul A. M.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2010-01-01

    The first crystal structure of Leishmania mexicana pyruvate kinase (LmPYK) obtained at a neutral pH. LmPYK was co-crystallized with the small molecule 1,3,6,8-pyrenetetrasulfonic acid, which provides a helpful intermolecular bridge between macromolecules. The inclusion of novel small molecules in crystallization experiments has provided very encouraging results and this method is now emerging as a promising alternative strategy for crystallizing ‘problematic’ biological macromolecules. These small molecules have the ability to promote lattice formation through stabilizing intermolecular interactions in protein crystals. Here, the use of 1,3,6,8-pyrenetetrasulfonic acid (PTS), which provides a helpful intermolecular bridge between Leishmania mexicana PYK (LmPYK) macromolecules in the crystal, is reported, resulting in the rapid formation of a more stable crystal lattice at neutral pH and greatly improved X-ray diffraction results. The refined structure of the LmPYK–PTS complex revealed the negatively charged PTS molecule to be stacked between positively charged (surface-exposed) arginine side chains from neighbouring LmPYK molecules in the crystal lattice

  14. Role of isoenzyme M2 of pyruvate kinase in urothelial tumorigenesis.

    Science.gov (United States)

    Zhou, Haiping; Wang, Xing; Mo, Lan; Liu, Yan; He, Feng; Zhang, Fenglin; Huang, Kuo-How; Wu, Xue-Ru

    2016-04-26

    The conversion of precancerous lesions to full-fledged cancers requires the affected cells to surpass certain rate-limiting steps. We recently showed that activation of HRAS proto-oncogene in urothelial cells of transgenic mice causes simple urothelial hyperplasia (SUH) which is persistent and whose transition to low-grade papillary urothelial carcinoma (UC) must undergo nodular urothelial hyperplasia (NUH). We hypothesized that NUH, which has acquired fibrovascular cores, plays critical roles in mesenchymal-to-epithelial signaling, breaching the barriers of urothelial tumor initiation. Using proteomics involving two-dimensional gel electrophoresis, immunoblotting with pan-phosphotyrosine antibody and MALDI-mass spectrometry, we identified isoform 2 of pyruvate kinase (PKM2) as the major tyrosine-phosphorylated protein switched on during NUH. We extended this finding using specimens from transgenic mice, human UC and UC cell lines, establishing that PKM2, but not its spliced variant PKM1, was over-expressed in low-grade and, more prominently, high-grade UC. In muscle-invasive UC, PKM2 was co-localized with cytokeratins 5 and 14, UC progenitor markers. Specific inhibition of PKM2 by siRNA or shRNA suppressed UC cell proliferation via increased apoptosis, autophagy and unfolded protein response. These results strongly suggest that PKM2 plays an important role in the genesis of low-grade non-invasive and high-grade invasive urothelial carcinomas.

  15. Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus

    International Nuclear Information System (INIS)

    Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko; Sakai, Hiroshi

    2005-01-01

    This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6 2 22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6 2 22, with unit-cell parameters a = b = 145.97, c = 118.03 Å

  16. SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Hung, Man-Hsin; Chu, Pei-Yi; Chen, Yao-Li; Chen, Li-Ju; Tsai, Ming-Hsien; Chen, Min-Husan; Shiau, Chung-Wai; Boo, Yin-Pin; Chen, Kuen-Feng

    2016-04-19

    Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

  17. Congenital insensitivity to pain with anhidrosis (CIPA): Novel mutations of the TRKA (NTRK1) gene, a putative uniparental disomy, and a linkage of the mutant TRKA and PKLR genes in a family with CIPA and pyruvate kinase deficiency

    NARCIS (Netherlands)

    Y. Indo (Yasuhiro); S. Mardy (Sek); Y. Miura (Yuichi); A. Moosa (Allie); E.A.R. Ismail (Essam A.); E. Toscano (Ennio); G. Andria (Generoso); V. Pavone (Vito); D.L. Brown (Deborah); A.S. Brooks (Alice); F. Endo (Fumio); I. Matsuda (Ichiro)

    2001-01-01

    textabstractCongenital insensitivity to pain with anhidrosis is an autosomal recessive hereditary disorder characterized by recurrent episodic fever, anhidrosis (inability to sweat), absence of reaction to noxious stimuli, self-mutilating behavior, and mental retardation. The human TRKA gene

  18. Effects of insulin on perfused liver from streptozotocin-diabetic and untreated rats: 13C NMR assay of pyruvate kinase flux

    International Nuclear Information System (INIS)

    Cohen, S.M.

    1987-01-01

    The effects of insulin in vitro on perfused liver from streptozotocin-diabetic rats and their untreated littermates during gluconeogenesis from either [3- 13 C]alanine + ethanol or [2- 13 C]pyruvate + NH 4 Cl + ethanol were studied by 13 C NMR. A 13 C NMR determination of the rate of pyruvate kinase flux under steady-state conditions of active gluconeogenesis was developed; this assay includes a check on the reuse of recycled pyruvate. The preparations studied provided gradations of pyruvate kinase flux within the confines of the assay's requirement of active gluconeogenesis. By this determination, the rate of pyruvate kinase flux was 0.74 +/- 0.04 of the gluconeogenic rate in liver from 24-h-fasted controls; in liver from 12-h fasted controls, relative pyruvate kinase flux increased to 1.0 +/- 0.2. In diabetic liver, this flux was undetectable by the authors NMR method. Insulin's hepatic influence in vitro was greatest in the streptozotocin model of type 1 diabetes: upon treatment of diabetic liver with 7 nM insulin in vitro, a partial reversal of many of the differences noted between diabetic and control liver was demonstrated by 13 C NMR. A major effect of insulin in vitro upon diabetic liver was the induction of a large increase in the rate of pyruvate kinase flux, bringing relative and absolute fluxes up to the levels measured in 24-h-fasted controls. By way of comparison, the effects of ischemia on diabetic liver were studied by 13 C NMR to test whether changes in allosteric effectors under these conditions could also increase pyruvate kinase flux. A large increase in this activity was demonstrated in ischemic diabetic liver

  19. miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Angela M Liu

    Full Text Available In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect, with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122 is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC. Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = <0.0001. In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2 is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

  20. An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

    Science.gov (United States)

    Yuan, Meng; McNae, Iain W; Chen, Yiyuan; Blackburn, Elizabeth A; Wear, Martin A; Michels, Paul A M; Fothergill-Gilmore, Linda A; Hupp, Ted; Walkinshaw, Malcolm D

    2018-05-10

    We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation K d is estimated to be ~0.9 µM with a slow dissociation rate (t 1/2 ~ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R-states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate. ©2018 The Author(s).

  1. Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

    International Nuclear Information System (INIS)

    Lodato, D.T.; Reed, G.H.

    1987-01-01

    The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EOR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17 O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the γ-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from 17 O regiospecifically incorporated into the γ-phosphate group of ATP. By contrast, 17 O in the α-phosphate or in the β-phosphate groups of ATP does not influence the spectrum. Experiments in 17 O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme-Mn(II)-oxalate-Mg(II)-ATP complex suggests a scheme for the normal reverse reaction of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the γ-phosphate of ATP

  2. The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

    Science.gov (United States)

    Lin, J W; Lu, H C; Chen, H Y; Weng, S F

    1997-10-09

    Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

  3. Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

    Science.gov (United States)

    Zoraghi, Roya; Campbell, Sara; Kim, Catrina; Dullaghan, Edie M; Blair, Lachlan M; Gillard, Rachel M; Reiner, Neil E; Sperry, Jonathan

    2014-11-01

    Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

    Czech Academy of Sciences Publication Activity Database

    Dowling, P.; Hughes, D. J.; Larkin, A.M.; Meiller, J.; Henry, M.; Meleady, P.; Lynch, V.; Pardini, B.; Naccarati, A.; Levý, M.; Vodička, Pavel; Neary, P.; Clynes, M.

    2015-01-01

    Roč. 441, feb. (2015), s. 133-141 ISSN 0009-8981 Institutional support: RVO:68378041 Keywords : biomarkers * colorectal cancer * proteomics * mass spectrometry * 14-3-3 proteins * pyruvate kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.799, year: 2015

  5. Comparative kinetic studies of Mn2+-activated and fructose-1,6-P-modified Mg2+-activated pyruvate kinase from Concholepas concholepas.

    Science.gov (United States)

    Carvajal, N; González, R; Morán, A; Oyarce, A M

    1985-01-01

    Initial velocity and product inhibition studies of Mn2+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzyme-PEP-ATP.

  6. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high-saturated fat diet

    OpenAIRE

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A.

    2012-01-01

    Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) might prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it might induce detrimental effects by inhibiting fatty acid oxidation. PPARα agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment with a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of...

  7. Data regarding the growth of Lactobacillus acidophilus NCFM on different carbohydrates and recombinant production of elongation factor G and pyruvate kinase

    Directory of Open Access Journals (Sweden)

    Hasan Ufuk Celebioglu

    2017-10-01

    Full Text Available The present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further and detailed interpretation of the data presented here, please see the research article “Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM” (Celebioglu et al., 2017 [1].

  8. Study of pyruvate decarboxylase and thiamine kinase from brewer's yeast by SERS

    Science.gov (United States)

    Maskevich, Sergei A.; Chernikevich, Ivan P.; Gachko, Gennedy A.; Kivach, Leonid N.; Strekal, Nataliya D.

    1993-06-01

    The Surface Enhanced Raman Scattering (SERS) spectra of holopyruvate decarboxylase (PDC) and thiamine kinase (ThK) adsorbed on silver electrode were obtained. In contrast to the Raman, the SERS spectrum of PDC contained no modes of tryptophan residues, it indicates a removal of this moiety from the surface. In the SERS spectrum of ThK the bands belonging to ligands bound to the protein were observed. A correlation between the SERS signal intensity and the enzymatic activity of the ThK separate fraction and found. The influence of amino acids on SERS spectra of thiamine (Th) was studied to determine the possible composition on microsurrounding of coenzyme.

  9. Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer.

    Science.gov (United States)

    Chao, Tai-Kuang; Huang, Tien-Shuo; Liao, Yu-Ping; Huang, Rui-Lan; Su, Po-Hsuan; Shen, Hueng-Yuan; Lai, Hung-Cheng; Wang, Yu-Chi

    2017-01-01

    Pyruvate kinase M2 (PKM2) regulates glycolysis and oxidative phosphorylation; however, the role of PKM2 in ovarian cancer remains largely unknown. We investigated whether ovarian cancer metabolism could provide insight into the development of therapeutic strategies. We performed immunohistochemical staining for PKM2 on a tissue microarray for multivariate analysis. It revealed that patients exhibiting higher PKM2 expression were significantly associated with malignancy groups (p < 0.001) and pathogenesis models (p < 0.001), had poor progression-free survival rates (p = 0.01) as compared with patients exhibiting lower PKM2 levels, and yielded a hazard ratio of death of 2.02 (95% confidence interval: 0.70-5.85). In cell lines, PKM2 inhibitor significantly inhibited the glycolytic rate according to cellular glucose consumption (p < 0.001). We also utilized Seahorse assays to assess metabolism-related cell-specific factors and the impact of PKM2 inhibitors. Energy shifts as per Seahorse analysis showed attenuation of the extracellular acidification rate (p < 0.05) and no significant difference in oxygen-consumption rate in SKOV3 cells. Treatment with PKM2 inhibitor suppressed ovarian cancer growth and cell migration in vitro and inhibited tumor growth without significant toxicity in a xenograft study. PKM2 inhibition disturbed Warburg effects and inhibited ovarian cancer cell growth. Targeting PKM2 may constitute a promising therapy for patients with ovarian cancer, and clinical trials involving shikonin are warranted.

  10. Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Tai-Kuang Chao

    Full Text Available Pyruvate kinase M2 (PKM2 regulates glycolysis and oxidative phosphorylation; however, the role of PKM2 in ovarian cancer remains largely unknown. We investigated whether ovarian cancer metabolism could provide insight into the development of therapeutic strategies. We performed immunohistochemical staining for PKM2 on a tissue microarray for multivariate analysis. It revealed that patients exhibiting higher PKM2 expression were significantly associated with malignancy groups (p < 0.001 and pathogenesis models (p < 0.001, had poor progression-free survival rates (p = 0.01 as compared with patients exhibiting lower PKM2 levels, and yielded a hazard ratio of death of 2.02 (95% confidence interval: 0.70-5.85. In cell lines, PKM2 inhibitor significantly inhibited the glycolytic rate according to cellular glucose consumption (p < 0.001. We also utilized Seahorse assays to assess metabolism-related cell-specific factors and the impact of PKM2 inhibitors. Energy shifts as per Seahorse analysis showed attenuation of the extracellular acidification rate (p < 0.05 and no significant difference in oxygen-consumption rate in SKOV3 cells. Treatment with PKM2 inhibitor suppressed ovarian cancer growth and cell migration in vitro and inhibited tumor growth without significant toxicity in a xenograft study. PKM2 inhibition disturbed Warburg effects and inhibited ovarian cancer cell growth. Targeting PKM2 may constitute a promising therapy for patients with ovarian cancer, and clinical trials involving shikonin are warranted.

  11. Pyruvate kinase M2 overexpression and poor prognosis in solid tumors of digestive system: evidence from 16 cohort studies.

    Science.gov (United States)

    Wu, Jiayuan; Hu, Liren; Chen, Manyu; Cao, Wenjun; Chen, Haicong; He, Taiping

    2016-01-01

    The expression of pyruvate kinase M2 (PKM2) has been linked to tumor formation and invasion. Specifically, the relationship between high PKM2 expression and prognosis has been evaluated in solid tumors of digestive system. However, the prognostic value of PKM2 remains controversial. A literature search of PubMed, Embase, and Cochrane databases was conducted until October 2015. The end point focused on overall survival (OS). The pooled hazard ratio (HR) or odds ratio and the 95% confidence intervals were calculated to correlate PKM2 overexpression with OS and clinicopathological characteristics by employing fixed- or random-effects models, depending on the heterogeneity of the included studies. We identified 18 cohorts in 16 studies involving 2,812 patients for this meta-analysis. Overall, the combined HR for OS in all tumor types was 1.74 (1.44-2.11; Pdigestive system, thereby suggesting that PKM2 might be an indicator of poor prognosis in digestive system cancers.

  12. In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

    Directory of Open Access Journals (Sweden)

    Ponnusamy Kalaiarasan

    Full Text Available Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs and 2 mutations of Human Pyruvate Kinase (PK M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431 could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

  13. SNPs within the beta myosin heavy chain (MYH7 and the pyruvate kinase muscle (PKM2 genes in horse

    Directory of Open Access Journals (Sweden)

    Vincenzo Russo

    2010-01-01

    Full Text Available Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

  14. Diagnostic value of fecal tumor M2-pyruvate kinase for CRC screening: a systematic review and meta-analysis.

    Science.gov (United States)

    Li, Rui; Liu, Jianjun; Xue, Huiping; Huang, Gang

    2012-10-15

    The measurement of fecal tumor M2-pyruvate kinase (PKM2), overexpressed in tumor cells, has been proposed as a novel tool for detecting colorectal cancer (CRC). However, the sensitivity and specificity of this test varied among studies. The aim of this meta-analysis was to determine the diagnostic accuracy of fecal PKM2 for CRC and to evaluate its utility in the CRC screening. It was compared to guaiac fecal occult blood test (gFOBT) or immunological fecal occult blood test (iFOBT). Through comprehensive literature search, 10 studies met the inclusion criteria and were included. Summary estimates for sensitivity and specificity were calculated by using the bivariate random effect model. The hierarchical summary receiver operating characteristic curve was also undertaken. The overall sensitivity and specificity of fecal PKM2 for detecting CRC were 79% (95% CI = 75-83%) and 81% (95% CI = 73-87%), respectively. The summary positive predictive value and negative predictive value were 74% (95% CI = 56-87%) and 86% (95% CI = 79-91%), respectively. The pooled diagnostic odds ratio was 16 (95% CI = 10-26). In head-to-head comparison, the diagnostic odds ratio of PKM2 and gFOBT for CRC were 10.167 (95% CI = 5.992-17.250) and 6.557 (95% CI = 3.467-12.403), respectively. The diagnostic odds ratio of PKM2 and iFOBT for CRC were 9.542 (95% CI = 5.893-15.452) and 67.248 (95% CI = 16.194-279.26), respectively. The fecal PKM2 test was a diagnostic tool with moderate sensitivity and specificity for detecting CRC. Its diagnostic efficiency was similar to that of gFOBT. Because of its relatively low specificity and positive predict value, fecal PKM2 was not recommended used alone as a screening tool for CRC. Copyright © 2012 UICC.

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

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    Rinnankoski-Tuikka Rita

    2012-06-01

    Full Text Available Abstract Background The expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4 gene expression via the estrogen-related receptor α (ERRα. We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle. Methods Insulin resistance was induced by a high-fat (HF diet for 19 weeks in C57BL/6 J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed. Results The HF mice were more insulin-resistant than the low-fat (LF -fed mice. Upregulation of PDK4 and ERRα mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria. Conclusions Our results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

  16. Distribution of phosphorylated metabolites and magnesium in the red cells of a patient with hyperactive pyruvate kinase

    International Nuclear Information System (INIS)

    Ouwerkerk, R.; van Echteld, C.J.; Staal, G.E.; Rijksen, G.

    1988-01-01

    The intracellular distribution of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) was studied in the red cells of a patient with a high-ATP syndrome by using 31P nuclear magnetic resonance. In this patient, red cell ATP was increased 2.5-fold, whereas 2,3-DPG was decreased fourfold due to the presence of a hyperactive pyruvate kinase. In oxygenated red cells, these abnormal concentrations were reflected to the same extent in all complexes in which ATP and 2,3-DPG take part. The diminished amount of 2,3-DPG bound to hemoglobin was almost completely replaced by ATP-hemoglobin complexes. Therefore, free hemoglobin was only slightly increased. In deoxygenated cells, the relative distribution of ATP and 2,3-DPG complexes was significantly disturbed. The main difference was a shift in the ratio of magnesium ATP (MgATP) over the ATP-hemoglobin complex; 74% of total ATP was complexed to hemoglobin (45% in normal cells), whereas the concentration of MgATP was only slightly increased with respect to normal. The shortage in 2,3-DPG bound to hemoglobin could partially be replenished by an increase in hemoglobin (Mg) ATP complexes. Therefore, the amount of uncomplexed hemoglobin raised from 15% in normal cells to 38% in the patient's cells. As a result, the oxygen-dissociation curve was only moderately shifted to the left. It is concluded that the regulatory role of 2,3-DPG in oxygen transport is taken over in part by (Mg) ATP in this patient. In both aerobic and anaerobic cells, the increase in magnesium bound to ATP, either free or bound to hemoglobin, exceeds the decrease in 2,3-DPG Mg complex. In spite of this, the amount of intracellular free Mg++ was normal or slightly lowered. This suggests the presence of a compensatory mechanism by which the amount of total cellular magnesium could be increased

  17. Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells

    International Nuclear Information System (INIS)

    Das, Mahua R.; Bag, Arup K.; Saha, Shekhar; Ghosh, Alok; Dey, Sumit K.; Das, Provas; Mandal, Chitra; Ray, Subhankar; Chakrabarti, Saikat; Ray, Manju; Jana, Siddhartha S.

    2016-01-01

    For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. PKM2

  18. Pyruvate kinase deficiency

    Science.gov (United States)

    ... anemia may need blood transfusions. Removing the spleen ( splenectomy ) may help reduce the destruction of red blood ... donor blood or plasma. Someone who had a splenectomy should receive the pneumococcal vaccine at recommended intervals. ...

  19. A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions

    DEFF Research Database (Denmark)

    Szyniarowski, Piotr; Corcelle-Termeau, Elisabeth; Farkas, Thomas

    2011-01-01

    , whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation...

  20. Effects of anoxia on the extra- and intracellular acid-base status in the land snail helix lucorum (L.): lack of evidence for a relationship between pyruvate kinase down-regulation and acid-base status

    Science.gov (United States)

    Michaelidis; Pallidou; Vakouftsi

    1999-06-01

    The aims of the present study were to describe a possible correlation between the regulation of the key glycolytic enzyme pyruvate kinase and the acid-base status in the haemolymph and in several other tissues of land snails during anoxia. To illustrate whether such a relationship exists, we determined (i) the acid-base variables in the haemolymph and tissues of the land snail Helix lucorum, (ii) the kinetic properties of pyruvate kinase from several tissues and (iii) the levels of the anaerobic end-products d-lactate and succinate in the haemolymph and tissues of aerobic and anoxic Helix lucorum. The results showed that the pH of haemolymph (pHe) decreased significantly over the first 20 h of anoxia and then recovered slowly towards control values. A similar pattern was observed for intracellular pH (pHi), which decreased significantly over the first 16 h of anoxia and slowly returned towards control levels. The reduction and recovery of pHi and pHe seem to reflect the rate of anaerobic metabolism. The main anaerobic end-products, d-lactate and succinate, accumulated rapidly during the initial stages of anoxia and more slowly as anoxia progressed. The decrease in the rate of accumulation of anaerobic end-products during prolonged anoxia was due to the conversion of tissue pyruvate kinase to a less active form. The results demonstrate a correlation between pyruvate kinase down-regulation and the recovery of acid-base status in the haemolymph and the tissues of land snails during anoxia.

  1. Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension Is Regulated Through a MicroRNA-124/PTBP1 (Polypyrimidine Tract Binding Protein 1)/Pyruvate Kinase Muscle Axis

    Czech Academy of Sciences Publication Activity Database

    Zhang, H.; Wang, D.; Li, M.; Plecitá-Hlavatá, Lydie; D'Alessandro, A.; Tauber, Jan; Riddle, S.; Kumar, S.; Flockton, A.; McKeon, B. A.; Frid, M. G.; Reisz, J. A.; Caruso, P.; El Kasmi, K. C.; Ježek, Petr; Morrell, N. W.; Hu, Ch.-J.; Stenmark, K. R.

    2017-01-01

    Roč. 136, č. 25 (2017), s. 2468-2485 ISSN 0009-7322 R&D Projects: GA MŠk(CZ) LH11055; GA MŠk(CZ) LH15071; GA ČR(CZ) GA16-04788S Institutional support: RVO:67985823 Keywords : hypoxia * metabolism * mitochondria * pyruvate kinase * shikonin * splicing factors * TEEP-46 Subject RIV: FC - Pulmology OBOR OECD: Respiratory systems Impact factor: 19.309, year: 2016

  2. Discovery and structure-activity relationship of novel 4-hydroxy-thiazolidine-2-thione derivatives as tumor cell specific pyruvate kinase M2 activators.

    Science.gov (United States)

    Li, Ridong; Ning, Xianling; Zhou, Shuo; Lin, Zhiqiang; Wu, Xingyu; Chen, Hong; Bai, Xinyu; Wang, Xin; Ge, Zemei; Li, Runtao; Yin, Yuxin

    2018-01-01

    Pyruvate kinase M2 isoform (PKM2) is a crucial protein responsible for aerobic glycolysis of cancer cells. Activation of PKM2 may alter aberrant metabolism in cancer cells. In this study, we discovered a 4-hydroxy-thiazolidine-2-thione compound 2 as a novel PKM2 activator from a random screening of an in-house compound library. Then a series of novel 4-hydroxy-thiazolidine-2-thione derivatives were designed and synthesized for screening as potent PKM2 activators. Among these, some compounds showed higher PKM2 activation activity than lead compound 2 and also exhibited significant anti-proliferative activities on human cancer cell lines at nanomolar concentration. The compound 5w was identified as the most potent antitumor agent, which showed excellent anti-proliferative effects with IC 50 values from 0.46 μM to 0.81 μM against H1299, HCT116, Hela and PC3 cell lines. 5w also showed less cytotoxicity in non-tumor cell line HELF compared with cancer cells. In addition, Preliminary pharmacological studies revealed that 5w arrests the cell cycle at the G2/M phase in HCT116 cell line. The best PKM2 activation by compound 5t was rationalized through docking studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high-saturated fat diet

    Science.gov (United States)

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A.

    2012-01-01

    SUMMARY Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) might prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it might induce detrimental effects by inhibiting fatty acid oxidation. PPARα agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment with a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild type and PDK4 knockout mice fed a high fat diet. As expected, treatment of wild type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, lowered blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid and a reduction in the capacity for fatty acid synthesis by PDK4 deficiency. PMID:22429297

  4. Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

    Science.gov (United States)

    Zhai, Zhengyuan; An, Haoran; Wang, Guohong; Luo, Yunbo; Hao, Yanling

    2015-11-19

    Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5'-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5'-TGTAAGCCCTAACA-3') upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.

  5. A systematic review and meta-analysis of the diagnostic accuracy of pyruvate kinase M2 isoenzymatic assay in diagnosing colorectal cancer.

    Science.gov (United States)

    Uppara, Mallikarjuna; Adaba, Franklin; Askari, Alan; Clark, Susan; Hanna, George; Athanasiou, Thanos; Faiz, Omar

    2015-02-13

    Screening programmes exist in many countries for colorectal cancer. In recent years, there has been a drive for a non-invasive screening marker of higher sensitivity and specificity. Stool-based pyruvate kinase isoenzyme M2 (M2-PK) is one such biomarker under investigation. The aim of this systematic review and meta-analysis is to determine the diagnostic accuracy, sensitivity and specificity of M2-PK as a screening tool in colorectal cancer. A literature search of Ovid Medline, EMBASE and Google Scholar was carried out. The search strategy was restricted to human subjects and studies published in English. Data on sensitivity and specificity were extracted and pooled. Statistical analysis was conducted using summary receiver operating characteristic (SROC) curve methodology. A total of eight studies were suitable for data synthesis and analysis. Our analysis showed a pooled sensitivity and specificity for M2-PK to be 79% (CI 73%-83%) and 80% (CI 73%-86%), respectively. The accuracy of M2-PK was 0.85(0.82-0.88). Faecal M2-PK assay has a relatively good sensitivity and specificity and high accuracy for screening colorectal cancer.

  6. 5´AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Lundsgaard, Annemarie; Jeppesen, Jacob

    2015-01-01

    after prolonged exercise and during the following six hours post exercise in 5´AMP activated protein kinase (AMPK)α2 and α1 knock-out (KO) and wild type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post...

  7. Small ubiquitin-like modifier 1 modification of pyruvate kinase M2 promotes aerobic glycolysis and cell proliferation in A549 human lung cancer cells

    Directory of Open Access Journals (Sweden)

    An S

    2018-04-01

    Full Text Available Shuxian An,1,* Liangqian Huang,2,3,* Ping Miao,1 Liang Shi,1 Mengqin Shen,1 Xiaoping Zhao,1 Jianjun Liu,1 Gang Huang1,3,4 1Department of Nuclear Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 2Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; 3Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine & Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 4Shanghai University of Medicine and Health Sciences, Shanghai, China *These authors contributed equally to this work Objective: Lung cancer is the leading cause of cancer-related death worldwide. Aerobic glycolysis is considered the seventh hallmark of cancer. The M2 isoform of pyruvate kinase (PKM2 is an important rate-limiting enzyme in glycolytic pathway, and is strongly expressed in several types of cancer. Thus, understanding the underlying mechanisms of regulation of PKM2 is of great value for targeted therapy for lung cancer.Patients and methods: Seventy-three lung adenocarcinoma patients were analyzed in our study. The expression levels of PKM2 were analyzed by immunohistochemistry on tissues. The effect of small ubiquitin-like modifier 1 (SUMO1 on PKM2 expression was investigated using Western blot assay and quantitative polymerase chain reaction. PKM2 SUMO1 modification was determined by in vitro and in vivo SUMOylation assays. 18F-deoxyglucose uptake and lactate production measurements were conducted to research the levels of glycolysis. The level of oxidative phosphorylation in cells was determined by cellular oxygen consumption rate measurements. Cell proliferation assays were carried out to confirm the growth ability of tumor cells.Results: PKM2 was overexpressed in lung adenocarcinoma patients based on immunohistochemical staining. Patients with high PKM2 expression had reduced

  8. Synergistic effects of baicalein with ciprofloxacin against NorA over-expressed methicillin-resistant Staphylococcus aureus (MRSA) and inhibition of MRSA pyruvate kinase.

    Science.gov (United States)

    Chan, Ben C L; Ip, Margaret; Lau, Clara B S; Lui, S L; Jolivalt, Claude; Ganem-Elbaz, Carine; Litaudon, Marc; Reiner, Neil E; Gong, Huansheng; See, Raymond H; Fung, K P; Leung, P C

    2011-09-01

    Baicalein, the active constituent derived from Scutellaria baicalensis Georgi., has previously been shown to significantly restore the effectiveness of β-lactam antibiotics and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA). With multiple therapeutic benefits, the antibacterial actions of baicalein may also be involved in overcoming other bacterial resistance mechanisms. The aim of the present study was to further investigate antibacterial activities of baicalein in association with various antibiotics against selected Staphylococcus aureus strains with known specific drug resistance mechanisms. A panel of clinical MRSA strains was used for further confirmation of the antibacterial activities of baicalein. The effect of baicalein on inhibiting the enzymatic activity of a newly discovered MRSA-specific pyruvate kinase (PK), which is essential for Staphylococcus aureus growth and survival was also examined. In the checkerboard dilution test and time-kill assay, baicalein at 16 μg/ml could synergistically restore the antibacterial actions of ciprofloxacin against the NorA efflux pump overexpressed SA-1199B, but not with the poor NorA substrate, pefloxacin. Moreover, synergistic effects were observed when baicalein was combined with ciprofloxacin against 12 out of 20 clinical ciprofloxacin resistant strains. For MRSA PK studies, baicalein alone could inhibit the enzymatic activity of MRSA PK in a dose-dependent manner. Our results demonstrated that baicalein could significantly reverse the ciprofloxacin resistance of MRSA possibly by inhibiting the NorA efflux pump in vitro. The inhibition of MRSA PK by baicalein could lead to a deficiency of ATP which might further contribute to the antibacterial actions of baicalein against MRSA. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

    Science.gov (United States)

    Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

    In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CS L ), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPC R ), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPC R (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CS L (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CS L mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD 660 ) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

  10. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high saturated fat diet.

    Science.gov (United States)

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A

    2012-05-01

    Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it may have detrimental effects by inhibiting fatty acid oxidation. Peroxisome proliferator-activated receptor α (PPARα) agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment using a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild-type and PDK4 knockout mice fed a high-fat diet. As expected, treatment of wild-type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, reduced blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid, and a reduction in the capacity for fatty acid synthesis as a result of PDK4 deficiency. Journal compilation © 2012 FEBS. No claim to original US government works.

  11. Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis

    Directory of Open Access Journals (Sweden)

    Tingjin Chen

    2017-11-01

    Full Text Available Abstract Background Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK, a crucial glycolytic enzyme, was characterized in this research. Results Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria, suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. Conclusions The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

  12. Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis.

    Science.gov (United States)

    Chen, Tingjin; Jiang, Hongye; Sun, Hengchang; Xie, Zhizhi; Ren, Pengli; Zhao, Lu; Dong, Huimin; Shi, Mengchen; Lv, Zhiyue; Wu, Zhongdao; Li, Xuerong; Yu, Xinbing; Huang, Yan; Xu, Jin

    2017-11-09

    Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research. Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

  13. Both high expression of pyruvate kinase M2 and vascular endothelial growth factor-C predicts poorer prognosis in human breast cancer.

    Science.gov (United States)

    Lin, Yang; Liu, Fangfang; Fan, Yu; Qian, Xiaolong; Lang, Ronggang; Gu, Feng; Gu, Jun; Fu, Li

    2015-01-01

    Pyruvate kinase M2 (PKM2) and vascular endothelial growth factor-C (VEGF-C) have been known to play an important role in tumorigenesis and tumor progression in breast cancer. However, the association between PKM2 and VEGF-C in breast cancer remains unclear. In the present study, a total of 218 specimens from breast cancer patients and 26 paired breast tumors with adjacent normal tissues as well as two breast cancer cell lines were enrolled to investigate the correlation between PKM2 and VEGF-C. We found that PKM2 and VEGF-C mRNA levels were both significantly increasing in breast tumors compared with adjacent normal tissues. Knockdown of PKM2 mRNA expression resulted in VEGF-C mRNA and protein down-regulated as well as cell proliferation inhibited. A positive correlation between PKM2 and VEGF-C expression was identified by immunohistochemical analyses of 218 specimens of patients with breast cancer (P=0.023). PKM2 high expression was significantly correlated with histological grade (P=0.030), lymph node stage (P=0.001), besides VEGF-C high expression was significantly associated with lymphovascular invasion (P=0.012). While combined high expression of PKM2 and VEGF-C was found to be associated with worse histological grade, more lymph node metastasis, more lymphovascular invasion, shorter progression free survival (PFS), and poorer overall survival (OS) in human breast cancer. The results of the present study suggested that PKM2 expression was correlated with VEGF-C expression, and combination of PKM2 and VEGF-C levels had the better prognostic significance in predicting the poor outcome of patients with breast cancer.

  14. ­Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression

    Directory of Open Access Journals (Sweden)

    Amanda M.S. Mattice

    2018-06-01

    Full Text Available Background Pyruvate kinase (PK is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. Methods To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (Trachemys scripta elegans. Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Results Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver and lysine methylation (by 43% in muscle and 70% in liver during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Discussion Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia.

  15. ­Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression

    Science.gov (United States)

    2018-01-01

    Background Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. Methods To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (Trachemys scripta elegans). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Results Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Discussion Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia. PMID:29900073

  16. -Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression.

    Science.gov (United States)

    Mattice, Amanda M S; MacLean, Isabelle A; Childers, Christine L; Storey, Kenneth B

    2018-01-01

    Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles ( Trachemys scripta elegans ). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I 50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I 50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia.

  17. Pyruvate Kinase M2 Is Required for the Expression of the Immune Checkpoint PD-L1 in Immune Cells and Tumors

    Directory of Open Access Journals (Sweden)

    Eva M. Palsson-McDermott

    2017-10-01

    Full Text Available Blocking interaction of the immune checkpoint receptor PD-1 with its ligand PD-L1 is associated with good clinical outcomes in a broad variety of malignancies. High levels of PD-L1 promote tumor growth by restraining CD8+ T-cell responses against tumors. Limiting PD-L1 expression and function is therefore critical for allowing the development of antitumor immune responses and effective tumor clearance. Pyruvate kinase isoform M2 (PKM2 is also a key player in regulating cancer as well as immune responses. PKM2 catalyzes the final rate-limiting step of glycolysis. Furthermore, PKM2 as a dimer translocates to the nucleus, where it stimulates hypoxia-inducible factor 1α (Hif-1α transactivation domain function and recruitment of p300 to the hypoxia response elements (HRE of Hif-1α target genes. Here, we provide the first evidence of a role for PKM2 in regulating the expression of PD-L1 on macrophages, dendritic cells (DCs, T cells, and tumor cells. LPS-induced expression of PD-L1 in primary macrophages was inhibited by the PKM2 targeting compound TEPP-46. Furthermore, RNA silencing of PKM2 inhibited LPS-induced PD-L1 expression. This regulation occurs through direct binding of PKM2 and Hif-1α to HRE sites on the PD-L1 promoter. Moreover, TEPP-46 inhibited expression of PD-L1 on macrophages, DCs, and T cells as well as tumor cells in a mouse CT26 cancer model. These findings broaden our understanding of how PKM2 may contribute to tumor progression and may explain the upregulation of PD-L1 in the tumor microenvironment.

  18. Caloric Restriction Mimetic 2-Deoxyglucose Alleviated Inflammatory Lung Injury via Suppressing Nuclear Pyruvate Kinase M2–Signal Transducer and Activator of Transcription 3 Pathway

    Directory of Open Access Journals (Sweden)

    Kai Hu

    2018-03-01

    Full Text Available Inflammation is an energy-intensive process, and caloric restriction (CR could provide anti-inflammatory benefits. CR mimetics (CRM, such as the glycolytic inhibitor 2-deoxyglucose (2-DG, mimic the beneficial effects of CR without inducing CR-related physiologic disturbance. This study investigated the potential anti-inflammatory benefits of 2-DG and the underlying mechanisms in mice with lipopolysaccharide (LPS-induced lethal endotoxemia. The results indicated that pretreatment with 2-DG suppressed LPS-induced elevation of tumor necrosis factor alpha and interleukin 6. It also suppressed the upregulation of myeloperoxidase, attenuated Evans blue leakage, alleviated histological abnormalities in the lung, and improved the survival of LPS-challenged mice. Treatment with 2-DG had no obvious effects on the total level of pyruvate kinase M2 (PKM2, but it significantly suppressed LPS-induced elevation of PKM2 in the nuclei. Prevention of PKM2 nuclear accumulation by ML265 mimicked the anti-inflammatory benefits of 2-DG. In addition, treatment with 2-DG or ML265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3. Inhibition of STAT3 by stattic suppressed LPS-induced inflammatory injury. Interestingly, posttreatment with 2-DG at the early stage post-LPS challenge also improved the survival of the experimental animals. This study found that treatment with 2-DG, a representative CRM, provided anti-inflammatory benefits in lethal inflammation. The underlying mechanisms included suppressed nuclear PKM2-STAT3 pathway. These data suggest that 2-DG might have potential value in the early intervention of lethal inflammation.

  19. Caloric Restriction Mimetic 2-Deoxyglucose Alleviated Inflammatory Lung Injury via Suppressing Nuclear Pyruvate Kinase M2-Signal Transducer and Activator of Transcription 3 Pathway.

    Science.gov (United States)

    Hu, Kai; Yang, Yongqiang; Lin, Ling; Ai, Qing; Dai, Jie; Fan, Kerui; Ge, Pu; Jiang, Rong; Wan, Jingyuan; Zhang, Li

    2018-01-01

    Inflammation is an energy-intensive process, and caloric restriction (CR) could provide anti-inflammatory benefits. CR mimetics (CRM), such as the glycolytic inhibitor 2-deoxyglucose (2-DG), mimic the beneficial effects of CR without inducing CR-related physiologic disturbance. This study investigated the potential anti-inflammatory benefits of 2-DG and the underlying mechanisms in mice with lipopolysaccharide (LPS)-induced lethal endotoxemia. The results indicated that pretreatment with 2-DG suppressed LPS-induced elevation of tumor necrosis factor alpha and interleukin 6. It also suppressed the upregulation of myeloperoxidase, attenuated Evans blue leakage, alleviated histological abnormalities in the lung, and improved the survival of LPS-challenged mice. Treatment with 2-DG had no obvious effects on the total level of pyruvate kinase M2 (PKM2), but it significantly suppressed LPS-induced elevation of PKM2 in the nuclei. Prevention of PKM2 nuclear accumulation by ML265 mimicked the anti-inflammatory benefits of 2-DG. In addition, treatment with 2-DG or ML265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 by stattic suppressed LPS-induced inflammatory injury. Interestingly, posttreatment with 2-DG at the early stage post-LPS challenge also improved the survival of the experimental animals. This study found that treatment with 2-DG, a representative CRM, provided anti-inflammatory benefits in lethal inflammation. The underlying mechanisms included suppressed nuclear PKM2-STAT3 pathway. These data suggest that 2-DG might have potential value in the early intervention of lethal inflammation.

  20. Microorganisms and methods for producing pyruvate, ethanol, and other compounds

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Jennifer L.; Zhang, Xiaolin

    2017-12-26

    Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

  1. Phosphorylation site on yeast pyruvate dehydrogenase complex

    International Nuclear Information System (INIS)

    Uhlinger, D.J.

    1986-01-01

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

  2. Combining structure-based pharmacophore modeling, virtual screening, and in silico ADMET analysis to discover novel tetrahydro-quinoline based pyruvate kinase isozyme M2 activators with antitumor activity

    Directory of Open Access Journals (Sweden)

    Chen C

    2014-09-01

    Full Text Available Can Chen,1,2,* Ting Wang,1,3,* Fengbo Wu,1,* Wei Huang,4 Gu He,1 Liang Ouyang,1 Mingli Xiang,1 Cheng Peng,4 Qinglin Jiang1,2 1State Key Laboratory of Biotherapy and Department of Pharmacy, West China Hospital, Sichuan University, Chengdu, 2College of Pharmacy and the First Affiliated Hospital, Chengdu Medical College, Chengdu, 3Department of Cardiology, Genenal Hospital of Chengdu Military Command, Chengdu, 4State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China*These authors contributed equally to this workAbstract: Compared with normal differentiated cells, cancer cells upregulate the expression of pyruvate kinase isozyme M2 (PKM2 to support glycolytic intermediates for anabolic processes, including the synthesis of nucleic acids, amino acids, and lipids. In this study, a combination of the structure-based pharmacophore modeling and a hybrid protocol of virtual screening methods comprised of pharmacophore model-based virtual screening, docking-based virtual screening, and in silico ADMET (absorption, distribution, metabolism, excretion and toxicity analysis were used to retrieve novel PKM2 activators from commercially available chemical databases. Tetrahydroquinoline derivatives were identified as potential scaffolds of PKM2 activators. Thus, the hybrid virtual screening approach was applied to screen the focused tetrahydroquinoline derivatives embedded in the ZINC database. Six hit compounds were selected from the final hits and experimental studies were then performed. Compound 8 displayed a potent inhibitory effect on human lung cancer cells. Following treatment with Compound 8, cell viability, apoptosis, and reactive oxygen species (ROS production were examined in A549 cells. Finally, we evaluated the effects of Compound 8 on mice xenograft tumor models in vivo. These results may provide important

  3. Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension Is Regulated Through a MicroRNA-124/PTBP1 (Polypyrimidine Tract Binding Protein 1)/Pyruvate Kinase Muscle Axis.

    Science.gov (United States)

    Zhang, Hui; Wang, Daren; Li, Min; Plecitá-Hlavatá, Lydie; D'Alessandro, Angelo; Tauber, Jan; Riddle, Suzette; Kumar, Sushil; Flockton, Amanda; McKeon, B Alexandre; Frid, Maria G; Reisz, Julie A; Caruso, Paola; El Kasmi, Karim C; Ježek, Petr; Morrell, Nicholas W; Hu, Cheng-Jun; Stenmark, Kurt R

    2017-12-19

    An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial

  4. Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

    Science.gov (United States)

    Caruso, Paola; Dunmore, Benjamin J; Schlosser, Kenny; Schoors, Sandra; Dos Santos, Claudia; Perez-Iratxeta, Carol; Lavoie, Jessie R; Zhang, Hui; Long, Lu; Flockton, Amanda R; Frid, Maria G; Upton, Paul D; D'Alessandro, Angelo; Hadinnapola, Charaka; Kiskin, Fedir N; Taha, Mohamad; Hurst, Liam A; Ormiston, Mark L; Hata, Akiko; Stenmark, Kurt R; Carmeliet, Peter; Stewart, Duncan J; Morrell, Nicholas W

    2017-12-19

    Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 ( BMPR2 ) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1 , and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Pulmonary vascular and

  5. Lack of association between PKLR rs3020781 and NOS1AP rs7538490 and type 2 diabetes, overweight, obesity and related metabolic phenotypes in a Danish large-scale study: case-control studies and analyses of quantitative traits

    DEFF Research Database (Denmark)

    Andreasen, Camilla Helene; Mogensen, Mette S.; Borch-Johnsen, Knut

    2008-01-01

    of the variants in relation to overweight and obesity. METHODS: PKLR rs3020781 and NOS1AP rs7538490 were genotyped, using TaqMan allelic discrimination, in a combined study sample comprising a total of 16,801 and 16,913 individuals, respectively. The participants were ascertained from four different study groups...

  6. Pyruvate metabolism: A therapeutic opportunity in radiation-induced skin injury

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hyun; Kang, Jeong Wook [Department of Radiation Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Lee, Dong Won [Department of Plastic Surgery, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Oh, Sang Ho [Department of Dermatology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Lee, Yun-Sil [College of Pharmacy & Division of Life and Pharmaceutical Sciences, Ewah Womans University, Seoul 120-750 (Korea, Republic of); Lee, Eun-Jung [Department of Radiation Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Cho, Jaeho, E-mail: jjhmd@yuhs.ac [Department of Radiation Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

    2015-05-08

    Ionizing radiation is used to treat a range of cancers. Despite recent technological progress, radiation therapy can damage the skin at the administration site. The specific molecular mechanisms involved in this effect have not been fully characterized. In this study, the effects of pyruvate, on radiation-induced skin injury were investigated, including the role of the pyruvate dehydrogenase kinase 2 (PDK2) signaling pathway. Next generation sequencing (NGS) identified a wide range of gene expression differences between the control and irradiated mice, including reduced expression of PDK2. This was confirmed using Q-PCR. Cell culture studies demonstrated that PDK2 overexpression and a high cellular pyruvate concentration inhibited radiation-induced cytokine expression. Immunohistochemical studies demonstrated radiation-induced skin thickening and gene expression changes. Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness and inflammatory cytokine expression. These findings indicated that regulation of the pyruvate metabolic pathway could provide an effective approach to the control of radiation-induced skin damage. - Highlights: • The effects of radiation on skin thickness in mice. • Next generation sequencing revealed that radiation inhibited pyruvate dehydrogenase kinase 2 expression. • PDK2 inhibited irradiation-induced cytokine gene expression. • Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness.

  7. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    Science.gov (United States)

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

  8. PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy.

    Science.gov (United States)

    Liu, Laura X; Rowe, Glenn C; Yang, Steven; Li, Jian; Damilano, Federico; Chan, Mun Chun; Lu, Wenyun; Jang, Cholsoon; Wada, Shogo; Morley, Michael; Hesse, Michael; Fleischmann, Bernd K; Rabinowitz, Joshua D; Das, Saumya; Rosenzweig, Anthony; Arany, Zoltan

    2017-12-08

    Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations, are not well understood. To determine the mechanisms underlying cardiac substrate use during pregnancy. We use here 13 C glucose, 13 C lactate, and 13 C fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not seem altered. Insulin signaling seems intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 (cluster of differentiation 36) and GLUT4 (glucose transporter type 4), are also unchanged. Rather, we find that the pregnancy hormone progesterone induces PDK4 (pyruvate dehydrogenase kinase 4) in cardiomyocytes and that elevated PDK4 levels in late pregnancy lead to inhibition of PDH (pyruvate dehydrogenase) and pyruvate flux into the tricarboxylic acid cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy. Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling. © 2017 American Heart Association, Inc.

  9. Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase

    Energy Technology Data Exchange (ETDEWEB)

    Noce, P S; Utter, M F

    1975-01-01

    Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn/sup 2 +/ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn/sup 2 +/, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.

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

    Science.gov (United States)

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

    2011-11-01

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

  11. Pyruvate reduces 4-aminophenol in vitro toxicity

    International Nuclear Information System (INIS)

    Harmon, R. Christopher; Kiningham, Kinsley K.; Valentovic, Monica A.

    2006-01-01

    Pyruvate has been observed to reduce the nephrotoxicity of some agents by maintaining glutathione status and preventing lipid peroxidation. This study examined the mechanism for pyruvate protection of p-aminophenol (PAP) nephrotoxicity. Renal cortical slices from male Fischer 344 rats were incubated for 30-120 min with 0, 0.1, 0.25 or 0.5 mM PAP in oxygenated Krebs buffer containing 0 or 10 mM pyruvate or glucose (1.28 or 5.5 mM). LDH leakage was increased above control by 0.25 and 0.5 mM PAP beginning at 60 min and by 0.1 mM PAP at 120 min. Pyruvate prevented an increase in LDH leakage at 60- and 120-min exposure to 0.1 and 0.25 mM PAP. Pyruvate also prevented a decline in ATP levels. Glucose (1.28 and 5.5 mM) provided less protection than pyruvate from PAP toxicity. Total glutathione levels were diminished by 0.1 and 0.25 mM PAP within 60 and 30 min, respectively. Pyruvate prevented the decline in glutathione by 0.1 mM PAP at both time periods and at 30 min for 0.25 mM PAP. Pyruvate reduced the magnitude of glutathione depletion by 0.25 mM PAP following a 60-min incubation. Glutathione disulfide (GSSG) levels in renal slices were increased at 60 min by exposure to 0.25 mM PAP, while pyruvate prevented increased GSSG levels by PAP. Pyruvate also reduced the extent of 4-hydroxynonenal (4-HNE)-adducted proteins present after a 90-min incubation with PAP. These results indicate that pyruvate provided protection for PAP toxicity by providing an energy substrate and reducing oxidative stress

  12. Dynamics of pyruvate metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Melchiorsen, Claus Rix; Jensen, Niels B.S.; Christensen, Bjarke

    2001-01-01

    The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end...... product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis...

  13. Cultivation of parasitic leptospires: effect of pyruvate.

    Science.gov (United States)

    Johnson, R C; Walby, J; Henry, R A; Auran, N E

    1973-07-01

    Sodium pyruvate (100 mug/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes.

  14. Pyruvate transport by thermogenic-tissue mitochondria.

    OpenAIRE

    Proudlove, M O; Beechey, R B; Moore, A L

    1987-01-01

    1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced...

  15. “Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

    Science.gov (United States)

    The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated...

  16. Ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of the HMGB1–RAGE and AKT pathways

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ping; Dai, Weiqi; Wang, Fan; Lu, Jie; Shen, Miao; Chen, Kan; Li, Jingjing; Zhang, Yan; Wang, Chengfen; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Guo, Chuan-Yong, E-mail: guochuanyong@hotmail.com; Xu, Ling, E-mail: xuling606@sina.com

    2014-01-24

    Highlights: • Ethyl pyruvate inhibits liver cancer. • Promotes apoptosis. • Decreased the expression of HMGB1, p-Akt. - Abstract: Ethyl pyruvate (EP) was recently identified as a stable lipophilic derivative of pyruvic acid with significant antineoplastic activities. The high mobility group box-B1 (HMGB1)–receptor for advanced glycation end-products (RAGE) and the protein kinase B (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. We tried to observe the effects of ethyl pyruvate on liver cancer growth and explored its effects in hepatocellular carcinoma model. In this study, three hepatocellular carcinoma cell lines were treated with ethyl pyruvate. An MTT colorimetric assay was used to assess the effects of EP on cell proliferation. Flow cytometry and TUNEL assays were used to analyze apoptosis. Real-time PCR, Western blotting and immunofluorescence demonstrated ethyl pyruvate reduced the HMGB1–RAGE and AKT pathways. The results of hepatoma orthotopic tumor model verified the antitumor effects of ethyl pyruvate in vivo. EP could induce apoptosis and slow the growth of liver cancer. Moreover, EP decreased the expression of HMGB1, RAGE, p-AKT and matrix metallopeptidase-9 (MMP9) and increased the Bax/Bcl-2 ratio. In conclusion, this study demonstrates that ethyl pyruvate induces apoptosis and cell-cycle arrest in G phase in hepatocellular carcinoma cells, plays a critical role in the treatment of cancer.

  17. Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.

    Science.gov (United States)

    Park, Jae Mo; Josan, Sonal; Jang, Taichang; Merchant, Milton; Watkins, Ron; Hurd, Ralph E; Recht, Lawrence D; Mayer, Dirk; Spielman, Daniel M

    2016-03-01

    MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate. © 2015 Wiley Periodicals, Inc.

  18. Role of pyruvate dehydrogenase inhibition in the development of hypertrophy in the hyperthyroid rat heart: a combined magnetic resonance imaging and hyperpolarized magnetic resonance spectroscopy study.

    Science.gov (United States)

    Atherton, Helen J; Dodd, Michael S; Heather, Lisa C; Schroeder, Marie A; Griffin, Julian L; Radda, George K; Clarke, Kieran; Tyler, Damian J

    2011-06-07

    Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output. This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic

  19. Cultivation of Parasitic Leptospires: Effect of Pyruvate

    Science.gov (United States)

    Johnson, R. C.; Walby, J.; Henry, R. A.; Auran, N. E.

    1973-01-01

    Sodium pyruvate (100 μg/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes. Images PMID:4580191

  20. Scanning mutagenesis of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

    Directory of Open Access Journals (Sweden)

    Nagib eAhsan

    2012-07-01

    Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

  1. Compartmented pyruvate in perfused working heart

    International Nuclear Information System (INIS)

    Buenger, R.

    1985-01-01

    Pyruvate compartmentation and lactate dehydrogenase (LDH) were studied in isolated perfused working guinea pig hearts. The mean intracellular pyruvate (Pyr) contents increased with perfusate Pyr (0-2 mM) but varied only slightly with glucose (0-10 mM) and additional insulin (0.04-5 U/l), respectively. With 5-10 mM glucose plus 5 U/l insulin, but not with Pyr or lactate (Lac) as substrates, a near equilibrium between the LDH and the glycerol-3-phosphate dehydrogenase seemed to exist. Evidence for an inhibitory effect of Pyr on the activity of the LDH system of the perfused hearts was not obtained. With [U- 14 C]glucose as sole substrate, the specific activity of coronary venous Lac was near half that of precursor glucose. 14 CO 2 production was thus in quantitative agreement with rates of pyruvate oxidation that were determined as glucose uptake minus (Pyr + Lac) release. In contrast, with 0.2 mM [1- 14 C]Pyr plus 5 mM glucose, the ratio of 14 CO 2 production to specific activity of Lac overestimated Pyr oxidation judged from myocardial substrate balances and O 2 uptake, respectively; here, at least three pools of [ 14 C]HCO-3 and [ 14 C]lac, respectively, were kinetically demonstrable during washout of trace amounts of 14 C-labeled Pyr. Evidently, the specific activity of Lac was equivalent to that of mitochondrial oxidized Pyr provided [ 14 C]glucose was the sole or major precursor of cellular pyruvate. However, exogenously applied [1- 14 C]Pyr of high specific activity seemed to induce intracellular formation of both a highly and lowly labeled Pyr; the latter Pyr compartment did not seem in ready equilibrium with the cell physiologically prevailing highly labeled Pyr pool

  2. Enterococcus faecalis phosphomevalonate kinase

    Science.gov (United States)

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni++ affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37°C. The activation energy was ~5.6 kcal/mol. Activity with Mn++, the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). Km values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 μmol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed. PMID:15802646

  3. Loss of Mitochondrial Pyruvate Carrier 2 in Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling

    Science.gov (United States)

    McCommis, Kyle S.; Chen, Zhouji; Fu, Xiaorong; McDonald, William G.; Colca, Jerry R.; Kletzien, Rolf F.; Burgess, Shawn C.; Finck, Brian N.

    2015-01-01

    SUMMARY Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite step for gluconeogenesis in hepatocytes, which is important for maintenance of normoglycemia during prolonged food deprivation, but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2−/−) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte pyruvate metabolism, labelled pyruvate conversion to TCA cycle intermediates and glucose, and glucose production from pyruvate. Unbiased metabolomic analyses of livers from fasted LS-Mpc2−/− mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2−/− hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import. PMID:26344101

  4. Ethyl pyruvate protects colonic anastomosis from ischemia-reperfusion injury.

    Science.gov (United States)

    Unal, B; Karabeyoglu, M; Huner, T; Canbay, E; Eroglu, A; Yildirim, O; Dolapci, M; Bilgihan, A; Cengiz, O

    2009-03-01

    Ethyl pyruvate is a simple derivative in Ca(+2)- and K(+)-containing balanced salt solution of pyruvate to avoid the problems associated with the instability of pyruvate in solution. It has been shown to ameliorate the effects of ischemia-reperfusion (I/R) injury in many organs. It has also been shown that I/R injury delays the healing of colonic anastomosis. In this study, the effect of ethyl pyruvate on the healing of colon anastomosis and anastomotic strength after I/R injury was investigated. Anastomosis of the colon was performed in 32 adult male Wistar albino rats divided into 4 groups of 8 individuals: (1) sham-operated control group (group 1); (2) 30 minutes of intestinal I/R by superior mesenteric artery occlusion (group 2); (3) I/R+ ethyl pyruvate (group 3), ethyl pyruvate was administered as a 50-mg/kg/d single dose; and (4) I/R+ ethyl pyruvate (group 4), ethyl pyruvate administration was repeatedly (every 6 hours) at the same dose (50 mg/kg). On the fifth postoperative day, animals were killed. Perianastomotic tissue hydroxyproline contents and anastomotic bursting pressures were measured in all groups. When the anastomotic bursting pressures and tissue hydroxyproline contents were compared, it was found that they were decreased in group 2 when compared with groups 1, 3, and 4 (P .05). Ethyl pyruvate significantly prevents the delaying effect of I/R injury on anastomotic strength and healing independent from doses of administration.

  5. Pyruvate carboxylase is expressed in human skeletal muscle

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable...

  6. Production and Recovery of Pyruvic Acid: Recent Advances

    Science.gov (United States)

    Pal, Dharm; Keshav, Amit; Mazumdar, Bidyut; Kumar, Awanish; Uslu, Hasan

    2017-12-01

    Pyruvic acid is an important keto-carboxylic acid and can be manufactured by both chemical synthesis and biotechnological routes. In the present paper an overview of recent developments and challenges in various existing technique for the production and recovery of pyruvic acid from fermentation broth or from waste streams has been presented. The main obstacle in biotechnological production of pyruvic acid is development of suitable microorganism which can provide high yield and selectivity. On the other hand, technical limitation in recovery of pyruvic acid from fermentation broth is that, it could not be separated as other carboxylic acid in the form of salts by addition of alkali. Besides, pyruvic acid cannot be crystallized. Commercial separation by distillation is very expensive because pyruvic acid decomposes at higher temperature. It is also chemically reactive due to its peculiar molecular structure and has tendency to polymerize. Thus, at high concentration the various type of reaction leads to lower yield of the product, and hence, conventional methods are not favorable. Alternate separation technologies viable to both synthetic and biological routes are the current research areas. Latest techniques such as reactive extraction is new to the field of recovery of pyruvic acid. Recent development and future prospects in downstream processing of biochemically produced pyruvic acids has been discussed in this review article.

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

    International Nuclear Information System (INIS)

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

    1980-01-01

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

  8. Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle

    DEFF Research Database (Denmark)

    Haagensen, L.; Jensen, D.H.; Gesser, Hans

    2008-01-01

    The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the pyr......The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP...... by the pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout...... myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase...

  9. Cerebrospinal fluid lactate and pyruvate concentrations and their ratio.

    Science.gov (United States)

    Zhang, Wan-Ming; Natowicz, Marvin R

    2013-05-01

    Determinations of cerebrospinal fluid (CSF) lactate and pyruvate concentrations and CSF lactate:pyruvate (L/P) ratios are important in several clinical settings, yet published normative data have significant limitations. We sought to determine a large dataset of stringently-defined normative data for CSF lactate and pyruvate concentrations and CSF L/P ratios. We evaluated data from 627 patients who had determinations of CSF lactate and/or CSF pyruvate from 2001 to 2011 at the Cleveland Clinic. Inclusion in the normal reference population required normal CSF cell counts, glucose and protein and routine serum chemistries and absence of progressive brain disorder, epilepsy, or seizure within 24h. Brain MRI, if done, showed no evidence of tumor, acute changes or basal ganglia abnormality. CSF cytology, CSF alanine and immunoglobulin levels, and oligoclonal band analysis were required to be normal, if done. Various inclusion/exclusion criteria were compared. 92 patients fulfilled inclusion/exclusion criteria for a reference population. The 95% central intervals (2.5%-97.5%) for CSF lactate and pyruvate levels were 1.01-2.09mM and 0.03-0.15mM, respectively, and 9.05-26.37 for CSF L/P. There were no significant gender-related differences of CSF lactate or pyruvate concentrations or of CSF L/P. Weak positive correlations between the concentration of CSF lactate or pyruvate and age were noted. Using stringent inclusion/exclusion criteria, we determined normative data for CSF lactate and pyruvate concentrations and CSF L/P ratios in a large, well-characterized reference population. Normalcy of routine CSF and blood analytes are the most important parameters in determining reference intervals for CSF lactate and pyruvate. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  10. Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

    Directory of Open Access Journals (Sweden)

    Alba Timón-Gómez

    Full Text Available Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

  11. Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

    Science.gov (United States)

    Timón-Gómez, Alba; Proft, Markus; Pascual-Ahuir, Amparo

    2013-01-01

    Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

  12. Effects of IL-6 on pyruvate dehydrogenase regulation in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Biensø, Rasmus Sjørup; Knudsen, Jakob Grunnet; Brandt, Nina

    2014-01-01

    Skeletal muscle regulates substrate choice according to demand and availability and pyruvate dehydrogenase (PDH) is central in this regulation. Circulating interleukin (IL)-6 increases during exercise and IL-6 has been suggested to increase whole body fat oxidation. Furthermore, IL-6 has been...... reported to increase AMP-activated protein kinase (AMPK) phosphorylation and AMPK suggested to regulate PDHa activity. Together, this suggests that IL-6 may be involved in regulating PDH. The aim of this study was to investigate the effect of a single injection of IL-6 on PDH regulation in skeletal muscle...... in fed and fasted mice. Fed and 16-18 h fasted mice were injected with either 3 ng · g(-1) recombinant mouse IL-6 or PBS as control. Fasting markedly reduced plasma glucose, muscle glycogen, muscle PDHa activity, as well as increased PDK4 mRNA and protein content in skeletal muscle. IL-6 injection did...

  13. In vitro bioconversion of chitin to pyruvate with thermophilic enzymes.

    Science.gov (United States)

    Honda, Kohsuke; Kimura, Keisuke; Ninh, Pham Huynh; Taniguchi, Hironori; Okano, Kenji; Ohtake, Hisao

    2017-09-01

    Chitin is the second most abundant organic compound on the planet and thus has been regarded as an alternative resource to petroleum feedstocks. One of the key challenges in the biological conversion of biomass-derived polysaccharides, such as cellulose and chitin, is to close the gap between optimum temperatures for enzymatic saccharification and microbial fermentation and to implement them in a single bioreactor. To address this issue, in the present study, we aimed to perform an in vitro, one-pot bioconversion of chitin to pyruvate, which is a precursor of a wide range of useful metabolites. Twelve thermophilic enzymes, including that for NAD + regeneration, were heterologously produced in Escherichia coli and semi-purified by heat treatment of the crude extract of recombinant cells. When the experimentally decided concentrations of enzymes were incubated with 0.5 mg mL -1 colloidal chitin (equivalent to 2.5 mM N-acetylglucosamine unit) and an adequate set of cofactors at 70°C, 0.62 mM pyruvate was produced in 5 h. Despite the use of a cofactor-balanced pathway, determination of the pool sizes of cofactors showed a rapid decrease in ATP concentration, most probably due to the thermally stable ATP-degrading enzyme(s) derived from the host cell. Integration of an additional enzyme set of thermophilic adenylate kinase and polyphosphate kinase led to the deceleration of ATP degradation, and the final product titer was improved to 2.1 mM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids

    DEFF Research Database (Denmark)

    Mourtzakis, Marina; Saltin, B.; Graham, T.

    2006-01-01

    During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline...... amino acid taken up during exercise and recovery. Alanine and glutamine were also associated...... with pyruvate metabolism, and they comprised 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism...

  15. Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

    Science.gov (United States)

    Jitrapakdee, S; Vidal-Puig, A; Wallace, J C

    2006-04-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

  16. Exogenous pyruvate facilitates cancer cell adaptation to hypoxia by serving as an oxygen surrogate.

    Science.gov (United States)

    Yin, Chengqian; He, Dan; Chen, Shuyang; Tan, Xiaoling; Sang, Nianli

    2016-07-26

    Molecular oxygen is the final electron acceptor in cellular metabolism but cancer cells often become adaptive to hypoxia, which promotes resistance to chemotherapy and radiation. The reduction of endogenous glycolytic pyruvate to lactate is known as an adaptive strategy for hypoxic cells. Whether exogenous pyruvate is required for hypoxic cell proliferation by either serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxic and ρ0 cells defective in electron transfer chain, we show that exogenous pyruvate is required to sustain proliferation of both cancer and non-cancer cells that cannot utilize oxygen. Particularly, we show that absence of pyruvate led to glycolysis inhibition and AMPK activation along with decreased NAD+ levels in ρ0 cells; and exogenous pyruvate increases lactate yield, elevates NAD+/NADH ratio and suppresses AMPK activation. Knockdown of lactate dehydrogenase significantly inhibits the rescuing effects of exogenous pyruvate. In contrast, none of pyruvate-derived metabolites tested (including acetyl-CoA, α-ketoglutarate, succinate and alanine) can replace pyruvate in supporting ρ0 cell proliferation. Knockdown of pyruvate carboxylase, pyruvate dehydrogenase and citrate synthase do not impair exogenous pyruvate to rescue ρ0 cells. Importantly, we show that exogenous pyruvate relieves ATP insufficiency and mTOR inhibition and promotes proliferation of hypoxic cells, and that well-oxygenated cells release pyruvate, providing a potential in vivo source of pyruvate. Taken together, our data support a novel pyruvate cycle model in which oxygenated cells release pyruvate for hypoxic cells as an oxygen surrogate. The pyruvate cycle may be targeted as a new therapy of hypoxic cancers.

  17. Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Møller, Kasper; Langkjær, Rikke Breinhold; Nielsen, Jens

    2004-01-01

    was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts...... is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae...

  18. Characterization of a C 4 maize pyruvate orthophosphate dikinase ...

    African Journals Online (AJOL)

    Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in plants that utilize the C4 photosynthetic pathway to fix CO2. The enzymatic reaction catalyzed by PPDK is critically controlled by light and is one of the rate-limiting steps of the C4 pathway. The intact maize (Zea mays) C4-PPDK gene, containing its own promoter, ...

  19. Amperometric pyruvate sensor based on a pyruvate dehydrogenase-immobilized carbon paste electrode containing vitamin K3 as a mediator

    Energy Technology Data Exchange (ETDEWEB)

    Miki, K. [Nara National College of Technology, Nara (Japan); Kinoshita, H. [Kawassui Women`s College, Nagasaki (Japan); Yamamoto, Y. [Kyoto Municipal Junior College of Nursing, Kyoto (Japan); Taniguchi, N. [Kyoto Research Center for Hygiene, Kyoto (Japan); Ikeda, T. [Kyoto University, Kyoto (Japan). Faculty of Agriculture

    1995-12-05

    Pyruvate dehydrogenase (PDH) was immobilized on the surface of a carbon paste electrode containing vitamin K3 (2-Methyl-1,4-naphthoquinone, VK), and the electrode surface was covered with a dialysis membrane. The enzyme electrode produced an anodic current starting from -0.2 V to reach a limiting current at +0.1 V vs. Ag/AgCl due to the enzyme-catalyzed oxidation of pyruvate in a phosphate buffer solution of pH 7.0. The current response to pyruvate depended on the amounts of both the immobilized-PDH and VK mixed in the carbon paste electrode at low amount of the enzyme and VK, and became independent at above 0.15 mg PDH and 0.65% (w/w) VK. The electrode with 0.15mg PDH and 0.65% (w/w) VK could be used as a pyruvate sensor to measure in the range of 2 ,{mu}M to 3mM. The response time was about 60 sec, and the current was independent of pH in the range of 5.7 - 7.2. The presence of L-ascorbic acid didn`t interfere with this measurement. Phosphate ion could also be determined with this electrode in a citrate buffer solution. 14 refs., 6 figs., 1 tab.

  20. Enzymatic synthesis of 11C-pyruvic acid and 11C-L-lactic acid

    International Nuclear Information System (INIS)

    Cohen, M.B.; Spolter, L.; Chang, C.C.; Cook, J.S.; Macdonald, N.S.

    1980-01-01

    L-Lactic acid is formed as the end product of glycolysis under anaerobic conditions in all cells, but this reaction is of special significance in the myocardium. L-Lactic acid is reversibly formed from and is in equilibrium with myocardial pyruvic acid, which is its sole metabolic pathway. 11 C-Pyruvic acid is synthesized from 11 C carbon dioxide using pyruvate-ferredoxin oxidoreductase and coenzymes. The 11 C-pyruvic acid is then converted to 11 -L-lactic acid by lactic acid dehydrogenase. The availability of 11 C-pyruvic acid and 11 C-L-lactic acid will permit the in vivo investigation of lactate metabolism. (author)

  1. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    The present review on casein kinases focuses mainly on the possible metabolic role of CK-2, with special emphasis on its behavior in pathological tissues. From these data at least three ways to regulate CK-2 activity emerge: (i) CK-2 activity changes during embryogenesis, being high at certain...

  2. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-15

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

  3. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  4. Role of isoenzyme M2 of pyruvate kinase in urothelial tumorigenesis

    OpenAIRE

    Zhou, Haiping; Wang, Xing; Mo, Lan; Liu, Yan; He, Feng; Zhang, Fenglin; Huang, Kuo-How; Wu, Xue-Ru

    2016-01-01

    The conversion of precancerous lesions to full-fledged cancers requires the affected cells to surpass certain rate-limiting steps. We recently showed that activation of HRAS proto-oncogene in urothelial cells of transgenic mice causes simple urothelial hyperplasia (SUH) which is persistent and whose transition to low-grade papillary urothelial carcinoma (UC) must undergo nodular urothelial hyperplasia (NUH). We hypothesized that NUH, which has acquired fibrovascular cores, plays critical role...

  5. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  6. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    OpenAIRE

    Vanderperre, Beno?t; Herzig, S?bastien; Krznar, Petra; H?rl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic p...

  7. An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

    OpenAIRE

    McCommis, Kyle S.; Hodges, Wesley T.; Bricker, Daniel K.; Wisidagama, Dona R.; Compan, Vincent; Remedi, Maria S.; Thummel, Carl S.; Finck, Brian N.

    2016-01-01

    Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-prod...

  8. The mitochondrial pyruvate carrier mediates high fat diet-induced increases in hepatic TCA cycle capacity

    OpenAIRE

    Rauckhorst, Adam J.; Gray, Lawrence R.; Sheldon, Ryan D.; Fu, Xiaorong; Pewa, Alvin D.; Feddersen, Charlotte R.; Dupuy, Adam J.; Gibson-Corley, Katherine N.; Cox, James E.; Burgess, Shawn C.; Taylor, Eric B.

    2017-01-01

    Objective: Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. T...

  9. Binding of ethyl pyruvate to bovine serum albumin: Calorimetric, spectroscopic and molecular docking studies

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, Mallika [Department of Chemistry, Miranda House, University of Delhi, Delhi 11007 (India); Mishra, Rashmi; Agarwala, Paban K. [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Ojha, Himanshu, E-mail: himanshu.drdo@gmail.com [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Singh, Bhawna [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Singh, Anju; Kukreti, Shrikant [Nucleic Acid Research Laboratory, Department of Chemistry, University of Delhi, Delhi 11007 (India)

    2016-06-10

    Highlights: • ITC study showed binding of ethyl pyruvate with BSA with high binding affinity. • Ethyl pyruvate binding caused conformation alteration of BSA. • Fluorescence quenching mechanism is static in nature. • Electrostatic, hydrogen bonding and hydrophobic forces involved in binding. • Docking confirmed role of electrostatic, hydrogen bonding and hydrophobic forces. - Abstract: Various in vitro and in vivo studies have shown the anti-inflammatory and anticancer potential role of ethyl pyruvate. Bio-distribution of drugs is significantly influenced by the drug-serum protein binding. Therefore, the binding mechanism of the ethyl pyruvate with bovine serum albumin was investigated using UV–vis absorption, fluorescence, circular dichroism, isothermal titration calorimetry and molecular docking techniques. Absorption and fluorescence quenching studies indicated the binding of ethyl pyruvate with protein. Circular dichroism spectra of bovine serum albumin confirmed significant change in the conformation of protein upon binding. Thermodynamic data confirmed that ethyl pyruvate binds to bovine serum albumin at the two different sites with high affinity. Binding of ethyl pyruvate to bovine serum albumin involves hydrogen bonding, van der Waal and hydrophobic interactions. Further, docking studies indicated that ethyl pyruvate could bind significantly at the three binding sites. The results will definitely contribute to the development of ethyl pyruvate as drug.

  10. Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis

    Directory of Open Access Journals (Sweden)

    F. Habarou

    2015-03-01

    Full Text Available Pyruvate carboxylase (PC is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A. Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1 and type B (patient 2. Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

  11. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast

    NARCIS (Netherlands)

    A.J. van Maris; J.M. Geertman; A. Vermeulen; M.K. Groothuizen; A.A. Winkler; M.D. Piper; J.P. van Dijken; J.T. Pronk

    2004-01-01

    textabstractThe absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects:

  12. Field dependence of T1 for hyperpolarized [1-13C]pyruvate

    DEFF Research Database (Denmark)

    Chattergoon, N.; Martnez-Santiesteban, F.; Handler, W. B.

    2013-01-01

    conformation and properties of the dissolution media such as buffer composition, solution pH, temperature and magnetic field. We have measured the magnetic field dependence of the spin–lattice relaxation time of hyperpolarized [1-13C]pyruvate using field-cycled relaxometry. [1-13C]pyruvate was hyperpolarized...

  13. Relations between fatty acid synthesis, pyruvate concentration and cell concentration of suspensions of isolated rat hepatocytes

    NARCIS (Netherlands)

    Beynen, A.C.; Geelen, M.J.H.

    1984-01-01

    1. 1. The cell concentration of suspensions of isolated rat hepatocytes affects both the rate of pyruvate accumulation in the incubation medium and the rate of fatty acid synthesis. 2. 2. At low cell concentrations pyruvate accumulation is directly related to the cell concentration but levels off

  14. Apparent rate constant mapping using hyperpolarized [1-(13) C]pyruvate

    DEFF Research Database (Denmark)

    Khegai, O.; Schulte, R. F.; Janich, M. A.

    2014-01-01

    Hyperpolarization of [1-13C]pyruvate in solution allows real-time measurement of uptake and metabolism using MR spectroscopic methods. After injection and perfusion, pyruvate is taken up by the cells and enzymatically metabolized into downstream metabolites such as lactate, alanine, and bicarbona...

  15. A Patient With Pyruvate Carboxylase Deficiency and Nemaline Rods on Muscle Biopsy

    DEFF Research Database (Denmark)

    Unal, Ozlem; Orhan, Diclehan; Ostergaard, Elsebet

    2013-01-01

    Nemaline rods are the pathologic hallmark of nemaline myopathy, but they have also been described as a secondary phenomenon in a variety of other disorders. Nemaline rods have not been reported in pyruvate carboxylase deficiency before. Here we present a patient with pyruvate carboxylase deficiency...

  16. The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel.

    NARCIS (Netherlands)

    Huberts, D.H.; Venselaar, H.; Vriend, G.; Veenhuis, M.; Klei, I.J. van der

    2010-01-01

    Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenulapolymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional

  17. Detection of myocardial ischemia before infarction, based on accumulation of labeled pyruvate

    International Nuclear Information System (INIS)

    Goldstein, R.A.; Klein, M.S.; Sobel, B.E.

    1980-01-01

    To determine whether ischemic, but not irreversibly injured myocardium, can be differentiated from normal tissue based on accumulation of labeled pyruvate, isolated hearts were perfused with buffer containing [ 14 C]pyruvate under conditions of normal or low flow. Fifteen minutes after the hearts were exposed to labeled material, myocardial radioactivity was fourfold greater in ischemic compared to control hearts, due to accumulation of label in sequestered lactate produced from the pyruvate. Open-chest rabbits subjected to coronary occlusion exhibited a 1.73:1 ratio of radioactivity in ischemic compared with normal myocardium 15 min after systemic injection of [ 14 C]pyruvate. The results obtained suggest that zones of myocardial ischemia should be detectable in vivo by positron tomography after systemic administration of [ 11 C]pyruvate as well

  18. Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer.

    Science.gov (United States)

    Sprowl-Tanio, Stephanie; Habowski, Amber N; Pate, Kira T; McQuade, Miriam M; Wang, Kehui; Edwards, Robert A; Grun, Felix; Lyou, Yung; Waterman, Marian L

    2016-01-01

    There is increasing evidence that oncogenic Wnt signaling directs metabolic reprogramming of cancer cells to favor aerobic glycolysis or Warburg metabolism. In colon cancer, this reprogramming is due to direct regulation of pyruvate dehydrogenase kinase 1 ( PDK1 ) gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1. Whether these genes are also regulated at the transcriptional level, and therefore a part of a core metabolic gene program targeted by oncogenic WNT signaling, is not known. Here, we identify monocarboxylate transporter 1 (MCT-1; encoded by SLC16A1 ) as a direct target gene supporting Wnt-driven Warburg metabolism. We identify and validate Wnt response elements (WREs) in the proximal SLC16A1 promoter and show that they mediate sensitivity to Wnt inhibition via dominant-negative LEF-1 (dnLEF-1) expression and the small molecule Wnt inhibitor XAV939. We also show that WREs function in an independent and additive manner with c-Myc, the only other known oncogenic regulator of SLC16A1 transcription. MCT-1 can export lactate, the byproduct of Warburg metabolism, and it is the essential transporter of pyruvate as well as a glycolysis-targeting cancer drug, 3-bromopyruvate (3-BP). Using sulforhodamine B (SRB) assays to follow cell proliferation, we tested a panel of colon cancer cell lines for sensitivity to 3-BP. We observe that all cell lines are highly sensitive and that reduction of Wnt signaling by XAV939 treatment does not synergize with 3-BP, but instead is protective and promotes rapid recovery. We conclude that MCT-1 is part of a core Wnt signaling gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism.

  19. Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

    Science.gov (United States)

    Agrimi, Gennaro; Mena, Maria C; Izumi, Kazuki; Pisano, Isabella; Germinario, Lucrezia; Fukuzaki, Hisashi; Palmieri, Luigi; Blank, Lars M; Kitagaki, Hiroshi

    2014-03-01

    Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  20. Mitochondrial Pyruvate Carrier 2 Hypomorphism in Mice Leads to Defects in Glucose-Stimulated Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Patrick A. Vigueira

    2014-06-01

    Full Text Available Carrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. Mitochondrial pyruvate carrier 2 (Mpc2 is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2Δ16 was viable but exhibited a reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2Δ16 mice. Additionally, compared with wild-type controls, Mpc2Δ16 mice exhibited normal insulin sensitivity but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic β cell glucose sensing.

  1. Single pyruvate intake induces blood alkalization and modification of resting metabolism in humans.

    Science.gov (United States)

    Olek, Robert A; Luszczyk, Marcin; Kujach, Sylwester; Ziemann, Ewa; Pieszko, Magdalena; Pischel, Ivo; Laskowski, Radoslaw

    2015-03-01

    Three separate studies were performed with the aim to 1) determine the effect of a single sodium pyruvate intake on the blood acid-base status in males and females; 2) compare the effect of sodium and calcium pyruvate salts and establish their role in the lipolysis rate; and 3) quantify the effect of single pyruvate intake on the resting energy metabolism. In all, 48 individuals completed three separate studies. In all the studies, participants consumed a single dose of pyruvate 0.1 g/kg 60 min before commencing the measurements. The whole blood pH, bicarbonate concentration, base excess or plasma glycerol, free fatty acids, glucose concentrations, or resting energy expenditure and calculated respiratory exchange ratio were determined. The analysis of variance for repeated measurements was performed to examine the interaction between treatment and time. The single dose of sodium pyruvate induced blood alkalization, which was more marked in the male than in the female participants. Following the ingestion of sodium or calcium pyruvate, the blood acid-base parameters were higher than in the placebo trial. Furthermore, 3-h postingestion glycerol was lower in both pyruvate trials than in placebo. Resting energy expenditure did not differ between the trials; however, carbohydrate oxidation was increased after sodium pyruvate ingestion. Pyruvate intake induced mild alkalization in a sex-dependent fashion. Moreover, it accelerated carbohydrate metabolism and delayed the rate of glycerol appearance in the blood, but had no effect on the resting energy expenditure. Furthermore, sodium salt seems to have had a greater effect on the blood buffering level than calcium salt. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Effects of pyruvate dose on in vivo metabolism and quantification of hyperpolarized 13C spectra

    DEFF Research Database (Denmark)

    Janich, M. A.; Menzel, M. I.; Wiesinger, F.

    2012-01-01

    Real‐time in vivo measurements of metabolites are performed by signal enhancement of [1‐13C]pyruvate using dynamic nuclear polarization, rapid dissolution and intravenous injection, acquisition of free induction decay signals and subsequent quantification of spectra. The commonly injected dose...... uptake and metabolic conversion. The goal of this study was to examine the effects of a [1‐13C]pyruvate bolus on metabolic conversion in vivo. Spectra were quantified by three different methods: frequency‐domain fitting with LCModel, time‐domain fitting with AMARES and simple linear least‐squares fitting...... in the time domain. Since the simple linear least‐squares approach showed bleeding artifacts and LCModel produced noisier time signals. AMARES performed best in the quantification of in vivo hyperpolarized pyruvate spectra. We examined pyruvate doses of 0.1–0.4 mmol/kg (body mass) in male Wistar rats...

  3. A pyruvate-buffered dialysis fluid induces less peritoneal angiogenesis and fibrosis than a conventional solution

    NARCIS (Netherlands)

    van Westrhenen, Roos; Zweers, Machteld M.; Kunne, Cindy; de Waart, Dirk R.; van der Wal, Allard C.; Krediet, Raymond T.

    2008-01-01

    BACKGROUND: Conventional lactate-buffered peritoneal dialysis (PD) fluids containing glucose and glucose degradation products are believed to contribute to the development of fibrosis and angiogenesis in the dialyzed peritoneum. To reduce potential negative effects of lactate, pyruvate was

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

    Science.gov (United States)

    Vogel, O; Hoehn, B; Henning, U

    1972-06-01

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

  5. Effect of hexoses on the levels of pyruvate decarboxylase in Mucor rouxii.

    OpenAIRE

    Barrera, C R; Corral, J

    1980-01-01

    Pyruvate decarboxylase activity in the dimorphic fungus Mucor rouxii increased 25- to 35-fold in yeastlike and mycelial cells grown in the presence of glucose as compared to the activity observed in mycelial cultures grown in the absence of glucose.

  6. Changes in myocardial lactate, pyruvate and lactate-pyruvate ratio during cardiopulmonary bypass for elective adult cardiac surgery: Early indicator of morbidity

    Directory of Open Access Journals (Sweden)

    P M Kapoor

    2011-01-01

    Full Text Available Background: Myocardial lactate assays have been established as a standard method to compare various myocardial protection strategies. This study was designed to test whether coronary sinus (CS lactates, pyruvate and lactate-pyruvate (LP ratio correlates with myocardial dysfunction and predict postoperative outcomes. Materials and Methods: This prospective observational study was conducted on 40 adult patients undergoing elective cardiac surgery with the aid of cardiopulmonary bypass (CPB. CS blood sampling was done for estimation of myocardial lactate (ML, pyruvate (MP and lactate-pyruvate ratio (MLPR namely: pre-CPB (T 1 , after removal of aortic cross clamp (T 2 and 30 minutes post-CPB (T 3 . Results: Baseline myocardial LPR strongly correlated with Troponin-I at T1 (s: 0.6. Patients were sub grouped according to the median value of myocardial lactate (2.9 at baseline T1 into low myocardial lactate (LML group, mean (2.39±0.4 mmol/l, n=19 and a high myocardial lactate (HML group, mean (3.65±0.9 mmol/l, n=21. A significant increase in PL, ML, MLPR and TropI occurred in both groups as compared to baseline. Patients in HML group had significant longer period of ICU stay. Patients with higher inotrope score had significantly higher ML (T2, T3. ML with a baseline value of 2.9 mmol/l had 70.83% sensitivity and 62.5% specificity (ROC area: 0.7109 Std error: 0.09 while myocardial pyruvate with a baseline value of 0.07 mmol/l has 79.17% sensitivity and 68.75% specificity (ROC area: 0.7852, Std error: 0.0765 for predicting inotrope requirement after CPB. Conclusion: CS lactate, pyruvate and LP ratio correlate with myocardial function and can predict postoperative outcome.

  7. Pyruvate incubation enhances glycogen stores and sustains neuronal function during subsequent glucose deprivation.

    Science.gov (United States)

    Shetty, Pavan K; Sadgrove, Matthew P; Galeffi, Francesca; Turner, Dennis A

    2012-01-01

    The use of energy substrates, such as lactate and pyruvate, has been shown to improve synaptic function when administered during glucose deprivation. In the present study, we investigated whether prolonged incubation with monocarboxylate (pyruvate or lactate) prior rather than during glucose deprivation can also sustain synaptic and metabolic function. Pyruvate pre-incubation(3-4h) significantly prolonged (>25 min) the tolerance of rat hippocampal slices to delayed glucose deprivation compared to control and lactate pre-incubated slices, as revealed by field excitatory post synaptic potentials (fEPSPs); pre-incubation with pyruvate also reduced the marked decrease in NAD(P)H fluorescence resulting from glucose deprivation. Moreover, pyruvate exposure led to the enhancement of glycogen stores with time, compared to glucose alone (12 μmol/g tissue at 4h vs. 3.5 μmol/g tissue). Prolonged resistance to glucose deprivation following exogenous pyruvate incubation was prevented by glycogenolysis inhibitors, suggesting that enhanced glycogen mediates the delay in synaptic activity failure. The application of an adenosine A1 receptor antagonist enhanced glycogen utilization and prolonged the time to synaptic failure, further confirming this hypothesis of the importance of glycogen. Moreover, tissue levels of ATP were also significantly maintained during glucose deprivation in pyruvate pretreated slices compared to control and lactate. In summary, these experiments indicate that pyruvate exposure prior to glucose deprivation significantly increased the energy buffering capacity of hippocampal slices, particularly by enhancing internal glycogen stores, delaying synaptic failure during glucose deprivation by maintaining ATP levels, and minimizing the decrease in the levels of NAD(P)H. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Pyruvate Decarboxylase Activity Assay in situ of Different Industrial Yeast Strains

    Directory of Open Access Journals (Sweden)

    Dorota Kręgiel

    2009-01-01

    Full Text Available Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1 is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.

  9. Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes.

    Science.gov (United States)

    Jung, Jong Gab; Choi, Sung-E; Hwang, Yoon-Jung; Lee, Sang-A; Kim, Eun Kyoung; Lee, Min-Seok; Han, Seung Jin; Kim, Hae Jin; Kim, Dae Jung; Kang, Yup; Lee, Kwan-Woo

    2011-10-15

    Elevated fatty acid levels have been thought to contribute to insulin resistance. Repression of the glucose transporter 4 (GLUT4) gene as well as impaired GLUT4 translocation may be a mediator for fatty acid-induced insulin resistance. This study was initiated to determine whether palmitate treatment repressed GLUT4 expression, whether glucose/fatty acid metabolism influenced palmitate-induced GLUT4 gene repression (PIGR), and whether attempts to prevent PIGR restored palmitate-induced impairment of glucose uptake (PIIGU) in C2 myotubes. Not only stimulators of fatty acid oxidation, such as bezafibrate, AICAR, and TOFA, but also TCA cycle substrates, such as pyruvate, leucine/glutamine, and α-ketoisocaproate/monomethyl succinate, significantly prevented PIGR. In particular, supplementing with pyruvate through methyl pyruvate resulted in nearly complete prevention of PIIGU, whereas palmitate treatment reduced the intracellular pyruvate level. These results suggest that pyruvate depletion plays a critical role in PIGR and PIIGU; thus, pyruvate supplementation may help prevent obesity-induced insulin resistance in muscle cells. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

  10. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarcha...

  11. Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra- to the extracellular space.

    Science.gov (United States)

    Reineri, Francesca; Daniele, Valeria; Cavallari, Eleonora; Aime, Silvio

    2016-08-01

    The use of [1-(13) C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra- and extracellular contributions to the (13) C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden 'quenching' of the extracellular metabolites' signal. The paramagnetic Gd-tetraazacyclododecane triacetic acid (Gd-DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the (13) C-carboxylate resonances of both pyruvate and lactate. When Gd-DO3A was added to an MCF-7 cellular culture, which had previously received a dose of hyperpolarized [1-(13) C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the (13) C-carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra- to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non-destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy

    Directory of Open Access Journals (Sweden)

    Miao Shen

    2013-01-01

    Full Text Available Background. Hepatic ischemia-reperfusion (I/R injury is a pivotal clinical problem occurring in many clinical conditions such as transplantation, trauma, and hepatic failure after hemorrhagic shock. Apoptosis and autophagy have been shown to contribute to cell death in hepatic I/R injury. Ethyl pyruvate, a stable and simple lipophilic ester, has been shown to have anti-inflammatory properties. In this study, the purpose is to explore both the effect of ethyl pyruvate on hepatic I/R injury and regulation of intrinsic pathway of apoptosis and autophagy. Methods. Three doses of ethyl pyruvate (20 mg/kg, 40 mg/kg, and 80 mg/kg were administered 1 h before a model of segmental (70% hepatic warm ischemia was established in Balb/c mice. All serum and liver tissues were obtained at three different time points (4 h, 8 h, and 16 h. Results. Alanine aminotransferase (ALT, aspartate aminotransferase (AST, and pathological features were significantly ameliorated by ethyl pyruvate (80 mg/kg. The expression of Bcl-2, Bax, Beclin-1, and LC3, which play an important role in the regulation of intrinsic pathway of apoptosis and autophagy, was also obviously decreased by ethyl pyruvate (80 mg/kg. Furthermore, ethyl pyruvate inhibited the HMGB1/TLR4/ NF-κb axis and the release of cytokines (TNF-α and IL-6. Conclusion. Our results showed that ethyl pyruvate might attenuate to hepatic I/R injury by inhibiting intrinsic pathway of apoptosis and autophagy, mediated partly through downregulation of HMGB1/TLR4/ NF-κb axis and the competitive interaction with Beclin-1 of HMGB1.

  13. [Diagnostic value of detection of blood levels of lactate, pyruvate and 2,3-diphosphoglycerate in children with diabetes mellitus].

    Science.gov (United States)

    Marchenko, L F; Baturin, A A; Terent'eva, E A

    1991-01-01

    Measurements were made of lactate, pyruvate and 2,3-diphosphoglycerate in 69 children admitted to the hospital in a state of diabetic ketoacidosis of different intensity. Depending on the intensity of metabolic abnormalities, the content of lactate and pyruvate was found to be increased, whereas that of 2,3-diphosphoglycerate to be lowered. Measurements of the content of lactate and the lactate/pyruvate ratio enables carrying out differential diagnosis between the ketoacidotic and lactacidotic varieties of diabetic coma.

  14. Enhanced pyruvate dehydrogenase activity improves cardiac outcomes in a murine model of cardiac arrest.

    Directory of Open Access Journals (Sweden)

    Lin Piao

    Full Text Available Post-ischemic changes in cellular metabolism alter myocardial and neurological function. Pyruvate dehydrogenase (PDH, the limiting step in mitochondrial glucose oxidation, is inhibited by increased expression of PDH kinase (PDK during ischemia/reperfusion injury. This results in decreased utilization of glucose to generate cellular ATP. Post-cardiac arrest (CA hypothermia improves outcomes and alters metabolism, but its influence on PDH and PDK activity following CA are unknown. We hypothesized that therapeutic hypothermia (TH following CA is associated with the inhibition of PDK activity and increased PDH activity. We further hypothesized that an inhibitor of PDK activity, dichloroacetate (DCA, would improve PDH activity and post-CA outcomes.Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 12-minute KCl-induced CA followed by cardiopulmonary resuscitation. Compared to normothermic (37°C CA controls, administering TH (30°C improved overall survival (72-hour survival rate: 62.5% vs. 28.6%, P<0.001, post-resuscitation myocardial function (ejection fraction: 50.9±3.1% vs. 27.2±2.0%, P<0.001; aorta systolic pressure: 132.7±7.3 vs. 72.3±3.0 mmHg, P<0.001, and neurological scores at 72-hour post CA (9.5±1.3 vs. 5.4±1.3, P<0.05. In both heart and brain, CA increased lactate concentrations (1.9-fold and 3.1-fold increase, respectively, P<0.01, decreased PDH enzyme activity (24% and 50% reduction, respectively, P<0.01, and increased PDK protein expressions (1.2-fold and 1.9-fold, respectively, P<0.01. In contrast, post-CA treatment with TH normalized lactate concentrations (P<0.01 and P<0.05 and PDK expressions (P<0.001 and P<0.05, while increasing PDH activity (P<0.01 and P<0.01 in both the heart and brain. Additionally, treatment with DCA (0.2 mg/g body weight 30 min prior to CA improved both myocardial hemodynamics 2 hours post-CA (aortic systolic pressure: 123±3 vs. 96±4 mmHg, P<0.001 and 72-hour survival rates

  15. Chronic pyruvate supplementation increases exploratory activity and brain energy reserves in young and middle-aged mice

    DEFF Research Database (Denmark)

    Koivisto, Hennariikka; Leinonen, Henri; Puurula, Mari

    2016-01-01

    to brain and thereby attenuate aging- or AD-related cognitive impairment. Mice received ~800 mg/kg/day Na-pyruvate in their chow for 2-6 months. In middle-aged wild-type mice and in 6.5-month-old APP/PS1 mice, pyruvate facilitated spatial learning and increased exploration of a novel odor. However......, in passive avoidance task for fear memory, the treatment group was clearly impaired. Independent of age, long-term pyruvate increased explorative behavior, which likely explains the paradoxical impairment in passive avoidance. We also assessed pyruvate effects on body weight, muscle force, and endurance...

  16. Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

    Science.gov (United States)

    Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

    2010-08-01

    Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

  17. An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

    Directory of Open Access Journals (Sweden)

    Kyle S. McCommis

    2016-08-01

    Full Text Available Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-producing cells. Results: In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, we show that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity. Conclusions: Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia. Keywords: Stimulus-coupled secretion, Insulin, β-Cell, Diabetes, Pyruvate, Mitochondria, Drosophila

  18. The mitochondrial pyruvate carrier mediates high fat diet-induced increases in hepatic TCA cycle capacity.

    Science.gov (United States)

    Rauckhorst, Adam J; Gray, Lawrence R; Sheldon, Ryan D; Fu, Xiaorong; Pewa, Alvin D; Feddersen, Charlotte R; Dupuy, Adam J; Gibson-Corley, Katherine N; Cox, James E; Burgess, Shawn C; Taylor, Eric B

    2017-11-01

    Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. The goal of this investigation was to test whether hepatocyte MPC disruption provides sustained protection from hyperglycemia during long-term HFD and the differential effects of hepatocyte MPC disruption on TCA cycle metabolism in NCD versus HFD conditions. We utilized long-term high fat feeding, serial measurements of postabsorptive blood glucose and metabolomic profiling and 13 C-lactate/ 13 C-pyruvate tracing to investigate the contribution of the MPC to hyperglycemia and altered hepatic TCA cycle metabolism during HFD-induced obesity. Hepatocyte MPC disruption resulted in long-term attenuation of hyperglycemia induced by HFD. HFD increased hepatic mitochondrial pyruvate utilization and TCA cycle capacity in an MPC-dependent manner. Furthermore, MPC disruption decreased progression of fibrosis and levels of transcript markers of inflammation. By contributing to chronic hyperglycemia, fibrosis, and TCA cycle expansion, the hepatocyte MPC is a key mediator of the pathophysiology induced in the HFD model of T2D. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  19. A new synthesis of [3-11C]pyruvic acid using alanine racemase

    International Nuclear Information System (INIS)

    Ikemoto, M.; Okamoto, E.; Sasaki, M.; Haradahira, T.; Omura, H.; Furuya, Y.; Suzuki, K.; Watanabe, Y.

    1998-01-01

    The synthesis of [3- 11 C]pyruvic acid was attempted by two reaction systems (A: alanine racemase and D-amino acid oxidase, B: alanine racemase and L-alanine dehydrogenase) utilizing a new thermostable enzyme, alanine racemase. Conversion rates from D,L-[3- 11 C]alanine to [3- 11 C]pyruvic acid were almost 100% in both methods. Similar results were obtained with immobilized enzymes packed in a single column. Furthermore, the same column could be used repeatedly without a remarkable decrease of the [3- 11 C]pyruvic acid yield. Various matrices were tested for the immobilizing enzyme, and Aminopropyl-CPG was concluded to be the most suitable since the loss of the enzyme activity was the least in the studied matrices

  20. Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate

    Science.gov (United States)

    Nelson, Sarah J.; Kurhanewicz, John; Vigneron, Daniel B.; Larson, Peder E. Z.; Harzstark, Andrea L.; Ferrone, Marcus; van Criekinge, Mark; Chang, Jose W.; Bok, Robert; Park, Ilwoo; Reed, Galen; Carvajal, Lucas; Small, Eric J.; Munster, Pamela; Weinberg, Vivian K.; Ardenkjaer-Larsen, Jan Henrik; Chen, Albert P.; Hurd, Ralph E.; Odegardstuen, Liv-Ingrid; Robb, Fraser J.; Tropp, James; Murray, Jonathan A.

    2014-01-01

    This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-13C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-fold enhancement in signal relative to conventional magnetic resonance (MR) imaging. When combined with the rapid acquisition of in vivo 13C MR data, it is possible to evaluate the distribution of agents such as [1-13C]pyruvate and its metabolic products lactate, alanine, and bicarbonate in a matter of seconds. Preclinical studies in cancer models have detected elevated levels of hyperpolarized [1-13C]lactate in tumor, with the ratio of [1-13C]lactate/[1-13C]pyruvate being increased in high-grade tumors and decreased after successful treatment. Translation of this technology into humans was achieved by modifying the instrument that generates the hyperpolarized agent, constructing specialized radio frequency coils to detect 13C nuclei, and developing new pulse sequences to efficiently capture the signal. The study population comprised patients with biopsy-proven prostate cancer, with 31 subjects being injected with hyperpolarized [1-13C]pyruvate. The median time to deliver the agent was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-13C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-13C]lactate/[1-13C]pyruvate in regions of biopsy-proven cancer. These findings will be valuable for noninvasive cancer diagnosis and treatment monitoring in future clinical trials. PMID:23946197

  1. Pyruvate administration reduces recurrent/moderate hypoglycemia-induced cortical neuron death in diabetic rats.

    Directory of Open Access Journals (Sweden)

    Bo Young Choi

    Full Text Available Recurrent/moderate (R/M hypoglycemia is common in type 1 diabetes patients. Moderate hypoglycemia is not life-threatening, but if experienced recurrently it may present several clinical complications. Activated PARP-1 consumes cytosolic NAD, and because NAD is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Pyruvate, however, can be metabolized in the absence of cytosolic NAD. We therefore hypothesized that pyruvate may be able to improve the outcome in diabetic rats subjected to insulin-induced R/M hypoglycemia by terminating hypoglycemia with glucose plus pyruvate, as compared with delivering just glucose alone. In an effort to mimic juvenile type 1 diabetes the experiments were conducted in one-month-old young rats that were rendered diabetic by streptozotocin (STZ, 50mg/kg, i.p. injection. One week after STZ injection, rats were subjected to moderate hypoglycemia by insulin injection (10 U/kg, i.p. without anesthesia for five consecutive days. Pyruvate (500 mg/kg was given by intraperitoneal injection after each R/M hypoglycemia. Three hours after last R/M hypoglycemia, zinc accumulation was evaluated. Three days after R/M hypoglycemia, neuronal death, oxidative stress, microglial activation and GSH concentrations in the cerebral cortex were analyzed. Sparse neuronal death was observed in the cortex. Zinc accumulation, oxidative injury, microglial activation and GSH loss in the cortex after R/M hypoglycemia were all reduced by pyruvate injection. These findings suggest that when delivered alongside glucose, pyruvate may significantly improve the outcome after R/M hypoglycemia by circumventing a sustained impairment in neuronal glucose utilization resulting from PARP-1 activation.

  2. Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

    Science.gov (United States)

    Plaga, W; Lottspeich, F; Oesterhelt, D

    1992-04-01

    An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

  3. Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

    Science.gov (United States)

    Wojtkowiak, Jonathan W; Cornnell, Heather C; Matsumoto, Shingo; Saito, Keita; Takakusagi, Yoichi; Dutta, Prasanta; Kim, Munju; Zhang, Xiaomeng; Leos, Rafael; Bailey, Kate M; Martinez, Gary; Lloyd, Mark C; Weber, Craig; Mitchell, James B; Lynch, Ronald M; Baker, Amanda F; Gatenby, Robert A; Rejniak, Katarzyna A; Hart, Charles; Krishna, Murali C; Gillies, Robert J

    2015-01-01

    Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to

  4. The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel

    NARCIS (Netherlands)

    Huberts, Daphne H. E. W.; Venselaar, Hanka; Vriend, Gert; Veenhuis, Marten; van der Klei, Ida J.

    Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenula polymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional

  5. Chronic pyruvate supplementation increases exploratory activity and brain energy reserves in young and middle-aged mice

    Directory of Open Access Journals (Sweden)

    Hennariikka eKoivisto

    2016-03-01

    Full Text Available Numerous studies have reported neuroprotective effects of pyruvate when given in systemic injections. Impaired glucose uptake and metabolism are found in Alzheimer's disease (AD and in AD mouse models. We tested whether dietary pyruvate supplementation is able to provide added energy supply to brain and thereby attenuate aging- or AD-related cognitive impairment. Mice received ~ 800 mg/kg/day Na-pyruvate in their chow for 2- 6 months. In middle-aged wild-type mice and in 6.5-month-old APP/PS1 mice, pyruvate facilitated spatial learning and increased exploration of a novel odor. However, in passive avoidance task for fear memory, the treatment group was clearly impaired. Independent of age, long-term pyruvate increased explorative behavior, which likely explains the paradoxical impairment in passive avoidance. We also assessed pyruvate effects on body weight, muscle force and endurance, and found no effects. Metabolic post-mortem assays revealed increased energy compounds in nuclear magnetic resonance spectroscopy as well as increased brain glycogen storages in the pyruvate group. Pyruvate supplementation may counteract aging-related behavioral impairment but its beneficial effect seems related to increased explorative activity rather than direct memory enhancement.

  6. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  7. Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

    DEFF Research Database (Denmark)

    Nellemann, B.; Vendelbo, M.H.; Nielsen, Thomas Svava

    2014-01-01

    Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting....

  8. Studies to enhance the hyperpolarization level in PHIP-SAH-produced C13-pyruvate

    Science.gov (United States)

    Cavallari, Eleonora; Carrera, Carla; Aime, Silvio; Reineri, Francesca

    2018-04-01

    The use of [1-13C]pyruvate, hyperpolarized by dissolution-Dynamic Nuclear Polarization (d-DNP), in in vivo metabolic studies has developed quickly, thanks to the imaging probe's diagnostic relevance. Nevertheless, the cost of a d-DNP polarizer is quite high and the speed of hyperpolarization process is relatively slow, meaning that its use is limited to few research laboratories. ParaHydrogen Induced Polarization Side Arm Hydrogenation (PHIP-SAH) (Reineri et al., 2015) is a cost effective and easy-to-handle method that produces 13C-MR hyperpolarization in [1-13C]pyruvate and other metabolites. This work aims to identify the main determinants of the hyperpolarization levels observed in C13-pyruvate using this method. By dissecting the various steps of the PHIP-SAH procedure, it has been possible to assess the role of several experimental parameters whose optimization must be pursued if this method is to be made suitable for future translational steps. The search for possible solutions has led to improvements in the polarization of sodium [1-13C]pyruvate from 2% to 5%. Moreover, these results suggest that observed polarization levels could be increased considerably by an automatized procedure which would reduce the time required for the work-up passages that are currently carried out manually. The results reported herein mean that the attainment of polarization levels suitable for the metabolic imaging applications of these hyperpolarized substrates show significant promise.

  9. Multi site Kinetic Modeling of 13C Metabolic MR Using [1-13C]Pyruvate

    International Nuclear Information System (INIS)

    Damian, P.A.G.; Sperl, J.I.; Janich, M.A.; Wiesinger, F.; Schulte, R.F.; Menzel, M.I.; Damian, P.A.G.; Damian, P.A.G.; Haase, A.; Janich, M.A.; Schwaiger, M.; Janich, M.A.; Khegai, O.; Glaser, S.J.

    2014-01-01

    Hyperpolarized 13 C imaging allows real-time in vivo measurements of metabolite levels. Quantification of metabolite conversion between [1- 13 C]pyruvate and downstream metabolites [1- 13 C]alanine, [1- 13 C]lactate, and [ 13 C] bicarbonate can be achieved through kinetic modeling. Since pyruvate interacts dynamically and simultaneously with its downstream metabolites, the purpose of this work is the determination of parameter values through a multi site, dynamic model involving possible biochemical pathways present in MR spectroscopy. Kinetic modeling parameters were determined by fitting the multi site model to time-domain dynamic metabolite data. The results for different pyruvate doses were compared with those of different two-site models to evaluate the hypothesis that for identical data the uncertainty of a model and the signal-to-noise ratio determine the sensitivity in detecting small physiological differences in the target metabolism. In comparison to the two-site exchange models, the multi site model yielded metabolic conversion rates with smaller bias and smaller standard deviation, as demonstrated in simulations with different signal-to-noise ratio. Pyruvate dose effects observed previously were confirmed and quantified through metabolic conversion rate values. Parameter interdependency allowed an accurate quantification and can therefore be useful for monitoring metabolic activity in different tissues

  10. Dissociative electron attachment and anion-induced dimerization in pyruvic acid

    Czech Academy of Sciences Publication Activity Database

    Zawadzki, Mateusz; Ranković, Miloš; Kočišek, Jaroslav; Fedor, Juraj

    2018-01-01

    Roč. 20, č. 10 (2018), s. 6838-6844 ISSN 1463-9076 R&D Projects: GA ČR GA17-04844S; GA ČR GJ16-10995Y Institutional support: RVO:61388955 Keywords : pyruvic acid * electron attachment * dimerization Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 4.123, year: 2016

  11. Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

    Science.gov (United States)

    Patterson, Jessica N; Cousteils, Katelyn; Lou, Jennifer W; Manning Fox, Jocelyn E; MacDonald, Patrick E; Joseph, Jamie W

    2014-05-09

    It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

  12. Magnetic resonance and fluorescence studies on pyruvate dehydrogenase complexes and their small molecular weight constituents

    NARCIS (Netherlands)

    Grande, H.J.

    1976-01-01

    The articles presented in this thesis do not describe at first glance one well-defined subject. They are, however, in fact connected by one central theme: the study of large enzyme aggregates by molecular physical methods. Chosen was the pyruvate dehydrogenase complex (PDC) because of its

  13. Pyruvate Oxidase Influences the Sugar Utilization Pattern and Capsule Production in Streptococcus pneumoniae

    NARCIS (Netherlands)

    Carvalho, Sandra M.; Farshchi Andisi, Vahid; Gradstedt, Henrik; Neef, Jolanda; Kuipers, Oscar P.; Neves, Ana R.; Bijlsma, Jetta J. E.

    2013-01-01

    Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence,

  14. Multisite Kinetic Modeling of 13C Metabolic MR Using [1-13C]Pyruvate

    Directory of Open Access Journals (Sweden)

    Pedro A. Gómez Damián

    2014-01-01

    Full Text Available Hyperpolarized 13C imaging allows real-time in vivo measurements of metabolite levels. Quantification of metabolite conversion between [1-13C]pyruvate and downstream metabolites [1-13C]alanine, [1-13C]lactate, and [13C]bicarbonate can be achieved through kinetic modeling. Since pyruvate interacts dynamically and simultaneously with its downstream metabolites, the purpose of this work is the determination of parameter values through a multisite, dynamic model involving possible biochemical pathways present in MR spectroscopy. Kinetic modeling parameters were determined by fitting the multisite model to time-domain dynamic metabolite data. The results for different pyruvate doses were compared with those of different two-site models to evaluate the hypothesis that for identical data the uncertainty of a model and the signal-to-noise ratio determine the sensitivity in detecting small physiological differences in the target metabolism. In comparison to the two-site exchange models, the multisite model yielded metabolic conversion rates with smaller bias and smaller standard deviation, as demonstrated in simulations with different signal-to-noise ratio. Pyruvate dose effects observed previously were confirmed and quantified through metabolic conversion rate values. Parameter interdependency allowed an accurate quantification and can therefore be useful for monitoring metabolic activity in different tissues.

  15. NH4+ triggers the release of astrocytic lactate via mitochondrial pyruvate shunting

    Science.gov (United States)

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T.; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L. Felipe

    2015-01-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K+ as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4+, a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4+ with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4+ and in the somatosensory cortex of anesthetized mice in response to i.v. NH4+. Unexpectedly, NH4+ had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4+ diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4+ is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4+ behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes. PMID:26286989

  16. NH4(+) triggers the release of astrocytic lactate via mitochondrial pyruvate shunting.

    Science.gov (United States)

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L Felipe

    2015-09-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K(+) as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4(+), a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4(+) with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4(+) and in the somatosensory cortex of anesthetized mice in response to i.v. NH4(+). Unexpectedly, NH4(+) had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4(+) diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4(+) is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4(+) behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes.

  17. Metabolic imaging of patients with prostate cancer using hyperpolarized [1-¹³C]pyruvate

    DEFF Research Database (Denmark)

    Nelson, Sarah J; Kurhanewicz, John; Vigneron, Daniel B

    2013-01-01

    This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-¹³C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-f...

  18. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    Science.gov (United States)

    Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  19. Hyperpolarized 1-13C Pyruvate Imaging of Porcine Cardiac Metabolism shift by GIK Intervention

    DEFF Research Database (Denmark)

    Søvsø Szocska Hansen, Esben; Tougaard, Rasmus Stilling; Mikkelsen, Emmeli

    to evaluate the general feasibility to detect an imposed shift in metabolic substrate utilization during metabolic modulation with glucose, insulin and potassium (GIK) infusion. This study demonstrates that hyperpolarized 13C-pyruvate, in a large animal, is a feasible method for cardiac studies, and...

  20. Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest

    DEFF Research Database (Denmark)

    Kiilerich, Kristian; Jørgensen, Stine Ringholm; Biensø, Rasmus Sjørup

    2011-01-01

    To test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH), 6 healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, the subjects completed an OGTT and a one-legged knee...

  1. Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis.

    Science.gov (United States)

    Charbonnier, Teddy; Le Coq, Dominique; McGovern, Stephen; Calabre, Magali; Delumeau, Olivier; Aymerich, Stéphane; Jules, Matthieu

    2017-10-03

    At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB ), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB We show that both glucose and malate, the preferred carbon sources for B. subtilis , trigger the binding of CcpA upstream of pftAB , which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB , which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis IMPORTANCE Pyruvate is a small-molecule metabolite ubiquitous in living cells. Several species also use it as a carbon source as well as excrete it into the environment. The bacterial systems for pyruvate import/export have yet to be discovered. Here, we identified in the model bacterium Bacillus subtilis the first import

  2. Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain.

    Science.gov (United States)

    Morken, Tora Sund; Brekke, Eva; Håberg, Asta; Widerøe, Marius; Brubakk, Ann-Mari; Sonnewald, Ursula

    2014-01-01

    Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-(13)C]glucose and were sacrificed 30 min later. Brain extracts were analysed using (1)H- and (13)C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-(13)C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-(13)C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-(13)C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.

  3. Protective effect of pyruvate against ethanol-induced apoptotic neurodegeneration in the developing rat brain.

    Science.gov (United States)

    Ullah, Najeeb; Naseer, Muhammad Imran; Ullah, Ikram; Lee, Hae Young; Koh, Phil Ok; Kim, Myeong Ok

    2011-12-01

    Exposure to alcohol during the early stages of brain development can lead to neurological disorders in the CNS. Apoptotic neurodegeneration due to ethanol exposure is a main feature of alcoholism. Exposure of developing animals to alcohol (during the growth spurt period in particular) elicits apoptotic neuronal death and causes fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS). A single episode of ethanol intoxication (at 5 g/kg) in a seven-day-old developing rat can activate the apoptotic cascade, leading to widespread neuronal death in the brain. In the present study, we investigated the potential protective effect of pyruvate against ethanol-induced neuroapoptosis. After 4h, a single dose of ethanol induced upregulation of Bax, release of mitochondrial cytochrome-c into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1), all of which promote apoptosis. These effects were all reversed by co-treatment with pyruvate at a well-tolerated dosage (1000 mg/kg). Histopathology performed at 24 and 48 h with Fluoro-Jade-B and cresyl violet stains showed that pyruvate significantly reduced the number of dead cells in the cerebral cortex, hippocampus and thalamus. Immunohistochemical analysis at 24h confirmed that ethanol-induced cell death is both apoptotic and inhibited by pyruvate. These findings suggest that pyruvate treatment attenuates ethanol-induced neuronal cell loss in the developing rat brain and holds promise as a safe therapeutic and neuroprotective agent in the treatment of neurodegenerative disorders in newborns and infants. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. 13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and of the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

    International Nuclear Information System (INIS)

    Cohen, S.M.

    1987-01-01

    13 C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13 C enrichments at the individual carbons of glutamate when [3- 13 C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13 C label into fatty acids was monitored in this liver preparation. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1- 13 C]acetyl-CoA (from [2- 13 C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3- 13 C]alanine plus [2- 13 C]ethanol, which are converted to [2- 13 C]acetyl-CoA. Thus, measurement of 13 C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids. In addition to obtaining the rate of lipogenesis, it was possible to distinguish the contributions of chain elongation from those of the de novo synthesis pathway and to estimate the average chain length of the 13 C-labeled fatty acids produced

  5. Effect of thiamine deficiency, pyrithiamine and oxythiamine on pyruvate metabolism in rat liver and brain in vivo

    International Nuclear Information System (INIS)

    Meghal, S.K.; O'Neal, R.M.; Koeppe, R.E.

    1977-01-01

    Rats were fed either a thiamine-deficient diet or diets containing pyrithiamine or oxythiamine. When symptoms of thiamine deficiency appeared, the animals were injected intraperitoneally with [2- 14 C] pyruvate six to twelve minutes prior to sacrifice. Free glutamic and aspartic acids were isolated from liver and brain and degraded. The results indicate that, in thiamine-deficient or oxythiamine-treated rats, pyruvate metabolism in liver and brain is similar to that in normal animals. In contrast, pyrithinamine drastically decreases the oxidative decarboxylation of pyruvate by rat liver. (auth.)

  6. Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: requirement for ADP.

    Science.gov (United States)

    Bulos, B A; Thomas, B J; Shukla, S P; Sacktor, B

    1984-11-01

    Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate ( + proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (Km = 8.0 microM) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled alpha-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (greater than 99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 microM), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 +/- 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent KI was 0.8 mM. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, alpha-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the

  7. Reprint of "How do components of real cloud water affect aqueous pyruvate oxidation?"

    Science.gov (United States)

    Boris, Alexandra J.; Desyaterik, Yury; Collett, Jeffrey L.

    2015-01-01

    Chemical oxidation of dissolved volatile or semi-volatile organic compounds within fog and cloud droplets in the atmosphere could be a major pathway for secondary organic aerosol (SOA) formation. This proposed pathway consists of: (1) dissolution of organic chemicals from the gas phase into a droplet; (2) reaction with an aqueous phase oxidant to yield low volatility products; and (3) formation of particle phase organic matter as the droplet evaporates. The common approach to simulating aqueous SOA (aqSOA) reactions is photo-oxidation of laboratory standards in pure water. Reactions leading to aqSOA formation should be studied within real cloud and fog water to determine whether additional competing processes might alter apparent rates of reaction as indicated by rates of reactant loss or product formation. To evaluate and identify the origin of any cloud water matrix effects on one example of observed aqSOA production, pyruvate oxidation experiments simulating aqSOA formation were monitored within pure water, real cloud water samples, and an aqueous solution of inorganic salts. Two analysis methods were used: online electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR-ToF-MS), and offline anion exchange chromatography (IC) with quantitative conductivity and qualitative ESI-HR-ToF-MS detection. The apparent rate of oxidation of pyruvate was slowed in cloud water matrices: overall measured degradation rates of pyruvate were lower than in pure water. This can be at least partially accounted for by the observed formation of pyruvate from reactions of other cloud water components. Organic constituents of cloud water also compete for oxidants and/or UV light, contributing to the observed slowed degradation rates of pyruvate. The oxidation of pyruvate was not significantly affected by the presence of inorganic anions (nitrate and sulfate) at cloud-relevant concentrations. Future bulk studies of aqSOA formation reactions using simplified

  8. Next-generation sequencing-based molecular diagnosis of chronic non-spherocytic hemolysis in erythrocytic enzymopathies

    Directory of Open Access Journals (Sweden)

    Manu Jamwal

    2017-10-01

    Full Text Available Mutations in genes encoding red blood cell enzymes are often inherited in an autosomal recessive manner and can lead to chronic nonspherocytic hemolytic anemia (CNSHA in homozygotes and compound heterozygotes. Usual clinical manifestations include jaundice, cholelithiasis and splenomegaly with normocytic normochromic hemolysis. Phenotypes range from fully-compensated hemolysis (without anemia to transfusion-dependent states. Definitive diagnosis requires biochemical testing of enzyme levels, which for rarer enzymes are often difficult and not easily available. Molecular diagnosis using a gene-by-gene approach is expensive, time-consuming and cumbersome. Targeted resequencing can expedite the molecular diagnosis in cases where the hemolysis remains unexplained after routine laboratory tests. Ten patients with clinical and laboratory evidence suggestive of hemolytic anemia, but negative family history, were enrolled. Various biochemical and molecular tests were used to exclude glucose-6-phosphate dehydrogenase (G6PD deficiency, thalassemias, hemoglobinopathies, autoimmune hemolysis, hereditary spherocytosis and pyruvate kinase (PKLR deficiency. Common G6PD and PKLR variants were excluded by molecular tests. DNA Libraries were prepared using TruSight One™ panel and sequenced on MiSeq™ Sequencing System. MiSeq Reporter™ and VariantStudio™ v2.1 were used for analysis, classification, and reporting of genomic variants reporting genomic variants. All 10 patients’ diagnoses were resolved by targeted resequencing: two had G6PD deficiency, two had glucose-6-phosphate isomerase (GPI deficiency and six unexpectedly had pyruvate kinase deficiency despite pyruvate kinase enzyme activity assays previously being normal in all. All the mutations were predicted deleterious by PolyPhen, SIFT, Provean, mutpred and Mutationtaster software. The mutations were validated in parents and/or siblings (where available to establish the mode of inheritance. Our

  9. Carbon-14 tracer studies in rat-liver perfusion experiments under conditions of gluconeogenesis from lactate and pyruvate

    International Nuclear Information System (INIS)

    Muellhofer, G.; Schwab, A.; Mueller, C.; Stetten, C. von; Gruber, E.

    1977-01-01

    The intracellular events in the metabolic pathway of gluconeogenesis from lactate and pyruvate in liver tissue were assumed to be understood. Nevertheless the results of several 14 C-tracer experiments gave rise to the postulation of still unknown intracellular interactions under this condition. A contribution was made to the solution of this problem by using different 14 C labelled tracers such as [1- 14 C]lactate or pyruvate and [2- 14 C]lactate or pyruvate. [ 14 C]bicarbonate and [1- 14 C]-octanoate in perfusion experiments with livers from rats under conditions of gluconeogenesis from lactate and pyruvate. The 14 C labelling patterns of intracellular metabolities such as malate, citrate, phosphoenolpyruvate, phosphoglycerate and newly synthesized glucose were analysed under different conditions. A comparison with values calculated by using metabolic models based on the generally accepted concepts of intracellular interactions showed some fundamental discrepancies which justify the postulation. (orig./MG) [de

  10. Investigating tumor perfusion and metabolism using multiple hyperpolarized 13C compounds: HP001, pyruvate and urea

    DEFF Research Database (Denmark)

    von Morze, Cornelius; Larson, Peder E.Z.; Hu, Simon

    2012-01-01

    The metabolically inactive hyperpolarized agents HP001 (bis-1,1-(hydroxymethyl)-[1-13C]cyclopropane-d8) and urea enable a new type of perfusion magnetic resonance imaging based on a direct signal source that is background-free. The addition of perfusion information to metabolic information obtained...... (T1=95 s ex vivo, 32 s in vivo at 3 T) using a pulse sequence with balanced steady-state free precession and ramped flip angle over time for efficient utilization of the hyperpolarized magnetization and three-dimensional echo-planar spectroscopic imaging of urea copolarized with [1-13C...... of separate dynamic HP001 imaging and copolarized pyruvate/urea imaging were compared. A strong and significant correlation (R=0.73, P=.02) detected between the urea and HP001 data confirmed the value of copolarizing urea with pyruvate for simultaneous assessment of perfusion and metabolism....

  11. Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism

    DEFF Research Database (Denmark)

    Perry, Rachel J; Borders, Candace B; Cline, Gary W

    2016-01-01

    /tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously...... at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only...... tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo....

  12. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    International Nuclear Information System (INIS)

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-01-01

    Potassium tellurite (K 2 TeO 3 ) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  13. Enzyme mechanisms for pyruvate-to-lactate flux attenuation: a study of Sherpas, Quechuas, and hummingbirds.

    Science.gov (United States)

    Hochachka, P W; Stanley, C; McKenzie, D C; Villena, A; Monge, C

    1992-10-01

    During incremental exercise to fatigue under hypobaric hypoxia, Andean Quechua natives form and accumulate less plasma lactate than do lowlanders under similar conditions. This phenomenon of low lactate accumulation despite hypobaric hypoxia, first discovered some half century ago, is known in Quechuas to be largely unaffected by acute exposure to hypoxia or by acclimatization to sea level conditions. Earlier Nuclear Magnetic Resonance (NMR) spectroscopy and metabolic biochemistry studies suggest that closer coupling of energy demand and energy supply in Quechuas allows given changes in work rate with relatively modest changes in muscle adenylate and phosphagen concentrations, thus tempering the activation of glycolytic flux to pyruvate--a coarse control mechanism operating at the level of overall pathway flux. Later studies of enzyme activities in skeletal muscles of Quechuas and of Sherpas have identified a finely-tuned control mechanism which by adaptive modifications of a few key enzymes apparently serves to specifically attenuate pyruvate flux to lactate.

  14. MCA Vmean and the arterial lactate-to-pyruvate ratio correlate during rhythmic handgrip

    DEFF Research Database (Denmark)

    Rasmussen, Peter; Plomgaard, Peter; Krogh-Madsen, Rikke

    2006-01-01

    /P ratio at two plasma lactate levels. MCA Vmean was determined by ultrasound Doppler sonography at rest, during 10 min of rhythmic handgrip exercise at approximately 65% of maximal voluntary contraction force, and during 20 min of recovery in seven healthy male volunteers during control...... and a approximately 15 mmol/l hyperglycemic clamp. Cerebral arteriovenous differences for metabolites were obtained by brachial artery and retrograde jugular venous catheterization. Control resting arterial lactate was 0.78 +/- 0.09 mmol/l (mean +/- SE) and pyruvate 55.7 +/- 12.0 micromol/l (L/P ratio 16.4 +/- 1......Regulation of cerebral blood flow during physiological activation including exercise remains unknown but may be related to the arterial lactate-to-pyruvate (L/P) ratio. We evaluated whether an exercise-induced increase in middle cerebral artery mean velocity (MCA Vmean) relates to the arterial L...

  15. Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain

    DEFF Research Database (Denmark)

    Zhang, Yiming; Liu, Guodong; Engqvist, Martin K. M.

    2015-01-01

    Background: A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole...... DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase...... further increased the maximum specific growth rate to 0.069 h-1. Conclusions: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing...

  16. Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein

    DEFF Research Database (Denmark)

    Schreiber, K; Boes, N; Escbach, M

    2006-01-01

    the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress...... proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression...... was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter...

  17. Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

    1997-03-01

    Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

  18. Sodium Pyruvate Reduced Hypoxic-Ischemic Injury to Neonatal Rat Brain

    OpenAIRE

    Pan, Rui; Rong, Zhihui; She, Yun; Cao, Yuan; Chang, Li-Wen; Lee, Wei-Hua

    2012-01-01

    Background Neonatal hypoxia-ischemia (HI) remains a major cause of severe brain damage and is often associated with high mortality and lifelong disability. Immature brains are extremely sensitive to hypoxia-ischemia, shown as prolonged mitochondrial neuronal death. Sodium pyruvate (SP), a substrate of the tricarboxylic acid cycle and an extracellular antioxidant, has been considered as a potential treatment for hypoxic-ischemic encephalopathy (HIE), but its effects have not been evaluated in ...

  19. Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

    Science.gov (United States)

    Li, Xiaoli; Li, Yaqing; Han, Gaoyang; Li, Xiaoran; Ji, Yasai; Fan, Zhirui; Zhong, Yali; Cao, Jing; Zhao, Jing; Mariusz, Goscinski; Zhang, Mingzhi; Wen, Jianguo; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice. PMID:27835892

  20. Beneficial effect of pyruvate therapy on Leigh syndrome due to a novel mutation in PDH E1α gene.

    Science.gov (United States)

    Koga, Yasutoshi; Povalko, Nataliya; Katayama, Koujyu; Kakimoto, Noriko; Matsuishi, Toyojiro; Naito, Etsuo; Tanaka, Masashi

    2012-02-01

    Leigh syndrome (LS) is a progressive untreatable degenerating mitochondrial disorder caused by either mitochondrial or nuclear DNA mutations. A patient was a second child of unconsanguineous parents. On the third day of birth, he was transferred to neonatal intensive care units because of severe lactic acidosis. Since he was showing continuous lactic acidosis, the oral supplementation of dichloroacetate (DCA) was introduced on 31st day of birth at initial dose of 50 mg/kg, followed by maintenance dose of 25 mg/kg/every 12 h. The patient was diagnosed with LS due to a point mutation of an A-C at nucleotide 599 in exon 6 in the pyruvate dehydrogenase E1α gene, resulting in the substitution of aspartate for threonine at position 200 (N200T). Although the concentrations of lactate and pyruvate in blood were slightly decreased, his clinical conditions were deteriorating progressively. In order to overcome the mitochondrial or cytosolic energy crisis indicated by lactic acidosis as well as clinical symptoms, we terminated the DCA and administered 0.5 g/kg/day TID of sodium pyruvate orally. We analyzed the therapeutic effects of DCA or sodium pyruvate in the patient, and found that pyruvate therapy significantly decreased lactate, pyruvate and alanine levels, showed no adverse effects such as severe neuropathy seen in DCA, and had better clinical response on development and epilepsy. Though the efficacy of pyruvate on LS will be evaluated by randomized double-blind placebo-controlled study design in future, pyruvate therapy is a possible candidate for therapeutic choice for currently incurable mitochondrial disorders such as LS. Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  1. Single Sodium Pyruvate Ingestion Modifies Blood Acid-Base Status and Post-Exercise Lactate Concentration in Humans

    Directory of Open Access Journals (Sweden)

    Robert A. Olek

    2014-05-01

    Full Text Available This study examined the effect of a single sodium pyruvate ingestion on a blood acid-base status and exercise metabolism markers. Nine active, but non-specifically trained, male subjects participated in the double-blind, placebo-controlled, crossover study. One hour prior to the exercise, subjects ingested either 0.1 g·kg−1 of body mass of a sodium pyruvate or placebo. The capillary blood samples were obtained at rest, 60 min after ingestion, and then three and 15 min after completing the workout protocol to analyze acid-base status and lactate, pyruvate, alanine, glucose concentrations. The pulmonary gas exchange, minute ventilation and the heart rate were measured during the exercise at a constant power output, corresponding to ~90% O2max. The blood pH, bicarbonate and the base excess were significantly higher after sodium pyruvate ingestion than in the placebo trial. The blood lactate concentration was not different after the ingestion, but the post-exercise was significantly higher in the pyruvate trial (12.9 ± 0.9 mM than in the placebo trial (10.6 ± 0.3 mM, p < 0.05 and remained elevated (nonsignificant after 15 min of recovery. The blood pyruvate, alanine and glucose concentrations, as well as the overall pulmonary gas exchange during the exercise were not affected by the pyruvate ingestion. In conclusion, the sodium pyruvate ingestion one hour before workout modified the blood acid-base status and the lactate production during the exercise.

  2. MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4

    OpenAIRE

    Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Espindola Camacho, Carolina; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A.L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.

    2016-01-01

    Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but ...

  3. Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Teddy Charbonnier

    2017-10-01

    Full Text Available At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB, the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB. We show that both glucose and malate, the preferred carbon sources for B. subtilis, trigger the binding of CcpA upstream of pftAB, which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB, which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis.

  4. Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

    OpenAIRE

    Compan V; Pierredon S; Vanderperre B; Krznar P; Marchiq I; Zamboni N; Pouyssegur J; Martinou JC

    2015-01-01

    The transport of pyruvate into mitochondria requires a specific carrier the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism and its activity is likely to play a key role in bioenergetics. Until now investigation of the MPC activity has been limited. However the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell popu...

  5. MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells

    OpenAIRE

    Vanderperre, Benoît; Cermakova, Kristina; Escoffier Breancon, Jessica; Kaba, Mayis; Bender, Tom; Nef, Serge; Martinou, Jean-Claude

    2016-01-01

    Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence...

  6. Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis

    Directory of Open Access Journals (Sweden)

    Stefan Christen

    2016-10-01

    Full Text Available Cellular proliferation depends on refilling the tricarboxylic acid (TCA cycle to support biomass production (anaplerosis. The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis in breast-cancer-derived lung metastases compared to their primary cancers using in vivo 13C tracer analysis. We discovered that lung metastases have higher PC-dependent anaplerosis compared to primary breast cancers. Based on in vitro analysis and a mathematical model for the determination of compartment-specific metabolite concentrations, we found that mitochondrial pyruvate concentrations can promote PC-dependent anaplerosis via enzyme kinetics. In conclusion, we show that breast cancer cells proliferating as lung metastases activate PC-dependent anaplerosis in response to the lung microenvironment.

  7. Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

    Science.gov (United States)

    Westerhold, Lauren E; Bridges, Lance C; Shaikh, Saame Raza; Zeczycki, Tonya N

    2017-07-11

    Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

  8. Stem Cell Metabolism in Cancer and Healthy Tissues: Pyruvate in the Limelight

    Directory of Open Access Journals (Sweden)

    Cyril Corbet

    2018-01-01

    Full Text Available Normal and cancer stem cells (CSCs share the remarkable potential to self-renew and differentiate into many distinct cell types. Although most of the stem cells remain under quiescence to maintain their undifferentiated state, they can also undergo cell divisions as required to regulate tissue homeostasis. There is now a growing evidence that cell fate determination from stem cells implies a fine-tuned regulation of their energy balance and metabolic status. Stem cells can shift their metabolic substrate utilization, between glycolysis and mitochondrial oxidative metabolism, during specification and/or differentiation, as well as in order to adapt their microenvironmental niche. Pyruvate appears as a key metabolite since it is at the crossroads of cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. This Review describes how metabolic reprogramming, focusing on pyruvate utilization, drives the fate of normal and CSCs by modulating their capacity for self-renewal, clonal expansion/differentiation, as well as metastatic potential and treatment resistance in cancer. This Review also explores potential therapeutic strategies to restore or manipulate stem cell function through the use of small molecules targeting the pyruvate metabolism.

  9. Increased production of pyruvic acid by Escherichia coli RNase G mutants in combination with cra mutations.

    Science.gov (United States)

    Sakai, Taro; Nakamura, Naoko; Umitsuki, Genryou; Nagai, Kazuo; Wachi, Masaaki

    2007-08-01

    The Escherichia coli RNase G is known as an endoribonuclease responsible for the 5'-end maturation of 16S rRNA and degradation of several specific mRNAs such as adhE and eno mRNAs. In this study, we found that an RNase G mutant derived from the MC1061 strain did not grow on a glucose minimal medium. Genetic analysis revealed that simultaneous defects of cra and ilvIH, encoding a transcriptional regulator of glycolysis/gluconeogenesis and one of isozymes of acetohydroxy acid synthase, respectively, were required for this phenomenon to occur. The results of additional experiments presented here indicate that the RNase G mutation, in combination with cra mutation, caused the increased production of pyruvic acid from glucose, which was then preferentially converted to valine due to the ilvIH mutation, resulting in depletion of isoleucine. In fact, the rng cra double mutant produced increased amount of pyruvate in the medium. These results suggest that the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine.

  10. Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

    Science.gov (United States)

    Su, Yu-Bin; Peng, Bo; Li, Hui; Cheng, Zhi-Xue; Zhang, Tian-Tuo; Zhu, Jia-Xin; Li, Dan; Li, Min-Yi; Ye, Jin-Zhou; Du, Chao-Chao; Zhang, Song; Zhao, Xian-Liang; Yang, Man-Jun; Peng, Xuan-Xian

    2018-02-13

    The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

  11. Study on the protective effect of ethyl pyruvate on mouse models of sepsis-induced lung injury

    International Nuclear Information System (INIS)

    Ti Dongdong; Deng Zihui; Xue Hui; Wang Luhuan; Lin Ji; Yan Guangtao

    2008-01-01

    Objective: To investigate the protective role of ethyl pyruvate on mouse models of lung injury from sepsis. Methods: Mouse sepsis models were established by cecal ligation-perforation. Four enzyme parameters related to synthesis of free radicals in lung homogenized fluids namely malonaldehyde (MDA), pyruvate acid, lactic acid and total anti-oxidative capacity (TAOC) were determined with spectrophotometry, and serum leptin levels were detected with radioimmunoassay at 3, 6, 9, 12h after operation in these models. Half of the models were treated with intraperitoneal injection of ethyl pyruvate (EP) (75mg/kg). Results: In the models treated with ethyl pyruvate injection, the activity of malonaldehyde, pyruvate acid, lactic acid and total anti-oxidative capacity were affected to certain extent, at some time frames but the results were not unanimously inhibitive or promotive. Serum leptin levels in EP injection models at 6h and 12h after sepsis were significantly higher than those in non-treated models. Conclusion: Ethyl pyruvate perhaps exerted its protective effect on sepsis-induced lung injury through increase of leptin levels in the models. (authors)

  12. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  13. From Phosphosites to Kinases

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations...... sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available...

  14. Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

    Science.gov (United States)

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-10-24

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex*

    Science.gov (United States)

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S.; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-01-01

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

  16. Hyperpolarized [1-(13) C]pyruvate MRI for noninvasive examination of placental metabolism and nutrient transport: A feasibility study in pregnant guinea pigs.

    Science.gov (United States)

    Friesen-Waldner, Lanette J; Sinclair, Kevin J; Wade, Trevor P; Michael, Banoub; Chen, Albert P; de Vrijer, Barbra; Regnault, Timothy R H; McKenzie, Charles A

    2016-03-01

    To test the feasibility of hyperpolarized [1-(13) C]pyruvate magnetic resonance imaging (MRI) for noninvasive examination of guinea pig fetoplacental metabolism and nutrient transport. Seven pregnant guinea pigs with a total of 30 placentae and fetuses were anesthetized and scanned at 3T. T1 -weighted (1) H images were obtained from the maternal abdomen. An 80 mM solution of hyperpolarized [1-(13) C]pyruvate (hereafter referred to as pyruvate) was injected into a vein in the maternal foot. Time-resolved 3D (13) C images were acquired starting 10 seconds after the beginning of bolus injection and every 10 seconds after to 50 seconds. The pregnant guinea pigs were recovered after imaging. Regions of interest (ROIs) were drawn around the maternal heart and each placenta and fetal liver in all slices in the (1) H images. These ROIs were copied to the (13) C images and were used to calculate the sum of the pyruvate and lactate signal intensities for each organ. The signal intensities were normalized by the volume of the organ and the maximum signal in the maternal heart. No adverse events were observed in the pregnant guinea pigs and natural pupping occurred at term (∼68 days). Pyruvate signal was observed in all 30 placentae, and lactate, a by-product of pyruvate metabolism, was also observed in all placentae. The maximum pyruvate and lactate signals in placentae occurred at 20 seconds. In addition to the observation of pyruvate and lactate signals in the placentae, both pyruvate and lactate signals were observed in all fetal livers. The maximum pyruvate and lactate signals in the fetal livers occurred at 10 seconds and 20 seconds, respectively. This work demonstrates the feasibility of using hyperpolarized [1-(13) C]pyruvate MRI to noninvasively examine fetoplacental metabolism and transport of pyruvate in guinea pigs. Hyperpolarized (13) C MRI may provide a novel method for longitudinal studies of fetoplacental abnormalities. © 2015 Wiley Periodicals, Inc.

  17. Combined Hyperpolarized 13C-pyruvate MRS and 18F-FDG PET (HyperPET) Estimates of Glycolysis in Canine Cancer Patients

    DEFF Research Database (Denmark)

    Hansen, Adam E.; Gutte, Henrik; Holst, Pernille

    2018-01-01

    13C Magnetic Resonance Spectroscopy (MRS) using hyperpolarized 13C-labeled pyruvate as a substrate offers a measure of pyruvate-lactate interconversion and is thereby a marker of the elevated aerobic glycolysis (Warburg effect) generally exhibited by cancer cells. Here, we aim to compare hyperpol......13C Magnetic Resonance Spectroscopy (MRS) using hyperpolarized 13C-labeled pyruvate as a substrate offers a measure of pyruvate-lactate interconversion and is thereby a marker of the elevated aerobic glycolysis (Warburg effect) generally exhibited by cancer cells. Here, we aim to compare...

  18. Comparison the effectiveness of pyruvic acid 50% and salicylic acid 30% in the treatment of acne

    Directory of Open Access Journals (Sweden)

    Fariba Jaffary

    2016-01-01

    Full Text Available Background: Acne vulgaris is a chronic inflammatory disease of the pilosebaceous follicles and one of the most common skin diseases. The peeling method has been recently found to be effective for acne treatment. This study aimed to compare the efficacy of pyruvic acid 50% and salicylic acid 30% peeling in the treatment of mild to moderate acne. Materials and Methods: In a prospective single-blinded clinical trial, 86 patients with acne were randomly assigned into two groups. In both groups, the routine treatment of acne (topical solution of erythromycin 4%, triclorocarban soap, and sunscreen were used twice a day for 8 weeks. In addition, salicylic acid 30% for the control group and pyruvic acid 50% for the case group were used. In both groups, acne severity index (ASI was calculated before and at week 2, 4, 6, and 8 of the treatment. Patient satisfaction was assessed at the end of the treatment. Side effects were recorded using a checklist. Results: In both groups, the reduction in the number of comedones, papules, and ASI were statistically significant (P < 0.001 in the course of treatment. However, it was not significant regarding the number of pustules (P = 0.09. None of the number of comedone, papules, pustules, and ASI was statistically different between study groups. Both treatment groups had similar side effects except for scaling in the fifth session, which was significantly lower in salicylic acid - treated patients (P = 0.015. Conclusion: Both pyruvic acid 50% and salicylic acid 30% are effective in the improvement of mild to moderate acne with no significant difference in efficacy and side effects.

  19. Comparison the effectiveness of pyruvic acid 50% and salicylic acid 30% in the treatment of acne.

    Science.gov (United States)

    Jaffary, Fariba; Faghihi, Gita; Saraeian, Sara; Hosseini, Sayed Mohsen

    2016-01-01

    Acne vulgaris is a chronic inflammatory disease of the pilosebaceous follicles and one of the most common skin diseases. The peeling method has been recently found to be effective for acne treatment. This study aimed to compare the efficacy of pyruvic acid 50% and salicylic acid 30% peeling in the treatment of mild to moderate acne. In a prospective single-blinded clinical trial, 86 patients with acne were randomly assigned into two groups. In both groups, the routine treatment of acne (topical solution of erythromycin 4%, triclorocarban soap, and sunscreen) were used twice a day for 8 weeks. In addition, salicylic acid 30% for the control group and pyruvic acid 50% for the case group were used. In both groups, acne severity index (ASI) was calculated before and at week 2, 4, 6, and 8 of the treatment. Patient satisfaction was assessed at the end of the treatment. Side effects were recorded using a checklist. In both groups, the reduction in the number of comedones, papules, and ASI were statistically significant ( P < 0.001) in the course of treatment. However, it was not significant regarding the number of pustules ( P = 0.09). None of the number of comedone, papules, pustules, and ASI was statistically different between study groups. Both treatment groups had similar side effects except for scaling in the fifth session, which was significantly lower in salicylic acid - treated patients ( P = 0.015). Both pyruvic acid 50% and salicylic acid 30% are effective in the improvement of mild to moderate acne with no significant difference in efficacy and side effects.

  20. Maturation of pig oocytes in vitro in a medium with pyruvate

    Directory of Open Access Journals (Sweden)

    H. Gonzales-Figueroa

    2005-06-01

    Full Text Available The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF and in SM + pFF + gonadotropins (eCG and hCG for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9% and SM + pFF (42.9% showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6% showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6% and SM + pFF (42.9 ± 3.7%. These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

  1. Protective Effect of Pyruvate Against Radiation-Induced Damage in Collagenized Tissues

    Science.gov (United States)

    Griko, Y. V.; Yan, Xiaoli

    2016-01-01

    Exposure to high doses of ionizing radiation produces both acute and late effects on the collagenized tissues and have profound effects on wound healing. Because of the crucial practical importance for new radioprotective agents, our study has been focused on evaluation of the efficacy of non-toxic naturally occurring compounds to protect tissue integrity against high-dose gamma radiation. Here, we demonstrate that molecular integrity of collagen may serve as a sensitive biological marker for quantitative evaluation of molecular damage to collagenized tissue and efficacy of radioprotective agents. Increasing doses of gamma radiation (0-50kGy) result in progressive destruction of the native collagen fibrils, which provide a structural framework, strength, and proper milieu for the regenerating tissue. The strategy used in this study involved the thermodynamic specification of all structural changes in collagenized matrix of skin, aortic heart valve, and bone tissue induced by different doses and conditions of g-irradiation. This study describes a simple biophysical approach utilizing the Differential Scanning Calorimetry (DSC) to characterize the structural resistance of the aortic valve matrix exposed to different doses of g-irradiation. It allows us to identify the specific response of each constituent as well as to determine the influence of the different treatments on the characteristic parameters of protein structure. We found that pyruvate, a substance that naturally occurs in the body, provide significant protection (up to 80%) from biochemical and biomechanical damage to the collagenized tissue through the effective targeting of reactive oxygen species. The recently discovered role of pyruvate in the cell antioxidant defense to O2 oxidation, and its essential constituency in the daily human diet, indicate that the administration of pyruvate-based radioprotective formulations may provide safe and effective protection from deleterious effects of ionizing

  2. A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

    Science.gov (United States)

    Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

    2016-04-01

    Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

  3. Creatine and creatine pyruvate reduce hypoxia-induced effects on phrenic nerve activity in the juvenile mouse respiratory system.

    Science.gov (United States)

    Scheer, Monika; Bischoff, Anna M; Kruzliak, Peter; Opatrilova, Radka; Bovell, Douglas; Büsselberg, Dietrich

    2016-08-01

    Adequate concentrations of ATP are required to preserve physiological cell functions and protect tissue from hypoxic damage. Decreased oxygen concentration results in ATP synthesis relying increasingly on the presence of phosphocreatine. The lack of ATP through hypoxic insult to neurons that generate or regulate respiratory function, would lead to the cessation of breathing (apnea). It is not clear whether creatine plays a role in maintaining respiratory phrenic nerve (PN) activity during hypoxic challenge. The aim of the study was to test the effects of exogenously applied creatine or creatine pyruvate in maintaining PN induced respiratory rhythm against the deleterious effects of severe hypoxic insult using Working Heart-Brainstem (WHB) preparations of juvenile Swiss type mice. WHB's were perfused with control perfusate or perfusate containing either creatine [100μM] or creatine pyruvate [100μM] prior to hypoxic challenge and PN activity recorded throughout. Results showed that severe hypoxic challenge resulted in an initial transient increase in PN activity, followed by a reduction in that activity leading to respiratory apnea. The results demonstrated that perfusing the WHB preparation with creatine or creatine pyruvate, significantly reduced the onset of apnea compared to control conditions, with creatine pyruvate being the more effective substance. Overall, creatine and creatine pyruvate each produced time-dependent degrees of protection against severe hypoxic-induced disturbances of PN activity. The underlying protective mechanisms are unknown and need further investigations. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Modeling of the pyruvate production with Escherichia coli: comparison of mechanistic and neural networks-based models.

    Science.gov (United States)

    Zelić, B; Bolf, N; Vasić-Racki, D

    2006-06-01

    Three different models: the unstructured mechanistic black-box model, the input-output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249-258 (2004); Zelić et al. Eng Life Sci 3:299-305 (2003); Zelić et al Biotechnol Bioeng 85:638-646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.

  5. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  6. Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain

    OpenAIRE

    Morken, Tora Sund; Brekke, Eva Mari Førland; Håberg, Asta; Widerøe, Marius; Brubakk, Ann-Mari; Sonnewald, Ursula

    2014-01-01

    Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity duri...

  7. Inborn Errors of Metabolism with Acidosis: Organic Acidemias and Defects of Pyruvate and Ketone Body Metabolism.

    Science.gov (United States)

    Schillaci, Lori-Anne P; DeBrosse, Suzanne D; McCandless, Shawn E

    2018-04-01

    When a child presents with high-anion gap metabolic acidosis, the pediatrician can proceed with confidence by recalling some basic principles. Defects of organic acid, pyruvate, and ketone body metabolism that present with acute acidosis are reviewed. Flowcharts for identifying the underlying cause and initiating life-saving therapy are provided. By evaluating electrolytes, blood sugar, lactate, ammonia, and urine ketones, the provider can determine the likelihood of an inborn error of metabolism. Freezing serum, plasma, and urine samples during the acute presentation for definitive diagnostic testing at the provider's convenience aids in the differential diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    Science.gov (United States)

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Effect of gamma radiation on the concentration of pyruvate and lactate in erythrocytes of healthy men after submaximal physical exercise

    International Nuclear Information System (INIS)

    Zagorski, T.; Dudek, I.; Berkan, L.; Chmielewski, H.; Kedziora, J.

    1993-01-01

    The aim of this work was to study the effect of gamma radiation and submaximal physical exercise on the concentration of final products of anaerobic glycolytic pathway in erythrocytes of healthy men. Twenty one men aged 20-22 were examined. They underwent physical exercise at doses of 2 w/kg body weight for 15 min. Erythrocytes were taken in the rest and after physical exercise and were exposed to gamma radiation (500 Gy doses) from 60 Co source. The concentration of pyruvate was estimated by Fermognost tests and the concentration of lactate by Boehringer Mannheim tests. The submaximal physical exercise was found to cause a significantly increased concentration of pyruvate and lactate in the non-radiated and irradiated erythrocytes. Gamma radiation at 500 Gy dose was found to increase concentration of pyruvate in erythrocytes (in the rest and after physical exercise) with simultaneous decrease of lactate concentration. (author). 17 refs, 1 tab

  10. Recovery of Pyruvic Acid using Tri-n-butylamine Dissolved in Non-Toxic Diluent (Rice Bran Oil)

    Science.gov (United States)

    Pal, Dharm; Keshav, Amit

    2016-04-01

    An attempt has been made to investigate the effectiveness of the vegetable oil based biocompatible solvent for the separation of pyruvic acid from fermentation broth, by using rice bran oil as natural, non-toxic diluent. Reactive extraction of pyruvic acid (0.1-0.5 k mol/m3) from aqueous solutions has been studied using tri-n-butylamine (TBA; 10-70 %) as an extractant dissolved in non toxic rice bran oil at T = 30 ± 1 °C. Results were presented in terms of distribution coefficient (Kd), extraction efficiency (E %), loading ratio (Z), and complexation constant (\\varphi_{α β }). Extraction equilibrium was interpreted using mass action modeling approach. Based on the extent of loading (Z < 0.5) only (1:1), pyruvic acid: TBA complex was proposed. Equilibrium complexation constant was evaluated to 1.22 m3/k mol. Results obtained are useful in understanding the extraction mechanism.

  11. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  12. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  13. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  14. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  15. Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Sandra M Carvalho

    Full Text Available Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

  16. Characterization of cDNAs encoding human pyruvate dehydrogenase α subunit

    International Nuclear Information System (INIS)

    Ho, Lap; Wexler, I.D.; Liu, Techung; Thekkumkara, T.J.; Patel, M.S.

    1989-01-01

    A cDNA clone (1,423 base pairs) comprising the entire coding region of the precursor form of the α subunit of pyruvate dehydrogenase (E 1 α) has been isolated from a human liver cDNA library in phage λgt11. 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 E 1 α 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 E 1 α 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 E 1 α cDNA resolves existing discrepancies among three previously reported human E 1 α cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients

  17. Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia.

    Science.gov (United States)

    Gass, Nathalie; Glagotskaia, Tatiana; Mellema, Stefan; Stuurman, Jeroen; Barone, Mario; Mandel, Therese; Roessner-Tunali, Ute; Kuhlemeier, Cris

    2005-08-01

    Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen-specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild-type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen-specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen-pistil interaction.

  18. n-Octyl gallate as inhibitor of pyruvate carboxylation and lactate gluconeogenesis.

    Science.gov (United States)

    Eler, Gabrielle Jacklin; Santos, Israel Souza; de Moraes, Amarilis Giaretta; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

    2015-04-01

    The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n-propyl gallate, n-pentyl gallate, and n-octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n-octyl gallate > n-pentyl gallate > n-propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects. © 2014 Wiley Periodicals, Inc.

  19. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

    Science.gov (United States)

    Cheng, Tzuling; Sudderth, Jessica; Yang, Chendong; Mullen, Andrew R.; Jin, Eunsook S.; Matés, José M.; DeBerardinis, Ralph J.

    2011-01-01

    Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how “glutamine-addicted” cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis. PMID:21555572

  20. Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide.

    Science.gov (United States)

    Verhoef, René; de Waard, Pieter; Schols, Henk A; Siika-aho, Matti; Voragen, Alphons G J

    2003-09-01

    The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.

  1. Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae.

    Science.gov (United States)

    Carvalho, Sandra M; Farshchi Andisi, Vahid; Gradstedt, Henrik; Neef, Jolanda; Kuipers, Oscar P; Neves, Ana R; Bijlsma, Jetta J E

    2013-01-01

    Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

  2. Ketogenic diet in pyruvate dehydrogenase complex deficiency: short- and long-term outcomes.

    Science.gov (United States)

    Sofou, Kalliopi; Dahlin, Maria; Hallböök, Tove; Lindefeldt, Marie; Viggedal, Gerd; Darin, Niklas

    2017-03-01

    Our aime was to study the short- and long-term effects of ketogenic diet on the disease course and disease-related outcomes in patients with pyruvate dehydrogenase complex deficiency, the metabolic factors implicated in treatment outcomes, and potential safety and compliance issues. Pediatric patients diagnosed with pyruvate dehydrogenase complex deficiency in Sweden and treated with ketogenic diet were evaluated. Study assessments at specific time points included developmental and neurocognitive testing, patient log books, and investigator and parental questionnaires. A systematic literature review was also performed. Nineteen patients were assessed, the majority having prenatal disease onset. Patients were treated with ketogenic diet for a median of 2.9 years. All patients alive at the time of data registration at a median age of 6 years. The treatment had a positive effect mainly in the areas of epilepsy, ataxia, sleep disturbance, speech/language development, social functioning, and frequency of hospitalizations. It was also safe-except in one patient who discontinued because of acute pancreatitis. The median plasma concentration of ketone bodies (3-hydroxybutyric acid) was 3.3 mmol/l. Poor dietary compliance was associated with relapsing ataxia and stagnation of motor and neurocognitive development. Results of neurocognitive testing are reported for 12 of 19 patients. Ketogenic diet was an effective and safe treatment for the majority of patients. Treatment effect was mainly determined by disease phenotype and attainment and maintenance of ketosis.

  3. 3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects.

    Science.gov (United States)

    El Sayed, S M; El-Magd, R M Abou; Shishido, Y; Chung, S P; Diem, T H; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-02-01

    Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1-18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H(2)O(2) scavenging effect to exogenous H(2)O(2), while lactate had no scavenging effect. 3BP induced H(2)O(2) production. Pyruvate protected against H(2)O(2)-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.

  4. Preparation of C-II labeled pyruvic acid for use in assessment of hypoxia in tumors. Project 4

    International Nuclear Information System (INIS)

    Anon.

    1986-01-01

    Of the three methods of synthesis of C-II-labeled pyruvic acid that we had proposed to investigate in order to determine the best and most appropriate synthesis of C-II-labeled pyruvate, the cold chemistry of Method A, via an isocyanide intermediate, has been verified. Similarly, the cold chemistry of Method B, via the 1,3-dithiane derivative, has been verified up to the deprotection and last step of the synthesis. The difficulties which have been encountered with the biochemistry of Method C from ribulose 1,5-diphosphate, have yet to be resolved. 12 refs., 6 figs

  5. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  6. Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1‐13C]pyruvate MRS studies by infusion of glucose, insulin and potassium

    DEFF Research Database (Denmark)

    Lauritzen, Mette Hauge; Laustsen, Christoffer; Butt, Sadia Asghar

    2013-01-01

    A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized 13C‐labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fas...

  7. Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions

    NARCIS (Netherlands)

    Vaartjes, W.J.; Breejen, J.N. den; Geelen, M.J.H.; Bergh, S.G. van den

    1980-01-01

    1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite

  8. A novel mechanism for the pyruvate protection against zinc-induced cytotoxicity: mediation by the chelating effect of citrate and isocitrate.

    Science.gov (United States)

    Sul, Jee-Won; Kim, Tae-Youn; Yoo, Hyun Ju; Kim, Jean; Suh, Young-Ah; Hwang, Jung Jin; Koh, Jae-Young

    2016-08-01

    Intracellular accumulation of free zinc contributes to neuronal death in brain injuries such as ischemia and epilepsy. Pyruvate, a glucose metabolite, has been shown to block zinc neurotoxicity. However, it is largely unknown how pyruvate shows such a selective and remarkable protective effect. In this study, we sought to find a plausible mechanism of pyruvate protection against zinc toxicity. Pyruvate almost completely blocked cortical neuronal death induced by zinc, yet showed no protective effects against death induced by calcium (ionomycin, NMDA) or ferrous iron. Of the TCA cycle intermediates, citrate, isocitrate, and to a lesser extent oxaloacetate, protected against zinc toxicity. We then noted with LC-MS/MS assay that exposure to pyruvate, and to a lesser degree oxaloacetate, increased levels of citrate and isocitrate, which are known zinc chelators. While pyruvate added only during zinc exposure did not reduce zinc toxicity, citrate and isocitrate added only during zinc exposure, as did extracellular zinc chelator CaEDTA, completely blocked it. Furthermore, addition of pyruvate after zinc exposure substantially reduced intracellular zinc levels. Our results suggest that the remarkable protective effect of pyruvate against zinc cytotoxicity may be mediated indirectly by the accumulation of intracellular citrate and isocitrate, which act as intracellular zinc chelators.

  9. Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

    Directory of Open Access Journals (Sweden)

    Yoichi Takakusagi

    Full Text Available BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2, with minimal effect under aerobic conditions (21% O2. Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3, significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the

  10. Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

    Science.gov (United States)

    Takakusagi, Yoichi; Matsumoto, Shingo; Saito, Keita; Matsuo, Masayuki; Kishimoto, Shun; Wojtkowiak, Jonathan W; DeGraff, William; Kesarwala, Aparna H; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Munasinghe, Jeeva P; Gillies, Robert J; Mitchell, James B; Hart, Charles P; Krishna, Murali C

    2014-01-01

    TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3)), significantly delayed tumor growth. Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

  11. The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

    Science.gov (United States)

    Rueda, Elda M; Johnson, Jerry E; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J; Sigel, Irena; Chaney, Shawnta Y; Fox, Donald A

    2016-01-01

    The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The

  12. Comparative analysis of LytS/LytTR-type histidine kinase/response regulator systems in γ-proteobacteria.

    Directory of Open Access Journals (Sweden)

    Stefan Behr

    Full Text Available Bacterial histidine kinase/response regulator systems operate at the interface between environmental cues and physiological states. Escherichia coli contains two LytS/LytTR-type histidine kinase/response regulator systems, BtsS/BtsR (formerly YehU/YehT and YpdA/YpdB, which have been identified as pyruvate-responsive two-component systems. Since they exhibit remarkable similarity, we analyzed their phylogenetic distribution within the γ-proteobacteria, and experimentally characterized them in a set of representative species. We found that BtsS/BtsR is the predominant LytS/LytTR-type two-component system among γ-proteobacteria, whereas YpdA/YpdB primarily appears in a supplementary role. Based on our observations in E. coli, we used the highly conserved DNA-binding motifs to test the in vivo functionality of both systems in various genera, including Salmonella, Enterobacter, Citrobacter, Xenorhabdus, Yersinia, Aeromonas and Vibrio. The results suggest that, in all cases tested, BtsS/BtsR and YpdA/YpdB respond to different levels of pyruvate in the environment.

  13. Simultaneous hyperpolarized 13C-pyruvate MRI and 18F-FDG-PET in cancer (hyperPET)

    DEFF Research Database (Denmark)

    Gutte, Henrik; Hansen, Adam E.; Henriksen, Sarah T.

    2015-01-01

    named this concept hyper PET. Intravenous injection of the hyperpolarized 13C-pyruvate results in an increase of 13C-lactate, 13C-alanine and 13CCO2 (13C-HCO3) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization (DNP) and use......In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and 18F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We have...... of 13C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of 13C-pyruvate to 13C-lactate. In this study, we combined it with 18F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified local recurrence...

  14. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Oud, B.; Flores, C.L.; Gancedo, C.; Zhang, X.; Trueheart, J.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.

    2012-01-01

    Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards

  15. Leigh syndrome associated with a deficiency of the pyruvate dehydrogenase complex: results of treatment with a ketogenic diet

    NARCIS (Netherlands)

    Wijburg, F. A.; Barth, P. G.; Bindoff, L. A.; Birch-Machin, M. A.; van der Blij, J. F.; Ruitenbeek, W.; TURNBULL, D. M.; Schutgens, R. B.

    1992-01-01

    A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive E 1 alpha and E 1 beta. Leigh syndrome was diagnosed on the

  16. Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites

    Science.gov (United States)

    Cooper, George; Reed, Chris; Nguyen, Dang; Carter, Malika; Wang, Yi

    2011-01-01

    Carbonaceous meteorites deliver a variety of organic compounds to Earth that may have played a role in the origin and/or evolution of biochemical pathways. Some apparently ancient and critical metabolic processes require several compounds, some of which are relatively labile such as keto acids. Therefore, a prebiotic setting for any such individual process would have required either a continuous distant source for the entire suite of intact precursor molecules and/or an energetic and compact local synthesis, particularly of the more fragile members. To date, compounds such as pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α-ketoglutaric acid (all members of the citric acid cycle) have not been identified in extraterrestrial sources or, as a group, as part of a “one pot” suite of compounds synthesized under plausibly prebiotic conditions. We have identified these compounds and others in carbonaceous meteorites and/or as low temperature (laboratory) reaction products of pyruvic acid. In meteorites, we observe many as part of three newly reported classes of compounds: keto acids (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid and homologs), and tricarboxylic acids. Laboratory syntheses using 13C-labeled reactants demonstrate that one compound alone, pyruvic acid, can produce several (nonenzymatic) members of the citric acid cycle including oxaloacetic acid. The isotopic composition of some of the meteoritic keto acids points to interstellar or presolar origins, indicating that such compounds might also exist in other planetary systems. PMID:21825143

  17. Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart

    NARCIS (Netherlands)

    Mariotti, E.; Orton, M. R.; Eerbeek, O.; Ashruf, J. F.; Zuurbier, C. J.; Southworth, R.; Eykyn, T. R.

    2016-01-01

    Hyperpolarized (13)C MR measurements have the potential to display non-linear kinetics. We have developed an approach to describe possible non-first-order kinetics of hyperpolarized [1-(13)C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass

  18. Serum Glutamic-Oxaloacetic Transaminase (GOT) and Glutamic-Pyruvic Transaminase (GPT) Levels in Children and Adolescents with Intellectual Disabilities

    Science.gov (United States)

    Lin, Jin-Ding; Lin, Pei-Ying; Chen, Li-Mei; Fang, Wen-Hui; Lin, Lan-Ping; Loh, Ching-Hui

    2010-01-01

    The elevated serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) rate among people with intellectual disabilities (ID) is unknown and have not been sufficiently studies. The present paper aims to provide the profile of GOT and GPT, and their associated relationship with other biochemical levels of children or…

  19. The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E.

    NARCIS (Netherlands)

    Boxma, B.; Voncken, F.L.M.; Jannink, S.A.; Alen, T.A. van; Akhmanova, A.S.; Weelden, S.W. van; Hellemond, J.J. van; Ricard, G.N.S.; Huynen, M.A.; Tielens, A.G.; Hackstein, J.H.P.

    2004-01-01

    Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of

  20. The effects of creatine pyruvate and creatine citrate on performance during high intensity exercise

    Directory of Open Access Journals (Sweden)

    Purpura Martin

    2008-02-01

    Full Text Available Abstract Background A double-blind, placebo-controlled, randomized study was performed to evaluate the effect of oral creatine pyruvate (Cr-Pyr and creatine citrate (Cr-Cit supplementation on exercise performance in healthy young athletes. Methods Performance during intermittent handgrip exercise of maximal intensity was evaluated before (pretest and after (posttest 28 days of Cr-Pyr (5 g/d, n = 16, Cr-Cit (5 g/d, n = 16 or placebo (pla, 5 g/d, n = 17 intake. Subjects performed ten 15-sec exercise intervals, each followed by 45 sec rest periods. Results Cr-Pyr (p Conclusion It is concluded that four weeks of Cr-Pyr and Cr-Cit intake significantly improves performance during intermittent handgrip exercise of maximal intensity and that Cr-Pyr might benefit endurance, due to enhanced activity of the aerobic metabolism.

  1. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    DEFF Research Database (Denmark)

    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  2. Protective effect of ethyl pyruvate on mice sperm parameters in phenylhydrazine induced hemolytic anemia.

    Science.gov (United States)

    Mozafari, Ali Akbar; Shahrooz, Rasoul; Ahmadi, Abbas; Malekinjad, Hassan; Mardani, Karim

    2016-01-01

    The aim of the present study was to assess the protective effect of ethyl pyruvate (EP) on sperm quality parameters, testosterone level and malondialdehyde (MDA) in phenylhydrazine (PHZ) treated mice. For this purpose, 32 NMRI mice with the age range of 8 to 10 weeks, weight average 26.0 ± 2.0 g, were randomly divided into four equal groups. The control group (1) received normal saline (0. 1 mL per day) by intraperitoneal injection (IP). Group 2 (PHZ group) was treated with initial dose of PHZ (8 mg 100 g(-1), IP) followed by 6 mg 100 g(-1) , IP every 48 hr. Group 3, (Group PHZ+EP) received PHZ (according to the previous prescription) with EP (40 mg kg(-1), daily, IP). Ethyl pyruvate group (4) received only EP (40 mg kg(-1), daily, IP). Treatment period was 35 days. After euthanasia, sperms from caudal region of epididymis were collected and the total mean sperm count, sperm viability, motility and morphology were determined. Testis tissue MDA and serum testosterone levels of all experimental groups were also evaluated. A considerable reduction in mean percentage of number, natural morphology of sperm, sperm motility and viability and serum testosterone concentration besides DNA injury increment among mice treating with PHZ in comparison with control group were observed. However, in PHZ+EP group the above mentioned parameters were improved. This study showed that PHZ caused induction of toxicity on sperm parameters and reduction of testosterone as well as the increment of MDA level and EP as an antioxidant could reduce destructive effects of PHZ on sperm parameters, testosterone level and lipid peroxidation.

  3. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

    Science.gov (United States)

    Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

    2017-08-30

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

  4. Mechanistic photodecarboxylation of pyruvic acid: Excited-state proton transfer and three-state intersection

    Science.gov (United States)

    Chang, Xue-Ping; Fang, Qiu; Cui, Ganglong

    2014-10-01

    Photodissociation dynamics of pyruvic acid experimentally differs from that of commonly known ketones. We have employed the complete active space self-consistent field and its multi-state second-order perturbation methods to study its photodissociation mechanism in the S0, T1, and S1 states. We have uncovered four nonadiabatic photodecarboxylation paths. (i) The S1 system relaxes via an excited-state intramolecular proton transfer (ESIPT) to a hydrogen-transferred tautomer, near which an S1/S0 conical intersection funnels the S1 to S0 state. Then, some trajectories continue completing the decarboxylation reaction in the S0 state; the remaining trajectories via a reverse hydrogen transfer return to the S0 minimum, from which a thermal decarboxylation reaction occurs. (ii) Due to a small S1 -T1 energy gap and a large S1/T1 spin-orbit coupling, an efficient S1 → T1 intersystem crossing process happens again near this S1/S0 conical intersection. When decaying to T1 state, a direct photodecarboxylation proceeds. (iii) Prior to ESIPT, the S1 system first decays to the T1 state via an S1 → T1 intersystem crossing; then, the T1 system evolves to a hydrogen-transferred tautomer. Therefrom, an adiabatic T1 decarboxylation takes place due to a small barrier of 7.7 kcal/mol. (iv) Besides the aforementioned T1 ESIPT process, there also exists a comparable Norrish type I reaction in the T1 state, which forms the ground-state products of CH3CO and COOH. Finally, we have found that ESIPT plays an important role. It closes the S1-T1 and S1-S0 energy gaps, effecting an S1/T1/S0 three-state intersection region, and mediating nonadiabatic photodecarboxylation reactions of pyruvic acid.

  5. Receptor-interacting protein (RIP) kinase family

    OpenAIRE

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

  6. Effects of in ovo feeding of creatine pyruvate on the hatchability, growth performance and energy status in embryos and broiler chickens.

    Science.gov (United States)

    Zhao, M M; Gao, T; Zhang, L; Li, J L; Lv, P A; Yu, L L; Gao, F; Zhou, G H

    2017-10-01

    The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the hatchability, growth performance and energy status of embryos and broilers (Arbor Acres) were investigated. Five treatments were arranged as non-injected treatment (Control), 0.6 ml physiological saline (0.75%) injected treatment (Saline), and IOF treatments injected with 0.6 ml physiological saline (0.75%) containing 3, 6 or 12 mg CrPyr (CrPyr3, CrPyr6 or CrPyr12) into the amnion per fertile egg on day 17.5 of incubation. After hatching, 80 male chicks from each treatment with similar weight close to the average BW of their pooled group were selected and randomly assigned into eight replicates of 10 chicks each. The results showed that the hatchability was not affected among groups, whereas the hatching weight of broilers in CrPyr12 was significantly higher than the control and saline groups (P0.05). Irrespective of dosage, the concentrations of creatine and phosphocreatine, and activities of creatine kinase in embryos were enhanced in CrPyr treatments at 19 E when compared with the control and saline groups (P<0.05). The activities of glucose-6-phosphatase in liver in CrPyr6 and CrPyr12 treatments were higher than the control and saline groups at 19 E (P<0.05). In conclusion, these results indicated that IOF of CrPyr, especially at the level of 12 mg/egg, could improve energy status of embryos and hatchlings, which was useful for enhancing hatching weight, BW and pectoral muscle weight until the end of the experiments at 21 days post-hatch in broilers.

  7. Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions

    OpenAIRE

    Vaartjes, W.J.; Breejen, J.N. den; Geelen, M.J.H.; Bergh, S.G. van den

    1980-01-01

    1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC. 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, c...

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

    Science.gov (United States)

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

    2018-03-23

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

  9. Mechanistic photodecarboxylation of pyruvic acid: Excited-state proton transfer and three-state intersection

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Xue-Ping; Fang, Qiu, E-mail: fangqiu917@bnu.edu.cn; Cui, Ganglong, E-mail: ganglong.cui@bnu.edu.cn [Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875 (China)

    2014-10-21

    Photodissociation dynamics of pyruvic acid experimentally differs from that of commonly known ketones. We have employed the complete active space self-consistent field and its multi-state second-order perturbation methods to study its photodissociation mechanism in the S{sub 0}, T{sub 1}, and S{sub 1} states. We have uncovered four nonadiabatic photodecarboxylation paths. (i) The S{sub 1} system relaxes via an excited-state intramolecular proton transfer (ESIPT) to a hydrogen-transferred tautomer, near which an S{sub 1}/S{sub 0} conical intersection funnels the S{sub 1} to S{sub 0} state. Then, some trajectories continue completing the decarboxylation reaction in the S{sub 0} state; the remaining trajectories via a reverse hydrogen transfer return to the S{sub 0} minimum, from which a thermal decarboxylation reaction occurs. (ii) Due to a small S{sub 1} −T{sub 1} energy gap and a large S{sub 1}/T{sub 1} spin-orbit coupling, an efficient S{sub 1} → T{sub 1} intersystem crossing process happens again near this S{sub 1}/S{sub 0} conical intersection. When decaying to T{sub 1} state, a direct photodecarboxylation proceeds. (iii) Prior to ESIPT, the S{sub 1} system first decays to the T{sub 1} state via an S{sub 1} → T{sub 1} intersystem crossing; then, the T{sub 1} system evolves to a hydrogen-transferred tautomer. Therefrom, an adiabatic T{sub 1} decarboxylation takes place due to a small barrier of 7.7 kcal/mol. (iv) Besides the aforementioned T{sub 1} ESIPT process, there also exists a comparable Norrish type I reaction in the T{sub 1} state, which forms the ground-state products of CH{sub 3}CO and COOH. Finally, we have found that ESIPT plays an important role. It closes the S{sub 1}-T{sub 1} and S{sub 1}-S{sub 0} energy gaps, effecting an S{sub 1}/T{sub 1}/S{sub 0} three-state intersection region, and mediating nonadiabatic photodecarboxylation reactions of pyruvic acid.

  10. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

    2015-01-01

    , and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

  11. Formation and utilization of acetoin, an unusual product of pyruvate metabolism by Ehrlich and AS30-D tumor mitochondria.

    Science.gov (United States)

    Baggetto, L G; Lehninger, A L

    1987-07-15

    [14C]Pyruvate was rapidly non-oxidatively decarboxylated by Ehrlich tumor mitochondria at a rate of 40 nmol/min/mg of protein in the presence or absence of ADP. A search for decarboxylation products led to significant amounts of acetoin formed when Ehrlich tumor mitochondria were incubated with 1 mM [14C] pyruvate in the presence of ATP. Added acetoin to aerobic tumor mitochondria was rapidly utilized in the presence of ATP at a rate of 65 nmol/min/mg of protein. Citrate has been found as a product of acetoin utilization and was exported from the tumor mitochondria. Acetoin has been found in the ascitic liquid of Ehrlich and AS30-D tumor-bearing animals. These unusual reactions were not observed in control rat liver mitochondria.

  12. Blood glucose, lactate, pyruvate, glycerol, 3-hydroxybutyrate and acetoacetate measurements in man using a centrifugal analyser with a fluorimetric attachment.

    Science.gov (United States)

    Harrison, J; Hodson, A W; Skillen, A W; Stappenbeck, R; Agius, L; Alberti, K G

    1988-03-01

    Methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood, using the Cobas Bio centrifugal analyser fitted with a fluorimetric attachment. Intra-assay and inter-assay coefficients of variation ranged from 1.9 to 7.9% and from 1.0 to 7.2% respectively. Correlation coefficients ranged from 0.96 to 0.99 against established continuous-flow and manual spectrophotometric methods. All seven metabolites can be measured using a single perchloric acid extract of 20 microliter of blood. The versatility of the assays is such that as little as 100 pmol pyruvate, 3-hydroxybutyrate or as much as 15 nmol glucose can be measured in the same 20 microliter extract.

  13. Pyruvic Oxime Nitrification and Copper and Nickel Resistance by a Cupriavidus pauculus, an Active Heterotrophic Nitrifier-Denitrifier

    OpenAIRE

    Ramirez, Miguel; Obrzydowski, Jennifer; Ayers, Mary; Virparia, Sonia; Wang, Meijing; Stefan, Kurtis; Linchangco, Richard; Castignetti, Domenic

    2014-01-01

    Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3–C(NOH)–COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a g...

  14. Performance during a strenuous swimming session is associated with high blood lactate: pyruvate ratio and hypoglycemia in fasted rats.

    Science.gov (United States)

    Travassos, P B; Godoy, G; De Souza, H M; Curi, R; Bazotte, R B

    2018-03-26

    The aim of this study was to investigate the effect of lactatemia elevation and glycemia reduction on strenuous swimming performance in fasted rats. Three rats were placed in a swimming tank at the same time. The first rat was removed immediately (control group) and the remaining ones were submitted to a strenuous swimming session. After the second rat was exhausted (Exh group), the third one was immediately removed from the water (Exe group). According to the period of time required for exhaustion, the rats were divided into four groups: low performance (3-7 min), low-intermediary performance (8-12 min), high-intermediary performance (13-17 min), and high performance (18-22 min). All rats were removed from the swimming tanks and immediately killed by decapitation for blood collection or anesthetized for liver perfusion experiments. Blood glucose, lactate, and pyruvate concentrations, blood lactate/pyruvate ratio, and liver lactate uptake and its conversion to glucose were evaluated. Exhaustion in low and low-intermediary performance were better associated with higher lactate/pyruvate ratio. On the other hand, exhaustion in high-intermediary and high performance was better associated with hypoglycemia. Lactate uptake and glucose production from lactate in livers from the Exe and Exh groups were maintained. We concluded that there is a time sequence in the participation of lactate/pyruvate ratio and hypoglycemia in performance during an acute strenuous swimming section in fasted rats. The liver had an important participation in preventing hyperlactatemia and hypoglycemia during swimming through lactate uptake and its conversion to glucose.

  15. Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide.

    OpenAIRE

    Thomas, A P; Halestrap, A P

    1981-01-01

    1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis...

  16. Application of mitochondrial pyruvate carrier blocker UK5099 creates metabolic reprogram and greater stem-like properties in LnCap prostate cancer cells in vitro

    OpenAIRE

    Zhong, Yali; Li, Xiaoran; Yu, Dandan; Li, Xiaoli; Li, Yaqing; Long, Yuan; Yuan, Yuan; Ji, Zhenyu; Zhang, Mingzhi; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2015-01-01

    Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly...

  17. Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

    OpenAIRE

    Stappenbeck, R.; Hodson, A. W.; Skillen, A. W.; Agius, L.; Alberti, K. G. M. M.

    1990-01-01

    Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were ...

  18. Crystallization and preliminary X-ray analysis of dihydrodipicolinate synthase from Clostridium botulinum in the presence of its substrate pyruvate

    International Nuclear Information System (INIS)

    Atkinson, Sarah C.; Dobson, Renwick C. J.; Newman, Janet M.; Gorman, Michael A.; Dogovski, Con; Parker, Michael W.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyzes an important step in lysine biosynthesis. Here, the crystallization and preliminary diffraction analysis to 1.2 Å resolution of DHDPS from C. botulinum in the presence of its substrate pyruvate is reported. In this paper, the crystallization and preliminary X-ray diffraction analysis to near-atomic resolution of DHDPS from Clostridium botulinum crystallized in the presence of its substrate pyruvate are presented. The enzyme crystallized in a number of forms using a variety of PEG precipitants, with the best crystal diffracting to 1.2 Å resolution and belonging to space group C2, in contrast to the unbound form, which had trigonal symmetry. The unit-cell parameters were a = 143.4, b = 54.8, c = 94.3 Å, β = 126.3°. The crystal volume per protein weight (V M ) was 2.3 Å 3 Da −1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%. The high-resolution structure of the pyruvate-bound form of C. botulinum DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  19. Pyruvate remediation of cell stress and genotoxicity induced by haloacetic acid drinking water disinfection by-products.

    Science.gov (United States)

    Dad, Azra; Jeong, Clara H; Pals, Justin A; Wagner, Elizabeth D; Plewa, Michael J

    2013-10-01

    Monohaloacetic acids (monoHAAs) are a major class of drinking water disinfection by-products (DBPs) and are cytotoxic, genotoxic, mutagenic, and teratogenic. We propose a model of toxic action based on monoHAA-mediated inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a target cytosolic enzyme. This model predicts that GAPDH inhibition by the monoHAAs will lead to a severe reduction of cellular ATP levels and repress the generation of pyruvate. A loss of pyruvate will lead to mitochondrial stress and genomic DNA damage. We found a concentration-dependent reduction of ATP in Chinese hamster ovary cells after monoHAA treatment. ATP reduction per pmol monoHAA followed the pattern of iodoacetic acid (IAA) > bromoacetic acid (BAA) > chloroacetic acid (CAA), which is the pattern of potency observed with many toxicological endpoints. Exogenous supplementation with pyruvate enhanced ATP levels and attenuated monoHAA-induced genomic DNA damage as measured with single cell gel electrophoresis. These data were highly correlated with the SN 2 alkylating potentials of the monoHAAs and with the induction of toxicity. The results from this study strongly support the hypothesis that GAPDH inhibition and the possible subsequent generation of reactive oxygen species is linked with the cytotoxicity, genotoxicity, teratogenicity, and neurotoxicity of these DBPs. Copyright © 2013 Wiley Periodicals, Inc.

  20. Coordination of manganous ion at the active site of pyruvate, phosphate dikinase: the complex of oxalate with the phosphorylated enzyme

    International Nuclear Information System (INIS)

    Kofron, J.L.; Ash, D.E.; Reed, G.H.

    1988-01-01

    Electron paramagnetic resonance spectroscopy has been used to investigate the structure of the complex of manganous ion with the phosphorylated form of pyruvate, phosphate dikinase (E/sub p/) and the inhibitor oxalate. Oxalate, an analogue of the enolate of pyruvate, is competitive with respect to pyruvate in binding to the phosphorylated form of the enzyme. Superhyperfine coupling between the unpaired electrons of Mn(I) and ligands specifically labeled with 17 O has been used to identify oxygen ligands to Mn(II) in the complex with oxalate and the phosphorylated form of the enzyme. Oxalate binds at the active site as a bidentate chelate with Mn(II). An oxygen from the 3'-N-phosphohistidyl residue of the protein is in the coordination sphere of Mn(II), and at least two water molecules are also bound to Mn(II) in the complex. Oxalate also binds directly to Mn(II) in a complex with nonphosphorylated enzyme. The structure for the E/sub p/-Mn(II)-oxalate complex implies that simultaneous coordination of a phospho group and of the attacking nucleophile to the divalent cation is likely an important factor in catalysis of this phospho-transfer reaction

  1. Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT--relationship to newly identified mitochondrial pyruvate carrier proteins.

    Directory of Open Access Journals (Sweden)

    Jerry R Colca

    Full Text Available Thiazolidinedione (TZD insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44 and BRP44 Like (BRP44L, which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

  2. Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis.

    Science.gov (United States)

    Gray, Lawrence R; Sultana, Mst Rasheda; Rauckhorst, Adam J; Oonthonpan, Lalita; Tompkins, Sean C; Sharma, Arpit; Fu, Xiaorong; Miao, Ren; Pewa, Alvin D; Brown, Kathryn S; Lane, Erin E; Dohlman, Ashley; Zepeda-Orozco, Diana; Xie, Jianxin; Rutter, Jared; Norris, Andrew W; Cox, James E; Burgess, Shawn C; Potthoff, Matthew J; Taylor, Eric B

    2015-10-06

    Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4

    Science.gov (United States)

    Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Camacho, Carolina Espindola; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A. L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.; Dubinett, Steven M.; Critchlow, Susan E.; Kurdistani, Siavash K.; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G.; Christofk, Heather R.

    2016-01-01

    SUMMARY Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors further supporting their use as anti-cancer therapeutics. PMID:26876179

  4. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

    Science.gov (United States)

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

    2014-01-01

    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

  5. A case of pyruvate dehydrogenase deficiency with low density areas in white matter noticed by CT scan

    International Nuclear Information System (INIS)

    Kimura, Akiko; Kyoya, Seizo; Matsushima, Akihiro; Irimichi, Hideki; Koike, Yoshiko.

    1985-01-01

    The patient was a 4-month-old boy, the first child of healthy, non-consanguineous patient. He was mildly asphyxiated at birth and developed severe convulsions at two days of age. At 4 months of age, he was referred to us because of infantile spasms and motor retardation. The EEG showed hypsarhythmia, ACTH and anticonvulsants were started, but his seizures were not controlled completely. At 8 months of age, the CT scan demonstrated a cerebral atrophy with enlarged ventricles and a diffuse low density of cerebral white matter, and lactic acidosis was first noticed. The glucose, glucagon, fructose, and alanine tolerance tests revealed almost normal responses in blood glucose levels and elevation of lactate levels above the initial value. Enzyme studies revealed a severe deficiency of pyruvate dehydrogenase complex and pyruvate dehydrogenase (E 1 ), and a normal activity of pyruvate carboxylase in liver obtained by biopsy. In biopsied muscle, mitochondria appeared normal. Treatment with thiamine, lipoic acid and anticonvulsants was not effective. The clinical picture of PDC deficiency has been correlated with the amount of the residual activity, and this case confirmed to the ''severe'' category. Several pathologic entities may be associated with PDHC deficiency, and CT findings in our case demonstrated the demyelinating condition. The precise relationship between the defect and the pathogenesis remains to be elucidated. (author)

  6. Improvement of ethanol yield from glycerol via conversion of pyruvate to ethanol in metabolically engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Yu, Kyung Ok; Jung, Ju; Ramzi, Ahmad Bazli; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2012-02-01

    The conversion of low-priced glycerol to higher value products has been proposed as a way to improve the economic viability of the biofuels industry. In a previous study, the conversion of glycerol to ethanol in a metabolically engineered strain of Saccharomyces cerevisiae was accomplished by minimizing the synthesis of glycerol, the main by-product in ethanol fermentation processing. To further improve ethanol production, overexpression of the native genes involved in conversion of pyruvate to ethanol in S. cerevisiae was successfully accomplished. The overexpression of an alcohol dehydrogenase (adh1) and a pyruvate decarboxylase (pdc1) caused an increase in growth rate and glycerol consumption under fermentative conditions, which led to a slight increase of the final ethanol yield. The overall expression of the adh1 and pdc1 genes in the modified strains, combined with the lack of the fps1 and gpd2 genes, resulted in a 1.4-fold increase (about 5.4 g/L ethanol produced) in fps1Δgpd2Δ (pGcyaDak, pGupCas) (about 4.0 g/L ethanol produced). In summary, it is possible to improve the ethanol yield by overexpression of the genes involved in the conversion of pyruvate to ethanol in engineered S. cerevisiae using glycerol as substrate.

  7. Regulation of Muscle Pyruvate Dehydrogenase Complex in Insulin Resistance: Effects of Exercise and Dichloroacetate

    Directory of Open Access Journals (Sweden)

    Dumitru Constantin-Teodosiu

    2013-10-01

    Full Text Available Since the mitochondrial pyruvate dehydrogenase complex (PDC controls the rate of carbohydrate oxidation, impairment of PDC activity mediated by high-fat intake has been advocated as a causative factor for the skeletal muscle insulin resistance, metabolic syndrome, and the onset of type 2 diabetes (T2D. There are also situations where muscle insulin resistance can occur independently from high-fat dietary intake such as sepsis, inflammation, or drug administration though they all may share the same underlying mechanism, i.e., via activation of forkhead box family of transcription factors, and to a lower extent via peroxisome proliferator-activated receptors. The main feature of T2D is a chronic elevation in blood glucose levels. Chronic systemic hyperglycaemia is toxic and can lead to cellular dysfunction that may become irreversible over time due to deterioration of the pericyte cell's ability to provide vascular stability and control to endothelial proliferation. Therefore, it may not be surprising that T2D's complications are mainly macrovascular and microvascular related, i.e., neuropathy, retinopathy, nephropathy, coronary artery, and peripheral vascular diseases. However, life style intervention such as exercise, which is the most potent physiological activator of muscle PDC, along with pharmacological intervention such as administration of dichloroacetate or L-carnitine can prove to be viable strategies for treating muscle insulin resistance in obesity and T2D as they can potentially restore whole body glucose disposal.

  8. Role of plastidic pyruvate dehydrogenase complex (pl. PDC) in chloroplast metabolism of spinach

    International Nuclear Information System (INIS)

    Schulze-Siebert, D.; Homeyer, U.; Schultz, G.

    1986-01-01

    Labeling experiments of chloroplasts in the light ( 14 CO 2 , 2- 14 C-pyruvate etc.) revealed that pl. PDC is predominantly involved in the synthesis of branched chain amino acids and pl. isoprenoids (carotenes, PQ, α-T). In this context, pl. phosphoglycerate mutase as missing link in the C 3 → C 2 metabolism of chloroplasts was identified by latency experiments. This indicates a direct pathway from Calvin cycle to pl. PDC. Using protoplasts, maximal rates in pl. PDC metabolism were obtained. On the other hand, mitochondrial PDC in protoplasts is mainly involved in fatty acid synthesis by known mechanism. Additionally, cytosolic-ER-isoprenoids were formed (e.g. sterols). When 14 CO 2 was simultaneously applied with unlabeled acetate to protoplasts in the light an isotopic dilution of fatty acids were found but not of pl. isoprenoids. This may indicate an partially channeling of pl. PDC and mevalonate pathway for pl. isoprenoid synthesis. Inhibitory studies with DCMU point in the same direction

  9. A catalyzing phantom for reproducible dynamic conversion of hyperpolarized [1-¹³C]-pyruvate.

    Science.gov (United States)

    Walker, Christopher M; Lee, Jaehyuk; Ramirez, Marc S; Schellingerhout, Dawid; Millward, Steven; Bankson, James A

    2013-01-01

    In vivo real time spectroscopic imaging of hyperpolarized ¹³C labeled metabolites shows substantial promise for the assessment of physiological processes that were previously inaccessible. However, reliable and reproducible methods of measurement are necessary to maximize the effectiveness of imaging biomarkers that may one day guide personalized care for diseases such as cancer. Animal models of human disease serve as poor reference standards due to the complexity, heterogeneity, and transient nature of advancing disease. In this study, we describe the reproducible conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate using a novel synthetic enzyme phantom system. The rate of reaction can be controlled and tuned to mimic normal or pathologic conditions of varying degree. Variations observed in the use of this phantom compare favorably against within-group variations observed in recent animal studies. This novel phantom system provides crucial capabilities as a reference standard for the optimization, comparison, and certification of quantitative imaging strategies for hyperpolarized tracers.

  10. A catalyzing phantom for reproducible dynamic conversion of hyperpolarized [1-¹³C]-pyruvate.

    Directory of Open Access Journals (Sweden)

    Christopher M Walker

    Full Text Available In vivo real time spectroscopic imaging of hyperpolarized ¹³C labeled metabolites shows substantial promise for the assessment of physiological processes that were previously inaccessible. However, reliable and reproducible methods of measurement are necessary to maximize the effectiveness of imaging biomarkers that may one day guide personalized care for diseases such as cancer. Animal models of human disease serve as poor reference standards due to the complexity, heterogeneity, and transient nature of advancing disease. In this study, we describe the reproducible conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate using a novel synthetic enzyme phantom system. The rate of reaction can be controlled and tuned to mimic normal or pathologic conditions of varying degree. Variations observed in the use of this phantom compare favorably against within-group variations observed in recent animal studies. This novel phantom system provides crucial capabilities as a reference standard for the optimization, comparison, and certification of quantitative imaging strategies for hyperpolarized tracers.

  11. Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate.

    Science.gov (United States)

    Kamminga, Tjerko; Slagman, Simen-Jan; Bijlsma, Jetta J E; Martins Dos Santos, Vitor A P; Suarez-Diez, Maria; Schaap, Peter J

    2017-10-01

    Mycoplasma hyopneumoniae is cultured on large-scale to produce antigen for inactivated whole-cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint-based genome-scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time-series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non-growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model-driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3-4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. Biotechnol. Bioeng. 2017;114: 2339-2347. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

    Science.gov (United States)

    Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

    2017-04-01

    Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

    Science.gov (United States)

    Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

    2017-07-14

    The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cells

    Science.gov (United States)

    Li, Xiaoran; Kan, Quancheng; Fan, Zhirui; Li, Yaqing; Ji, Yasai; Zhao, Jing; Zhang, Mingzhi; Grigalavicius, Mantas; Berge, Viktor; Goscinski, Mariusz Adam; M. Nesland, Jahn; Suo, Zhenhe

    2017-01-01

    One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro. PMID:28624784

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

    Science.gov (United States)

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

    2002-01-01

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

  16. Ameliorative effect of ethyl pyruvate in neuropathic pain induced by chronic constriction injury of sciatic nerve

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    Varsha J. Bansode

    2014-01-01

    Full Text Available Objective: The present study was designed to investigate the ameliorative effects of ethyl pyruvate (EP in chronic constriction injury (CCI-induced painful neuropathy in rats. Materials and Methods: EP 50 and 100 mg/kg was administered for 21 consecutive days starting from the day of surgery. The effects of EP in the paw pressure, acetone drop, and tail heat immersion tests were assessed, reflecting the degree of mechanical hyperalgesia, cold allodynia, and spinal thermal sensation, respectively. Axonal degeneration of the sciatic nerve was assessed histopathologically. The levels of thiobarbituric acid reactive species, reduced glutathione (GSH, catalase (CAT, and superoxide dismutase (SOD were determined to assess oxidative stress. Key Findings: Administration of 50 and 100 mg/kg EP attenuated the reduction of nociceptive threshold in the paw pressure, acetone drop, and tail heat immersion tests. EP 100 mg/kg significantly attenuated reactive changes in histopathology and increase in oxidative stress. Conclusion: EP 100 mg/kg showed beneficial activity against nerve trauma-induced neuropathy. Hence, it can be used as a better treatment option in neuropathic pain (NP. The observed antinociceptive effects of EP may possibly be attributed to its antioxidant and anti-inflammatory activity.

  17. Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk

    2016-02-01

    Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

  18. Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

    Science.gov (United States)

    Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

    2017-12-01

    Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

  19. Functional Characterization of Waterlogging and Heat Stresses Tolerance Gene Pyruvate decarboxylase 2 from Actinidia deliciosa

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    Hui-Ting Luo

    2017-11-01

    Full Text Available A previous report showed that both Pyruvate decarboxylase (PDC genes were significantly upregulated in kiwifruit after waterlogging treatment using Illumina sequencing technology, and that the kiwifruit AdPDC1 gene was required during waterlogging, but might not be required during other environmental stresses. Here, the function of another PDC gene, named AdPDC2, was analyzed. The expression of the AdPDC2 gene was determined using qRT-PCR, and the results showed that the expression levels of AdPDC2 in the reproductive organs were much higher than those in the nutritive organs. Waterlogging, NaCl, and heat could induce the expression of AdPDC2. Overexpression of kiwifruit AdPDC2 in transgenic Arabidopsis enhanced resistance to waterlogging and heat stresses in five-week-old seedlings, but could not enhance resistance to NaCl and mannitol stresses at the seed germination stage and in early seedlings. These results suggested that the kiwifruit AdPDC2 gene may play an important role in waterlogging resistance and heat stresses in kiwifruit.

  20. Brain Glycogenolysis, Adrenoceptors, Pyruvate Carboxylase, Na+,K+-ATPase and Marie E. Gibbs’ Pioneering Learning Studies

    Directory of Open Access Journals (Sweden)

    Leif eHertz

    2013-04-01

    Full Text Available The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., JCBFM, in press. According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present ‘perspective’ suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either i activation of astrocytic receptors, perhaps especially beta-adrenergic, or ii even small increases in extracellular K+ concentration above its normal resting level. It discusses i the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; ii the established role of K+-stimulated glycogenolysis for K+ uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K+ homeostasis in the brain in vivo; and iii the plausible role of transmitter-induced glycogenolysis, stimulating Na+,K+-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K+ concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP.

  1. Acute hypertensive stress imaged by cardiac hyperpolarized [1-C]pyruvate magnetic resonance

    DEFF Research Database (Denmark)

    Tougaard, Rasmus Stilling; Hansen, Esben Søvsø Szocska; Laustsen, Christoffer

    2018-01-01

    PURPOSE: Deranged metabolism is now recognized as a key causal factor in a variety of heart diseases, and is being studied extensively. However, invasive methods may alter metabolism, and conventional imaging techniques measure tracer uptake but not downstream metabolism. These challenges may...... be overcome by hyperpolarized MR, a noninvasive technique currently crossing the threshold into human trials. The aim of this study was to image metabolic changes in the heart in response to endogastric glucose bolus and to acute hypertension. METHODS: Five postprandial pigs were scanned with hyperpolarized.......008) and ejection fraction decreased from 54 ± 2% to 47 ± 6% (P = 0.03) The hemodynamic changes were accompanied by increases in the hyperpolarized [1-13C]pyruvate MR derived ratios of lactate/alanine (from 0.58 ± 0.13 to 0.78 ± 0.06, P = 0.03) and bicarbonate/alanine (from 0.55 ± 0.12 to 0.91 ± 0.14, P = 0...

  2. Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

    International Nuclear Information System (INIS)

    Voss, Jarrod E.; Scally, Stephen W.; Taylor, Nicole L.; Dogovski, Con; Alderton, Malcolm R.; Hutton, Craig A.; Gerrard, Juliet A.; Parker, Michael W.; Dobson, Renwick C. J.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyses an important step in lysine biosynthesis. Here, the expression, purification, crystallization and preliminary diffraction analysis to 2.15 Å resolution of DHDPS from B. anthracis soaked with the substrate pyruvate are reported. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  3. Accuracies of fecal calprotectin, lactoferrin, M2-pyruvate kinase, neopterin and zonulin to predict the response to infliximab in ulcerative colitis.

    Science.gov (United States)

    Frin, Anne-Claire; Filippi, Jérôme; Boschetti, Gilles; Flourie, Bernard; Drai, Jocelyne; Ferrari, Patricia; Hebuterne, Xavier; Nancey, Stéphane

    2017-01-01

    Fecal markers might predict the response to anti-TNFα in ulcerative colitis (UC). To compare the performance of fecal calprotectin (fCal), lactoferrin (fLact), M2-PK (fM2-PK), neopterin (fNeo), and zonulin (fZon) to predict the response to therapy in active UC patients. Disease activity from 31 consecutive patients with an active UC, treated with infliximab (IFX) was assessed by the Mayo score at baseline and at week 14 and by the partial Mayo score at W52 and stool samples collected for fecal marker measurements at W0, W2, and W14. At W14, 19 patients (61%) were responders to IFX induction. The median levels of fCal, fLact and fM2-PK drop dramatically from baseline to W14 in clinical responders. At W2, fM2-PK, fLact and fCal levels predicted accurately the response to IFX induction. At W14, fLact, fCal, and fM2-PK were individually reliable markers to predict sustained response at W52. The performances of fNeo and fZon were weaker in this setting. The performance of fM2-PK at W2 to predict response to induction therapy with IFX was superior to that of fLact and fCal, whereas monitoring fLact was the best tool to predict adequately the course of the disease at one year under maintenance IFX in UC. Copyright © 2016. Published by Elsevier Ltd.

  4. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

    Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

    2017-01-01

    The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

  5. Attenuation of Methotrexate-Induced Embryotoxicity and Oxidative Stress by Ethyl Pyruvate

    Directory of Open Access Journals (Sweden)

    Najafi Gholamreza

    2016-07-01

    Full Text Available Background Methotrexate (MTX, as an anti-folate agent, is widely used in the treatment of rheumatic disorders and malignant tumors, however it damages reproductive sys- tem in mice. The aim of this research was to study the effects of ethyl pyruvate (EP on embryo development and oxidative stress changes in the testis of mice treated with MTX. Materials and Methods In this experimental study, thirty-two adult male Naval Medical Research Institute mice, with average weight of 26 ± 2 g, were divided into four groups. The first group (control received distilled water (0.1 ml/mice/day, while the second group was intraperitoneally (IP treated with 20 mg/kg MTX once per week. The third group was IP treated with 40 mg/kg/day EP, and the fourth group was IP treated with both 20 mg/kg MTX and 40 mg/kg/day EP for 30 days. At the end of treatment fertilization rate and embryonic development were evaluated. Differences between these groups were assessed by ANOVA using the SPSS software package for Windows with a Tukey-Kramer multiple post-hoc comparison test. Results MTX treatment caused significant (P<0.05 increase in malondialdehyde (MDA and reduced catalase (CAT, as well as leading to in vitro fertilization (IVF and embryonic development. The improved effects of EP on the IVF were determined by the reduced level of MDA (index of oxidative stress and significant increased level of CAT (a key antioxidant. We observed significant increase in fertilization rate and embryonic development in the treated group with both MTX and EP. Conclusion It is suggested that EP can be useful in ameliorating testicular damages and embryotoxicity induced by MTX. These effects could be attributed to its antioxidant properties.

  6. The effect of ethyl pyruvate on oxidative stress in intestine and bacterial translocation after thermal injury.

    Science.gov (United States)

    Karabeyoğlu, Melih; Unal, Bülent; Bozkurt, Betül; Dolapçi, Iştar; Bilgihan, Ayşe; Karabeyoğlu, Işil; Cengiz, Omer

    2008-01-01

    Thermal injury causes a breakdown in the intestinal mucosal barrier due to ischemia reperfusion injury, which can induce bacterial translocation (BT), sepsis, and multiple organ failure in burn patients. The aim of this study was to investigate the effect of ethyl pyruvate (EP) on intestinal oxidant damage and BT in burn injury. Thirty-two rats were randomly divided into four groups. The sham group was exposed to 21 degrees C water and injected intraperitoneal with saline (1 mL/100 g). The sham + EP group received EP (40 mg/kg) intraperitoneally 6 h after the sham procedure. The burn group was exposed to thermal injury and given intraperitoneal saline injection (1 mL/100 g). The burn + EP group received EP (40 mg/kg) intraperitoneally 6 h after thermal injury. Twenty-four hours later, tissue samples were obtained from mesenteric lymph nodes, spleen, and liver for microbiological analysis and ileum samples were harvested for biochemical analysis. Thermal injury caused severe BT in burn group. EP supplementation decreased BT in mesenteric lymph nodes and spleen in the burn + EP group compared with the burn group (P < 0.05). Also, burn caused BT in liver, but this finding was not statistically significant among all groups. Thermal injury caused a statistically significant increase in malondialdehyde and myeloperoxidase levels, and EP prevented this effects in the burn + EP group compared with the burn group (P < 0.05). Our data suggested that EP can inhibit the BT and myeloperoxidase and malondialdehyde production in intestine following thermal injury, suggesting anti-inflammatory and anti-oxidant properties of EP.

  7. Physical exercise reduces pyruvate carboxylase (PCB) and contributes to hyperglycemia reduction in obese mice.

    Science.gov (United States)

    Muñoz, Vitor Rosetto; Gaspar, Rafael Calais; Crisol, Barbara Moreira; Formigari, Guilherme Pedron; Sant'Ana, Marcella Ramos; Botezelli, José Diego; Gaspar, Rodrigo Stellzer; da Silva, Adelino S R; Cintra, Dennys Esper; de Moura, Leandro Pereira; Ropelle, Eduardo Rochete; Pauli, José Rodrigo

    2018-07-01

    The present study evaluated the effects of exercise training on pyruvate carboxylase protein (PCB) levels in hepatic tissue and glucose homeostasis control in obese mice. Swiss mice were distributed into three groups: control mice (CTL), fed a standard rodent chow; diet-induced obesity (DIO), fed an obesity-inducing diet; and a third group, which also received an obesity-inducing diet, but was subjected to an exercise training protocol (DIO + EXE). Protocol training was carried out for 1 h/d, 5 d/wk, for 8 weeks, performed at an intensity of 60% of exhaustion velocity. An insulin tolerance test (ITT) was performed in the last experimental week. Twenty-four hours after the last physical exercise session, the animals were euthanized and the liver was harvested for molecular analysis. Firstly, DIO mice showed increased epididymal fat and serum glucose and these results were accompanied by increased PCB and decreased p-Akt in hepatic tissue. On the other hand, physical exercise was able to increase the performance of the mice and attenuate PCB levels and hyperglycemia in DIO + EXE mice. The above findings show that physical exercise seems to be able to regulate hyperglycemia in obese mice, suggesting the participation of PCB, which was enhanced in the obese condition and attenuated after a treadmill running protocol. This is the first study to be aimed at the role of exercise training in hepatic PCB levels, which may be a novel mechanism that can collaborate to reduce the development of hyperglycemia and type 2 diabetes in DIO mice.

  8. Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Ansaya Thonpho

    Full Text Available Pyruvate carboxylase (PC is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an activator element(s for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y, as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

  9. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  10. Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogues

    Science.gov (United States)

    Lietzan, Adam D.; St. Maurice, Martin

    2014-01-01

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates. PMID:24157795

  11. GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles.

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    Arseny S Khakhalin

    Full Text Available In the developing mammalian brain, gamma-aminobutyric acid (GABA is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl(- in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl(- concentrations, and therefore a more depolarized GABA receptor (GABAR reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.

  12. Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

    Science.gov (United States)

    Choutko, Alexandra; Eichenberger, Andreas P; Gunsteren, Wilfred F; Dolenc, Jožica

    2013-01-01

    The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. PMID:23595942

  13. Exploration of swapping enzymatic function between two proteins: a simulation study of chorismate mutase and isochorismate pyruvate lyase.

    Science.gov (United States)

    Choutko, Alexandra; Eichenberger, Andreas P; van Gunsteren, Wilfred F; Dolenc, Jožica

    2013-06-01

    The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. © 2013 The Protein Society.

  14. Pyruvate dehydrogenase complexes from the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum, helminths exhibiting anaerobic mitochondrial metabolism.

    Science.gov (United States)

    Diaz, F; Komuniecki, R W

    1994-10-01

    The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from 2 parasitic helminths exhibiting anaerobic mitochondrial metabolism, the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum. The P. equorum PDC yielded 7 major bands when separated by SDS-PAGE. The bands of 72, 55-53.5, 41 and 36 kDa corresponded to E2, E3, E1 alpha and E1 beta, respectively. The complex also contained additional unidentified proteins of 43 and 45 kDa. Incubation of the complex with [2-14C]pyruvate resulted in the acetylation of only E2. These results suggest that the P. equorum PDC lacks protein X and exhibits an altered subunit composition, as has been described previously for the PDC of the related nematode, Ascaris suum. In contrast, the D. caninum PDC yielded only four major bands after SDS-PAGE of 59, 58, 39 and 34 kDa, which corresponded to E3, E2, E1 alpha and E1 beta, respectively. Incubation of the D. caninum complex with [2-14C]pyruvate resulted in the acetylation of E2 and a second protein which comigrated with E3, suggesting that the D. caninum complex contained protein X and had a subunit composition similar to PDCs from other eukaryotic organisms. Both helminth complexes appeared less sensitive to inhibition by elevated NADH/NAD+ ratios than complexes isolated from aerobic organisms, as would be predicted for PDCs from organisms exploiting microaerobic habitats. These results suggest that although these helminths have similar anaerobic mitochondrial pathways, they contain significantly different PDCs.

  15. Effects of hypoxia and pyruvate infusion on myocardial fatty acid oxidation measured with 123I heptadecanoic acid

    International Nuclear Information System (INIS)

    Comans, E.F.I.; Visser, F.C.; Elzinga, Gijs

    1993-01-01

    Radio-iodinated fatty acids like 123 I heptadecanoic acid (HDA) can be used for the non-invasive delineation of myocardial non-esterified fatty acid (FA) metabolism. In this study the quantitative value of HDA was assessed for the measurement of myocardial FA oxidation. In an isolated saline perfused rat heart preparation myocardial time-activity curves were made during control perfusion and after inhibition of FA oxidation by hypoxia and infusion of 10.0 mM pyruvate, respectively. Control experiments were performed using 1- 14 C palmitate as the 'golden standard' for myocardial FA oxidation. Myocardial HDA oxidation was calculated from the amplitude of the third exponential term of the time-activity curve. During control perfusion no differences were observed between the calculated oxygen equivalents (from HDA oxidation) and the measured (A-V oxygen content difference) and the estimated ( 14 CO 2 production) values. Inhibition of palmitate oxidation with pyruvate was accurately detected with HDA. During hypoxic perfusion, an overestimation of palmitate oxidation was calculated on the basic of HDA oxidation. Infusion of pyruvate did not influence the time constants of the time-activity curves, whereas during hypoxic perfusion an increase of the time constant of the third exponential term was observed, probably caused by the presence of back-diffusion of non-metabolized HDA. We conclude that HDA can be used as a quantitative tool for the measurement of myocardial FA oxidation under various metabolic conditions. During periods of a decreased oxygen availability back-diffusion of FA needs to be taken into account for the interpretation of the myocardial time-activity curves. (author)

  16. Gluconeogenesis is associated with high rates of tricarboxylic acid and pyruvate cycling in fasting northern elephant seals.

    Science.gov (United States)

    Champagne, Cory D; Houser, Dorian S; Fowler, Melinda A; Costa, Daniel P; Crocker, Daniel E

    2012-08-01

    Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ∼95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.

  17. Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

    Science.gov (United States)

    Xu, Tao; Mayer, Dirk; Gu, Meng; Yen, Yi-Fen; Josan, Sonal; Tropp, James; Pfefferbaum, Adolf; Hurd, Ralph; Spielman, Daniel

    2011-10-01

    With signal-to-noise ratio enhancements on the order of 10,000-fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1-(13)C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1-(13)C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time-to-peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small-flip-angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral-based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo (13)C labeling of lactate and alanine after a bolus injection of [1-(13)C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis-Menten-like formulation, with the resulting estimated apparent maximal reaction velocity V(max) and apparent Michaelis constant K(M) being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis-Menten kinetics, it is conceptually different. In this study, we focus on the (13)C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport

  18. Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria

    Directory of Open Access Journals (Sweden)

    Masome Zeinali

    2016-09-01

    Full Text Available Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined. Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase from Zymomonas mobilis bacteria was isolated and cloned at E. coli bacteria by freeze and thaw method. For gene cloning, we used specific primers of pdc and PCR reaction and then pdc gene isolated and pET 28a plasmid double digested with (Sal I and Xho I enzymes. Digestion Products were ligated by T4 DNA ligase in 16 °C for 16 hours. Results: Results of bacteria culture showed that a few colonies containing pET 28a plasmid could grow. Result of colony pcr of pdc gene with specific primers revealed 1700 bp bands in 1% agarose gel electrophoresis. The results of PCR with T7 promotor forward primer and pdc revers primer have proved the accurate direction of integration of pdc gene into plasmid and revealed 1885 bp band. Double digestion of recombinant plasmid with SalI and XhoI enzymes revealed same bands. Finally, RT showed the expected band of 1700 bp that implies the desired gene expression in the samples. Discussion and conclusion: Due to the increased production of ethanol via pyruvate decarboxylase gene cloning in expression plasmids with a strong promoter upstream of the cloning site can conclude that, pyruvate decarboxylase cloning as a key gene would be useful and according to beneficial properties of E. coli bacteria, transfering the gene to bacteria appears to be reasonable.

  19. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

  20. A Comparison between Radiolabeled Fluorodeoxyglucose Uptake and Hyperpolarized 13C-Labeled Pyruvate Utilization as Methods for Detecting Tumor Response to Treatment

    Directory of Open Access Journals (Sweden)

    Timothy H. Witney

    2009-06-01

    Full Text Available Detection of early tumor responses to treatment can give an indication of clinical outcome. Positron emission tomography measurements of the uptake of the glucose analog, [18F] 2-fluoro-2-deoxy-d-glucose (FDG, have demonstrated their potential for detecting early treatment response in the clinic. We have shown recently that 13C magnetic resonance spectroscopy and spectroscopic imaging measurements of the uptake and conversion of hyperpolarized [1-13C]pyruvate into [1-13C]lactate can be used to detect treatment response in a murine lymphoma model. The present study compares these magnetic resonance measurements with changes in FDG uptake after chemotherapy. A decrease in FDG uptake was found to precede the decrease in flux of hyperpolarized 13C label between pyruvate and lactate, both in tumor cells in vitro and in tumors in vivo. However, the magnitude of the decrease in FDG uptake and the decrease in pyruvate to lactate flux was comparable at 24 hours after drug treatment. In cells, the decrease in FDG uptake was shown to correlate with changes in plasma membrane expression of the facilitative glucose transporters, whereas the decrease in pyruvate to lactate flux could be explained by an increase in poly(ADP-ribose polymerase activity and subsequent depletion of the NAD(H pool. These results show that measurement of flux between pyruvate and lactate may be an alternative to FDG-positron emission tomography for imaging tumor treatment response in the clinic.

  1. The progression from a lower to a higher invasive stage of bladder cancer is associated with severe alterations in glucose and pyruvate metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Conde, Vanessa R. [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Oliveira, Pedro F. [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine, Abel Salazar Institute of Biomedical Sciences, University of Porto – UMIB/ICBAS/UP (Portugal); Nunes, Ana R.; Rocha, Cátia S. [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Ramalhosa, Elsa; Pereira, José A. [Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of Bragança (Portugal); Alves, Marco G., E-mail: alvesmarc@gmail.com [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Silva, Branca M., E-mail: bmcms@ubi.pt [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal)

    2015-07-01

    Cancer cells present a particular metabolic behavior. We hypothesized that the progression of bladder cancer could be accompanied by changes in cells glycolytic profile. We studied two human bladder cancer cells, RT4 and TCCSUP, in which the latter represents a more invasive stage. The levels of glucose, pyruvate, alanine and lactate in the extracellular media were measured by Proton Nuclear Magnetic Resonance. The protein expression levels of glucose transporters 1 (GLUT1) and 3 (GLUT3), monocarboxylate transporter 4 (MCT4), phosphofructokinase-1 (PFK1), glutamic-pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) were determined. Our data showed that glucose consumption and GLUT3 levels were similar in both cell lines, but TCCSUP cells displayed lower levels of GLUT1 and PFK expression. An increase in pyruvate consumption, concordant with the higher levels of lactate and alanine production, was also detected in TCCSUP cells. Moreover, TCCSUP cells presented lower protein expression levels of GPT and LDH. These results illustrate that bladder cancer progression is associated with alterations in cells glycolytic profile, namely the switch from glucose to pyruvate consumption in the more aggressive stage. This may be useful to develop new therapies and to identify biomarkers for cancer progression. - Highlights: • Metabolic phenotype of less and high invasive bladder cancer cells was studied. • Bladder cancer progression involves alterations in cells glycolytic profile. • More invasive bladder cancer cells switch from glucose to pyruvate consumption. • Our results may help to identify metabolic biomarkers of bladder cancer progression.

  2. Growth of Campylobacter incubated aerobically in fumarate-pyruvate media or media supplemented with dairy, meat, or soy extracts and peptones.

    Science.gov (United States)

    Hinton, Arthur

    2016-09-01

    The ability of Campylobacter to grow aerobically in media supplemented with fumarate-pyruvate or with dairy, meat, or soy extracts or peptones was examined. Optical densities (OD) of Campylobacter cultured in basal media, media supplemented with fumarate-pyruvate or with 1.0, 2.5, 5.0, or 7.5% beef extract was measured. Growth was also compared in media supplemented with other extracts or peptones. Finally, cfu/mL of Campylobacter recovered from basal media or media supplemented with fumarate-pyruvate, casamino acids, beef extract, soytone, or beef extract and soytone was determined. Results indicated that OD of cultures grown in media supplemented with fumarate-pyruvate or with 5.0 or 7.5% beef extract were higher than OD of isolates grown in basal media or media supplemented with lower concentrations of beef extract. Highest OD were produced by isolates grown in media supplemented with beef extract, peptone from meat, polypeptone, proteose peptone, or soytone. Also, more cfu/mL were recovered from media with fumarate-pyruvate, beef extract, soytone, or beef extract-soytone than from basal media or media with casamino acids. Findings indicate that media supplemented with organic acids, vitamins, and minerals and media supplemented with extracts or peptones containing these metabolites can support aerobic growth of Campylobacter. Published by Elsevier Ltd.

  3. Metabolic fate of glucose in rats with traumatic brain injury and pyruvate or glucose treatments: A NMR spectroscopy study.

    Science.gov (United States)

    Shijo, Katsunori; Sutton, Richard L; Ghavim, Sima S; Harris, Neil G; Bartnik-Olson, Brenda L

    2017-01-01

    Administration of sodium pyruvate (SP; 9.08 μmol/kg, i.p.), ethyl pyruvate (EP; 0.34 μmol/kg, i.p.) or glucose (GLC; 11.1 μmol/kg, i.p.) to rats after unilateral controlled cortical impact (CCI) injury has been reported to reduce neuronal loss and improve cerebral metabolism. In the present study these doses of each fuel or 8% saline (SAL; 5.47 nmoles/kg) were administered immediately and at 1, 3, 6 and 23 h post-CCI. At 24 h all CCI groups and non-treated Sham injury controls were infused with [1,2 13 C] glucose for 68 min 13 C nuclear magnetic resonance (NMR) spectra were obtained from cortex + hippocampus tissues from left (injured) and right (contralateral) hemispheres. All three fuels increased lactate labeling to a similar degree in the injured hemisphere. The amount of lactate labeled via the pentose phosphate and pyruvate recycling (PPP + PR) pathway increased in CCI-SAL and was not improved by SP, EP, and GLC treatments. Oxidative metabolism, as assessed by glutamate labeling, was reduced in CCI-SAL animals. The greatest improvement in oxidative metabolism was observed in animals treated with SP and fewer improvements after EP or GLC treatments. Compared to SAL, all three fuels restored glutamate and glutamine labeling via pyruvate carboxylase (PC), suggesting improved astrocyte metabolism following fuel treatment. Only SP treatments restored the amount of [4 13 C] glutamate labeled by the PPP + PR pathway to sham levels. Milder injury effects in the contralateral hemisphere appear normalized by either SP or EP treatments, as increases in the total pool of 13 C lactate and labeling of lactate in glycolysis, or decreases in the ratio of PC/PDH labeling of glutamine, were found only for CCI-SAL and CCI-GLC groups compared to Sham. The doses of SP, EP and GLC examined in this study all enhanced lactate labeling and restored astrocyte-specific PC activity but differentially affected neuronal metabolism after CCI injury. The restoration of

  4. Neonatal pyruvate dehydrogenase deficiency due to a R302H mutation in the PDHA1 gene: MRI findings

    International Nuclear Information System (INIS)

    Soares-Fernandes, Joao P.; Ribeiro, Manuel; Magalhaes, Zita; Rocha, Jaime F.; Teixeira-Gomes, Roseli; Cruz, Romeu; Leijser, Lara M.

    2008-01-01

    Pyruvate dehydrogenase (PDH) deficiency is one of the most common causes of congenital lactic acidosis. Correlations between the genetic defect and neuroimaging findings are lacking. We present conventional and diffusion-weighted MRI findings in a 7-day-old male neonate with PDH deficiency due to a mosaicism for the R302H mutation in the PDHA1 gene. Corpus callosum dysgenesis, widespread increased diffusion in the white matter, and bilateral subependymal cysts were the main features. Although confirmation of PDH deficiency depends on specialized biochemical analyses, neonatal MRI plays a role in evaluating the pattern and extent of brain damage, and potentially in early diagnosis and clinical decision making. (orig.)

  5. Coupling between the blood lactate-to-pyruvate ratio and MCA Vmean at the onset of exercise in humans

    DEFF Research Database (Denmark)

    Rasmussen, Peter; Madsen, Camilla A; Nielsen, Henning B

    2009-01-01

    Activation-induced increase in cerebral blood flow is coupled to enhanced metabolic activity, maybe with brain tissue redox state and oxygen tension as key modulators. To evaluate this hypothesis at the onset of exercise in humans, blood was sampled at 0.1 to 0.2 Hz from the radial artery and right...... internal jugular vein, while middle cerebral artery mean flow velocity (MCA V(mean)) was recorded. Both the arterial and venous lactate-to-pyruvate ratio increased after 10 s (P capillary...... state and oxygenation as potential modulators of an increase in cerebral blood flow at the onset of exercise....

  6. Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

    Science.gov (United States)

    Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

    2012-02-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

  7. Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

  8. Decreased expression of pyruvate dehydrogenase A1 predicts an unfavorable prognosis in ovarian carcinoma.

    Science.gov (United States)

    Li, Yaqing; Huang, Ruixia; Li, Xiaoli; Li, Xiaoran; Yu, Dandan; Zhang, Mingzhi; Wen, Jianguo; Goscinski, Mariusz Adam; Trope, Claes G; Nesland, Jahn M; Suo, Zhenhe

    2016-01-01

    Pyruvate dehydrogenase A1 (PDHA1) serves as a gate-keeper enzyme link between glycolysis and the mitochondrial citric acid cycle. The inhibition of PDHA1 in cancer cells can result in an increased Warburg effect and a more aggressive phenotype in cancer cells. This study was conducted to investigate the expression of PDHA1 in ovarian cancer and the correlation between PDHA1 expression and the prognosis of patients. The PDHA1 protein expression in 3 ovarian cancer cell lines (OVCAR-3, SKOV-3 and ES-2) and 248 surgically removed ovarian carcinoma samples was immunocytochemically examined. Statistical analyses were performed to evaluate the correlations between PDHA1 expression and the clinicopathological characteristics of the patients as well as the predictive value of PDHA1. The results showed the presence of variable expression of PDHA1 in the three ovarian cancer cell lines. Of the 248 ovarian cancer tissue specimens, 45 cases (18.1%) were negative in tumor cells for PDHA1, 162 cases (65.3%) displayed a low expression level, and 41 cases (16.5%) had a relatively high PDHA1 staining. The expression of PDHA1 was associated with the histological subtype ( P =0.004) and FIGO stage ( P =0.002). The median OS time in the PDHA1 negative group, low expression group and high expression group were 0.939 years, 1.443 years and 9.900 years, respectively. The median PFS time in the above three groups were 0.287 years, 0.586 years and 9.900 years, respectively. Furthermore, the high expression of PDHA1 in ovarian carcinoma cells was significantly associated with better OS and PFS by statistical analyses. Multivariate analyses showed that PDHA1 expression was also an independent prognostic factor for higher OS in ovarian cancer patients (HR=0.705, 95% CI 0.541-0.918, P =0.01). Our study indicated that the decreased expression of PDHA1 might be an independent prognostic factor in unfavorable outcomes.

  9. Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci.

    Science.gov (United States)

    Cheng, Xingqun; Redanz, Sylvio; Cullin, Nyssa; Zhou, Xuedong; Xu, Xin; Joshi, Vrushali; Koley, Dipankar; Merritt, Justin; Kreth, Jens

    2018-01-15

    Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H 2 O 2 , which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans H 2 O 2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H 2 O 2 production by S. sanguinis and S. gordonii S. sanguinis H 2 O 2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H 2 O 2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H 2 O 2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H 2 O 2 IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H 2 O 2 Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival

  10. Expression of pyruvate dehydrogenase is an independent prognostic marker in gastric cancer

    Science.gov (United States)

    Sun, Xu-Ren; Sun, Zhe; Zhu, Zhi; Guan, Hai-Xia; Li, Chen-Yan; Zhang, Jun-Yan; Zhang, Yi-Ning; Zhou, Huan; Zhang, Hui-Jing; Xu, Hui-Mian; Sun, Ming-Jun

    2015-01-01

    AIM: To investigate the expression and prognostic role of pyruvate dehydrogenase (PDH) in gastric cancer (GC). METHODS: This study included 265 patients (194 male, 71 female, mean age 59 years (range, 29-81 years) with GC who underwent curative surgery at the First Affiliated Hospital of China Medical University from January 2006 to May 2007. All patients were followed up for more than 5 years. Patient-derived paraffin embedded GC specimens were collected for tissue microarrays (TMAs). We examined PDH expression by immunohistochemistry in TMAs containing tumor tissue and matched non-neoplastic mucosa. Immunoreactivity was evaluated independently by two researchers. Overall survival (OS) rates were determined using the Kaplan-Meier estimator. Correlations with other clinicopathologic factors were evaluated by two-tailed χ2 tests or a two-tailed t-test. The Cox proportional-hazard model was used in univariate analysis and multivariate analysis to identify factors significantly correlated with prognosis. RESULTS: Immunohistochemistry showed that 35.47% of total cancer tissue specimens had cytoplasmic PDH staining. PDH expression was much higher in normal mucosa specimens (75.09%; P = 0.001). PDH expression was correlated with Lauren grade (70.77% in intestinal type vs 40.0% in diffuse type; P = 0.001), lymph node metastasis (65.43% with no metastasis vs 51.09% with metastasis; P = 0.033), lymphatic invasion (61.62% with no invasion vs 38.81% with invasion; P = 0.002), histologic subtypes (70.77% in intestinal type vs 40.0% in diffuse type; P = 0.001) and tumor-node-metastasis (TNM) stage (39% in poorly differentiated vs 65.91% in well differentiated and 67.11% in moderately differentiated; P = 0.001) in GC. PDH expression in cancer tissue was significantly associated with higher OS (P < 0.001). The multivariate analysis adjusted for age, Lauren classification, TNM stage, lymph node metastasis, histological type, tumor size, depth of invasion and lymphatic invasion

  11. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  12. Signaling network of the Btk family kinases.

    Science.gov (United States)

    Qiu, Y; Kung, H J

    2000-11-20

    The Btk family kinases represent new members of non-receptor tyrosine kinases, which include Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Tec. They are characterized by having four structural modules: PH (pleckstrin homology) domain, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and kinase (Src homology 1) domain. Increasing evidence suggests that, like Src-family kinases, Btk family kinases play central but diverse modulatory roles in various cellular processes. They participate in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors and integrins. They are regulated by many non-receptor tyrosine kinases such as Src, Jak, Syk and FAK family kinases. In turn, they regulate many of major signaling pathways including those of PI3K, PLCgamma and PKC. Both genetic and biochemical approaches have been used to dissect the signaling pathways and elucidate their roles in growth, differentiation and apoptosis. An emerging new role of this family of kinases is cytoskeletal reorganization and cell motility. The physiological importance of these kinases was amply demonstrated by their link to the development of immunodeficiency diseases, due to germ-line mutations. The present article attempts to review the structure and functions of Btk family kinases by summarizing our current knowledge on the interacting partners associated with the different modules of the kinases and the diverse signaling pathways in which they are involved.

  13. Effects of adrenergic agents on intracellular ca(2+) homeostasis and metabolism of glucose in astrocytes with an emphasis on pyruvate carboxylation, oxidative decarboxylation and recycling

    DEFF Research Database (Denmark)

    Obel, Linea Lykke Frimodt; Andersen, Karen M H; Bak, Lasse Kristoffer

    2012-01-01

    and oxidative decarboxylation in astrocytic glucose metabolism. Importantly, pyruvate carboxylation was best visualized at 10 min of incubation. The abundance and pattern of labeling in lactate and alanine indicated not only an extensive activity of malic enzyme (initial step for pyruvate recycling) but also...... a high degree of compartmentalization of the pyruvate pool. Stimulating with 1 µM NE had no effect on labeling patterns and glycogen metabolism, whereas 100 µM NE increased glutamate labeling and decreased labeling in alanine, the latter supposedly due to dilution from degradation of non-labeled glycogen....... It is suggested that further experiments uncovering the correlation between adrenergic and glutamatergic pathways should be performed in order to gain further insight into the role of astrocytes in brain function and dysfunction, the latter including excitotoxicity....

  14. Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

    Science.gov (United States)

    Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

    2008-11-01

    Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

  15. Intraperitoneal lactate/pyruvate ratio and the level of glucose and glycerol concentration differ between patients surgically treated for upper and lower perforations of the gastrointestinal tract

    DEFF Research Database (Denmark)

    Sabroe, Jonas E; Axelsen, Anne R; Ellebæk, Mark B

    2017-01-01

    collected every 4th hour for up to 7 postoperative days. Samples were analysed for concentrations of glucose, lactate, pyruvate and glycerol. RESULTS: Microdialysis results showed that patients with upper gastrointestinal tract lesions had significantly higher levels of postoperative intraperitoneal glucose...... and glycerol concentrations, as well as lower lactate/pyruvate ratios and lactate/glucose ratios. In the group with perforation of the lower gastrointestinal tract, those patients with a complicated course showed lower levels of postoperative intraperitoneal glucose concentration and glycerol concentration...... and higher lactate/pyruvate ratios and lactate/glucose ratios than those patients with an uncomplicated course. CONCLUSION: Patients with upper and lower gastrointestinal tract lesions showed differences in postoperative biomarker levels. A difference was also seen between patients with complicated...

  16. Receptor-interacting protein (RIP) kinase family

    Science.gov (United States)

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

  17. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  19. Molecular identification and characterization of the pyruvate decarboxylase gene family associated with latex regeneration and stress response in rubber tree.

    Science.gov (United States)

    Long, Xiangyu; He, Bin; Wang, Chuang; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-02-01

    In plants, ethanolic fermentation occurs not only under anaerobic conditions but also under aerobic conditions, and involves carbohydrate and energy metabolism. Pyruvate decarboxylase (PDC) is the first and the key enzyme of ethanolic fermentation, which branches off the main glycolytic pathway at pyruvate. Here, four PDC genes were isolated and identified in a rubber tree, and the protein sequences they encode are very similar. The expression patterns of HbPDC4 correlated well with tapping-simulated rubber productivity in virgin rubber trees, indicating it plays an important role in regulating glycometabolism during latex regeneration. HbPDC1, HbPDC2 and HbPDC3 had striking expressional responses in leaves and bark to drought, low temperature and high temperature stresses, indicating that the HbPDC genes are involve in self-protection and defense in response to various abiotic and biotic stresses during rubber tree growth and development. To understand ethanolic fermentation in rubber trees, it will be necessary to perform an in-depth study of the regulatory pathways controlling the HbPDCs in the future. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. Mitochondrial pyruvate carrier function is negatively linked to Warburg phenotype in vitro and malignant features in esophageal squamous cell carcinomas

    Science.gov (United States)

    Li, Yaqing; Li, Xiaoran; Kan, Quancheng; Zhang, Mingzhi; Li, Xiaoli; Xu, Ruiping; Wang, Junsheng; Yu, Dandan; Goscinski, Mariusz Adam; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2017-01-01

    Aerobic glycolysis is one of the emerging hallmarks of cancer cells. In this study, we investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with MPC blocker UK5099 and the metabolic alteration as well as aggressive features of esophageal squamous carcinoma. It was found that blocking pyruvate transportation into mitochondria attenuated mitochondrial oxidative phosphorylation (OXPHOS) and triggered aerobic glycolysis, a feature of Warburg effect. In addition, the HIF-1α expression and ROS production were also activated upon UK5099 application. It was further revealed that the UK5099-treated cells became significantly more resistant to chemotherapy and radiotherapy, and the UK5099-treated tumor cells also exhibited stronger invasive capacity compared to the parental cells. In contrast to esophageal squamous epithelium cells, decreased MPC protein expression was observed in a series of 157 human squamous cell carcinomas, and low/negative MPC1 expression predicted an unfavorable clinical outcome. All these results together revealed the potential connection of altered MPC expression/activity with the Warburg metabolic reprogramming and tumor aggressiveness in cell lines and clinical samples. Collectively, our findings highlighted a therapeutic strategy targeting Warburg reprogramming of human esophageal squamous cell carcinomas. PMID:27911865

  1. Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy

    International Nuclear Information System (INIS)

    CaJacob, C.A.; Frey, P.A.; Hainfeld, J.F.; Wall, J.S.; Yang, H.

    1985-01-01

    Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes are 2.06 X 10(5) for the dimeric pyruvate dehydrogenase (E1), 1.15 X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and 2.20 X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions. The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2)

  2. Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production.

    Science.gov (United States)

    Melo, Nadiele T M; Mulder, Kelly C L; Nicola, André Moraes; Carvalho, Lucas S; Menino, Gisele S; Mulinari, Eduardo; Parachin, Nádia S

    2018-02-16

    Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris , a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris . To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

  3. miR-378 Activates the Pyruvate-PEP Futile Cycle and Enhances Lipolysis to Ameliorate Obesity in Mice

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    2016-03-01

    Full Text Available Obesity has been linked to many health problems, such as diabetes. However, there is no drug that effectively treats obesity. Here, we reveal that miR-378 transgenic mice display reduced fat mass, enhanced lipolysis, and increased energy expenditure. Notably, administering AgomiR-378 prevents and ameliorates obesity in mice. We also found that the energy deficiency seen in miR-378 transgenic mice was due to impaired glucose metabolism. This impairment was caused by an activated pyruvate-PEP futile cycle via the miR-378-Akt1-FoxO1-PEPCK pathway in skeletal muscle and enhanced lipolysis in adipose tissues mediated by miR-378-SCD1. Our findings demonstrate that activating the pyruvate-PEP futile cycle in skeletal muscle is the primary cause of elevated lipolysis in adipose tissues of miR-378 transgenic mice, and it helps orchestrate the crosstalk between muscle and fat to control energy homeostasis in mice. Thus, miR-378 may serve as a promising agent for preventing and treating obesity in humans.

  4. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  5. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  6. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  7. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  8. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae.

    Science.gov (United States)

    Oud, Bart; Flores, Carmen-Lisset; Gancedo, Carlos; Zhang, Xiuying; Trueheart, Joshua; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2012-09-15

    Pyruvate-decarboxylase negative (Pdc⁻) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc⁻S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc⁻ strains cannot grow on high glucose concentrations and require C₂-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Genetic analysis of a Pdc⁻ strain previously evolved to overcome these deficiencies revealed a 225 p in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc⁻ strain enabled growth on 20 g l⁻¹ glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h⁻¹) similar to that of the evolved Pdc⁻ strain (0.23 h⁻¹). Furthermore, the reverse engineered Pdc⁻ strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h⁻¹) than the evolved strain (0.20 h⁻¹). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc⁻S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc⁻ strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C₂-compound auxotrophy. In this study we have discovered and characterised a

  9. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Oud Bart

    2012-09-01

    Full Text Available Abstract Background Pyruvate-decarboxylase negative (Pdc- strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Results Genetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v ethanol at a maximum specific growth rate (0.24 h-1 similar to that of the evolved Pdc- strain (0.23 h-1. Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1 than the evolved strain (0.20 h-1. The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy. Conclusions In this study we have discovered and

  10. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-01-01

    is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

  11. Measuring Kinase Activity-A Global Challenge.

    Science.gov (United States)

    Cann, Marissa L; McDonald, Ian M; East, Michael P; Johnson, Gary L; Graves, Lee M

    2017-11-01

    The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Simultaneous hyperpolarized 13C-pyruvate MRI and 18F-FDG-PET in cancer (hyperPET)

    DEFF Research Database (Denmark)

    Borgwardt, Henrik Gutte; Hansen, Adam Espe; Henriksen, Sarah T.

    2015-01-01

    have named this concept hyper PET. Intravenous injection of the hyperpolarized (13)C-pyruvate results in an increase of (13)C-lactate, (13)C-alanine and (13)C-CO2 ((13)C-HCO3) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization......In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and (18)F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We...... (DNP) and use of (13)C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of (13)C-pyruvate to (13)C-lactate. In this study, we combined it with (18)F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified...

  13. Interaction between the thyroid hormone receptor and co-factors on the promoter of the gene encoding phospho enol pyruvate carboxykinase

    NARCIS (Netherlands)

    Schmidt, E. D.; van Beeren, M.; Glass, C. K.; Wiersinga, W. M.; Lamers, W. H.

    1993-01-01

    Using transient transfection studies we localized a thyroid hormone-responsive element on the promoter of the rat phospho-enol pyruvate carboxykinase gene between 355 and 174 bp upstream of the transcription start site. DNAse 1 footprinting analysis within this region showed that a 28 bp fragment at

  14. The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    DEFF Research Database (Denmark)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao

    2016-01-01

    deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates...

  15. omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

    Science.gov (United States)

    Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

    2004-04-01

    Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.

  16. ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

    Science.gov (United States)

    Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

    2004-01-01

    Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and Vmax for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and Vmax for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion. PMID:15066855

  17. Studies on the formation of lactate and pyruvate from glucose in cultured skin fibroblasts: implications for detection of respiratory chain defects

    NARCIS (Netherlands)

    Wijburg, F. A.; Feller, N.; Scholte, H. R.; Przyrembel, H.; Wanders, R. J.

    1989-01-01

    We investigated the time course of the formation of lactate and pyruvate from glucose in cultured skin fibroblasts from controls, from a patient with a cytochrome c oxidase deficiency and from controls treated with inhibitors of the individual respiratory chain complexes. Fibroblasts from the

  18. Functional pyruvate formate lyase pathway expressed with two different electron donors in Saccharomyces cerevisiae at aerobic growth

    DEFF Research Database (Denmark)

    Zhang, Yiming; Dai, Zongjie; Krivoruchko, Anastasia

    2015-01-01

    pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc-MTH1-ΔT ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression...

  19. Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.

    Science.gov (United States)

    Delgado, María Graciela; Oliva, Carlos; López, Estefanía; Ibacache, Andrés; Galaz, Alex; Delgado, Ricardo; Barros, L Felipe; Sierralta, Jimena

    2018-01-19

    The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

  20. Computer-assisted study on the reaction between pyruvate and ylide in the pathway leading to lactyl-ThDP.

    Science.gov (United States)

    Alvarado, Omar; Jaña, Gonzalo; Delgado, Eduardo J

    2012-08-01

    In this study the formation of the lactyl-thiamin diphosphate intermediate (L-ThDP) is addressed using density functional theory calculations at X3LYP/6-31++G(d,p) level of theory. The study includes potential energy surface scans, transition state search, and intrinsic reaction coordinate calculations. Reactivity is analyzed in terms of Fukui functions. The results allow to conclude that the reaction leading to the formation of L-ThDP occurs via a concerted mechanism, and during the nucleophilic attack on the pyruvate molecule, the ylide is in its AP form. The calculated activation barrier for the reaction is 19.2 kcal/mol, in agreement with the experimental reported value.

  1. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

  2. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

    Science.gov (United States)

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

  3. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

    Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

  4. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    Science.gov (United States)

    2012-01-01

    Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

  5. Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

    Science.gov (United States)

    Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

    2011-01-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

  6. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    Directory of Open Access Journals (Sweden)

    Milanovic Vesna

    2012-02-01

    Full Text Available Abstract Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1 and alcohol dehydrogenase (Adh1 were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation

  7. Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

    Science.gov (United States)

    Plaga, W; Frank, R; Knappe, J

    1988-12-15

    Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

  8. Triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass

    Science.gov (United States)

    Olson, Aaron K.; Bouchard, Bertrand; Ning, Xue-Han; Isern, Nancy; Rosiers, Christine Des

    2012-01-01

    Triiodothyronine (T3) supplementation improves clinical outcomes in infants after cardiac surgery using cardiopulmonary bypass by unknown mechanisms. We utilized a translational model of infant cardiopulmonary bypass to test the hypothesis that T3 modulates pyruvate entry into the citric acid cycle (CAC), thereby providing the energy support for improved cardiac function after ischemia-reperfusion (I/R). Neonatal piglets received intracoronary [2-13Carbon(13C)]pyruvate for 40 min (8 mM) during control aerobic conditions (control) or immediately after reperfusion (I/R) from global hypothermic ischemia. A third group (I/R-Tr) received T3 (1.2 μg/kg) during reperfusion. We assessed absolute CAC intermediate levels and flux parameters into the CAC through oxidative pyruvate decarboxylation (PDC) and anaplerotic carboxylation (PC) using [2-13C]pyruvate and isotopomer analysis by gas and liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy. When compared with I/R, T3 (group I/R-Tr) increased cardiac power and oxygen consumption after I/R while elevating flux of both PDC and PC (∼4-fold). Although neither I/R nor I/R-Tr modified absolute CAC levels, T3 inhibited I/R-induced reductions in their molar percent enrichment. Furthermore, 13C-labeling of CAC intermediates suggests that T3 may decrease entry of unlabeled carbons at the level of oxaloacetate through anaplerosis or exchange reaction with asparate. T3 markedly enhances PC and PDC fluxes, thereby providing potential substrate for elevated cardiac function after reperfusion. This T3-induced increase in pyruvate fluxes occurs with preservation of the CAC intermediate pool. Our labeling data raise the possibility that T3 reduces reliance on amino acids for anaplerosis after reperfusion. PMID:22180654

  9. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

    Science.gov (United States)

    Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

    2016-12-01

    Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

  10. A rice kinase-protein interaction map.

    Science.gov (United States)

    Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

    2009-03-01

    Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

  11. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based drug discovery approach, we have identified small-molecule histidine-kinase

  12. dependent/calmodulin- stimulated protein kinase from moss

    Indian Academy of Sciences (India)

    Unknown

    stimulated protein kinase; CDPK, calmodulin domain-like protein kinase; KM14, 14 amino acid synthetic peptide; .... used were obtained from Sigma Chemical Company, USA, ..... Plant chimeric Ca2+/Calmodulin-dependent protein kinase.

  13. The Protein Kinase RSK Family - Roles in Prostate Cancer

    National Research Council Canada - National Science Library

    Lannigan, Deborah

    2006-01-01

    The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined...

  14. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

    Science.gov (United States)

    Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

    2008-09-05

    The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

  15. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  16. TYROSINE KINASE INHIBITORS AND PREGNANCY

    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese

    2014-04-01

    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  17. Computational Modelling of the Metabolic States Regulated by the Kinase Akt

    Directory of Open Access Journals (Sweden)

    Ettore eMosca

    2012-11-01

    Full Text Available Signal transduction pathways and gene regulation determine a major reorganization of metabolic activities in order to support cell proliferation. Protein Kinase B (PKB, also known as Akt, participates in the PI3K/Akt/mTOR pathway, a master regulator of aerobic glycolysis and cellular biosynthesis, two activities shown by both normal and cancer proliferating cells. Not surprisingly considering its relevance for cellular metabolism, Akt/PKB is often found hyperactive in cancer cells. In the last decade, many efforts have been made to improve the understanding of the control of glucose metabolism and the identification of a therapeutic window between proliferating cancer cells and proliferating normal cells. In this context, we have modelled the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production and nucleotide biosynthesis. We used a computational model in order to compare two metabolic states generated by the specific variation of the metabolic fluxes regulated by the activity of the PI3K/Akt/mTOR pathway. One of the two states represented the metabolism of a growing cancer cell characterised by aerobic glycolysis and cellular biosynthesis, while the other state represented the same metabolic network with a reduced glycolytic rate and a higher mitochondrial pyruvate metabolism, as reported in literature in relation to the activity of the PI3K/Akt/mTOR. Some steps that link glycolysis and pentose phosphate pathway revealed their importance for controlling the dynamics of cancer glucose metabolism.

  18. The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

    Directory of Open Access Journals (Sweden)

    Cole Johnson

    2014-03-01

    Full Text Available The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.

  19. Investigation of the biosynthesis of acetyl-CoA and oxaloacetic acid from pyruvic acid and the quantitative evaluation of incorporated 13C-labeled l-alanine in Arthrobacter hyalinus

    International Nuclear Information System (INIS)

    Katsumi Iida

    2014-01-01

    Studies on the contribution to acetyl-CoA and oxaloacetic acid from the pyruvic acid transformation from l-alanine in Arthrobacter hyalinus were conducted by means of feeding experiments with l-[1- 13 C]alanine and l-[3- 13 C]alanine, followed by an analysis of the labeling patterns of coproporphyrinogen III using 13 C NMR spectroscopy. The results demonstrated that l-alanine was transformed via pyruvic acid to both acetyl-CoA and oxaloacetic acid. Additionally, the quantitative analysis indicated that pyruvic acid was transformed to acetyl-CoA and oxaloacetic acid in the ratio of 1:0.8. (author)

  20. Whole-protein alanine-scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism.

    Science.gov (United States)

    Tang, Qingling; Fenton, Aron W

    2017-09-01

    Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play "key" roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid sidechains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (Fru-1,6-BP; activator) and alanine (inhibitor). Each nonalanine/nonglycine residue of hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using a limited number of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms. © 2017 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Archana B Siva

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

  2. Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex*

    Science.gov (United States)

    Wang, Junjie; Nemeria, Natalia S.; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-01-01

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s−1, comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP). PMID:24742683

  3. Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

    Science.gov (United States)

    Wang, Junjie; Nemeria, Natalia S; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-05-30

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP). © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Effect of L-carnitine and pyruvate on equine sperm maintained at 5 ºC and 15 ºC during 24 h: preliminary results

    Directory of Open Access Journals (Sweden)

    Avila G

    2016-12-01

    Full Text Available The aim of this study was to evaluate if the addition of L-carnitine and pyruvate to two semen transport extenders (Kenney and Kenney modified by Tyrodes is able to maintain sperm parameters for 24 h at 5 ºC and 15 ºC. Semen was obtained from 3 stallions (n=3; r=2 and at time 0 and after 24 h of cooling, the following parameters evaluated: total and progressive motility (CASA, viability and acrosome status (FITC-PNA-PI, membrane function (HOS, and DNA with Toluidine Blue stain (TB and the Sperm Chromatin Dispersion assay (SCD. Each temperature was individually analyzed using a factorial design with a 5% significance level. No interactions were observed. For the moment, the Kenney extender with the addition of L-carnitine and pyruvate showed the best results for maintaining most sperm parameters for 24 h at both 5 ºC and 15 ºC.

  5. Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment.

    Science.gov (United States)

    Stappenbeck, R; Hodson, A W; Skillen, A W; Agius, L; Alberti, K G

    1990-01-01

    Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 mul of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture.

  6. Protein Kinase A in Cancer

    International Nuclear Information System (INIS)

    Caretta, Antonio; Mucignat-Caretta, Carla

    2011-01-01

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors

  7. Protein Kinase A in Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Caretta, Antonio; Mucignat-Caretta, Carla, E-mail: carla.mucignat@unipd.it [Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova (Italy)

    2011-02-28

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors.

  8. Protective effect of indole-3-pyruvate against ultraviolet b-induced damage to cultured HaCaT keratinocytes and the skin of hairless mice.

    Directory of Open Access Journals (Sweden)

    Reiji Aoki

    Full Text Available Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr, 4-hydroxyphenylpyruvate (HPPyr, and indole-3-pyruvate (IPyr against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm2 and maintained with or without test compounds (1-25 mM.In addition, the dorsal skin of hairless mice (HR-1 was treated with test compounds (10 μmol and exposed to UVB light (1 J/cm2 twice [corrected]. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β and interleukin 6 (IL-6. IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2 expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.

  9. Preparation, characterization and thermal behaviour study of 4-dimethyl amino benzal pyruvate of lanthanides (III) and yttrium (III) in solid state

    International Nuclear Information System (INIS)

    Miyano, M.H.

    1990-01-01

    Solid state compounds involving Ln and DMBP, where Ln trivalent lanthanides (except promethium) and yttrium; DMBP 4-dimethyl amino benzylidene pyruvate, were prepared by addition of ligand to the corresponding metal ions chlorides, both in aqueous solution. The precipitates were washed with distilled water and dried at 40 0 C in a forced circulation oven. Complexometry with EDTA, thermogravimetry (TG), differential thermal analysis (DTA), infra-red absorption and X-ray diffraction have been used in the study of these compounds. (author)

  10. Increased Interstitial Concentrations of Glutamate and Pyruvate in Vastus Lateralis of Women with Fibromyalgia Syndrome Are Normalized after an Exercise Intervention - A Case-Control Study.

    Directory of Open Access Journals (Sweden)

    Björn Gerdle

    Full Text Available Fibromyalgia syndrome (FMS is associated with central alterations, but controversies exist regarding the presence and role of peripheral factors. Microdialysis (MD can be used in vivo to study muscle alterations in FMS. Furthermore for chronic pain conditions such as FMS, the mechanisms for the positive effects of exercise are unclear. This study investigates the interstitial concentrations of algesics and metabolites in the vastus lateralis muscle of 29 women with FMS and 28 healthy women before and after an exercise intervention.All the participants went through a clinical examination and completed a questionnaire. In addition, their pressure pain thresholds (PPTs in their upper and lower extremities were determined. For both groups, MD was conducted in the vastus lateralis muscle before and after a 15-week exercise intervention of mainly resistance training of the lower limbs. Muscle blood flow and interstitial muscle concentrations of lactate, pyruvate, glutamate, glucose, and glycerol were determined.FMS was associated with significantly increased interstitial concentrations of glutamate, pyruvate, and lactate. After the exercise intervention, the FMS group exhibited significant decreases in pain intensity and in mean interstitial concentrations of glutamate, pyruvate, and glucose. The decrease in pain intensity in FMS correlated significantly with the decreases in pyruvate and glucose. In addition, the FMS group increased their strength and endurance.This study supports the suggestion that peripheral metabolic and algesic muscle alterations are present in FMS patients and that these alterations contribute to pain. After an exercise intervention, alterations normalized, pain intensity decreased (but not abolished, and strength and endurance improved, all findings that suggest the effects of exercise are partially peripheral.

  11. Technique development of 3D dynamic CS-EPSI for hyperpolarized 13 C pyruvate MR molecular imaging of human prostate cancer.

    Science.gov (United States)

    Chen, Hsin-Yu; Larson, Peder E Z; Gordon, Jeremy W; Bok, Robert A; Ferrone, Marcus; van Criekinge, Mark; Carvajal, Lucas; Cao, Peng; Pauly, John M; Kerr, Adam B; Park, Ilwoo; Slater, James B; Nelson, Sarah J; Munster, Pamela N; Aggarwal, Rahul; Kurhanewicz, John; Vigneron, Daniel B

    2018-03-25

    The purpose of this study was to develop a new 3D dynamic carbon-13 compressed sensing echoplanar spectroscopic imaging (EPSI) MR sequence and test it in phantoms, animal models, and then in prostate cancer patients to image the metabolic conversion of hyperpolarized [1- 13 C]pyruvate to [1- 13 C]lactate with whole gland coverage at high spatial and temporal resolution. A 3D dynamic compressed sensing (CS)-EPSI sequence with spectral-spatial excitation was designed to meet the required spatial coverage, time and spatial resolution, and RF limitations of the 3T MR scanner for its clinical translation for prostate cancer patient imaging. After phantom testing, animal studies were performed in rats and transgenic mice with prostate cancers. For patient studies, a GE SPINlab polarizer (GE Healthcare, Waukesha, WI) was used to produce hyperpolarized sterile GMP [1- 13 C]pyruvate. 3D dynamic 13 C CS-EPSI data were acquired starting 5 s after injection throughout the gland with a spatial resolution of 0.5 cm 3 , 18 time frames, 2-s temporal resolution, and 36 s total acquisition time. Through preclinical testing, the 3D CS-EPSI sequence developed in this project was shown to provide the desired spectral, temporal, and spatial 5D HP 13 C MR data. In human studies, the 3D dynamic HP CS-EPSI approach provided first-ever simultaneously volumetric and dynamic images of the LDH-catalyzed conversion of [1- 13 C]pyruvate to [1- 13 C]lactate in a biopsy-proven prostate cancer patient with full gland coverage. The results demonstrate the feasibility to characterize prostate cancer metabolism in animals, and now patients using this new 3D dynamic HP MR technique to measure k PL , the kinetic rate constant of [1- 13 C]pyruvate to [1- 13 C]lactate conversion. © 2018 International Society for Magnetic Resonance in Medicine.

  12. Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

    Science.gov (United States)

    Darr, Christa R; Varner, Dickson D; Teague, Sheila; Cortopassi, Gino A; Datta, Sandipan; Meyers, Stuart A

    2016-08-01

    Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity. © 2016 by the Society for the Study of Reproduction, Inc.

  13. Fluorimetric methods for the measurement of intermediate metabolites (lactate, pyruvate, alanine, β-hydroxybutyrate, glycerol) using a COBAS FARA centrifugal analyser

    OpenAIRE

    Monti, L. D.; Sandoli, P. E.; Costa, S.; Phan, V. C.; Piatti, P. M.

    1993-01-01

    Intermediate products of the metabolism of glucose, fat and amino-acid are important in the evaluation of such metabolic disorders as diabetes mellitus, liver disease and metabolic acidosis. In the present study, methods for the measurement of intermediate metabolites (lactate, pyruvate, alanine, β-hydroxybutyrate and glycerol) have been adapted to a fast centrifugal analyzer: the COBAS FARA. Correlation coeffcients rangedfrom 0.90 to 0.99, compared to established manual spectrophotometric me...

  14. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

    2009-01-01

    of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

  15. Isoprenoid biosynthesis and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Henneman, L.

    2011-01-01

    Mevalonaat Kinase Deficiëntie (MKD) is een aangeboren ziekte geassocieerd met heftige koortsaanvallen die drie tot vier dagen aanhouden en gepaard gaan met koude rillingen, gewrichtsklachten, huiduitslag, hoofdpijn, duizeligheid, buikpijn, braken en diarree. De koortsaanvallen treden gemiddeld eens

  16. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  17. MAP kinase cascades in Arabidopsis innate immunity

    DEFF Research Database (Denmark)

    Rasmussen, Magnus Wohlfahrt; Roux, Milena Edna; Petersen, Morten

    2012-01-01

    Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs) by host transmembrane pattern recognition receptors which trigger MAPK-dependent innate ...

  18. Protein Kinases in Human Breast Carcinoma

    National Research Council Canada - National Science Library

    Cane, William

    1998-01-01

    .... Rak is a novel nuclear tyrosine that our group has identified in breast cancer tissues and cell lines that has structural homology to the Src tyrosine kinase, with SH2 and SH3 domains at its amino terminus...

  19. Flux control analysis of mitochondrial oxidative phosphorylation in rat skeletal muscle: pyruvate and palmitoyl-carnitine as substrates give different control patterns

    DEFF Research Database (Denmark)

    Fritzen, Anette J; Grunnet, Niels; Quistorff, Bjørn

    2007-01-01

    was associated with the ADP-generating system, i.e., 0.58 +/- 0.05 with pyruvate, but significantly lower, 0.40 +/- 0.05, with palmitoyl-carnitine as substrate. The flux control coefficients of complex I, III and IV, the ATP synthase, the ATP/ADP carrier and the P(i) carrier were 0.070 +/- 0.03, 0.083 +/- 0.......04, 0.054 +/- 0.01, 0.11 +/- 0.03, 0.090 +/- 0.03 and 0.026 +/- 0.01, respectively, with pyruvate as substrate. With palmitoyl-carnitine all control coefficients were significantly different, except for the P(i) carrier (i.e., 0.024 +/- 0.001, 0.036 +/- 0.01, 0.052 +/- 0.02, 0.020 +/- 0.002, 0.034 +/- 0.......02 and 0.012 +/- 0.002, respectively), probably caused by the shift from NADH to FADH(2) oxidation. The sum of flux control coefficients was not significantly different from unity with pyruvate, while only 0.58 with palmitoyl-carnitine, indicating significant control contributions from the enzymes involved...

  20. Chemical protection against radiation effects on Serum transaminase and the levels of glutamic and pyruvic acids following gamma irradiation of rats

    International Nuclear Information System (INIS)

    Mahdy, A.M.; EL-Kashef, H.S.

    1988-01-01

    The present study been carried out to evaluate the radioprotective efficiency of urea and vitamin E for protecting certain enzymatic systems from deleterious radiation effects. The activities of serum transaminase; aspartate aminotransferase (A S T) and alanine aminotransferase (A L T); as well as their relative substrates; glutamic and pyruvic acid levels; were selected for this study. The results indicated that whole body gamma irradiation at the dose of 7 Gy caused an evident elevation in the activities of both A S T and A L T and in the level of pyruvic acid at the experiment period (first,third,seventh and tenth days post irradiation). On the other hand the free glutamic acid level decreased at all post irradiation days. The variation in both enzymatic activities, pyruvic and glutamic acid levels became less pronounced in rats treated with either urea or vitamin E as chemical radioprotectors before whole body gamma irradiation. The results showed that the two agents are good radioprotectors, with respect to these parameters under investigation

  1. Probing early tumor response to radiation therapy using hyperpolarized [1-¹³C]pyruvate in MDA-MB-231 xenografts.

    Directory of Open Access Journals (Sweden)

    Albert P Chen

    Full Text Available Following radiation therapy (RT, tumor morphology may remain unchanged for days and sometimes weeks, rendering anatomical imaging methods inadequate for early detection of therapeutic response. Changes in the hyperpolarized [1-¹³C]lactate signals observed in vivo following injection of pre-polarized [1-¹³C]pyruvate has recently been shown to be a marker for tumor progression or early treatment response. In this study, the feasibility of using ¹³C metabolic imaging with [1-¹³C]pyruvate to detect early radiation treatment response in a breast cancer xenograft model was demonstrated in vivo and in vitro. Significant decreases in hyperpolarized [1-¹³C]lactate relative to [1-¹³C]pyruvate were observed in MDA-MB-231 tumors 96 hrs following a single dose of ionizing radiation. Histopathologic data from the treated tumors showed higher cellular apoptosis and senescence; and changes in the expression of membrane monocarboxylate transporters and lactate dehydrogenase B were also observed. Hyperpolarized ¹³C metabolic imaging may be a promising new tool to develop novel and adaptive therapeutic regimens for patients undergoing RT.

  2. Simultaneous Hyperpolarized 13C-Pyruvate MRI and 18F-FDG PET (HyperPET) in 10 Dogs with Cancer

    DEFF Research Database (Denmark)

    Gutte, Henrik; Hansen, Adam E; Larsen, Majbrit M E

    2015-01-01

    with biopsy-verified spontaneous malignant tumors were included for imaging. All dogs underwent a protocol of simultaneous (18)F-FDG PET, anatomic MR, and hyperpolarized dynamic nuclear polarization with (13)C-pyruvate imaging. The data were acquired using a combined clinical PET/MR imaging scanner. We found...... that combined (18)F-FDG PET and (13)C-pyruvate MRS imaging was possible in a single session of approximately 2 h. A continuous workflow was obtained with the injection of (18)F-FDG when the dogs was placed in the PET/MR scanner. (13)C-MRS dynamic acquisition demonstrated in an axial slab increased (13)C......With the introduction of combined PET/MR spectroscopic (MRS) imaging, it is now possible to directly and indirectly image the Warburg effect with hyperpolarized (13)C-pyruvate and (18)F-FDG PET imaging, respectively, via a technique we have named hyperPET. The main purpose of this present study...

  3. Pyruvic oxime nitrification and copper and nickel resistance by a Cupriavidus pauculus, an active heterotrophic nitrifier-denitrifier.

    Science.gov (United States)

    Ramirez, Miguel; Obrzydowski, Jennifer; Ayers, Mary; Virparia, Sonia; Wang, Meijing; Stefan, Kurtis; Linchangco, Richard; Castignetti, Domenic

    2014-01-01

    Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3-C(NOH)-COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a genus (Cupriavidus) known for its resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus designated as C. pauculus strain UM1) was examined for its ability to perform heterotrophic nitrification in the presence of Cu(2+) and Ni(2+) and to metabolize the xenobiotic phenol. The bacterium heterotrophically nitrified well when either 1 mM Cu(2+) or 0.5 mM Ni(2+) was present in either enriched or minimal medium. The organism also used phenol as a sole carbon source in either the presence or absence of 1 mM Cu(2+) or 0.5 mM Ni(2+). The ability of this isolate to perform a number of different metabolisms, its noteworthy resistance to copper and nickel, and its potential use as a bioremediation agent are discussed.

  4. Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

    Science.gov (United States)

    Sandulache, Vlad C; Chen, Yunyun; Lee, Jaehyuk; Rubinstein, Ashley; Ramirez, Marc S; Skinner, Heath D; Walker, Christopher M; Williams, Michelle D; Tailor, Ramesh; Court, Laurence E; Bankson, James A; Lai, Stephen Y

    2014-01-01

    Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

  5. Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

    Directory of Open Access Journals (Sweden)

    Vlad C Sandulache

    Full Text Available Ionizing radiation (IR cytotoxicity is primarily mediated through reactive oxygen species (ROS. Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP magnetic resonance spectroscopy (MRS and spectroscopic imaging (MRSI can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC, an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

  6. Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism.

    Science.gov (United States)

    Plaga, W; Vielhaber, G; Wallach, J; Knappe, J

    2000-01-21

    The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.

  7. Preventing intraperitoneal adhesions with ethyl pyruvate and hyaluronic acid/carboxymethylcellulose: a comparative study in an experimental model.

    Science.gov (United States)

    Caglayan, E Kıyak; Caglayan, K; Erdogan, N; Cinar, H; Güngör, B

    2014-10-01

    To compare the effectiveness of ethyl pyruvate (EP) with that of hyaluronic acid+carboxymethyl cellulose (Seprafilm) for the prevention of intraperitoneal adhesions. Seprafilm has been shown to be effective in many experimental and clinical studies. Thirty rats were divided into three groups at random, and uterine horn abrasion was performed by laparotomy. One group received no treatment (control group), one group received a single intraperitoneal dose of EP 50mg/kg (EP group), and a 2×1-cm patch of Seprafilm was applied in the third group (Seprafilm group). All rats were killed 14 days after surgery. Macroscopic and histopathological evaluation were performed by a surgeon and a pathologist who were blinded to group allocation. Histopathologically, inflammation, fibroblastic activity, foreign body reaction, collagen proliferation, vascular proliferation, Masson-Trichrome score, matrix metalloproteinase-2 score and vascular endothelial growth factor score were studied. Median macroscopic intraperitoneal adhesion scores for the control, EP and Seprafilm groups were 2.8, 1.2 and 1.1, respectively. Multiple comparisons between groups showed a significant difference (p0.05). After histopathological evaluation, significant differences in all parameters were found between the groups (p0.0167). In comparison with the untreated control group, EP and Seprafilm were found to reduce the formation of intraperitoneal adhesions. No significant difference was found between EP and Seprafilm. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Pyruvic Oxime Nitrification and Copper and Nickel Resistance by a Cupriavidus pauculus, an Active Heterotrophic Nitrifier-Denitrifier

    Directory of Open Access Journals (Sweden)

    Miguel Ramirez

    2014-01-01

    Full Text Available Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2 and nitrous oxide (N2O while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3–C(NOH–COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a genus (Cupriavidus known for its resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus designated as C. pauculus strain UM1 was examined for its ability to perform heterotrophic nitrification in the presence of Cu2+ and Ni2+ and to metabolize the xenobiotic phenol. The bacterium heterotrophically nitrified well when either 1 mM Cu2+ or 0.5 mM Ni2+ was present in either enriched or minimal medium. The organism also used phenol as a sole carbon source in either the presence or absence of 1 mM Cu2+ or 0.5 mM Ni2+. The ability of this isolate to perform a number of different metabolisms, its noteworthy resistance to copper and nickel, and its potential use as a bioremediation agent are discussed.

  9. Ror receptor tyrosine kinases: orphans no more

    OpenAIRE

    Green, Jennifer L.; Kuntz, Steven G.; Sternberg, Paul W.

    2008-01-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either act...

  10. Mediator kinase module and human tumorigenesis.

    Science.gov (United States)

    Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

    2015-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

  11. Fibronectin phosphorylation by ecto-protein kinase

    International Nuclear Information System (INIS)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

    1988-01-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

  12. The PIM kinases in hematological cancers.

    Science.gov (United States)

    Alvarado, Yesid; Giles, Francis J; Swords, Ronan T

    2012-02-01

    The PIM genes represent a family of proto-oncogenes that encode three different serine/threonine protein kinases (PIM1, PIM2 and PIM3) with essential roles in the regulation of signal transduction cascades, which promote cell survival, proliferation and drug resistance. PIM kinases are overexpressed in several hematopoietic tumors and support in vitro and in vivo malignant cell growth and survival, through cell cycle regulation and inhibition of apoptosis. PIM kinases do not have an identified regulatory domain, which means that these proteins are constitutively active once transcribed. They appear to be critical downstream effectors of important oncoproteins and, when overexpressed, can mediate drug resistance to available agents, such as rapamycin. Recent crystallography studies reveal that, unlike other kinases, they possess a hinge region, which creates a unique binding pocket for ATP, offering a target for an increasing number of potent small-molecule PIM kinase inhibitors. Preclinical studies in models of various hematologic cancers indicate that these novel agents show promising activity and some of them are currently being evaluated in a clinical setting. In this review, we profile the PIM kinases as targets for therapeutics in hematologic malignancies.

  13. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

    Directory of Open Access Journals (Sweden)

    Daniel Thomas

    Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

  14. Phosphate-Catalyzed Hydrogen Peroxide Formation from Agar, Gellan, and κ-Carrageenan and Recovery of Microbial Cultivability via Catalase and Pyruvate.

    Science.gov (United States)

    Kawasaki, Kosei; Kamagata, Yoichi

    2017-11-01

    Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H 2 O 2 ) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659-7666, 2014, https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H 2 O 2 formation in agar. The H 2 O 2 formation was pH dependent: H 2 O 2 was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H 2 O 2 formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H 2 O 2 after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H 2 O 2 scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H 2 O 2 from PT medium, these observations indicate that although H 2 O 2 is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved. IMPORTANCE The majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H 2 O

  15. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  16. A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  17. A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  18. Role of adiponectin/phosphatidylinositol 3-kinase/protein kinase B ...

    African Journals Online (AJOL)

    The adiponectin/phosphatidylinositol 3-kinase/protein kinase B (ADP/PI3k/Akt) signal transduction pathway has an important role in promoting cell survival. This study was designed to determine if the ADP/PI3K/Akt signaling pathway has a role in the mechanism of ischemia–reperfusion injury in vivo. Sprague–Dawley rats ...

  19. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  20. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ting-Lei Gu

    Full Text Available Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23 of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

  1. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    Science.gov (United States)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  2. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    Directory of Open Access Journals (Sweden)

    Konrad Kubiński

    2017-02-01

    Full Text Available The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors.

  3. syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction

    Science.gov (United States)

    El-Hillal, O.; Kurosaki, T.; Yamamura, H.; Kinet, J.-P.; Scharenberg, A. M.

    1997-01-01

    Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well. PMID:9050880

  4. The pyruvic acid analog 3-bromopyruvate interferes with the tetrazolium reagent MTS in the evaluation of cytotoxicity.

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Vali, Mustafa

    2010-04-01

    3-Bromopyruvate (3BrPA) is a pyruvate analog known for its alkylating property. Recently, several reports have documented the antiglycolytic and anticancer effects of 3BrPA and its potential for therapeutic applications. 3BrPA-mediated cytotoxicity has been evaluated in vitro by various methods including tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based assays such as MTT, MTS, and so on. However, growing body of evidences has shown that tetrazolium reagent may interfere with the test compounds. In this study, we investigated whether the tetrazolium reagent interferes with the assessment of 3BrPA cytotoxicity. The results of the tetrazolium-based MTS assay were compared with 3 distinct cell viability detection methods, that is, Trypan Blue staining, ATP depletion, and Annexin V staining in 2 different cell lines, Vx-2 and HepG2. The MTS assay data showed false positive results by indicating increased cell viability at 1 mM and 2 mM 3BrPA whereas the other cell viability assays demonstrated that both Vx-2 and HepG2 cells are not viable at the same treatment conditions. In order to validate the direct interaction of 3BrPA with MTS reagent, we tested cell-free media incubated with different concentrations of 3BrPA. The results of cell-free media showed an increase in absorbance in a dose-dependent manner confirming the interaction of MTS with 3BrPA. Thus, our data clearly demonstrate that 3BrPA interferes with the accuracy of MTS-based cytotoxicity evaluation. Hence, we suggest that employing multiple methods of biochemical as well as morphological cytotoxicity assays is critical to evaluate 3BrPA-mediated cell death.

  5. H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells.

    Science.gov (United States)

    Ju, YoungJun; Untereiner, Ashley; Wu, Lingyun; Yang, Guangdong

    2015-11-01

    Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H(2)S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H(2)S S-sulfhydration is now proposed as a major mechanism for H(2)S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H(2)S and its implication in gluconeogenesis in the liver have been unknown. Gene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. Exogenously applied H(2)S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H(2)S-induced gluconeogenesis. H(2)S had no effect on the expressions of PC mRNA and protein, while H(2)S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H(2)S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H(2)S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. CSE/H(2)S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. Tissue-specific regulation of CSE/H(2)S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants

    Directory of Open Access Journals (Sweden)

    David Morales-Alamo

    2018-03-01

    Full Text Available Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH activation and reactive nitrogen and oxygen species (RNOS in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (PIO2 = 75 mmHg or room air (PIO2 = 143 mmHg. Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO2. Immediately after the sprints, Ser293- and Ser300-PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively. However, 30 min into recovery Ser293-PDH-E1α phosphorylation reached pre-exercise values while Ser300-PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser293-PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser293 and Ser300-PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo (r = 0.74, P < 0.001; n = 18, but not after antioxidants ingestion (r = 0.35, P = 0.15. In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser293 re-phosphorylates at a faster rate than Ser300-PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms.

  7. Mechanism of polyphosphate kinase from Propionibacterium shermanii

    International Nuclear Information System (INIS)

    Robinson, N.A.

    1986-01-01

    Polyphosphate kinase, which catalyzes the reaction shown below, is one of two enzymes which have been reported to catalyze the synthesis of polyphosphate. Purification performed by ammonium sulfate precipitation (0-40% fraction) was followed by chromatography. The enzyme represents 70% of the protein in the hydroxylapatite pool and is stable at this level of purity. The subunit molecular weight was determined by SDS polyacrylamide gel analysis, (83,000 +/- 3000), nondenaturing polyacrylamide gel electrophoresis, (80,000 and 86,000 daltons), gel filtration (Biogel A 0.5m column was 85,000 +/- 4000.) Polyphosphate kinase appears to be a monomeric enzyme of ∼83,000 daltons. Four assays were developed for polyphosphate kinase. Basic proteins such as polylysine stimulate the synthesis of polyphosphate, these proteins cause precipitation of polyphosphate kinase from relatively impure enzyme extracts: Synthesized polyphosphate interacts noncovalently with the basic protein-enzyme precipitate. Efficient synthesis of polyphosphate requires the addition of either phosphate or short chain polyphosphate. Synthesis did occur at 1/10 the rate when neither of these two compounds were included. Initiation, elongation, and termination events of polyphosphate synthesis were examined. Short chain polyphosphate acts as a primer, with [ 32 P] short-chain polyphosphate incorporation into long chain polyphosphate by the kinase

  8. Radioimmunoassay of bovine heart protein kinase

    International Nuclear Information System (INIS)

    Fleischer, N.; Rosen, O.M.; Reichlin, M.

    1976-01-01

    Immunization of guinea pigs with bovine cardiac cAMP-dependent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) resulted in the development of precipitating antibodies to the cAMP-binding subunit of the enzyme. Both the phosphorylated and nonphosphorylated cAMP-binding protein of the protein kinase reacted with the antiserum. A radioimmunoassay was developed that detects 10 ng of holoenzyme and permits measurement of enzyme concentrations in bovine cardiac muscle. Bovine liver, kidney, brain, and skeletal muscle contain protein kinases which are immunologically identical to those found in bovine cardiac muscle. However, the proportion of immunoreactive enzyme activity differed for each tissue. All of the immunologically nonreactive enzyme in skeletal muscle and heart was separable from immunoreactive enzyme by chromatography on DEAE-cellulose. Rat tissues and pig heart contained protein kinase activity that cross reacted immunologically in a nonparallel fashion with bovine cardiac enzyme. These results indicate that cAMP-dependent protein kinases within and between species are immunologically heterogeneous

  9. The target landscape of clinical kinase drugs.

    Science.gov (United States)

    Klaeger, Susan; Heinzlmeir, Stephanie; Wilhelm, Mathias; Polzer, Harald; Vick, Binje; Koenig, Paul-Albert; Reinecke, Maria; Ruprecht, Benjamin; Petzoldt, Svenja; Meng, Chen; Zecha, Jana; Reiter, Katrin; Qiao, Huichao; Helm, Dominic; Koch, Heiner; Schoof, Melanie; Canevari, Giulia; Casale, Elena; Depaolini, Stefania Re; Feuchtinger, Annette; Wu, Zhixiang; Schmidt, Tobias; Rueckert, Lars; Becker, Wilhelm; Huenges, Jan; Garz, Anne-Kathrin; Gohlke, Bjoern-Oliver; Zolg, Daniel Paul; Kayser, Gian; Vooder, Tonu; Preissner, Robert; Hahne, Hannes; Tõnisson, Neeme; Kramer, Karl; Götze, Katharina; Bassermann, Florian; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Walch, Axel; Greif, Philipp A; Schneider, Sabine; Felder, Eduard Rudolf; Ruland, Juergen; Médard, Guillaume; Jeremias, Irmela; Spiekermann, Karsten; Kuster, Bernhard

    2017-12-01

    Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  10. Janus kinase inhibitors: jackpot or potluck?

    Directory of Open Access Journals (Sweden)

    Pavithran Keechilat

    2012-06-01

    Full Text Available The reports of a unique mutation in the Janus kinase-2 gene (JAK2 in polycythemia vera by several independent groups in 2005 quickly spurred the development of the Janus kinase inhibitors. In one of the great victories of translational research in recent times, the first smallmolecule Janus kinase inhibitor ruxolitinib entered a phase I trial in 2007. With the approval of ruxolitinib by the US Federal Drug Administration in November 2011 for high-risk and intermediate-2 risk myelofibrosis, a change in paradigm has occurred in the management of a subset of myeloproliferative neoplasms (MPN: primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Whereas the current evidence for ruxolitinib only covers high-risk and intermediate-2 risk myelofibrosis, inhibitors with greater potency are likely to offer better disease control and survival advantage in patients belonging to these categories, and possibly to the low-risk and intermediate-1 risk categories of MPN as well. But use of the Janus kinase inhibitors also probably has certain disadvantages, such as toxicity, resistance, withdrawal phenomenon, non-reversal of histology, and an implausible goal of disease clone eradication, some of which could offset the gains. In spite of this, Janus kinase inhibitors are here to stay, and for use in more than just myeloproliferative neoplasms.

  11. Protocols for the Design of Kinase-focused Compound Libraries.

    Science.gov (United States)

    Jacoby, Edgar; Wroblowski, Berthold; Buyck, Christophe; Neefs, Jean-Marc; Meyer, Christophe; Cummings, Maxwell D; van Vlijmen, Herman

    2018-05-01

    Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring

    Czech Academy of Sciences Publication Activity Database

    Andrs, M.; Kobarecny, J.; Jun, D.; Hodný, Zdeněk; Bartek, Jiří; Kuca, K.

    2015-01-01

    Roč. 58, č. 1 (2015), s. 41-71 ISSN 0022-2623 R&D Projects: GA MŠk(CZ) CZ.1.07/2.3.00/30.0044 Grant - others:University Hospital Hradec Kralove(CZ) 00179906; Faculty of Military Health Sciences, University of Defence(CZ) SV/FVZ201402 Institutional support: RVO:68378050 Keywords : DEPENDENT PROTEIN-KINASE * STRAND BREAK REPAIR * SELECTIVE PI3K-BETA INHIBITORS * TELANGIECTASIA MUTATED KINASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.589, year: 2015

  13. Protein Kinases in Shaping Plant Architecture.

    Science.gov (United States)

    Wu, Juan; Wang, Bo; Xin, Xiaoyun; Ren, Dongtao

    2018-02-13

    Plant architecture, the three-dimensional organization of the plant body, includes the branching pattern and the size, shape, and position of organs. Plant architecture is genetically controlled and is influenced by environmental conditions. The regulations occur at most of the stages from the first division of the fertilized eggs to the final establishment of plant architecture. Among the various endogenous regulators, protein kinases and their associated signaling pathways have been shown to play important roles in regulating the process of plant architecture establishment. In this review, we summarize recent progress in the understanding of the mechanisms by which plant architecture formation is regulated by protein kinases, especially mitogen-activated protein kinase (MAPK). Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. The Role of PAS Kinase in PASsing the Glucose Signal

    Directory of Open Access Journals (Sweden)

    Julianne H. Grose

    2010-06-01

    Full Text Available PAS kinase is an evolutionarily conserved nutrient responsive protein kinase that regulates glucose homeostasis. Mammalian PAS kinase is activated by glucose in pancreatic beta cells, and knockout mice are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet. Yeast PAS kinase is regulated by both carbon source and cell integrity stress and stimulates the partitioning of glucose toward structural carbohydrate biosynthesis. In our current model for PAS kinase regulation, a small molecule metabolite binds the sensory PAS domain and activates the enzyme. Although bona fide PAS kinase substrates are scarce, in vitro substrate searches provide putative targets for exploration.

  15. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    International Nuclear Information System (INIS)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

  16. 2-Aminobenzimidazoles as potent Aurora kinase inhibitors.

    Science.gov (United States)

    Zhong, Min; Bui, Minna; Shen, Wang; Baskaran, Subramanian; Allen, Darin A; Elling, Robert A; Flanagan, W Michael; Fung, Amy D; Hanan, Emily J; Harris, Shannon O; Heumann, Stacey A; Hoch, Ute; Ivy, Sheryl N; Jacobs, Jeffrey W; Lam, Stuart; Lee, Heman; McDowell, Robert S; Oslob, Johan D; Purkey, Hans E; Romanowski, Michael J; Silverman, Jeffrey A; Tangonan, Bradley T; Taverna, Pietro; Yang, Wenjin; Yoburn, Josh C; Yu, Chul H; Zimmerman, Kristin M; O'Brien, Tom; Lew, Willard

    2009-09-01

    This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.

  17. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  18. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

    Science.gov (United States)

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

    The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

  19. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

    Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

    1990-01-01

    Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

  20. Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

    DEFF Research Database (Denmark)

    Niefind, K; Raaf, J; Issinger, Olaf-Georg

    2009-01-01

    the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP......Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...

  1. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders. Several strategies .... family, the β-adrenergic receptor kinase (βARK), the ribosomal S6 ..... urinary bladder smooth muscle cells. While no ...

  2. Creatine kinase activity is associated with blood pressure

    NARCIS (Netherlands)

    Brewster, Lizzy M.; Mairuhu, Gideon; Bindraban, Navin R.; Koopmans, Richard P.; Clark, Joseph F.; van Montfrans, Gert A.

    2006-01-01

    BACKGROUND: We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular

  3. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Mikkelsen, N.E.; Clausen, A.R.

    2009-01-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution...

  4. Deoxyribonucleoside kinases in mitochondrial DNA depletion.

    Science.gov (United States)

    Saada-Reisch, Ann

    2004-10-01

    Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.

  5. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  6. Plant PA signaling via diacylglycerol kinase

    NARCIS (Netherlands)

    Arisz, S.A.; Testerink, C.; Munnik, T.

    2009-01-01

    Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential

  7. Nonorthologous gene displacement of phosphomevalonate kinase

    NARCIS (Netherlands)

    Houten, S. M.; Waterham, H. R.

    2001-01-01

    Phosphomevalonate kinase (PMK; EC 2.7.4.2) catalyzes the phosphorylation of 5-phosphomevalonate into 5-diphosphomevalonate, an essential step in isoprenoid biosynthesis via the mevalonate pathway. So far, two nonorthologous genes encoding PMK have been described, the Saccharomyces cerevisiae ERG8

  8. Casein kinase-2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1992-01-01

    Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form...

  9. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

  10. Kinase-Centric Computational Drug Development

    NARCIS (Netherlands)

    Kooistra, Albert J.; Volkamer, Andrea

    2017-01-01

    Kinases are among the most studied drug targets in industry and academia, due to their involvement in a majority of cellular processes and, upon dysregulation, in a variety of diseases including cancer, inflammation, and autoimmune disorders. The high interest in this druggable protein family

  11. Kinases involved in Rec8 phosphorylation revealed

    Czech Academy of Sciences Publication Activity Database

    Anger, Martin

    2010-01-01

    Roč. 9, č. 14 (2010), s. 2708-2708 ISSN 1538-4101 Institutional research plan: CEZ:AV0Z50450515 Keywords : kinases * Rec8 * meisosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.999, year: 2010

  12. Gene regulation by MAP kinase cascades

    DEFF Research Database (Denmark)

    Fiil, Berthe Katrine; Petersen, Klaus; Petersen, Morten

    2009-01-01

    Mitogen-activated protein kinase (MAPK) cascades are signaling modules that transduce extracellular stimuli to a range of cellular responses. Research in yeast and metazoans has shown that MAPK-mediated phosphorylation directly or indirectly regulates the activity of transcription factors. Plant ...

  13. Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets.

    Science.gov (United States)

    Lai, Justine Y Q; Langston, Steven; Adams, Ruth; Beevers, Rebekah E; Boyce, Richard; Burckhardt, Svenja; Cobb, James; Ferguson, Yvonne; Figueroa, Eva; Grimster, Neil; Henry, Andrew H; Khan, Nawaz; Jenkins, Kerry; Jones, Mark W; Judkins, Robert; Major, Jeremy; Masood, Abid; Nally, James; Payne, Helen; Payne, Lloyd; Raphy, Gilles; Raynham, Tony; Reader, John; Reader, Valérie; Reid, Alison; Ruprah, Parminder; Shaw, Michael; Sore, Hannah; Stirling, Matthew; Talbot, Adam; Taylor, Jess; Thompson, Stephen; Wada, Hiroki; Walker, David

    2005-05-01

    This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an 'in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection. Copyright (c) 2004 Wiley Periodicals, Inc.

  14. Kinase detection with gallium nitride based high electron mobility transistors.

    Science.gov (United States)

    Makowski, Matthew S; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-07-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing.

  15. Diversity, classification and function of the plant protein kinase superfamily

    OpenAIRE

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase r...

  16. p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng

    2013-01-01

    204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...... MEK activity in a kinase independent manner, probably by serving as a scaffold to facilitate interaction of c-Raf....

  17. The Roles of Protein Kinases in Learning and Memory

    Science.gov (United States)

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  18. Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study

    Science.gov (United States)

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2018-01-01

    Abstract Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5′-kinase fusion genes, combinatorial effects between 3′-KDR kinases and their 5′-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3′-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of ‘effective’ (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3′-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs’ clinical implications. PMID:28013235

  19. Determination of pyruvate and lactate as potential biomarkers of embryo viability in assisted reproduction by capillary electrophoresis with contactless conductivity detection.

    Science.gov (United States)

    Mádr, Aleš; Celá, Andrea; Klejdus, Bořivoj; Pelcová, Marta; Crha, Igor; Žáková, Jana; Glatz, Zdeněk

    2015-06-01

    Human-assisted reproduction is increasing in importance due to the constantly rising number of couples suffering from infertility issue. A key step in in vitro fertilization is the proper assessment of embryo viability in order to select the embryo with the highest likelihood of resulting in a pregnancy. This study proposes a method based on CE with contactless conductivity detection for the determination of pyruvate and lactate in spent culture media used in human-assisted reproduction. A fused-silica capillary of 64.0 cm total length and 50 μm inner diameter was used. The inner capillary wall was modified by the coating of successive layers of the ionic polymers polybrene and dextran sulfate to reverse EOF. The BGE was composed of 10 mM MES/lithium hydroxide, pH 6.50. The sample was injected by pressure 50 mbar for 18 s, separation voltage was set to -24 kV, and capillary temperature to 15°C. The presented method requires only 2 μL of the culture medium, with LODs for pyruvate and lactate of 0.03 and 0.02 μM, respectively. The results demonstrated the method's suitability for the analysis of spent culture media to support embryo viability assessment by light microscopy, providing information about key metabolites of the energy metabolism of a developing embryo. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as non-invasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate

    Science.gov (United States)

    Matsumoto, Shingo; Saito, Keita; Yasui, Hironobu; Morris, H. Douglas; Munasinghe, Jeeva P.; Lizak, Martin; Merkle, Hellmut; Ardenkjaer-Larsen, Jan Henrik; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Koretsky, Alan P.; Mitchell, James B.; Krishna, Murali C.

    2012-01-01

    The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation, and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate (3-BP) blocks glycolysis pathway by inhibiting hypoxia inducible enzymes, and enhanced cytotoxicity of 3-BP under hypoxic conditions has been reported in vitro. However, the efficacy of 3-BP was substantially attenuated in hypoxic tumor regions (pO2 < 10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 (MCT1) is the major transporter for pyruvate and the analog 3-BP in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of MCT1 in vivo. Expression of MCT1 was enhanced in moderately hypoxic (8–15 mmHg) tumor regions, but down regulated in severely hypoxic (< 5 mmHg) tumor regions. These results emphasize the importance of non-invasive imaging biomarkers to confirm the action of hypoxia-activated drugs. PMID:22692861

  1. Assessing the survival of MRC5 and a549 cell lines upon exposure to pyruvic Acid, sodium citrate and sodium bicarbonate - biomed 2013.

    Science.gov (United States)

    Farah, Ibrahim O; Lewis, Veshell L; Ayensu, Wellington K; Cameron, Joseph A

    2013-01-01

    Lung cancer is among the most prevalent and deadly cancers in United States. In general, cancer cells are known to exhibit higher rates of glycolysis in comparison to normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, it was our hypothesis that upon exposure to organic inhibitors of glycolysis, cancer cells would not survive normally and that their growth and viability would be vastly decreased; essential glycolytic ATP production will be exhausted to the point of collapsing energy utilization. Furthermore, we hypothesize that no negative effect would be seen with exposures to organic inhibitors for normal lung cells. The human lung fibroblast MRC-5 and the human A549 alveolar epithelial cell lines were used as in vitro models of normal lung and lung cancers respectively. Using standard methods, both cell lines were maintained and exposed to pyruvic acid, sodium citrate and sodium bicarbonate reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging for parallel morphological displays of any changes in the course of their vitality and metabolic activities. Our results indicate that exposure of both cell lines to these organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Pyruvic acid, sodium citrate and sodium bicarbonate showed statistically significant (pcancer biotherapeutics.

  2. Insulin-induced inhibition of gluconeogenesis genes, including glutamic pyruvic transaminase 2, is associated with reduced histone acetylation in a human liver cell line.

    Science.gov (United States)

    Honma, Kazue; Kamikubo, Michiko; Mochizuki, Kazuki; Goda, Toshinao

    2017-06-01

    Hepatic glutamic pyruvic transaminase (GPT; also known as alanine aminotransferase) is a gluconeogenesis enzyme that catalyzes conversions between alanine and pyruvic acid. It is also used as a blood biomarker for hepatic damage. In this study, we investigated whether insulin regulates GPT expression, as it does for other gluconeogenesis genes, and if this involves the epigenetic modification of histone acetylation. Human liver-derived HepG2 cells were cultured with 0.5-100nM insulin for 8h, and the mRNA expression of GPT, glutamic-oxaloacetic transaminase (GOT), γ-glutamyltransferase (GGT), PCK1, G6PC and FBP1 was measured. We also investigated the extent of histone acetylation around these genes. Insulin suppressed the mRNA expression of gluconeogenesis genes (GPT2, GOT1, GOT2, GGT1, GGT2, G6PC, and PCK1) in HepG2 cells in a dose-dependent manner. mRNA levels of GPT2, but not GPT1, were decreased by insulin. Histone acetylation was also reduced around GPT2, G6PC, and PCK1 in response to insulin. The expression of GPT2 and other gluconeogenesis genes such as G6PC and PCK1 was suppressed by insulin, in association with decreases in histone H3 and H4 acetylation surrounding these genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Carbon-14 tracer studies in the metabolism of isolated rat-liver parenchymal cells under conditions of gluconeogenesis from lactate and pyruvate

    International Nuclear Information System (INIS)

    Muellhofer, G.; Mueller, C.; Stetten, C. von; Gruber, E.

    1977-01-01

    In rat liver perfusion experiments under conditions of gluconeogenesis from lactate and pyruvate, 14 C-labeling patterns of metabolites with (1- 14 C)-labeled and (2- 14 C)-labeled lactate or pyruvate. [ 14 C]bicarbonate and [1- 14 C]octanoate as tracers have been obtained which do not agree with generally assumed reaction schemes. The experiments have been repeated with incubations of isolated rat-liver parenchymal cells. The results demonstrate that the discrepancies between expected and analysed 14 C-labeling patterns of metabolites were still existent. From this observation, it may be concluded that 14 C-labelling patterns of metabolites are indicative for the existence of still unknown metabolic relationships in liver parenchymal cells. Furthermore, the results of our experiments prove that conclusions based on the exclusive analysis of metabolite levels are of limited value for studying intracellular events, because of uncharacterized compartmentations, which become evident in 14 C-tracer studies. It cannot be answered by our studies whether the apparent existence of differently labelled species of citrate, oxoglutarate, or acetyl-CoA represent intracellular compartmentation, or whether it is the result of metabolic heterogeneity of liver parenchym cells. (orig.) [de

  4. Application of mitochondrial pyruvate carrier blocker UK5099 creates metabolic reprogram and greater stem-like properties in LnCap prostate cancer cells in vitro

    Science.gov (United States)

    Zhong, Yali; Li, Xiaoran; Yu, Dandan; Li, Xiaoli; Li, Yaqing; Long, Yuan; Yuan, Yuan; Ji, Zhenyu; Zhang, Mingzhi; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2015-01-01

    Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly higher proportion of side population (SP) fraction and expressed higher levels of stemness markers Oct3/4 and Nanog. Chemosensitivity examinations revealed that the UK5099 treated cells became more resistant to chemotherapy compared to the non-treated cells. These results demonstrate probably an intimate connection between metabolic reprogram and stem-like phenotype of LnCap cells in vitro. We propose that MPC blocker (UK5099) application may be an ideal model for Warburg effect studies, since it attenuates mitochondrial OXPHOS and increases aerobic glycolysis, a phenomenon typically reflected in the Warburg effect. We conclude that impaired mitochondrial OXPHOS and upregulated glycolysis are related with stem-like phenotype shift in prostatic cancer cells. PMID:26413751

  5. Ethyl pyruvate ameliorates hepatic injury following blunt chest trauma and hemorrhagic shock by reducing local inflammation, NF-kappaB activation and HMGB1 release.

    Science.gov (United States)

    Wagner, Nils; Dieteren, Scott; Franz, Niklas; Köhler, Kernt; Mörs, Katharina; Nicin, Luka; Schmidt, Julia; Perl, Mario; Marzi, Ingo; Relja, Borna

    2018-01-01

    The treatment of patients with multiple trauma including blunt chest/thoracic trauma (TxT) and hemorrhagic shock (H) is still challenging. Numerous studies show detrimental consequences of TxT and HS resulting in strong inflammatory changes, organ injury and mortality. Additionally, the reperfusion (R) phase plays a key role in triggering inflammation and worsening outcome. Ethyl pyruvate (EP), a stable lipophilic ester, has anti-inflammatory properties. Here, the influence of EP on the inflammatory reaction and liver injury in a double hit model of TxT and H/R in rats was explored. Female Lewis rats were subjected to TxT followed by hemorrhage/H (60 min, 35±3 mm Hg) and resuscitation/R (TxT+H/R). Reperfusion was performed by either Ringer`s lactated solution (RL) alone or RL supplemented with EP (50 mg/kg). Sham animals underwent all surgical procedures without TxT+H/R. After 2h, blood and liver tissue were collected for analyses, and survival was assessed after 24h. Resuscitation with EP significantly improved haemoglobin levels and base excess recovery compared with controls after TxT+H/R, respectively (ptrauma and hemorrhagic shock is associated with NF-κB. In particular, the beneficial anti-inflammatory effects of ethyl pyruvate seem to be regulated by the HMGB1/NF-κB axis in the liver, thereby, restraining inflammatory responses and liver injury after double hit trauma in the rat.

  6. Relationship between exposure duration, carboxyhemoglobin, blood glucose, pyruvate and lactate and the severity of intoxication in 39 cases of acute carbon monoxide poisoning in man

    Energy Technology Data Exchange (ETDEWEB)

    Sokal, J.A.; Kralkowska, E.

    1985-08-01

    The relationship between exposure duration, COHb, blood glucose, pyruvate and lactate and the severity of intoxication was investigated in a group of 39 cases of acute CO poisoning treated in the Clinical Toxicology Center in Lodz, Poland. On the basis of clinical criteria the patients were classified into cases of mild, moderate, severe and very severe CO poisoning. COHb and carbohydrate metabolites were estimated in venous blood taken immediately after admission of the patient to hospital prior to treatment. The severity of intoxication did not correlate with blood COHb; variation in exposure duration seems to be responsible for this phenomenon. Severe and very severe poisonings were associated with longer exposures and were accompanied by a markedly higher blood lactate level, compared to mild and moderate cases. Blood pyruvate depended less than lactate on the severity of intoxication. Blood glucose depended neither on exposure duration nor on the severity of intoxication. Among the carbohydrate metabolic parameters studied, blood lactate determination can be helpful in the evaluation of the severity of CO poisoning in man.

  7. Coordinate cis-[Cr(C2O4(pm(OH22]+ Cation as Molecular Biosensor of Pyruvate’s Protective Activity Against Hydrogen Peroxide Mediated Cytotoxity

    Directory of Open Access Journals (Sweden)

    Lech Chmurzyński

    2008-08-01

    Full Text Available In this paper instrumental methods of carbon dioxide (CO2 detection in biological material were compared. Using cis-[Cr(C2O4(pm(OH22]+ cation as a specific molecular biosensor and the stopped-flow technique the concentrations of CO2 released from the cell culture medium as one of final products of pyruvate decomposition caused by hydrogen peroxide were determined. To prove the usefulness of our method of CO2 assessment in the case of biological samples we investigated protective properties of exogenous pyruvate in cultured osteosarcoma 143B cells exposed to 1 mM hydrogen peroxide (H2O2 added directly to culture medium. Pyruvic acid is well known scavenger of H2O2 and, moreover, a molecule which is recognized as one of the major mediator of oxidative stress detected in many diseases and pathological situations like ischemiareperfusion states. The pyruvate's antioxidant activity is described as its rapid reaction with H2O2,which causes nonenzymatic decarboxylation of pyruvate and releases of CO2, water and acetate as final products. In this work for the first time we have correlated the concentration of CO2 dissolved in culture medium with pyruvate's oxidant-scavenging abilities. Moreover, the kinetics of the reaction between aqueous solution of CO2 and coordinate ion, cis-[Cr(C2O4(pm(OH22]+ was analysed. The results obtained enabled determination of the number of steps of the reaction studied. Based on the kinetic equations, rate constants were determined for each step.

  8. Roles of Apicomplexan protein kinases at each life cycle stage.

    Science.gov (United States)

    Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya

    2012-06-01

    Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling.

    Science.gov (United States)

    Nyati, Shyam; Schinske-Sebolt, Katrina; Pitchiaya, Sethuramasundaram; Chekhovskiy, Katerina; Chator, Areeb; Chaudhry, Nauman; Dosch, Joseph; Van Dort, Marcian E; Varambally, Sooryanarayana; Kumar-Sinha, Chandan; Nyati, Mukesh Kumar; Ray, Dipankar; Walter, Nils G; Yu, Hongtao; Ross, Brian Dale; Rehemtulla, Alnawaz

    2015-01-06

    Transforming growth factor-β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion. Copyright © 2015, American Association for the Advancement of Science.

  10. Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

    Science.gov (United States)

    Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

    2008-01-01

    Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

  11. Tyrosine kinase inhibitors: Multi-targeted or single-targeted?

    Science.gov (United States)

    Broekman, Fleur; Giovannetti, Elisa; Peters, Godefridus J

    2011-02-10

    Since in most tumors multiple signaling pathways are involved, many of the inhibitors in clinical development are designed to affect a wide range of targeted kinases. The most important tyrosine kinase families in the development of tyrosine kinase inhibitors are the ABL, SCR, platelet derived growth factor, vascular endothelial growth factor receptor and epidermal growth factor receptor families. Both multi-kinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which are related to potential resistance mechanisms, pharmacokinetics, selectivity and tumor environment. In different malignancies various tyrosine kinases are mutated or overexpressed and several resistance mechanisms exist. Pharmacokinetics is influenced by interindividual differences and differs for two single targeted inhibitors or between patients treated by the same tyrosine kinase inhibitor. Different tyrosine kinase inhibitors have various mechanisms to achieve selectivity, while differences in gene expression exist between tumor and stromal cells. Considering these aspects, one type of inhibitor can generally not be preferred above the other, but will depend on the specific genetic constitution of the patient and the tumor, allowing personalized therapy. The most effective way of cancer treatment by using tyrosine kinase inhibitors is to consider each patient/tumor individually and to determine the strategy that specifically targets the consequences of altered (epi)genetics of the tumor. This strategy might result in treatment by a single multi kinase inhibitor for one patient, but in treatment by a couple of single kinase inhibitors for other patients.

  12. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  13. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Witters, L.A.; Bacon, G.W.

    1985-01-01

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

  14. Src family kinases in chronic kidney disease.

    Science.gov (United States)

    Wang, Jun; Zhuang, Shougang

    2017-09-01

    Src family kinases (SFKs) belong to nonreceptor protein tyrosine kinases and have been implicated in the regulation of numerous cellular processes, including cell proliferation, differentiation, migration and invasion, and angiogenesis. The role and mechanisms of SFKs in tumorgenesis have been extensively investigated, and some SFK inhibitors are currently under clinical trials for tumor treatment. Recent studies have also demonstrated the importance of SFKs in regulating the development of various fibrosis-related chronic diseases (e.g., idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, and systemic sclerosis). In this article, we summarize the roles of SFKs in various chronic kidney diseases, including glomerulonephritis, diabetic nephropathy, human immunodeficiency virus-associated nephropathy, autosomal dominant form of polycystic kidney disease, and obesity-associated kidney disease, and discuss the mechanisms involved. Copyright © 2017 the American Physiological Society.

  15. Tyrosine kinase signalling in breast cancer

    International Nuclear Information System (INIS)

    Hynes, Nancy E

    2000-01-01

    Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors to signalling molecules on neighbouring cells. Receptors of the tyrosine kinase family play an important role in the integration and interpretation of these external stimuli, allowing a cell to respond appropriately to its environment. The activation of receptor tyrosine kinases (RTKs) is tightly controlled, allowing a normal cell to correctly integrate its external environment with internal signal transduction pathways. In contrast, due to numerous molecular alterations arising during the course of malignancy, a tumour is characterized by an abnormal response to its environment, which allows cancer cells to evade the normal mechanisms controlling cellular proliferation. Alterations in the expression of various RTKs, in their activation, and in the signalling molecules lying downstream of the receptors play important roles in the development of cancer. This topic is the major focus of the thematic review section of this issue of Breast Cancer Research

  16. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    International Nuclear Information System (INIS)

    Boura, Evzen; Nencka, Radim

    2015-01-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine

  17. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  18. Ror receptor tyrosine kinases: orphans no more.

    Science.gov (United States)

    Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

    2008-11-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

  19. Aurora kinase inhibitors: Progress towards the clinic

    Czech Academy of Sciences Publication Activity Database

    Kollareddy, M.; Zheleva, D.; Dzubak, P.; Brahmkshatriya, Pathik; Lepšík, Martin; Hajduch, M.

    2012-01-01

    Roč. 30, č. 6 (2012), s. 2411-2432 ISSN 0167-6997 Grant - others:GA ČR(CZ) GA301/08/1649; GA ČR(CZ) GD303/09/H048 Program:GA; GD Institutional research plan: CEZ:AV0Z40550506 Keywords : Aurora kinases * cancer * inhibitors Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2012

  20. MAP kinases in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Coskun, Mehmet; Olsen, Jørgen; Seidelin, Jakob Benedict

    2011-01-01

    The mammalian family of mitogen-activated protein kinases (MAPKs) is activated by diverse extracellular and intracellular stimuli, and thereby they play an essential role in connecting cell-surface receptors to changes in transcriptional programs. The MAPK signaling pathways regulate a wide range...... these signaling pathways have been exploited for the development of therapeutics and discuss the current knowledge of potential MAPK inhibitors and their anti-inflammatory effects in clinical trials related to IBD....

  1. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Nencka, Radim

    2015-01-01

    Roč. 337, č. 2 (2015), s. 136-145 ISSN 0014-4827 R&D Projects: GA ČR GJ15-21030Y; GA MŠk LO1302; GA ČR GA15-09310S EU Projects: European Commission(XE) 333916 - STARPI4K Institutional support: RVO:61388963 Keywords : phosphatidylinositol 4-kinase * inhibitor * crystal structure * virus Subject RIV: CC - Organic Chemistry Impact factor: 3.378, year: 2015

  2. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  3. Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

    Science.gov (United States)

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-08-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

  4. CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

    Science.gov (United States)

    Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

    2000-09-12

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

  5. CIKS, a connection to IκB kinase and stress-activated protein kinase

    Science.gov (United States)

    Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

    2000-01-01

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

  6. The Pim kinases: new targets for drug development.

    Science.gov (United States)

    Swords, Ronan; Kelly, Kevin; Carew, Jennifer; Nawrocki, Stefan; Mahalingam, Devalingam; Sarantopoulos, John; Bearss, David; Giles, Francis

    2011-12-01

    The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.

  7. PAK4 crystal structures suggest unusual kinase conformational movements.

    Science.gov (United States)

    Zhang, Eric Y; Ha, Byung Hak; Boggon, Titus J

    2018-02-01

    In order for protein kinases to exchange nucleotide they must open and close their catalytic cleft. These motions are associated with rotations of the N-lobe, predominantly around the 'hinge region'. We conducted an analysis of 28 crystal structures of the serine-threonine kinase, p21-activated kinase 4 (PAK4), including three newly determined structures in complex with staurosporine, FRAX486, and fasudil (HA-1077). We find an unusual motion between the N-lobe and C-lobe of PAK4 that manifests as a partial unwinding of helix αC. Principal component analysis of the crystal structures rationalizes these movements into three major states, and analysis of the kinase hydrophobic spines indicates concerted movements that create an accessible back pocket cavity. The conformational changes that we observe for PAK4 differ from previous descriptions of kinase motions, and although we observe these differences in crystal structures there is the possibility that the movements observed may suggest a diversity of kinase conformational changes associated with regulation. Protein kinases are key signaling proteins, and are important drug targets, therefore understanding their regulation is important for both basic research and clinical points of view. In this study, we observe unusual conformational 'hinging' for protein kinases. Hinging, the opening and closing of the kinase sub-domains to allow nucleotide binding and release, is critical for proper kinase regulation and for targeted drug discovery. We determine new crystal structures of PAK4, an important Rho-effector kinase, and conduct analyses of these and previously determined structures. We find that PAK4 crystal structures can be classified into specific conformational groups, and that these groups are associated with previously unobserved hinging motions and an unusual conformation for the kinase hydrophobic core. Our findings therefore indicate that there may be a diversity of kinase hinging motions, and that these may

  8. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

    Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

  9. Ribosomal S6 Kinase Cooperates with Casein Kinase 2 to Modulate the Drosophila Circadian Molecular Oscillator

    Science.gov (United States)

    Akten, Bikem; Tangredi, Michelle M.; Jauch, Eike; Roberts, Mary A.; Ng, Fanny; Raabe, Thomas; Jackson, F. Rob

    2009-01-01

    There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the MAPK pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the Casein Kinase 2 regulatory subunit (CK2β), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism. PMID:19144847

  10. High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity : application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

    NARCIS (Netherlands)

    Stijntjes, G.J.; te Koppele, J.M.; Vermeulen, N P

    1992-01-01

    An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid,

  11. Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

    OpenAIRE

    Deprez, J; Bertrand, L; Alessi, D R; Krause, U; Hue, L; Rider, M H

    2000-01-01

    A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC...

  12. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

    2012-01-01

    , but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα......GDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest...

  13. Assessment of real-time myocardial uptake and enzymatic conversion of hyperpolarized [1-¹³C]pyruvate in pigs using slice selective magnetic resonance spectroscopy

    DEFF Research Database (Denmark)

    Menichetti, Luca; Frijia, Francesca; Flori, Alessandra

    2012-01-01

    . We applied a numerical approach for spectral analysis and kinetic fitting (LSFIT/KIMOfit), making a comparison with a well-known jMRUI/AMARES analysis and γ-variate function, and we estimated the apparent conversion rate of hyperpolarized [1-¹³C]pyruvate into its downstream metabolites [1-¹³C......]lactate, [1-¹³C]alanine and [¹³C]bicarbonate in a 3 T MR scanner. We detected an increase in the apparent kinetic constants (k(PX) ) for bicarbonate and lactate of two-fold during dobutamine infusion. These data correlate with the double product (rate-pressure product), an indirect parameter of cardiac oxygen...

  14. Differentiating inflamed and normal lungs by the apparent reaction rate constants of lactate dehydrogenase probed by hyperpolarized (13)C labeled pyruvate.

    Science.gov (United States)

    Xu, He N; Kadlececk, Stephen; Shaghaghi, Hoora; Zhao, Huaqing; Profka, Harilla; Pourfathi, Mehrdad; Rizi, Rahim; Li, Lin Z

    2016-02-01

    Clinically translatable hyperpolarized (HP) (13)C-NMR can probe in vivo enzymatic reactions, e.g., lactate dehydrogenase (LDH)-catalyzed reaction by injecting HP (13)C-pyruvate into the subject, which is converted to (13)C labeled lactate by the enzyme. Parameters such as (13)C-lactate signals and lactate-to-pyruvate signal ratio are commonly used for analyzing the HP (13)C-NMR data. However, the biochemical/biological meaning of these parameters remains either unclear or dependent on experimental settings. It is preferable to quantify the reaction rate constants with a clearer physical meaning. Here we report the extraction of the kinetic parameters of the LDH reaction from HP (13)C-NMR data and investigate if they can be potential predictors of lung inflammation. Male Sprague-Dawley rats (12 controls, 14 treated) were used. One dose of bleomycin (2.5 U/kg) was administered intratracheally to the treatment group. The lungs were removed, perfused, and observed by the HP-NMR technique, where a HyperSense dynamic nuclear polarization system was used to generate the HP (13)C-pyruvate for injecting into the lungs. A 20 mm (1)H/(13)C dual-tuned coil in a 9.4-T Varian vertical bore NMR spectrometer was employed to acquire the (13)C spectral data every 1 s over a time period of 300 s using a non-selective, 15-degree radiofrequency pulse. The apparent rate constants of the LDH reaction and their ratio were quantified by applying ratiometric fitting analysis to the time series data of (13)C labeled pyruvate and lactate. The apparent forward rate constant kp =(3.67±3.31)×10(-4) s(-1), reverse rate constant kl =(4.95±2.90)×10(-2) s(-1), rate constant ratio kp /kl =(7.53±5.75)×10(-3) for the control lungs; kp =(11.71±4.35)×10(-4) s(-1), kl =(9.89±3.89)×10(-2) s(-1), and kp /kl =(12.39±4.18)×10(-3) for the inflamed lungs at the 7(th) day post treatment. Wilcoxon rank-sum test showed that the medians of these kinetic parameters of the 7-day cohort were significantly

  15. A comparative study of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the saliva of diabetic and normal patients.

    Science.gov (United States)

    Verma, M; Metgud, R; Madhusudan, A S; Verma, N; Saxena, M; Soni, A

    2014-10-01

    Diabetes has been reported to affect salivary glands adversely in humans and experimental models. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) are salivary enzymes that also are widely distributed in animal tissues. We determined GOT and GPT levels in saliva samples of 100 type 1 and 30 type 2 diabetic patients using reflectance spectrophotometry and compared them to 30 age and sex matched healthy controls. Statistically significant differences were observed in the mean values of GOT and GPT in type 1 diabetics compared to type 2 and control groups. Significantly higher GOT levels were found in the 1-20 year age group of type 1 diabetics. Our findings suggest that salivary gland damage is due to the same immunological attack that affects pancreatic β cells and results in type 1 diabetes.

  16. Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

    Directory of Open Access Journals (Sweden)

    Yu Fangyou

    2010-11-01

    Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

  17. ¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

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    Dany J V Beste

    2011-07-01

    Full Text Available Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA. Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

  18. Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

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    McDonald, Tanya S; Borges, Karin

    2017-07-01

    To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U- 13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U- 13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U- 13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from

  19. Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C4 compounds.

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    Tsuji, Yoshinori; Suzuki, Iwane; Shiraiwa, Yoshihiro

    2012-06-01

    Pyruvate carboxylase (PYC) catalyzes the β-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic β-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.

  20. Beneficial effect of feeding a ketogenic diet to mothers on brain development in their progeny with a murine model of pyruvate dehydrogenase complex deficiency.

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    Pliss, Lioudmila; Jatania, Urvi; Patel, Mulchand S

    2016-06-01

    Pyruvate dehydrogenase complex (PDC) deficiency is a major inborn error of oxidative metabolism of pyruvate in the mitochondria causing congenital lactic acidosis and primarily structural and functional abnormalities of the central nervous system. To provide an alternate source of acetyl-CoA derived from ketone bodies to the developing brain, a formula high in fat content is widely employed as a treatment. In the present study we investigated efficacy of a high-fat diet given to mothers during pregnancy and lactation on lessening of the impact of PDC deficiency on brain development in PDC-deficient female progeny. A murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene was employed in this study. Maternal consumption of a high-fat diet during pregnancy and lactation had no effect on number of live-birth, body growth, tissue PDC activity levels, as well as the in vitro rates of glucose oxidation and fatty acid biosynthesis by the developing brain of PDC-deficient female offspring during the postnatal age 35 days, as compared to the PDC-deficient progeny born to dams on a chow diet. Interestingly, brain weight was normalized in PDC-deficient progeny of high fat-fed mothers with improvement in impairment in brain structure deficit whereas brain weight was significantly decreased and was associated with greater cerebral structural defects in progeny of chow-fed mothers as compared to control progeny of mothers fed either a chow or high fat diet. The findings provide for the first time experimental support for beneficial effects of a ketogenic diet during the prenatal and early postnatal periods on the brain development of PDC-deficient mammalian progeny.