176 resultados para PYRUVATE-DEHYDROGENASE


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The Bacillus subtilis strain 168 chromosomal region extending from 109 degrees to 112 degrees has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and beta-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase alpha-subunit (FdhA), protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.

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PURPOSE: We have investigated the expression and regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer. EXPERIMENTAL DESIGN: Clinical gastric adenocarcinoma samples were analyzed by immunohistochemistry and quantitative real-time PCR for protein and mRNA expression of 15-PGDH and for methylation status of 15-PGDH promoter. The effects of interleukin-1beta (IL-1beta) and epigenetic mechanisms on 15-PGDH regulation were assessed in gastric cancer cell lines. RESULTS: In a gastric cancer cell line with a very low 15-PGDH expression (TMK-1), the 15-PGDH promoter was methylated and treatment with a demethylating agent 5-aza-2'-deoxycytidine restored 15-PGDH expression. In a cell line with a relatively high basal level of 15-PGDH (MKN-28), IL-1beta repressed expression of 15-PGDH mRNA and protein. This effect of IL-1beta was at least in part attributed to inhibition of 15-PGDH promoter activity. SiRNA-mediated knockdown of 15-PGDH resulted in strong increase of prostaglandin E(2) production in MKN-28 cells and increased cell growth of these cells by 31% in anchorage-independent conditions. In clinical gastric adenocarcinoma specimens, 15-PGDH mRNA levels were 5-fold lower in gastric cancer samples when compared with paired nonneoplastic tissues (n = 26) and 15-PGDH protein was lost in 65% of gastric adenocarcinomas (n = 210). CONCLUSIONS: 15-PGDH is down-regulated in gastric cancer, which could potentially lead to accelerated tumor progression. Importantly, our data indicate that a proinflammatory cytokine linked to gastric carcinogenesis, IL-1beta, suppresses 15-PGDH expression at least partially by inhibiting promoter activity of the 15-PGDH gene.

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Axons, and particularly regenerating axons, have high metabolic needs in order to maintain critical functions such as axon transport and membrane depolarization. Though some of the required energy likely comes form extracellular glucose and ATP generated in the soma, we and others hypothesize that some of the energy may be supplied by lactate. Unlike glucose that requires glycolytic enzymes to produce pyruvate, lactate can be converted directly to pyruvate by lactate dehydrogenase and transported into mitochondria for oxidative metabolism. In order to be transported into or out of cells, lactate requires specific monocarboxylate transporters (MCTs), the most abundant of which is MCT1. If MCT1 and lactate are critical for nerve function and regeneration, we hypothesize that MCT1 heterozygote null mice, which appear phenotypically normal despite having approximately 40% MCT1 as compared to wildtype littermate mice, would have reduced capacity for repair following nerve injury. To investigate this, adult MCT1 heterozygote null mice or wild-type mice underwent unilateral sciatic nerve crush in the proximal thigh. We found that regeneration of the sciatic nerve, as measured by recovery of compound muscle action potentials (CMAP) in the lateral plantar muscles following proximal sciatic nerve stimulation, was delayed from a median of 21 days in wildtype mice to 38.5 days in MCT1 heterozygote mice. In fact, half of the MCT1 heterozygote null mice had no recovery of CMAP by the endpoint of the study at 42 days, while all of the wild-type mice had recovered. In addition, the maximal amplitude of CMAP recovery in MCT1 heterozygote mull mice was reduced from a mean of 3 mV to 0.5 mV. As would be expected, the denervated gastrocnemius muscle of MCT1 heterozygote null mice remained atrophic at 42 days compared to wild-type mice. Our experiments show that lactate supplied through MCT1 is necessary for nerve regeneration. Experiments are underway to determine whether loss of MCT1 prevents nerve regrowth directly due to reduced energy supply to axons or indirectly by dysfunctional Schwann cells normally dependent on lactate supply through MCT1.

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High aldehyde dehydrogenase (ALDH) activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDH(br)) cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDH(br) cells in the heart has not been evaluated so far. We have characterized ALDH(br) cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDH(br). ALDH(very-br) cells were more frequent in neonatal hearts than adult. ALDH(br) cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDH(very-br) cells, intermediate in ALDH(br) cells, and lowest in ALDH(dim) cells. ALDH1A2 expression was highest in ALDH(very-br) cells, intermediate in ALDH(dim) cells, and lowest in ALDH(br) cells. ALDH1A3 and ALDH2 expression was detectable in ALDH(very-br) and ALDH(br) cells, unlike ALDH(dim) cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDH(br) cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDH(br) cells, unlike ALDH(dim) cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α -actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDH(br) cells declined with cell passage. In conclusion, the cardiac-derived ALDH(br) population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDH(br) cells remains to be evaluated.

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(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.

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Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-Coenzyme A (CoA) during fermentation. The aims of our study were: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate-bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate-bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down-stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.

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A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

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The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is selectively expressed in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor. A diminished activity causes salt-sensitive hypertension. The mechanism of the variable and distinct 11β-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) expression in the cortical collecting duct is poorly understood. Here, we analyzed for the first time whether the 11β-HSD2 expression is modulated by microRNAs (miRNAs). In silico analysis revealed 53 and 27 miRNAs with potential binding sites on human or rat HSD11B2 3'-untranslated region. A reporter assay demonstrated 3'-untranslated region-dependent regulation of human and rodent HSD11B2. miRNAs were profiled from cortical collecting ducts and proximal convoluted tubules. Bioinformatic analyses showed a distinct clustering for cortical collecting ducts and proximal convoluted tubules with 53 of 375 miRNAs, where 13 were predicted to bind to the rat HSD11B2 3'-untranslated region. To gain insight into potentially relevant miRNAs in vivo, we investigated 2 models with differential 11β-HSD2 activity linked with salt-sensitive hypertension. (1) Comparing Sprague-Dawley with low and Wistar rats with high 11β-HSD2 activity revealed rno-miR-20a-5p, rno-miR-19b-3p, and rno-miR-190a-5p to be differentially expressed. (2) Uninephrectomy lowered 11β-HSD2 activity in the residual kidney with differentially expressed rno-miR-19b-3p, rno-miR-29b-3p, and rno-miR-26-5p. In conclusion, miRNA-dependent mechanisms seem to modulate 11β-HSD2 dosage in health and disease states.

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Metaphyseal chondromatosis with hydroxyglutaric aciduria (MC-HGA) is a generalized skeletal dysplasia, accompanied by urinary excretion of D-2- hydroxyglutarate (HGA), and variable cerebral involvement. By wholeexome sequencing 2 unrelated patients with MC-HGA, we have found mutations in isocitrate dehydrogenase 1 (IDH1) at codon 132, as apparent somatic mosaicism. IDH1 is a key enzyme of the Krebs cycle, which converts isocitrate into alpha-ketoglutarate (a-KG). Mutations at IDH1 Arg132 residue have originally been identified in different tumour types (isolated gliomas, leukemias, and chondrosarcomas). These mutations trans-specify the enzyme activity resulting in HGA accumulation and a-KG depletion. This induces activation of hypoxia-inducible factor 1-alpha (HIF-1a), an important regulator of chondrocyte proliferation at the growth plate. Differently from Arg132 somatic mutations found in isolated tumours, themutation in our patientsmust have occurred very early in embryogenesis to cause a generalized dysplasia with involvement of all long bones metaphyses and mutation detectability in blood. Identical mutations have subsequently been identified in chondromas excised from patients with multiple chondromatosis (Ollier disease). Tissue distribution of themutationmay explain variable cerebral involvement and the susceptibility to develop tumours in other organs. The postulated pathophysiology ofMC-HGA points out the link between Krebs cycle, hypoxia sensing and bone growth.

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Microtubule-associated protein 1B, MAP1B, is a major cytoskeletal protein during brain development and one of the largest brain MAPs associated with microtubules and microfilaments. Here, we identified several proteins that bind to MAP1B via immunoprecipitation with a MAP1B-specific antibody, by one and two-dimensional gel electrophoresis and subsequent mass spectrometry identification of precipitated proteins. In addition to tubulin and actin, a variety of proteins were identified. Among these proteins were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 8, dihydropyrimidinase related proteins 2 and 3, protein-L-isoaspartate O-methyltransferase, beta-spectrin, and clathrin protein MKIAA0034, linking either directly or indirectly to MAP1B. In particular, GAPDH, a key glycolytic enzyme, was bound in large quantity to the heavy chain of MAP1B in adult brain tissue. In vitro binding studies confirmed a direct binding of GAPDH to MAP1B. In PC12 cells, GAPDH was found in cytoplasm and nuclei and partially co-localized with MAP1B. It disappeared from the cytoplasm under oxidative stress or after a disruption of cytoskeletal elements after colcemid or cytochalasin exposure. GAPDH may be essential in the local energy provision of cytoskeletal structures and MAP1B may help to keep this key enzyme close to the cytoskeleton.

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L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

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In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.