RESUMO
The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.
Assuntos
Acil-CoA Desidrogenase/genética , Cupriavidus necator/genética , Enoil-CoA Hidratase/genética , Óleos de Plantas/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/genética , Acil-CoA Desidrogenase/metabolismo , Arabinose/genética , Arabinose/metabolismo , Caprilatos/metabolismo , Cupriavidus necator/metabolismo , Ácidos Decanoicos/metabolismo , Enoil-CoA Hidratase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Hidroxibutiratos/metabolismo , Plasmídeos/genética , Poli-Hidroxialcanoatos/metabolismo , Regiões Promotoras Genéticas/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transcrição Gênica/genéticaRESUMO
BACKGROUND/AIMS: Experimental and clinical studies have shown the direct toxic effects of cigarette smoke (CS) on the myocardium, independent of vascular effects. However, the underlying mechanisms are not well known. METHODS: Wistar rats were allocated to control (C) and cigarette smoke (CS) groups. CS rats were exposed to cigarette smoke for 2 months. RESULTS: After that morphometric, functional and biochemical parameters were measured. The echocardiographic study showed enlargement of the left atria, increase in the left ventricular systolic volume and reduced systolic function. Within the cardiac metabolism, exposure to CS decreased beta hydroxy acyl coenzyme A dehydrogenases and citrate synthases and increased lactate dehydrogenases. Peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were expressed similarly in both groups. CS increased serum lipids and myocardial triacylglycerols (TGs). These data suggest that impairment in fatty acid oxidation and the accumulation of cardiac lipids characterize lipotoxicity. CS group exhibited increased oxidative stress and decreased antioxidant defense. Finally, the myocyte cross-sectional area and active Caspase 3 were increased in the CS group. CONCLUSION: The cardiac remodeling that was observed in the CS exposure model may be explained by abnormalities in energy metabolism, including lipotoxicity and oxidative stress.
Assuntos
Cardiomiopatias/sangue , Miocárdio/metabolismo , Estresse Oxidativo , Fumar/efeitos adversos , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/patologia , Citrato (si)-Sintase/biossíntese , Ecocardiografia , Enoil-CoA Hidratase/biossíntese , Lactato Desidrogenases/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/biossíntese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ratos , Fatores de Transcrição/biossíntese , Triglicerídeos/sangueRESUMO
It has recently been shown that the anti-mycobacterial pro-drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build-up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that 'de novo' biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC-resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti-mycobacterial compound.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Enoil-CoA Hidratase/genética , Mycobacterium kansasii/enzimologia , Mycobacterium tuberculosis/enzimologia , Tioacetazona/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/metabolismo , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Dados de Sequência Molecular , Mutação , Mycobacterium kansasii/química , Mycobacterium kansasii/efeitos dos fármacos , Mycobacterium kansasii/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Óperon , Alinhamento de SequênciaRESUMO
Newborn was referred with diagnosis of neonatal epilepsy. Medical team could suspect and confirm D-bifunctional peroxisomal enzymatic deficiency diagnosis. It was made by family antecedents, severe neonatal hypotonia, uncontrolled neonatal seizures, craniofacial dysmorphic features, psychomotor retardation, neuronal migration defect and a positive peroxisomal panel. The full study in skin fibroblasts involved enzyme analysis, complementation studies and DNA analysis. The accumulation of very long chain fatty acids, partial deficiency in phytanic acid oxidation, and abnormal morphology of peroxisomes was consistent with a defect in peroxisomal fatty acid oxidation, involving D-bifunctional protein. It is very important to make a diagnosis of this innate error of metabolism in order to give preconceptional genetic counseling, to identify recurrence risk and to perform mutation analysis for the D-bifunctional protein gene, and to offer the prenatal diagnosis.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Enoil-CoA Hidratase/deficiência , Isomerases/deficiência , Doenças Metabólicas/diagnóstico , Humanos , Recém-Nascido , Masculino , Complexos Multienzimáticos/deficiência , Enzima Bifuncional do PeroxissomoRESUMO
Fetal abnormalities including chylous ascites, polyhydramnios, claw hands, and hammer toes were identified in an infant who had a missense mutation R106P and a 52bp deletion in the gene for a peroxisomal beta-oxidation enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase, D-bifunctional protein. The patient had psychomotor retardation and craniofacial dysmorphism and died at 7 months of age. The patient had atypical fetal manifestations of this enzyme deficiency.