RESUMO
BACKGROUND: Pyruvate dehydrogenase complex (PDHc) deficiencies are an important cause of primary lactic acidosis. Most cases result from mutations in the X-linked gene for the pyruvate dehydrogenase E1α subunit (PDHA1) while a few cases result from mutations in genes for E1ß (PDHB), E2 (DLAT), E3 (DLD) and E3BP (PDHX) subunits or PDH-phosphatase (PDP1). AIM: To report molecular characterization of 82 PDHc-deficient patients and analyze structural effects of novel missense mutations in PDHA1. METHODS: PDHA1 variations were investigated first, by exon sequencing using a long range PCR product, gene dosage assay and cDNA analysis. Mutation scanning in PDHX, PDHB, DLAT and DLD cDNAs was further performed in unsolved cases. Novel missense mutations in PDHA1 were located on the tridimensional model of human E1 protein to predict their possible functional consequences. RESULTS: PDHA1 mutations were found in 30 girls and 35 boys. Three large rearrangements, including two contiguous gene deletion syndrome were identified. Novel missense, frameshift and splicing mutations were also delineated and a nonsense mutation in a mosaic male. Mutations p.Glu75Ala, p.Arg88Ser, p.Arg119Trp, p.Gly144Asp, p.Pro217Arg, p.Arg235Gly, p.Tyr243Cys, p.Tyr243Ser, p.Arg245Gly, p.Pro250Leu, p.Gly278Arg, p.Met282Val, p.Gly298Glu in PDHA1 were predicted to impair active site channel conformation or subunit interactions. Six out of the seven patients with PDHB mutations displayed the recurrent p.Met101Val mutation; 9 patients harbored PDHX mutations and one patient DLD mutations. CONCLUSION: We provide an efficient stepwise strategy for mutation screening in PDHc genes and expand the growing list of PDHA1 mutations analyzed at the structural level.
Assuntos
Substituição de Aminoácidos , Piruvato Desidrogenase (Lipoamida)/genética , Doença da Deficiência do Complexo de Piruvato Desidrogenase/genética , Adolescente , Sequência de Bases , Domínio Catalítico , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Dosagem de Genes , Humanos , Ligação de Hidrogênio , Mutação INDEL , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , Análise de Sequência de DNARESUMO
PURPOSE: The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin-1 (hBest1) which is a Ca(2+)-sensitive chloride channel. This study was performed to identify disease-specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl(-) channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. METHODS: Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK-293 cells and the associated Cl(-) current was examined using whole-cell patch clamp analysis. RESULTS: Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non-functional. Furthermore, the Q293H mutant inhibited the function of wild-type bestrophin-1 channels in a dominant negative manner. CONCLUSIONS: This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease-linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.
Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Proteínas Mutantes/genética , Idade de Início , Substituição de Aminoácidos , Bestrofinas , Linhagem Celular , Criança , Pré-Escolar , Canais de Cloreto , Cloretos/metabolismo , Análise Mutacional de DNA , Éxons/genética , Feminino , Genes Dominantes , Humanos , Transporte de Íons/genética , Rim , Degeneração Macular/diagnóstico , Masculino , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Técnicas de Patch-Clamp , Linhagem , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Análise de Sequência de DNA , Relação Estrutura-Atividade , TransfecçãoRESUMO
Alterations in the energy metabolism of cancer cells have been reported for many years. However, the deleterious mechanisms involved in these deficiencies have not yet been clearly proved. The main goal of this study was to decipher the harmful mechanisms responsible for the respiratory chain deficiencies in the course of diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis, where mitochondrial DNA abnormalities had been previously reported. The respiratory activity of freshly isolated hepatoma mitochondria, assessed by oxygen consumption experiments and enzymatic assays, presented a severe complex I deficiency 19 months after DENA treatment, and later on, in addition, a defective complex III activity. Since respiratory complex subunits are encoded by both nuclear and mitochondrial genes, we checked whether the respiratory chain defects were due to impaired synthesis processes. The specific immunodetection of complex I failed to show any alterations in the steady-state levels of both nuclear and mitochondrial encoded subunits in the hepatomas. Moreover, in vitro protein synthesis experiments carried out on freshly isolated hepatoma mitochondria did not bring to light any modifications in the synthesis of the mitochondrial subunits of the respiratory complexes, whatever the degree of tumor progression. Finally, Southern blot analysis of mitochondrial DNA did not show any major mitochondrial DNA rearrangements in DENA-induced hepatomas. Because the synthetic processes of respiratory complexes did not seem to be implicated in the respiratory chain impairment, these deficiencies could be partly ascribed to a direct toxic impact of highly reactive molecules on these complexes, thus impairing their function. The mitochondrial respiratory chain is an important generator of noxious, reactive oxygen free radicals such as superoxide and H2O2, which are normally catabolized by powerful antioxidant scavengers. Nineteen months after DENA treatment, a general collapse of the antioxidant enzymatic system was demonstrated in the hepatomas, as recurrently observed in cancer cells. This oxidant versus antioxidant imbalance was characterized by the establishment of oxidative stress in the course of hepatocarcinogenesis, as partly shown by the important decrease of glutamine synthetase activity, an enzyme whose function is highly sensitive to oxidant reactions. This disequilibrium would result in a net increase of the steady-state concentration of superoxide generated between respiratory complexes I and III in the mitochondria. Once generated, superoxide would likely inactivate complexes I and III via oxidant reactions on their superoxide-sensitive [4Fe, 4S] clusters. The role of mitochondrial respiratory chain impairment in chemical carcinogenesis and/or the persistence of the cancerous state is further discussed.
Assuntos
Dietilnitrosamina/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Animais , Antioxidantes/metabolismo , Southern Blotting , DNA Mitocondrial/análise , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Sequestradores de Radicais Livres , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Mitocôndrias Hepáticas/enzimologia , Proteínas de Neoplasias/biossíntese , Polarografia , Ratos , Ratos Sprague-DawleyRESUMO
Cultured skin fibroblasts from four patients with Leigh syndrome and cytochrome c oxidase deficiency were studied. Mitochondrial DNA (mtDNA) analysis excluded large-scale deletions and known point mutations associated with Leigh syndrome. The COX activities were reduced to 18-44% of healthy probands, when measured in the presence of laurylmaltoside. COX activity from patients was shown to be more temperature sensitive than COX activity from control cells. In order to determine the subunit composition of COX immunoblotting studies were performed using mono- and polyclonal antibodies to distinct subunits. A monoclonal antibody to subunit IV crossreacted with two unknown proteins of higher apparent molecular weight in mitochondria from three patients, but not in mitochondria from control and the fourth patient. Quantification of immunoreactivity revealed a decrease of subunits II/III and IV parallel to the determined enzyme activity. In contrast, a variable amount of subunit VIIa (and/or VIIb) was found in mitochondria from different patients. The results indicate a defective COX holoenzyme complex in patients with Leigh syndrome and suggest different molecular origins of the defect.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Encefalomiopatias Mitocondriais/enzimologia , Sequência de Bases , Western Blotting , Primers do DNA/química , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/imunologia , Feminino , Fibroblastos/enzimologia , Temperatura Alta , Humanos , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Desnaturação Proteica , SíndromeRESUMO
Results from crosses between Goto-Kakizaki (GK) rats, which exhibit spontaneous non-insulin-dependent diabetes mellitus (NIDDM), and outbred nondiabetic Wistar rats have demonstrated an effect of maternal inheritance on diabetes in offspring of the first generation (F1). At 6 weeks of age, F1 offspring of sex-directed crosses exhibited plasma glucose values intermediate between GK and Wistar parents. Hyperglycemia in F1 rats born of female GK rats (F1GK) was more marked than in those born of female Wistar (F1W) rats. At 3 months of age, F1 rats showed a marked impairment of both glucose tolerance and insulin secretion, which was intermediate between GK and Wistar rats. Glucose intolerance was more pronounced in F1GK rats than in F1W. By contrast, insulin secretion in F1W rats was more deteriorated than in F1GK rats. No deletion in mitochondrial DNA was observed in the GK rats, which decreased the possibility of a mitochondrial inheritance effect as an explanation of our findings. These data support a polygenic model in diabetes inheritance of NIDDM and suggest that, in addition to genetic factors, a perturbed maternal metabolism can contribute to its inheritance.
Assuntos
DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Ratos Endogâmicos/genética , Animais , Sequência de Bases , Glicemia/metabolismo , Cruzamentos Genéticos , Primers do DNA , DNA Mitocondrial/análise , Diabetes Mellitus Tipo 2/sangue , Feminino , Hemoglobinas Glicadas/análise , Insulina/sangue , Masculino , Dados de Sequência Molecular , Gravidez , Ratos , Ratos Wistar/genética , Deleção de Sequência , Fatores SexuaisRESUMO
Expression of the inducible isoform of nitric oxide synthase (iNOS) is stimulated by cytokines in human epithelial cells. This work indicates that incubation of human umbilical cord endothelial cells with combinations of interleukin-1beta, tumor necrosis factor alpha, and interferon-gamma stimulated the synthesis of iNOS mRNA, as detected by reverse transcriptase-polymerase chain reaction. It is important to note that 50, 100, and 200 microM hydrogen peroxide was able to stimulate iNOS directly. Furthermore, 100 microM H2O2 enhanced synthesis of the oxidation products, nitrite (NO2-) and nitrate (NO3-) at 12 and 36 h. iNOS protein, detected by Western blot analysis, as well as L-citrulline levels, were also increased. When endothelial cell monolayers were incubated for 1 h with 100 microM H2O2 and subsequently with cytokines, iNOS mRNA was further augmented. Under the same conditions, we regularly observed an inhibition (25%) of intercellular adhesion molecule-1 (ICAM-1/CD54) expression. The latter was reversed when the NOS inhibitor N(G)-monomethyl-L-arginine was added, as shown by flow cytometry. These data suggest a specific effect of endogenous hydroperoxides on the biosynthesis and processing of the human endothelial iNOS isoform. We propose that H2O2 induces a temporary NO-dependent modulation of adhesion molecule expression to limit the tissue destruction that accompanies the vascular recruitment of leukocytes.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Óxido Nítrico Sintase/metabolismo , Células Cultivadas , Citrulina/metabolismo , Sinergismo Farmacológico , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Interferon gama/farmacologia , Interleucina-1/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolissacarídeos/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Oxirredução/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.
Assuntos
Complexo I de Transporte de Elétrons/genética , Doença de Leigh/etiologia , Doença de Leigh/genética , Mutação/genética , NADH Desidrogenase/genética , Subunidades Proteicas/genética , Criança , Complexo I de Transporte de Elétrons/deficiência , Evolução Fatal , Humanos , Proteínas Ferro-Enxofre/deficiência , Proteínas Ferro-Enxofre/genética , Doença de Leigh/enzimologia , Doença de Leigh/patologia , Masculino , NADH Desidrogenase/deficiência , Subunidades Proteicas/deficiênciaRESUMO
Cytochrome c oxidase (COX) deficiency is one of the major causes of Leigh Syndrome (LS), a fatal encephalopathy of infancy or childhood, characterized by symmetrical lesions in the basal ganglia and brainstem. Mutations in the nuclear genes encoding COX subunits have not been found in patients with LS and COX deficiency, but mutations have been identified in SURF1. SURF1 encodes a factor involved in COX biogenesis. To date, 30 different mutations have been reported in 40 unrelated patients. We aim to provide an overview of all known mutations in SURF1, and to propose a common nomenclature. Twelve of the mutations were insertion/deletion mutations in exons 1, 4, 6, 8, and 9; 10 were missense/nonsense mutations in exons 2, 4, 5, 7, and 8; and eight were detected at splicing sites in introns 3 to 7. The most frequent mutation was 312_321del 311_312insAT which was found in 12 patients out of 40. Twenty mutations have been described only once. We also list all polymorphisms discovered to date.
Assuntos
Deficiência de Citocromo-c Oxidase , Doença de Leigh/genética , Mutação/genética , Proteínas/genética , Terminologia como Assunto , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Éxons/genética , Frequência do Gene , Testes Genéticos , Humanos , Íntrons/genética , Doença de Leigh/diagnóstico , Doença de Leigh/enzimologia , Proteínas de Membrana , Proteínas Mitocondriais , Dados de Sequência Molecular , Polimorfismo Genético/genética , Proteínas/química , Sítios de Splice de RNA/genéticaRESUMO
Type I oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by the reduction or the absence of tyrosinase (TYR) activity in melanocytes of the skin, hair and eyes. Here we report an analysis of 45 patients with OCA. We found five novel mutations in the tyrosinase gene involved in the pathogenesis of oculocutaneous albinism type IA or type IB (OCA-1A/B) in five unrelated patients. Three mutations are missense mutations (G109R, P205T and H256Y) and two are nucleotide deletions (336-337delCA and 678-680delAGG). One patient is homozygous for the previously known V275F mutation but has an extremely mild OCA phenotype and has no eye features typical of OCA. In several patients we discovered only one or even no mutation in the coding sequence of the TYR gene. Thus, this disease may also result from mutations in non coding regions of the gene or in another gene involved in the biosynthesis of melanin. Hum Mutat 17:352, 2001.
Assuntos
Albinismo/enzimologia , Albinismo/genética , Monofenol Mono-Oxigenase/genética , Mutação/genética , Albinismo/classificação , Animais , Análise Mutacional de DNA , Éxons/genética , Feminino , Genes Recessivos/genética , Heterozigoto , Humanos , Masculino , Melaninas/biossíntese , Melaninas/genética , Mutação de Sentido Incorreto/genética , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
ABSTRACT We report five novel VMD2 mutations in Best's macular dystrophy patients (S16F, I73N, R92H, V235L, and N296S). An SSCP analysis of the VMD2 11 exons revealed electrophoretic mobility shifts exclusively in exons 2, 3, 4, 6 and 8. Direct sequencing indicated that these shifts are caused by mono-allelic transition in exons 2, 4, 6, 8 and transversion in exons 3 and 6. Five novel "silent" polymorphisms are also reported: 213T>C, 323C>A, 1514A>G, 1661C>T, and 1712T>C. Hum Mutat 17:235, 2001.
Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Sequência de Bases , Bestrofinas , Canais de Cloreto , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Degeneração Macular/patologia , Masculino , Mutação , Mutação de Sentido Incorreto , LinhagemRESUMO
We used phosphorus NMR spectroscopy to study 16 patients with muscular enzyme deficiencies affecting glycogenolysis and glycolysis. Study of phosphomonoester (Pm) kinetics and intracellular pH during exercise and recovery provided criteria for the distinction of these metabolic myopathies by NMR spectroscopy. The Pm peak was undetectable in patients lacking debrancher enzyme or phosphorylase. By contrast, in phosphofructokinase (PFK) or phosphoglycerate kinase (PGK) deficiency, the Pm peak was larger than that of inorganic phosphate in exercise, whereas it was always smaller in normal subjects. During recovery, the disappearance of Pm was slower in PGK than in PFK deficiency.
Assuntos
Doença de Depósito de Glicogênio Tipo III/diagnóstico , Doença de Depósito de Glicogênio Tipo V/diagnóstico , Doença de Depósito de Glicogênio/diagnóstico , Fosfofrutoquinase-1/deficiência , Fosfoglicerato Quinase/deficiência , Adolescente , Adulto , Diagnóstico Diferencial , Feminino , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Compostos Organofosforados/metabolismo , Fosfatos/metabolismo , Fósforo , Esforço FísicoRESUMO
Two brothers, aged 27 and 20 months, born from consanguineous healthy parents, presented with cardiomyopathy, lactic acidosis and carnitine abnormalities in serum and muscle, without clinical evidence of muscle involvement. The histochemical reaction for cytochrome c oxidase (COX) activity was negative in all muscle fibres, although the holoenzyme and subunits were present at a normal level, as shown by immunocytochemistry. The COX activity was, respectively, 5 and 25% of control values, in muscle biopsies. Partial deficiency of complex IV was confirmed in fresh isolated muscle mitochondria from patient 2 and was associated with a defect of complex I. Patient 1 died at age 3 yr 6 months. Partial improvement of cardiomyopathy in patient 2 was obtained under carnitine therapy, but seizures occurred and CT scan and magnetic resonance imaging (MRI) revealed thalamic hypodensity. Thus, the disorder appears to be progressive despite the clinical stabilization of the cardiomyopathy. This further demonstrates the complexity and clinical heterogeneity of combined respiratory chain complex deficiencies.
Assuntos
Cardiomiopatias/genética , Deficiência de Citocromo-c Oxidase , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Musculares/metabolismo , Músculos/patologia , NAD(P)H Desidrogenase (Quinona)/deficiência , Biópsia , Cardiomiopatias/enzimologia , Cardiomiopatias/patologia , Carnitina/metabolismo , Consanguinidade , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Lactente , Masculino , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/ultraestrutura , Músculos/ultraestrutura , NAD(P)H Desidrogenase (Quinona)/metabolismo , Consumo de Oxigênio , Valores de Referência , Succinato Desidrogenase/análiseRESUMO
This article reports a new MERRF family. The mother, regarded as suffering from Ramsay-Hunt Syndrome, and her three daughters, had the same clinical pattern: myoclonic epilepsy and ataxia. Two daughters were studied on morphological, biochemical and molecular genetic levels. Muscle biopsies showed ragged-red fibres and mitochondrial vasculopathy. Arterioles were strongly SDH-reactive and COX-negative. By electron microscopy, abnormal mitochondria were observed in skeletal muscle fibres, in smooth muscle fibres of intramuscular vessels and in sweat gland epithelium. The study of the respiratory chain showed complex IV and I + IV deficiency, respectively. Mitochondrial tRNA (lys) mutation at position 8344 was pointed out as previously reported in the MERRF syndrome.
Assuntos
Síndrome MERRF/genética , Síndrome MERRF/patologia , Mitocôndrias Musculares/patologia , Músculos/patologia , Mutação Puntual , RNA de Transferência de Lisina/genética , Adolescente , Adulto , Idade de Início , Biópsia , Feminino , Humanos , Masculino , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Mitocôndrias Musculares/ultraestrutura , Músculos/irrigação sanguínea , Pele/patologia , Pele/ultraestruturaRESUMO
NADH laser fluorimetry and mitochondrial oxigraphy were used to study myocardial oxidative energy metabolism during cardiac allograft rejection. Heterotopic cardiac transplantation was performed on Lewis rats; allografts (with Fischer rat donors) were compared with isografts (with Lewis rat donors). In vivo and in vitro assays were performed six days after transplantation. Myocardial NADH fluorescence was recorded in vivo from grafted hearts, at baseline; during brief, complete ischemia; and during reperfusion. Oxygen consumption of mitochondria isolated from both native and grafted hearts was determined. Neither baseline levels nor maximum ischemic levels of NADH fluorescence (F0 = k[NADH]) were found to be significantly different between allografts (0.45 +/- 0.05 to 0.87 +/- 0.10) and isografts (0.45 +/- 0.04 to 1.11 +/- 0.05). During recovery, the rate of fluorescence decrease was significantly lower in allografts than in isografts (0.024 +/- 0.001 vs. 0.038 +/- 0.002 delta F0.s-1, P less than 10(-3], indicating a lower rate of NADH reoxidation. In the presence of malate and glutamate substrates, mitochondrial O2 consumption was significantly lower in allografts than in isografts (30 +/- 9 vs. 100 +/- 15 nanoatoms O2. min-1.mg prot-1, P less than 10(-2]. These results indicate that mitochondrial oxidative metabolism was impaired during the rejection process. Such energy production disturbances may contribute to the dysfunction of rejecting hearts.
Assuntos
Rejeição de Enxerto/fisiologia , Transplante de Coração/efeitos adversos , Mitocôndrias Cardíacas/metabolismo , Animais , Lasers , Masculino , Miocárdio/citologia , Miocárdio/metabolismo , NAD/metabolismo , Oxirredução , Oxigênio/metabolismo , Fosforilação , Ratos , Ratos Endogâmicos Lew , Espectrometria de Fluorescência , Transplante HomólogoRESUMO
Gunn rats are characterized by hereditary hyperbilirubinemia and a decrease, when compared to Wistar rats, in hepatic heme pool size which could result from an alteration of mitochondrial functions. Unconjugated bilirubin present in Gunn rat liver did not modify either the ultrastructural morphology or oxidative metabolism of the mitochondria as compared to those in Wistar rat liver. However, 5-aminolevulinic acid synthase activity is reduced by nearly 40% in Gunn rat liver mitochondria, thus explaining the reduced size of the hepatic heme pool.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/enzimologia , Animais , Heme/biossíntese , Hiperbilirrubinemia/metabolismo , Fígado/ultraestrutura , Masculino , Mitocôndrias Hepáticas/ultraestrutura , Ratos , Ratos Gunn , Ratos WistarRESUMO
UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24 h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitonin-permeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.
Assuntos
Respiração Celular/efeitos da radiação , Mitocôndrias/efeitos da radiação , Trifosfato de Adenosina/metabolismo , Antioxidantes , Linhagem Celular , Respiração Celular/fisiologia , Humanos , Líquido Intracelular/metabolismo , Membranas Intracelulares/fisiologia , Membranas Intracelulares/efeitos da radiação , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Potenciais da Membrana/efeitos da radiação , Mitocôndrias/fisiologia , Superóxido Dismutase/metabolismo , Raios UltravioletaRESUMO
The present study was carried out to determine whether a selective decrease of mitochondrial (mt) DNA-encoded cytochrome c oxidase (CCO) subunits occurs in zidovudine myopathy, as expected with a compound known to induce selective mtDNA depletion. Fourteen HIV-infected patients with zidovudine myopathy were studied. Thirteen had partial CCO deficiency assessed by histochemistry. Western blot analysis of CCO subunits (II/III, IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, and VIIc) was performed on muscle biopsy samples. We evaluated the mtDNA-encoded subunits to nuclear DNA-encoded subunits ratio with the II/III to IV ratio. Patients had either a selective decrease of mtDNA-encoded CCO subunits (3 patients), or an overall decrease affecting both mtDNA-and nuclear DNA-encoded subunits (5 patients), or a normal expression of CCO subunits (6 patients). Positive correlations could not be established between the pattern of expression of CCO subunits and total zidovudine intake, degree of inflammation, and percentages of ragged-red fibers or CCO-deficient fibers. The finding of a decrease of both mtDNA- and nuclear DNA-encoded CCO subunits suggests that a factor additional to zidovudine could be implicated in the pathogenesis of the myopathy, at least in some patients. New insights into the pathogenesis of zidovudine myopathy might come from the use of more sensitive methods, including evaluation of CCO subunits in single fibers.
Assuntos
Núcleo Celular , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Mitocôndrias Musculares/efeitos dos fármacos , Miopatias Mitocondriais/induzido quimicamente , Proteínas Musculares/biossíntese , Zidovudina/efeitos adversos , Adulto , Deficiência de Citocromo-c Oxidase , Complexo IV da Cadeia de Transporte de Elétrons/genética , Indução Enzimática , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/enzimologia , Miopatias Mitocondriais/enzimologia , Miopatias Mitocondriais/genética , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Músculos/patologia , ATPases Translocadoras de Prótons/biossíntese , ATPases Translocadoras de Prótons/genéticaRESUMO
We report histochemical, immunocytochemical, biochemical and molecular studies of skeletal muscle from a 23-year-old man with Kearns-Sayre syndrome. Southern blot analysis revealed a 4.7 kb heteroplasmic deletion of the mitochondrial DNA mapping within genes coding for subunits of complexes I, IV and V of the respiratory chain and for tRNA. Cytochrome c oxidase activity was decreased by 30% in isolated muscle mitochondria, without alteration of the Km. Histochemical and immunocytochemical correlation studies for cytochrome c oxidase revealed a lack of activity in 34% of individual muscle fibers including all the typical ragged-red fibers and a low percentage of immunodeficient fibers.
Assuntos
Deficiência de Citocromo-c Oxidase , DNA Mitocondrial/metabolismo , Síndrome de Kearns-Sayre/metabolismo , Músculos/metabolismo , Oftalmoplegia/metabolismo , Adulto , Humanos , Imuno-Histoquímica , Síndrome de Kearns-Sayre/patologia , Masculino , Músculos/patologiaRESUMO
Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is a neuromuscular disorder characterized by mitochondrial myopathy and progressive myoclonus epilepsy. A heteroplasmic A to G transition mutation in the mitochondrial encoded tRNA(Lys) gene at nucleotide pair 8344 has been suggested to be linked to the MERRF-syndrome. We have investigated biochemically and histochemically muscle biopsies and studied the mitochondrial genomes of hair, blood and muscle tissue of a family including three cases of MERRF-syndrome as well as unaffected relatives within the maternal lineage. Sequence analysis of the mtDNAs, performed after amplification by the polymerase chain reaction (PCR), confirmed the A to G transition mutation in the tRNA(Lys) gene at position 8344. The additional point mutation at nucleotide pair 750 in the 12 S rRNA gene, which was also found by Shoffner et al. (1990), however, was absent in all investigated tissues. Quantitative analysis of the percentage of mutated mtDNA by mispairing PCR (Seibel et al., 1990) revealed variable contents in different tissues and individuals, including unaffected family members. Mitochondrial protein synthesis in cultured fibroblasts from MERRF patients revealed diminished incorporation of 35S-methionine into lysine-containing peptides.
Assuntos
DNA Mitocondrial/genética , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Mitocôndrias Musculares/metabolismo , Mutação , RNA de Transferência de Lisina/genética , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Citrato (si)-Sintase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Epilepsias Mioclônicas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Músculos/patologia , Conformação de Ácido Nucleico , Consumo de Oxigênio , Valores de Referência , Succinato Citocromo c Oxirredutase/metabolismo , SíndromeRESUMO
We report severe coenzyme Q10 deficiency of muscle in a 4-year-old boy presenting with progressive muscle weakness, seizures, cerebellar syndrome, and a raised cerebro-spinal fluid lactate concentration. State-3 respiratory rates of muscle mitochondria with glutamate, pyruvate, palmitoylcarnitine, and succinate as respiratory substrates were markedly reduced, whereas ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine were oxidized normally. The activities of complexes I, II, III and IV of the electron transport chain were normal, but the activities of complexes I+III and II+III, both systems requiring coenzyme Q10 as an electron carrier, were dramatically decreased. These results suggested a defect in the mitochondrial coenzyme Q10 content. This was confirmed by the direct assessment of coenzyme Q10 level by high-performance liquid chromatography in patient's muscle homogenate and isolated mitochondria, revealing levels of 16% and 6% of the control values, respectively. We did not find any impairment of the respiratory chain either in a lymphoblastoid cell line or in skin cultured fibroblasts from the patient, suggesting that the coenzyme Q10 depletion was tissue-specific. This is a new case of a muscle deficiency of mitochondrial coenzyme Q in a patient suffering from an encephalomyopathy.