RESUMO
Two nuclear genes, ACTN3, encoding for the α-actinin skeletal muscle isoform 3, and ACE encoding the angiotensin-converting enzyme, have both been associated with quantitative physical performance traits in the general population. The purpose of our study was to assess the association between the two nuclear gene variants, R577X (rs1815739) in ACTN3 and I/D (rs4340) in ACE, with elite athletes performance and the effect of training on the mitochondrial DNA (mtDNA) content in peripheral blood. We evaluated the genotypes and frequencies of ACTN3 R577X and ACE I/D polymorphisms between soccer players (n = 43) and healthy non-athletic controls (n = 128). Total DNA was extracted from peripheral blood samples using the standard procedure. The genotypes were assessed by PCR-RFLP analysis and mtDNA cellular content by RT-PCR. The soccer players showed a tendency to a prevalence of ACTN3RR and ACEDD genotypes both independently and in co-occurrence. The effect of physical training on the mitochondrial DNA content in the athletic population was reflected strikingly in its increase in peripheral blood. Based on our results, we suggest that the analysis of ACTN3 and ACE genotypes could predict talent in the soccer field and that knowledge of the genetic variants could determine types and training times for soccer players. In addition, the novelty of this work, never before described in the sports literature, is that the increase of mitochondrial content can be correlated with the training load, suggesting that the mtDNA copy number may be considered a viable bioenergetics biomarker.
Assuntos
Actinina/genética , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Peptidil Dipeptidase A/genética , Resistência Física/genética , Polimorfismo de Nucleotídeo Único , Futebol/fisiologia , Adulto , Atletas , Metabolismo Energético/genética , Expressão Gênica , Genoma Mitocondrial , Genótipo , Humanos , MasculinoRESUMO
Large-scale deletions of mitochondrial DNA (mtDNA) are associated with a subgroup of mitochondrial encephalomyopathies. We studied seven patients with Kearns-Sayre syndrome or isolated ocular myopathy who harboured a sub-population of partially-deleted mitochondrial genomes in skeletal muscle. Variable cytochrome c oxidase (COX) deficiencies and reduction of mitochondrially-encoded polypeptides were found in affected muscle fibres, but while many COX-deficient fibres had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. Our results suggest that a specific ratio between mutant and wild-type mitochondrial genomes is the most important determinant of a focal respiratory chain deficiency, even though absolute copy numbers may vary widely.
Assuntos
DNA Mitocondrial/genética , Síndrome de Kearns-Sayre/genética , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologia , Músculos Oculomotores/patologia , Deleção de Sequência , Southern Blotting , Deficiência de Citocromo-c Oxidase , Sondas de DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Síndrome de Kearns-Sayre/enzimologia , Síndrome de Kearns-Sayre/patologia , Síndrome MELAS/genética , Síndrome MERRF/genética , Miopatias Mitocondriais/enzimologia , Músculos Oculomotores/enzimologia , Reação em Cadeia da Polimerase/métodos , RNA/análise , RNA/genética , RNA Mitocondrial , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismoRESUMO
Molecular genetics, biochemistry, immunology and morphology, are being applied in a coordinated fashion to unveil the molecular basis of the mitochondrial encephalomyopathies. Mutations of mitochondrial DNA (mtDNA) have been found in well characterized clinical groups of these disorders. New and old morphologic methods have been applied to investigate muscle biopsies from patients with mtDNA mutations. Important observations have been made on the cellular localization of normal and mutated mtDNA and on the expression of mtDNA-encoded polypeptides. These observations have provided insight into the pathogenesis of respiratory chain enzyme deficiency at the level of individual muscle fibers. Application of immunocytochemical and in situ hybridization techniques at the electron microscopic level will extend these studies to the level of individual mitochondria.
Assuntos
DNA Mitocondrial/genética , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Corantes Fluorescentes , Humanos , Hibridização In Situ , Encefalomiopatias Mitocondriais/enzimologia , Mutação , Succinato Desidrogenase/análise , Succinato Desidrogenase/biossínteseRESUMO
Alterations of mitochondrial (mt) nucleic acid metabolism in methylmalonic aciduria (MMA) were studied in two cell lines from skin fibroblasts of patients with mitochondrial (GM00595) or cytosolic (GM10011) defects in the biosynthesis pathways of cobalamin coenzymes. The mtDNA level increased two-fold in GM00595 cells, which carry a mt defect in the adenosylcobalamin synthesis, whereas no appreciable change was found in GM10011 cells. The content of the two rRNAs 16S and 12S mtRNAs, normalized for the mtDNA copy number, decreased by 70% and 50% in GM00595 and GM10011, respectively. The normalized content of ND1, ND2 and CO I mRNAs decreased in GM00595, but was unchanged in GM10011. Respiratory chain complex activities measured in these two cell lines were not different from control activities. These data suggest that the maintenance of the mt function is due to doubling of mtDNA and that this compensatory response takes place only in those cells in which the greater reduction of the level of rRNA might have brought the content of these transcripts below the threshold value for optimal expression of the mt genome.
Assuntos
Cobamidas/biossíntese , DNA Mitocondrial/metabolismo , Fibroblastos/metabolismo , RNA/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Linhagem Celular , Respiração Celular/genética , Respiração Celular/fisiologia , DNA Mitocondrial/genética , Transporte de Elétrons/genética , Transporte de Elétrons/fisiologia , Fibroblastos/química , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Ácido Metilmalônico/urina , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , RNA/genética , RNA Mitocondrial , Transcrição Gênica/genéticaRESUMO
The effect of acetyl-L-carnitine on the quantity of the messenger RNA for the subunit I of cytochrome oxidase in the liver mitochondria of hypothyroid rat was measured by Northern blot and solution hybridization. Three hours after pre-treatment of hypothyroid rat with acetyl-L-carnitine, the level of the transcript increased strongly. This effect was also obtained when acetyl-L-carnitine was administered to T3 pre-treated hypothyroid rats. These results add further evidence to the suggestion that acetyl-L-carnitine is able to stimulate mitochondrial transcription under altered metabolic conditions.
Assuntos
Acetilcarnitina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Hipotireoidismo/genética , Mitocôndrias Hepáticas/fisiologia , Animais , DNA Mitocondrial/metabolismo , Expressão Gênica , Hipotireoidismo/enzimologia , Masculino , Hibridização de Ácido Nucleico , Ratos , Ratos Endogâmicos , Tri-Iodotironina/farmacologiaRESUMO
A cAMP-dependent protein kinase (PKA) is localized in mammalian mitochondria with the catalytic site at the matrix side of the membrane where it phosphorylates a number of proteins. One of these is the 18 kDa(IP) subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene. Mitochondria have a Ca(2+)-inhibited phosphatase, which dephosphorylates the 18 kDa phosphoprotein of complex I. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18 kDa protein is associated with stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome.
Assuntos
AMP Cíclico/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Sequência de Bases , Complexo I de Transporte de Elétrons , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The authors report on a 34-year-old woman who had developed severe weakness and reduction in grip strength in both upper and lower limbs. Laboratory blood tests revealed increased levels of muscle enzyme. The presence of progressive bilateral ptosis and external ophthalmoplegia raised the suspicion of a mitochondrial disease, subsequently confirmed by deltoid biopsy and genetic analysis of mitochondrial DNA that showed a deletion indicative of Kearns-Sayre syndrome. In this report we emphasise the need for a differential diagnosis between myositis and other myopathies, particularly the mitochondrial ones.
Assuntos
Síndrome de Kearns-Sayre/diagnóstico , Polimiosite/diagnóstico , Adulto , Biópsia por Agulha , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Síndrome de Kearns-Sayre/patologia , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/patologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Polimiosite/patologia , Índice de Gravidade de DoençaRESUMO
The effect of acetyl-L-carnitine on succinate oxidation, adenine nucleotide pool and lipid composition of synaptic and 'free', non-synaptic, mitochondria in cerebral hemispheres of senescent rats has been studied. Fisher rats (24- or 28-month-old) were treated with acetyl-L-carnitine (300 mg/kg body wt., intraperitoneally (i.p.)) 3 h before being killed. Oxygen consumption was measured using succinate as a substrate; adenine nucleotides and lipids were analyzed by high performance liquid chromatography (HPLC). Acetyl-L-carnitine reverses, in synaptic mitochondria, the age-related decrease in the respiratory control ratio due to a higher state 4 respiration rate. Administration of acetyl-L-carnitine to senescent rats does not affect the total adenine nucleotide pool of synaptic and non-synaptic mitochondria which was unchanged with age. Finally, pretreatment of senescent rats with acetyl-L-carnitine brings the cholesterol and phospholipid contents of synaptic mitochondria, reduced in senescent rats, to the adult level; pretreatment of adult rats has no such effect. Altogether these results suggest that acetyl-L-carnitine is able to reverse age-related deficits of brain mitochondria.
Assuntos
Acetilcarnitina/farmacologia , Envelhecimento/genética , DNA Mitocondrial/genética , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , DNA Mitocondrial/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Proteínas do Tecido Nervoso/biossíntese , RNA/biossíntese , RNA Mensageiro/metabolismo , RNA Mitocondrial , Ratos , Ratos Endogâmicos F344 , Sinaptossomos/metabolismo , Fatores de TempoRESUMO
In addition to sporadic or maternally-inherited mutations of the mitochondrial genome, abnormalities of mtDNA can be transmitted as mendelian traits. The latter are believed to be caused by mutations in still unknown nuclear genes, which deleteriously interact with the mitochondrial genome. Two groups of mtDNA-related mendelian disorders are known: those associated with mtDNA large-scale rearrangements and those characterized by severe reduction of the mtDNA copy number. The most frequent presentation of the first group of disorders is an adult-onset encephalomyopathy, defined clinically by the syndrome of progressive external ophthalmoplegia "plus", genetically by autosomal dominant transmission of the trait, and molecularly by the presence of multiple deletions of mtDNA. The second group of disorders comprises early-onset, organ-specific syndromes, associated with mtDNA depletion, that are presumably transmitted as autosomal recessive traits. Linkage analysis and search for candidate genes are two complementary strategies to clarify the molecular basis of these disorders of the nuclear-mitochondrial intergenomic signalling.
Assuntos
Núcleo Celular/genética , Doenças Genéticas Inatas/genética , Mitocôndrias/genética , Núcleo Celular/metabolismo , DNA Mitocondrial , Deleção de Genes , Humanos , Mitocôndrias/metabolismo , Encefalomiopatias Mitocondriais/genética , Linhagem , Transdução de Sinais , SíndromeRESUMO
A quantitative analysis of the frequency of the supercoiled mitochondrial DNA molecules containing the D-loop in rat heart and cerebral hemispheres, at different ages, is presented. Both tissues of aged animals exhibit a remarkable reduction in the content of super-coiled D-loop containing molecules compared to the adults. This alteration could be responsible for the age-dependent reduction of mitochondrial DNA transcription previously observed in rat brain and heart.
Assuntos
Envelhecimento/metabolismo , Química Encefálica/fisiologia , DNA Mitocondrial/metabolismo , DNA Super-Helicoidal/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias/metabolismo , Animais , Southern Blotting , Eletroforese em Gel de Poliacrilamida , Masculino , Conformação de Ácido Nucleico , RatosRESUMO
A specific mitochondrial DNA mutation at position 5460 in the ND2 gene of the human mitochondrial genome was recently reported to exist in 10 of 19 patients with Alzheimer's disease, implying an association between this mtDNA mutation and the occurrence of the disease. We have analyzed tissues from 15 patients with Alzheimer's disease for the presence of the ND2 mutation, and have not been able to confirm these findings. We believe that this mutation is not specifically associated with Alzheimer's disease, but rather, is a neutral polymorphism present in the population.
Assuntos
Doença de Alzheimer/genética , DNA Mitocondrial/genética , Genoma Humano , Mutação , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Encéfalo/fisiopatologia , DNA/genética , DNA/isolamento & purificação , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Mapeamento por RestriçãoRESUMO
A system for studying RNA synthesis in isolated mitochondria from rat brain was set up to investigate the mechanisms responsible for the age-dependent reduction of mtRNA content. In the presence of an appropriate incubation buffer both synaptic and non-synaptic mitochondria from cerebral hemispheres were able to synthesize and process mtRNA in a way quantitatively and qualitatively similar to the in vivo transcription. The comparison of the electrophoretic pattern of mtRNAs synthesized by adult and senescent rat showed, in the senescent rat, a 50% reduction in the mtRNA synthesis rate relative to the adult value. This indicates that the age-dependent decrease of the mtRNA content is linked to a lower efficiency of the mt transcription.
Assuntos
Envelhecimento/genética , Encéfalo/metabolismo , RNA/biossíntese , Animais , Densitometria , Masculino , RNA Mitocondrial , Ratos , Ratos Endogâmicos , Transcrição GênicaRESUMO
Isolated rat liver mitochondria in the presence of an appropriate incubation buffer are able to support DNA transcription and RNA processing in a way qualitatively and quantitatively similar to that used by intact cells. This system is also able to synthesize an RNA species of 155-175 nucleotides which probably corresponds to the 7S RNA, a type of RNA found so far only in growing cells. The role of this RNA in the mitochondrial replication and transcription processes, in relation to the cell metabolism, is discussed.
Assuntos
Mitocôndrias Hepáticas/metabolismo , RNA Mensageiro/biossíntese , Animais , Northern Blotting , Eletroforese em Gel de Ágar , Endonucleases , Humanos , Técnicas In Vitro , RNA Nuclear Pequeno/genética , Ratos , Ratos Endogâmicos , Mapeamento por Restrição , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.
Assuntos
Envelhecimento/metabolismo , Química Encefálica , Encéfalo/ultraestrutura , Lipídeos/análise , Mitocôndrias/química , Sinapses/ultraestrutura , Animais , Colesterol/análise , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Masculino , Mitocôndrias/metabolismo , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
A quantitative study on the effect of senescence on mitochondrial DNA expression has been carried out by measuring the levels of the 12S rRNA and of the mRNA for the subunit I of cytochrome oxidase in several tissues of adult and senescent rats. The concentration of both RNA species/mitochondrial DNA molecule is significantly reduced in senescent brain and heart, as opposed to the respective adult tissues. No appreciable variation occurs in the liver. A 1-h pretreatment with acetyl-L-carnitine brings back the level of senescent brain and heart transcripts to that of adult tissues. The same treatment of adult rats does not cause significant changes in mitochondrial RNA content. These results suggest that the age-dependent impairment of both heavy-strand mitochondrial DNA transcription units is related to altered environmental conditions which acetyl-L-carnitine, a substance which acts by stimulating, directly or indirectly, the energy metabolism, is able to remove.
Assuntos
Acetilcarnitina/farmacologia , Envelhecimento/genética , Carnitina/análogos & derivados , DNA/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Northern Blotting , Encéfalo/efeitos dos fármacos , DNA/isolamento & purificação , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Hibridização de Ácido Nucleico , Plasmídeos , RNA Mensageiro/isolamento & purificação , RNA Ribossômico/genética , RNA Ribossômico/isolamento & purificação , RNA de Transferência/genética , RNA de Transferência/isolamento & purificação , Ratos , Ratos Endogâmicos F344RESUMO
Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.
Assuntos
Códon sem Sentido , Doença de Leigh/genética , NADH NADPH Oxirredutases/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , DNA Complementar , Complexo I de Transporte de Elétrons , Eletroforese em Gel Bidimensional , Feminino , Humanos , Recém-Nascido , Dados de Sequência Molecular , NADH Desidrogenase , NADH NADPH Oxirredutases/químicaRESUMO
We have successfully applied a strategy based on the "cyberscreening" of the expressed sequence tags database using yeast protein sequences as "probes" to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes. In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-c oxidase. Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides. Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains. Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by an in vitro import and protease-protection assay. These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues. Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease. However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes.