RESUMO
BACKGROUND: The Rett Syndrome (RTT) brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected. RESULTS: Using microarrays and quantitative PCR, the mRNA expression profiles of these two neuroanatomical regions were compared in postmortem brain tissue from RTT patients and normal controls. A subset of genes was differentially expressed in the frontal cortex of RTT brains, some of which are known to be associated with neurological disorders (clusterin and cytochrome c oxidase subunit 1) or are involved in synaptic vesicle cycling (dynamin 1). RNAi-mediated knockdown of MeCP2 in vitro, followed by further expression analysis demonstrated that the same direction of abnormal expression was recapitulated with MeCP2 knockdown, which for cytochrome c oxidase subunit 1 was associated with a functional respiratory chain defect. Chromatin immunoprecipitation (ChIP) analysis showed that MeCP2 associated with the promoter regions of some of these genes suggesting that loss of MeCP2 function may be responsible for their overexpression. CONCLUSIONS: This study has shed more light on the subset of aberrantly expressed genes that result from MECP2 mutations. The mitochondrion has long been implicated in the pathogenesis of RTT, however it has not been at the forefront of RTT research interest since the discovery of MECP2 mutations. The functional consequence of the underexpression of cytochrome c oxidase subunit 1 indicates that this is an area that should be revisited.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Doenças Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/metabolismo , Síndrome de Rett/genética , Adolescente , Adulto , Criança , Pré-Escolar , Clusterina/genética , Dinamina I/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Metabolismo Energético/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia , Adulto JovemRESUMO
The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.
Assuntos
Antiporters/química , Antiporters/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Antiporters/genética , Proteínas de Bactérias/genética , Cisteína/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fluoresceínas/metabolismo , Glicina/genética , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Plasmídeos , Estrutura Secundária de Proteína/genética , Solventes , Staphylococcus aureus/química , Staphylococcus aureus/genética , Resistência a Tetraciclina/genéticaRESUMO
Skewed X chromosome inactivation (XCI) has been implicated in modulating the severity of Rett syndrome (RTT), although studies by different groups have yielded conflicting results. In this study we have characterised the XCI pattern in various neuroanatomical regions of nine RTT brains and non-neural tissue in two of these patients to determine whether or not variable XCI patterns occur in different brain regions or somatic tissues of the same patient. The mean XCI patterns for frontal and occipital cortex were compared between RTT and control subjects, and showed no significant differences when comparing RTT frontal to control frontal cortex or RTT occipital to control occipital cortex. However, one RTT subject displayed variability across the different neuroanatomical regions of the brain and skewing in some non-neural tissues. This observation adds another dimension to the epigenetic factors that may contribute to the phenotype in RTT. It also mandates that caution should be exercised in factoring XCI, including assumptions based on the blood XCI pattern, into the development of phenotype-genotype correlations.
