RESUMO
BACKGROUND: Insights into human brain diseases may emerge from tissue obtained after operations on patients. However techniques requiring transduction of transgenes carried by viral vectors cannot be applied to acute human tissue. NEW METHOD: We show that organotypic culture techniques can be used to maintain tissue from patients with three different neurological syndromes for several weeks in vitro. Optimized viral vector techniques and promoters for transgene expression are described. RESULTS: Region-specific differences in neuronal form, firing pattern and organization as well as pathological activities were maintained over 40-50â¯days in culture. Both adeno-associated virus and lentivirus based vectors were persistently expressed from â¼10â¯days after application, providing 30-40â¯days to exploit genetically expressed constructs. Different promoters, including hSyn, e/hSyn, CMV and CaMKII, provided cell-type specific transgene expression. The Ca probe GCaMP let us explore epileptogenic synchrony and a FRET-based probe was used to follow activity of the kinase mTORC1. COMPARISON WITH EXISTING METHODS: The use of a defined culture medium, with low concentrations of amino acids and no growth factors, permitted organotypic culture of tissue from humans aged 3-62 years. Epileptic activity was maintained and excitability changed relatively little until â¼6 weeks in culture. CONCLUSIONS: Characteristic morphology and region-specific neuronal activities are maintained in organotypic culture of tissue from patients diagnosed with mesial temporal lobe epilepsy, cortical dysplasia and cortical glioblastoma. Viral vector techniques permit expression of probes for long-term measurements of multi-cellular activity and intra-cellular signaling.
Assuntos
Encefalopatias/metabolismo , Encefalopatias/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Imagem Óptica , Técnicas de Cultura de Tecidos/métodos , Adolescente , Adulto , Encefalopatias/cirurgia , Criança , Pré-Escolar , Meios de Cultura , Epilepsia/metabolismo , Epilepsia/patologia , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Imagem Óptica/métodos , Técnicas de Cultura de Órgãos/métodos , Imagens com Corantes Sensíveis à Voltagem/métodos , Adulto JovemRESUMO
To direct the synthesis and secretion of recombinant human interleukin-11 (rhIL-11) in chicken HD11 cells, a plasmid targeting the c-lysozyme gene has been constructed which contains the mature cytokine cDNA in frame with the lysozyme leader sequence. The upregulation of rhIL-11 mediated by LPS proves the knock-in of hIL-11 cDNA in the lysozyme gene. The bioactivity of the expressed protein is demonstrated and quantified with the hIL-11 dependent 7TD1 and B9 cell lines. The electrophoretic mobility, receptor binding properties and growth promoting effect of the chicken-derived cytokine are identical to those of a rhIL-11 expressed in Escherichia coli. These results describe the secretion of a biologically active rhIL-11 expressed by an avian cellular machinery.
Assuntos
Técnicas Genéticas , Interleucina-11/biossíntese , Interleucina-11/química , Proteínas Recombinantes/química , Regiões 5' não Traduzidas/química , Animais , Ligação Competitiva , Western Blotting , Linhagem Celular , Proliferação de Células , Galinhas , Citocinas/metabolismo , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Genoma , Humanos , Interleucina-11/metabolismo , Cinética , Lipopolissacarídeos/metabolismo , Modelos Genéticos , Muramidase/química , Muramidase/genética , Plasmídeos/metabolismo , Recombinação Genética , Transfecção , Regulação para CimaRESUMO
Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.