RESUMO
A screening procedure was established to identify Corynebacterium glutamicum transposon mutants with an altered L-glutamate excretion behaviour. By this microtiter plate-based approach seven non- or less excreting C. glutamicum strains and two hyper-excreters were found. The subsequently carried out molecular analysis of a hyper-producing clone led to the identification of the gltS gene, which codes for the sodium-coupled secondary L-glutamate uptake system in C. glutamicum. Characterization of a gltS deletion strain revealed that this transporter has a weak but significant impact on L-glutamate production induced by biotin limitation in the wild type. Obviously, GltS leads to the re-uptake of excreted L-glutamate causing a futile cycle. In accord with this hypothesis, the overexpression of gltS decreased L-glutamate production.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Proteínas de Bactérias/genética , Corynebacterium/genética , Genes Bacterianos , Ácido Glutâmico/metabolismo , Simportadores/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Bactérias/metabolismo , Biotina/metabolismo , Corynebacterium/metabolismo , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Marcação de Genes , Mutagênese Insercional , Proteínas Recombinantes de Fusão/metabolismo , Sódio/metabolismo , Simportadores/metabolismoRESUMO
To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.