RESUMO
Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.
Assuntos
Pichia/genética , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mutants resistant to 6-thioguanine were selected from CHO cells which were either temperature sensitive or proline requiring. These mutants were stable and had low levels of hypoxanthine guanine phosphoribosyl transferase (HGPRT). Hybrids were selected which were heteroallelic at the hgprt locus and complementation between the mutants used was not observed. Interallelic recombination at this locus would generate hgprt+ cells which could be selected in Littlefield's HAT medium. Selection experiments with hybrids containing three different pairs of mutants yielded no recombinants among populations of 4 x 106 - 2 x 107 cells. After treatment with the recombinagen mitomycin C, 3 putative recombinants were detected amongst 1.4 x 107 surviving cells from one hybrid. One of these strains was examined and shown to have a normal level of HGPRT and its heterozygosity at this locus was demonstrated by the segregation of colonies resistant to 6-thioguanine. It cannot be excluded that the rare hgprt+ colonies seen arose by mutation rather than by recombination. Mitotic allelic recombination therefore appears to be a much less frequent event in CHO cells than it is in lower eukaryotes. It is possible that mitotic recombination is effectively suppressed in mammalian cells to prevent the expression of deleterious recessive mutants.
Assuntos
Alelos , Células Híbridas , Mitose , Recombinação Genética , Troca Genética , Resistência a Medicamentos , Hipoxantina Fosforribosiltransferase/metabolismo , Mitomicinas/farmacologia , Prolina/metabolismo , Tioguanina/farmacologiaRESUMO
The commitment theory may explain both the finite lifespan of diploid fibroblasts and the apparent immortality of transformed lines. Potentially immortal cells are assumed on division to generate with some fixed probability cells committed to senesce after a specific number of divisions. During the period between commitment and senescence, cells are assumed to maintain normal growth so that the uncommitted cells are diluted by committed ones and may ultimately be lost in subculturing. A number of predictions of this model are described and experiments strongly supporting the theory are reported. We conclude that the limited growth of diploid fibroblasts is, in effect, an artifact of normal culturing procedures.
Assuntos
Sobrevivência Celular , Modelos Biológicos , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Células Clonais/fisiologia , Fibroblastos , Humanos , Processos EstocásticosRESUMO
Immunological and enzymatic assessments of lactate dehydrogenase in human lung fibroblasts strongly suggest that altered proteins accumulate in ageing cells. This supports but does not prove the error catastrophe theory of all death.
Assuntos
Senescência Celular/fisiologia , Diploide , Fibroblastos/citologia , Fibroblastos/enzimologia , L-Lactato Desidrogenase/metabolismo , Modelos Biológicos , Biossíntese de Proteínas , Aminoácidos/metabolismo , Animais , Contagem de Células , Morte Celular , Desoxirribonucleases/metabolismo , Etionina/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fluoruracila/farmacologia , Humanos , L-Lactato Desidrogenase/genética , Pulmão , Metionina/metabolismo , Ribonucleases/metabolismoRESUMO
Diploid human fibroblasts accumulate heat labile enzymes during the final stages of their life-span in culture. The RNA base analogue 5-FU induces premature senescence, which is preceded by the appearance of altered enzyme. These observations support Orgel's "error catastrophe" theory of ageing.
