RESUMEN
We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.
Asunto(s)
Antígenos CD4/metabolismo , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Virus de la Leucemia Murina de Moloney , Receptores CXCR4/metabolismo , Animales , Antígenos CD4/genética , Marcación de Gen , Vectores Genéticos/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Ratones , Virus de la Leucemia Murina de Moloney/genética , Receptores CXCR4/genéticaRESUMEN
Programmed cell death regulates a number of biological phenomena, and the apoptotic signal must itself be tightly controlled to avoid inappropriate cell death. We established a genetic screen to search for molecules that inhibit the apoptotic signal from the Fas receptor. Here we report the isolation of a gene, LFG, that protects cells uniquely from Fas but not from the mechanistically related tumor necrosis factor alpha death signal. LFG is widely distributed, but remarkably is highly expressed in the hippocampus. LFG can bind to the Fas receptor, but does not regulate Fas expression or interfere with binding of an agonist antibody. Furthermore LFG does not inhibit binding of FADD to Fas.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/genética , Receptor fas/fisiología , Secuencia de Aminoácidos , Antígenos CD/fisiología , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , Cartilla de ADN , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Proteínas de la Membrana , Datos de Secuencia Molecular , Pruebas de Precipitina , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Homología de Secuencia de AminoácidoRESUMEN
We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli beta-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.
Asunto(s)
Marcación de Gen , Vectores Genéticos , Virus de la Leucemia Murina de Moloney/genética , Animales , Secuencia de Bases , Quimera/genética , Cartilla de ADN/genética , ADN Complementario/genética , Escherichia coli/enzimología , Escherichia coli/genética , Terapia Genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Receptores de LDL/genética , beta-Galactosidasa/genéticaRESUMEN
Ligation of DNA after replication and repair is a prerequisite for the preservation of DNA and chromosome structure and function. Biochemical studies with Bloom's syndrome cells have revealed an abnormal DNA ligase I activity. However, genetic analysis has not revealed any differences in transcript levels or in the cDNA sequences of DNA ligase I between Bloom's syndrome and normal cells. Another human cell line, 46BR, derived from an immunodeficient patient, also has an abnormal DNA ligase I. This cell line has recently been demonstrated to harbour two different missense mutations, one at each allele of DNA ligase I. These mutations resulted in a decreased ability of partially purified cell extracts to form an enzyme-adenylate reaction intermediate. We show that 46BR hypersensitivity to an alkylating agent, ethyl methanesulphonate, and to the polyADP-ribose polymerase inhibitor 3-aminobenzamide, is rescued by transfection of wild-type DNA ligase I sequences. This provides additional genetic evidence that the defect in 46BR is at the DNA ligase I locus.
Asunto(s)
Síndrome de Bloom/genética , ADN Ligasas/deficiencia , ADN Ligasas/genética , Reparación del ADN , Mutación , Benzamidas/toxicidad , Síndrome de Bloom/enzimología , Línea Celular Transformada , ADN Ligasa (ATP) , Metanosulfonato de Etilo/toxicidad , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Immunoblotting , Kanamicina Quinasa , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Fenotipo , Fosfotransferasas/genética , Plásmidos , TransfecciónRESUMEN
Very high plasma concentrations of factor-VIII-related antigen (RAG) (VIII-RAG) were found in renal allograft recipients during periods of nephrotoxicity induced by cyclosporine. In eight recipients, who were investigated at weekly intervals, levels of factor-VIII-RAG fell toward normal as the dose of cyclosporine was reduced. Plasma levels of C-reactive protein, an acute phase reactant protein, were never raised in these recipients. These findings are further evidence that toxic doses of cyclosporine are associated with vascular injury.