RESUMO
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.
Assuntos
Antígenos CD4/metabolismo , Vetores Genéticos , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Vírus da Leucemia Murina de Moloney , Receptores CXCR4/metabolismo , Animais , Antígenos CD4/genética , Marcação de Genes , Vetores Genéticos/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , HIV-1/fisiologia , Células HeLa , Humanos , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Receptores CXCR4/genéticaRESUMO
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.
Assuntos
Apoptose/genética , Proteínas de Transporte/genética , Receptor fas/fisiologia , Sequência de Aminoácidos , Antígenos CD/fisiologia , Proteínas Reguladoras de Apoptose , Sequência de Bases , Western Blotting , Linhagem Celular , Clonagem Molecular , Primers do DNA , Regulação para Baixo , Imunofluorescência , Células HeLa , Humanos , Proteínas de Membrana , Dados de Sequência Molecular , Testes de Precipitina , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Marcação de Genes , Vetores Genéticos , Vírus da Leucemia Murina de Moloney/genética , Animais , Sequência de Bases , Quimera/genética , Primers do DNA/genética , DNA Complementar/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Terapia Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Receptores de LDL/genética , beta-Galactosidase/genéticaRESUMO
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.
Assuntos
Síndrome de Bloom/genética , DNA Ligases/deficiência , DNA Ligases/genética , Reparo do DNA , Mutação , Benzamidas/toxicidade , Síndrome de Bloom/enzimologia , Linhagem Celular Transformada , DNA Ligase Dependente de ATP , Metanossulfonato de Etila/toxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Immunoblotting , Canamicina Quinase , Testes de Mutagenicidade , Mutagênicos/toxicidade , Fenótipo , Fosfotransferases/genética , Plasmídeos , TransfecçãoRESUMO
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.
