RESUMO
Remarkable progress has been made recently in identifying a new gene family related to the capsaicin (vanilloid) receptor, VR1. Using a combination of in silico analysis of expressed sequence tag (EST) databases and conventional molecular cloning, we have isolated a novel vanilloid-like receptor, which we call VRL-2, from human kidney. The translated gene shares 46% and 43% identity with VR1 and VRL-1, respectively, and maps to chromosome 12q23-24.1, a locus associated with bipolar affective disorder. VRL-2 mRNA was most strongly expressed in the trachea, kidney, and salivary gland. An affinity-purified antibody against a peptide incorporating the COOH terminal of the receptor localized VRL-2 immunolabel in the distal tubules of the kidney, the epithelial linings of both trachea and lung airways, serous cells of submucosal glands, and mononuclear cells. Unlike VR1 and VRL-1, VRL-2 was not detected in cell bodies of dorsal root ganglia (DRG) or sensory nerve fibers. However, VRL-2 was found on sympathetic and parasympathetic nerve fibers, such as those innervating the arrector pili smooth muscle in skin, sweat glands, intestine, and blood vessels. At least four vanilloid receptor-like genes exist, the newest member, VRL-2 is found in airway and kidney epithelia and in the autonomic nervous system.
Assuntos
Proteínas de Transporte de Cátions , Canais Iônicos , Receptores de Droga/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , RNA/genética , RNA/metabolismo , Mapeamento de Híbridos Radioativos , Ratos , Receptores de Droga/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPV , Distribuição TecidualRESUMO
The gene encoding human eukaryotic initiation factor 4A (EIF4A1) is located on chromosome 17p13, 667 bp upstream from the gene encoding the macrophage endosomal protein CD68. The EIF4AI gene contains 10 intervening sequences with the 1397-bp first intron containing a CpG-rich methylation-free island. Sequences capable of enhancing gene expression reside between positions -69 and -371 and positions -504 and -1100 of the EIF4AI 5' flanking sequence and within introns 1, 2, 3, 7, and 9. In macrophage cell lines, EIF4A1 expression vectors give sustained high-level reporter gene expression to levels 10 times higher than that obtained using the human cytomegalovirus immediate-early gene promoter/enhancer. Sequences of the human EIF4AI gene may find application in the development of new vectors for gene therapy and genetic vaccination.
Assuntos
Genes Reporter , Fatores de Iniciação de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Regiões 5' não Traduzidas/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Cromossomos Humanos Par 17/genética , Ilhas de CpG , Fator de Iniciação 4A em Eucariotos , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
The carbocyclic nucleoside 1592U89 is a selective inhibitor of the human immunodeficiency virus (HIV), targeting the reverse transcriptase (RT). In vitro selection studies were undertaken to generate resistant variants with both HIV type 1 (HIV-1) wild-type strain HIV-1(HXB2) and 3'-azido-3'-deoxythymidine (AZT)-resistant strain HIV-1(RTMC). At least two or three mutations in RT were required to produce a 10-fold reduction in susceptibility. The first RT mutation selected was at codon 184, methionine (M) to valine (V), for HIV-1(HXB2) and HIV-1(RTMC), conferring two- and fivefold resistance, respectively. Two additional mutations were selected with HIV-1(HXB2), either leucine (L) 74 to V and lysine (K) 65 to arginine (R) (first-passage series) or L74 to V and tyrosine (Y) 115 to phenylalanine (F) (second-passage series). Cloned variants, obtained from the 1592U89 selection, were either double RT mutants 65R/184V and 74V/184V or triple RT mutant 74V/115Y/184V. Molecular clones were constructed with single, double, and triple combinations of these mutations for resistance analysis with different RT inhibitors. Each individual mutation conferred only low-level resistance (two- to fourfold) to 1592U89 in the HXB2 background. Double mutants containing the 184V mutation and triple mutants showed slightly greater levels of resistance to 1592U89 (7- to 11-fold). Some of the 1592U89-resistant variants were cross-resistant with 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and (-)-2'-deoxy-3'-thiacytidine, but none were resistant to 2',3'-didehydro-3'-deoxythymidine or AZT.
