RESUMEN
A comprehensive study of changes in messenger RNA (mRNA) levels in human neutrophils following exposure to bacteria is described. Within 2 hours there are dramatic changes in the levels of several hundred mRNAs including those for a variety of cytokines, receptors, apoptosis-regulating products, and membrane trafficking regulators. In addition, there are a large number of up-regulated mRNAs that appear to represent a common core of activation response genes that have been identified as early-response products to a variety of stimuli in a number of other cell types. The activation response of neutrophils to nonpathogenic bacteria is greatly altered by exposure to Yersinia pestis, which may be a major factor contributing to the virulence and rapid progression of plague. Several gene clusters were created based on the patterns of gene induction caused by different bacteria. These clusters were consistent with those found by a principal components analysis. A number of the changes could be interpreted in terms of neutrophil physiology and the known functions of the genes. These findings indicate that active regulation of gene expression plays a major role in the neutrophil contribution to the cellular inflammatory response. Interruption of these changes by pathogens, such as Y pestis, could be responsible, at least in part, for the failure to contain infections by highly virulent organisms.
Asunto(s)
Escherichia coli/fisiología , Regulación de la Expresión Génica , Neutrófilos/metabolismo , ARN Mensajero/biosíntesis , Yersinia pestis/fisiología , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , ADN Complementario/genética , Endopeptidasas/biosíntesis , Endopeptidasas/genética , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Humanos , Inflamación , Neutrófilos/microbiología , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , ARN Ribosómico/biosíntesis , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Especificidad de la Especie , Técnica de Sustracción , Transcripción Genética , Activación Transcripcional , Virulencia , Yersinia pestis/clasificación , Yersinia pestis/patogenicidadRESUMEN
We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.
Asunto(s)
ADN Complementario/aislamiento & purificación , Neoplasias de la Próstata/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Cartilla de ADN , Presentación de Datos , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de la Próstata/patología , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Proteínas Tirosina Quinasas Receptoras/genética , Mapeo Restrictivo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
We have developed an approach to study changes in gene expression by selective PCR amplification and display of 3' end restriction fragments of double-stranded cDNAs. This method produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on a gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA data bases. Applying this method, we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in this study, as many as 3-4% represented mRNAs that are upregulated, while approximately 2% were down-regulated within 4 hr of activation of Jurkat T cells. These and other results suggest that this approach is suitable for the systematic, expeditious, and nearly exhaustive elucidation of subtle changes in the patterns of gene expression in cells with altered physiologic states.
Asunto(s)
ADN Complementario/genética , Expresión Génica , Activación de Linfocitos/genética , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T/metabolismo , Secuencia de Bases , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , ARN/biosíntesis , ADN Polimerasa Dirigida por ARN/metabolismo , Reproducibilidad de los ResultadosRESUMEN
In searching for a candidate mechanism for the immunosuppressive as well as fibrogenic consequences of cyclosporine usage, we have explored the hypothesis that cyclosporine stimulates transcription of transforming growth factor-beta 1 (TGF-beta 1), a multifunctional cytokine endowed with immunosuppressive and fibrogenic properties. Our results demonstrate that cyclosporine (i) stimulates TGF-beta 1 promoter-dependent transcription of chloramphenicol acetyl transferase gene in transiently transfected human A-549 cells, (ii) stimulates the synthesis of TGF-beta 1 RNA transcripts in human T cells, and (iii) permits the expression/emergence of DNA regulatory proteins (retinoblastoma control factor-1 (RCF-1) and RCF-2) that bind and regulate TGF-beta 1 promoter activity. Our studies demonstrate for the first time that cyclosporine stimulates TGF-beta 1 gene transcription and suggest a novel mechanism of action of cyclosporine.
Asunto(s)
Ciclosporina/farmacología , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/genética , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Linfocitos T/metabolismoRESUMEN
A mAb, OT145, recognizes a TCR allotype encoded by one of two alleles of the V beta 6.7 gene. The peptide products of the two V beta 6.7 alleles differ because of nonconservative amino acid substitutions at positions 38 and 72. V beta 6.7a encodes ser38 and gly72, whereas V beta 6.7b encodes arg38 and glu72. We show here that the binding of mAb OT145 ot the beta-chain of TCR is lost when residue 72 of V beta 6.7a is mutated from gly to glu. The binding of OT145 is not affected by mutation of residue 38 from ser to arg. Thus, OT145 recognizes an epitope related to position 72. Residue 72 of the beta-chain of the TCR is located at a putative superantigen-binding site.