RESUMEN

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Asunto(s)

Animales , Conejos , Ratas , AMP Cíclico , Regulación Enzimológica de la Expresión Génica , Peptidil-Dipeptidasa A , Regiones Promotoras Genéticas , Secuencia de Bases , Células Cultivadas , Células Endoteliales , Datos de Secuencia Molecular , Elementos de Respuesta , Transfección

RESUMEN

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Asunto(s)

AMP Cíclico/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Peptidil-Dipeptidasa A/genética , Regiones Promotoras Genéticas/fisiología , Animales , Secuencia de Bases , Células Cultivadas , AMP Cíclico/genética , Células Endoteliales , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Conejos , Ratas , Elementos de Respuesta/genética , Elementos de Respuesta/fisiología , Transfección

RESUMEN

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C-->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C-->G) mutation. Furthermore, this is the first time that the -195 (C-->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

Asunto(s)

Hemoglobina Fetal/genética , Genes Reporteros , Globinas/genética , Hemoglobinopatías/genética , Mutación , Mutación Puntual/genética , Adulto , Cartilla de ADN , Expresión Génica , Globinas/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Reacción en Cadena de la Polimerasa , Transfección , beta-Galactosidasa/metabolismo

RESUMEN

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression

Asunto(s)

Humanos , Adulto , Hemoglobina Fetal/genética , Genes Reporteros , Globinas/genética , Hemoglobinopatías/genética , Técnicas In Vitro , Mutación , beta-Galactosidasa/metabolismo , Cartilla de ADN , Expresión Génica , Globinas/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Mutación Puntual , Reacción en Cadena de la Polimerasa , Transfección

RESUMEN

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Asunto(s)

Genoma Bacteriano , Plantas/microbiología , Pseudomonadaceae/genética , Análisis de Secuencia de ADN , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Transporte Biológico , Mapeo Cromosómico , Citrus/microbiología , Reparación del ADN , ADN Bacteriano , Metabolismo Energético , Datos de Secuencia Molecular , Plantas Tóxicas , Biosíntesis de Proteínas , Pseudomonadaceae/metabolismo , Pseudomonadaceae/patogenicidad , Nicotiana/microbiología , Transcripción Genética , Virulencia/genética

RESUMEN

Available evidence for oxidative stress after angioplasty is indirect or ambiguous. We sought to characterize the pattern, time course, and possible sources of free radical generation early after arterial balloon injury. Ex vivo injury performed in arterial rings in buffer with lucigenin yielded a massive oxygen-dependent peak of luminescence that decayed exponentially and was proportional to the degree of injury. Signals for injured vs. control arteries were 207. 1 +/- 17.9 (n = 13) vs 4.1 +/- 0.7 (n = 22) cpm x 10(3)/mg/min (p <. 001). Data obtained with 0.25 mmol/l lucigenin were validated with 0. 005-0.05 mmol/l lucigenin or the novel superoxide-sensitive probe coelenterazine (5 micromol/l). Gentle removal of endothelium prior to injury scarcely affected the amount of luminescence. Lucigenin signals were amplified 5- to 20-fold by exogenous NAD(P)H, and were >85% inhibited by diphenyliodonium (DPI, a flavoenzyme inhibitor). Antagonists of several other potential free radical sources, including xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport, were without effect. Overdistension of intact rabbit iliac arteries in vivo (n = 7) induced 72% fall in intracellular reduced glutathione and 68% increase in oxidized glutathione, so that GSH/GSSG ratio changed from 7.93 +/- 2.14 to 0. 81 +/- 0.16 (p <.005). There was also 28.7% loss of the glutathione pool. Further studies were performed with electron paramagnetic resonance spectroscopy. Rabbit aortas submitted to ex vivo overdistension in the presence of the spin trap DEPMPO (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide, 100 mmol/l, n = 5) showed formation of radical adduct spectra, abolished by DPI or superoxide dismutase. Computer simulation indicated a mixture of hydroxyl and carbon-centered radical adducts, likely due to decay of superoxide adduct. Electrical mobility shift assays for NF-kappaB activation were performed in nuclear protein extracts from intact or previously injured rabbit aortas. Balloon injury induced early NF-kappaB activation, which was decreased by DPI. In conclusion, our data show unambiguously that arterial injury induces an immediate profound vascular oxidative stress. Such redox imbalance is likely accounted for by activation of vessel wall NAD(P)H oxidoreductase(s), generating radical species potentially involved in tissue repair.

Asunto(s)

Cateterismo/efectos adversos , Endotelio Vascular/lesiones , Imidazoles , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Especies Reactivas de Oxígeno , Acridinas/metabolismo , Animales , Compuestos de Bifenilo/farmacología , Óxidos N-Cíclicos , Inhibidores de la Ciclooxigenasa/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Endotelio Vascular/enzimología , Radicales Libres , Glutatión/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Mediciones Luminiscentes , Masculino , Metaloporfirinas/farmacología , NAD/metabolismo , NADP/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/farmacología , Compuestos Onio/farmacología , Oxidación-Reducción , Estrés Oxidativo , Consumo de Oxígeno , Pirazinas/farmacología , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Marcadores de Spin , Superóxido Dismutasa/farmacología , Superóxidos/metabolismo , Transcripción Genética , Cicatrización de Heridas , Xantina Oxidasa/farmacología

RESUMEN

To shed light on mechanisms of angiotensin-converting enzyme (ACE) upregulation, we used a rabbit endothelial cell model to characterize intracellular pathways of beta-adrenergic stimulation. In these cells, ACE activity is increased by isoproterenol (ISO). The stably transfected 1273-bp ACE promoter is stimulated by ISO in the presence of isobutyl methylxanthine. This effect is abolished by propranolol. Promoter stimulation is mimicked by cholera toxin, forskolin, and 8BrcAMP, but not by 8BrcGMP. Promoter stimulation by ISO and isobutyl methylxanthine is blocked by protein kinase A inhibitors, indicating that beta-adrenergic stimulation of the ACE gene depends on phosphorylation of protein kinase A targets. Activation by cAMP, resistance to phorbol ester, and lack of synergism between cAMP and phorbol ester suggest that promoter regulation is due to cAMP responsive element rather than to activating protein-2 sequences. Okadaic acid potentiation of 8BrcAMP induction indicated that promoter activation by cAMP is regulated by phosphatases controlling activation of typical cAMP responsive element regulated genes. In summary, beta-adrenergic activation of rat ACE promoter is specific; uses G(s) proteins, adenylyl cyclase, protein kinase A; and probably includes cAMP responsive element-like sequences.

Asunto(s)

Agonistas Adrenérgicos beta/farmacología , AMP Cíclico/metabolismo , Endotelio Vascular/metabolismo , Isoproterenol/farmacología , Peptidil-Dipeptidasa A/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Células Cultivadas , Combinación de Medicamentos , Inducción Enzimática/fisiología , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Conejos , Ratas , Ratas Endogámicas WKY , Receptores Adrenérgicos beta/fisiología

RESUMEN

Protein A is a cell wall linked protein of Staphylococcus aureus that binds mammalian IgG. Although protein A displays high size heterogeneity among strains, cloning and sequencing of its gene from two strains had not shown a large difference in size. Here we report a third protein A gene sequence that shows a size variation relative to these two, due to deletions on both one IgG binding domain and a cell wall binding domain (region X). By analysis of the three sequences we were able to delineate a hypothetic model for region X domain evolution and discussed the origin of genetic variability within and without strains.

Asunto(s)

Variación Genética , Proteína Estafilocócica A/genética , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Evolución Biológica , Clonación Molecular , Inmunoglobulina G/metabolismo , Datos de Secuencia Molecular , Mutación , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico