RESUMEN
The transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase (pac) gene was studied by modifying DNA sequences responsible for promoter activation by cyclic AMP receptor protein (CRP). The nucleotide sequence of the 5'-flanking region of the pac gene contains putative tandem CRP binding sites positioned at -69/-70 and at -111/-112 with respect to the transcriptional start site. Our results obtained with either point mutations or insertion or deletion mutants (each of which rotated the helix structure at the CRP binding site one-half turn) showed significant decrease of penicillin amidase (PA) activity, suggesting the CRP as a major activator. In this study, the evidence for the importance of spacing between tandem binding sites for CRP as well as for their location related to the promoter core sequence has been provided. Involvement of integration host factor (IHF) as an additional regulatory protein in the pac gene transcription regulation was also analyzed. It is shown that activation of the pac gene transcription is elevated by IHF.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Penicilina Amidasa/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , Escherichia coli/genética , Genes Reporteros , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Penicilina Amidasa/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción GenéticaRESUMEN
Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Penicilina Amidasa/genética , Fenilacetatos/metabolismo , Secuencia de Bases , Escherichia coli/enzimología , Genes Reporteros , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Acute exacerbations of asthma are frequently caused by viral infections, but the inflammatory mechanisms in virus-induced asthma are poorly understood. The aim of the present study was to determine whether viral infection in acute asthma was associated with increased sputum neutrophil degranulation and increased cellular lysis and whether these changes are related to clinical severity. Adults (n=49) presenting to the emergency department with acute asthma were examined for infection by means of sputum direct-fluorescence antigen detection, sputum culture, and sputum polymerase chain reaction for Mycoplasma, Chlamydia and Legionella pneumophila, and all common respiratory viruses. Subjects infected with one of these agents were classed as having an infective exacerbation. Spirometry and sputum induction were performed on presentation and 4-5 weeks later. Thirty-seven subjects (76%) had virus infection and acute asthma. Those with virus infection had increased sputum neutrophils (p<0.05) and increased neutrophil elastase (p<0.05), this was related to increased elevated sputum lactate dehydrogenase (LDH). Subjects with noninfective asthma had an increase in the proportion of sputum eosinophils. Both groups had elevated sputum eosinophil cationic protein (ECP) concentrations. Higher levels of sputum LDH and ECP were associated with a longer hospital stay. Virus infection and acute asthma is associated with neutrophilic inflammation, cell lysis and more severe clinical disease.
Asunto(s)
Asma/etiología , Asma/fisiopatología , Neutrófilos/fisiología , Ribonucleasas , Virosis/complicaciones , Adolescente , Adulto , Anciano , Proteínas Sanguíneas/análisis , Degranulación de la Célula/fisiología , Proteínas en los Gránulos del Eosinófilo , Femenino , Humanos , Inflamación , L-Lactato Deshidrogenasa/análisis , Masculino , Persona de Mediana Edad , Esputo/química , Esputo/citologíaRESUMEN
The effects of respiratory viral infection on the time course of chronic obstructive pulmonary disease (COPD) exacerbation were examined by monitoring changes in systemic inflammatory markers in stable COPD and at exacerbation. Eighty-three patients with COPD (mean [SD] age, 66.6 [7.1] yr, FEV(1), 1.06 [0.61] L) recorded daily peak expiratory flow rate and any increases in respiratory symptoms. Nasal samples and blood were taken for respiratory virus detection by culture, polymerase chain reaction, and serology, and plasma fibrinogen and serum interleukin-6 (IL-6) were determined at stable baseline and exacerbation. Sixty-four percent of exacerbations were associated with a cold occurring up to 18 d before exacerbation. Seventy-seven viruses (39 [58.2%] rhinoviruses) were detected in 66 (39.2%) of 168 COPD exacerbations in 53 (64%) patients. Viral exacerbations were associated with frequent exacerbators, colds with increased dyspnea, a higher total symptom count at presentation, a longer median symptom recovery period of 13 d, and a tendency toward higher plasma fibrinogen and serum IL-6 levels. Non-respiratory syncytial virus (RSV) respiratory viruses were detected in 11 (16%), and RSV in 16 (23.5%), of 68 stable COPD patients, with RSV detection associated with higher inflammatory marker levels. Respiratory virus infections are associated with more severe and frequent exacerbations, and may cause chronic infection in COPD. Prevention and early treatment of viral infections may lead to a decreased exacerbation frequency and morbidity associated with COPD.