RESUMO
We used an assay to measure quinolone sensitivity as a shift in the position of the cleavage-religation equilibrium. This assay was found to be useful in identifying the primary target of a quinolone drug and assessing the effect of quinolone resistance-conferring mutations.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Mutação/genética , Quinolonas/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Replacement of the alpha4 helix of ParC with that of GyrA increased the stability of topoisomerase IV-quinolone-DNA ternary complexes. This mutant topoisomerase IV-mediated cell killing was more efficient than topoisomerase IV-mediated cell killing in Escherichia coli. Thus, the alpha4 helix plays critical roles in determining the stability and the cytotoxicity of ternary complexes.
Assuntos
DNA Girase/genética , DNA Topoisomerase IV/química , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Escherichia coli/genética , Quinolonas/química , Anti-Infecciosos/farmacologia , Clonagem Molecular , DNA Girase/química , DNA Bacteriano/química , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Mutação/genética , Norfloxacino/farmacologiaRESUMO
Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ETA receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-beta 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-epsilon-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte "surface" mimicking the negative inotropic effects of bath-applied PKC activators and "deep" release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such "detubulated" cardiac myocytes showed no positive inotropic response to 20 nM ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-beta 1-PKC-epsilon signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.
