RESUMEN
To survive, Salmonella enterica serovar Typhimurium (S. Typhimurium) must sense signals found in phagocytic cells and modulate gene expression. In the present work, we evaluated the expression and cross-regulation of the transcription factors MarA, Rob, and SoxS in response to NaOCl. We generated strains ΔsoxS and ΔmarA, which were 20 times more sensitive to NaOCl as compared to the wild-type strain; while Δrob only 5 times. Subsequently, we determined that marA and soxS transcript and protein levels were increased while those of rob decreased in a wild-type strain treated with NaOCl. To assess if changes in S. Typhimurium after exposure to NaOCl were due to a cross-regulation, as in Escherichia coli, we evaluated the expression of marA, soxS, and rob in the different genetic backgrounds. The positive regulation observed in the wild-type strain of marA and soxS was retained in the Δrob strain. As in the wild-type strain, rob was down-regulated in the ΔmarA and ΔsoxS treated with NaOCl; however, this effect was decreased. Since rob was down-regulated by both factors, we generated a ΔmarA ΔsoxS strain finding that the negative regulation was abolished, confirming our hypothesis. Electrophoretic mobility shift assays using MarA and SoxS confirmed an interaction with the promoter of rob.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Hipoclorito de Sodio/farmacología , Factores de Transcripción/genética , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Mutación , Oxidantes/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Salmonella typhimurium/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OmpW of Salmonella enterica serovar Typhimurium has been described as a minor porin involved in osmoregulation, and is also affected by environmental conditions. Biochemical and genetic evidence from our laboratory indicates that OmpW is involved in efflux of and resistance towards paraquat (PQ), and its expression has been shown to be activated in response to oxidative stress. In this study we have explored ompW expression in response to PQ. Primer extension and transcriptional fusions showed that its expression was induced in the presence of PQ. In silico analyses suggested a putative binding site for the SoxS transcriptional factor at the ompW regulatory region. Electrophoretic mobility shift assays (EMSAs) and footprinting experiments showed that SoxS binds at a region that starts close to -54 and ends at about -197 upstream of the transcription start site. Transcriptional fusions support the relevance of this region in ompW activation. The SoxS site is in the forward orientation and its location suggests that the ompW gene has a class I SoxS-dependent promoter.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Salmonella typhimurium/metabolismo , Transactivadores/metabolismo , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Herbicidas/farmacología , Paraquat/farmacología , Regiones Promotoras Genéticas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Análisis de Secuencia de ADN , Activación Transcripcional/efectos de los fármacosRESUMEN
OBJECTIVE: To study the effects of cannabinoid, glutamate, and dopamine agonists and antagonists on the calcium current rat sympathetic neurons. METHODS: Calcium current was recorded using the whole-cell variant of the patch-clamp technique. After expression in neuronal membranes of the cannabinoid CB1, glutamate mGluR2, or dopamine D1 receptor (by microinjection of the levant receptor's cDNA into the neuron's nucleus) agonists' and antagonists' effects were observed. RESULTS: Applications of agonists of the expressed receptor (0.1-10 microM) decreased the calcium current. The calcium current was increased after application of cannabinoid antagonists (AM251 and AM630); these compounds thus act as inverse agonists in this preparation. Glutamate and dopamine antagonists had no effects on the calcium current by themselves. Combined application of cannabinoids and dopamine, but not glutamate, agonists produced a decrement in the calcium current that was bigger than either of the effects seen when one agonist was applied alone. CONCLUSIONS: These results suggest that cannabinoid with dopamine receptors have an interactive inhibitory effect on the calcium current in this preparation, indicating that within the nervous system, receptor interactions may be important in the regulation of ion-channel functions.
Asunto(s)
Calcio/metabolismo , Neuronas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Masculino , Neuronas/citología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/metabolismoRESUMEN
The aim of this study was to apply receiver operating characteristic (ROC) analysis to the microplate Alamar blue assay, a recently developed alternative for drug susceptibility testing of mycobacteria. As this is a quantitative assay, its performance can be determined by ROC analysis, in which the area under the ROC curve represents a summary of test performance (the higher the area, the better the test's performance). Sixty isolates of Mycobacterium tuberculosis were tested by the microcolorimetric assay against six twofold dilutions of streptomycin, isoniazid, rifampin, and ethambutol. For each isolate, the susceptibility pattern was simultaneously established by the agar proportion method, the result of which represented the gold standard value for the ROC analysis. The critical concentration, area under the curve, and P value for each drug were determined by ROC curve analysis. The results of the assay were obtained in an average of 8 days of incubation. The performance of the assay was excellent for all four drugs: the area under the curves was >0.97, the P values were 0.000, and sensitivity was 94%, specificity 97%, predictive value for resistance >/=92%, predictive value for susceptibility 97%, and test efficiency 97%. According to ROC analysis, the microplate Alamar blue assay is a reliable method for determination of drug-susceptibility. Rapidity and cost efficiency are two additional qualities that make this test an excellent alternative for the drug susceptibility testing of Mycobacterium tuberculosis. The ROC curve analysis is a robust statistical approach for evaluating the performance of new quantitative methods for determination of drug sensitivity of Mycobacterium tuberculosis isolates.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Agar , Antibióticos Antituberculosos/farmacología , Farmacorresistencia Microbiana , Etambutol/farmacología , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Probabilidad , Curva ROC , Rifampin/farmacología , Sensibilidad y Especificidad , Estreptomicina/farmacologíaRESUMEN
In adult rabbit ventricular preparations, action potential duration is significantly increased when stimulation frequency is increased from 0.1 to 1.0 Hz. In neonatal preparations, a similar change in stimulation frequency produced no significant increase in action potential duration. To identify the ionic basis for this difference, we studied different outward currents in single myocytes from papillary muscle and from epicardial tissue of adult and neonatal rabbits. The densities of the outward currents in neonatal cells were about one-half of the current density in adult cells. The density of the voltage-activated transient outward current (I(to1)) was smaller in cells from papillary muscle than in cells from epicardium in adult and newborn rabbits. We found major differences in the kinetic behavior of I(to1) between adult and neonatal cells: 1) the rate of apparent inactivation was faster in neonatal cells, and 2) the recovery from inactivation was significantly faster in neonatal cells, with a time constant of 113 vs. 1,356 ms. We propose that this marked difference in the recovery from inactivation of I(to1) is the basis for the difference in frequency dependence of action potential duration.