RESUMEN
MexZ is a transcriptional regulator of the mexXY multidrug transporter operon, which confers aminoglycoside resistance on Pseudomonas aeruginosa. Highly purified MexZ showed direct binding with a specific site of the mexZ-mexX intergenic DNA when probed by a gel retardation assay. Both in vitro chemical cross-linking experiments and an in vivo two-hybrid expression system showed that the active form of MexZ, which is capable of binding the intergenic DNA, appeared to be a dimer. These results explain the mechanism by which MexZ represses transcription of the mexXY operon, but do not explain the substrate-induced hyperproduction of MexXY. The presence of inducer antibiotic in the gel-retardation assay mixture failed to detect altered MexZ-probe DNA interaction suggesting the possible involvement of an additional regulator.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Intergénico/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Secuencia de Aminoácidos , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , ADN Bacteriano/metabolismo , Dimerización , Ensayo de Cambio de Movilidad Electroforética , Datos de Secuencia Molecular , Operón , Unión Proteica , Pseudomonas aeruginosa/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Instead of the original intermediate dose (0.5 g/m2), a modified intermediate-dose 1-beta-D-arabinofuranosylcytosine (ara-C) therapy (1 g/m2, 1-h intravenous infusion) was pharmacologically studied in 11 leukemic patients. PATIENTS AND METHODS: The concentrations of ara-C and its inactive metabolite, 1-beta-D-arabinofuranosyluracil (ara-U) in the plasma, urine and cerebrospinal fluid were determined using high performance liquid chromatography. RESULTS: The plasma ara-C reached a peak (61.2 +/- 52.7 microM) that far surpassed the saturating level for intracellular activation of the drug. The plasma ara-U reached its peak (139.8 +/- 40.0 microM) and was maintained with a half-life of 277 +/- 76 min. About 60% of the administered drug was recovered, mainly as ara-U in urine within 12 h. In contrast to the original intermediate dose, the therapeutic ara-C levels (above 0.4 microM) persisted in the central nervous system. The therapy salvaged one relapsed leukemia patient with central nervous system involvement. CONCLUSION: Modified intermediate-dose ara-C provides a sufficient plasma ara-C level with a concomitant therapeutic concentration in the cerebrospinal fluid.