RESUMO
We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE). Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol. Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels. S1 nuclease converted supercoiled plasmids into full-length linear molecules. Large plasmids migrated as discrete bands that were readily observed after ethidium staining. Their sizes were reliably estimated by comparison with linear DNA markers. Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic. S1-PFGE detected megaplasmids up to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for characterized megaplasmids. Eight new plasmids between 102 and 316 kb were discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously.
Assuntos
Bactérias/genética , Eletroforese em Gel de Campo Pulsado , Plasmídeos , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Enterobacteriaceae/genética , Fator F , Peso Molecular , Pseudomonas/genética , Rhizobium/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Staphylococcus/genéticaRESUMO
Fourteen patients with mania unresponsive to lithium were treated with verapamil in an open study. None of the eight patients treated for acute episodes of mania or hypomania showed improvement, although four patients experienced dysphoria, with two of the four switching into depression. Four patients were treated with verapamil prophylactically; one showed a mild improvement and one, a clear positive response. Two patients with persistent pharmacological hypomanias tolerated antidepressants and became euthymic while on verapamil. A case of pharmacological hypomania successfully treated with verapamil is presented in detail. A controlled trial using verapamil to prevent pharmacological mania/hypomania is indicated.