RESUMEN
Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus, in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant (Δdus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The Δdus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA(Phe) transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA(Phe) purified from the Δdus strain and tRNA(Phe) transcript revealed that other tRNA modifications are required for D formation at high temperatures.
Asunto(s)
Proteínas Bacterianas/química , Oxidorreductasas/química , ARN de Transferencia/química , Thermus thermophilus/enzimología , Uridina/análogos & derivados , Proteínas Bacterianas/aislamiento & purificación , Pruebas de Enzimas , Oxidación-Reducción , Oxidorreductasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Temperatura , Uridina/biosíntesisRESUMEN
Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Oncogénicas Virales/genética , Papillomaviridae/fisiología , ARN Interferente Pequeño/administración & dosificación , Neoplasias del Cuello Uterino/terapia , Animales , Femenino , Regulación Viral de la Expresión Génica , Silenciador del Gen , Células HeLa , Humanos , Inyecciones Intralesiones , Ratones , Ratones Desnudos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Trasplante Heterólogo , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , beta-Galactosidasa/metabolismoRESUMEN
The roles of protein kinase C (PKC) isoforms in cholinergic potentiation of glucose-induced insulin secretion were investigated in rat pancreatic islets. Western-blot analysis showed the presence of PKC-alpha, betaII, delta, epsilon, eta, and zeta, but not PKC-betaI, gamma, or iota, in the islets. Carbachol (CCh) caused translocations of PKC-alpha, betaII, delta, and epsilon from the cytosol to the plasma membrane. CCh facilitated 7-mM glucose-induced insulin secretion from isolated rat islets. The CCh-stimulated insulin secretion was significantly suppressed by the generic PKC inhibitor chelerythrine. In contrast, Go 6976, an inhibitor of conventional PKC isoforms, had no effect on the insulin secretion stimulated by CCh, although it significantly inhibited that induced by phorbol 12-myristate 13-acetate. These results suggest that the novel PKC isoforms activated by CCh, i.e., PKC-delta and/or epsilon, participate in the stimulatory effect of CCh on insulin secretion.