RESUMO
Recently we reported that neurotensin-SPDP-poly-L-lysine (NT-vector) is able to bind plasmid DNA (NT-polyplex) and polyfect cells expressing the high-affinity neurotensin receptor (NTRH) although with low transfecting efficiency: in vitro, 6.5+/-1.5%, and in vivo, 5+/-4%. In this work, we attempted to increase the transfecting efficiency by integrating the hemagglutinin HA2 fusogenic peptide and the Vp1 nuclear localization signal of SV40 to the NT-polyplex (fusogenic-karyophilic-NT-polyplex). Confocal microscopy and flow cytometry analysis showed that the fusogenic-karyophilic-NT-polyplex produced mostly nuclear localization of the plasmid DNA in NTRH-bearing N1E-115 cells. About 50% of N1E-115 cells internalized and expressed the reporter gene when the plasmid DNA was transferred by the fusogenic-karyophilic-NT-polyplex. Although to a less extent, the addition of each viral peptide separately to NT-polyplex (fusogenic-NT-polyplex or karyophilic-NT-polyplex) improved polyfection. Fusogenic-NT-polyplex produced 22.41+/-5.96% of internalization and 20.35+/-0.82% of expression in N1E-115 cells, whereas karyophilic-NT-polyplex yielded 13.75+/-3.88% and 10.94+/-2.04%, respectively. Basal internalization and expression were detected in N1E-115 cells in the presence of 100 nM SR-48692 and in NTRH-lacking cells. The fusogenic-karyophilic-NT-polyplex was microinjected into the substantia nigra to test its ability for gene transfer in vivo. Fusogenic-karyophilic-NT-polyplex internalization was observed within dopamine neurons only. Reporter gene expression was observed in a high proportion of dopamine neurons up to 60 days after NT-polyfection. Both internalization and expression were prevented by SR-48692. Our results show that the fusogenic-karyophilic-NT-polyplex is a highly efficient and specific gene vector and encourage its use to transfer gene of physiological interest to NTRH-bearing neurons.
Assuntos
Proteínas do Capsídeo/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hemaglutininas Virais/genética , Neurotensina/genética , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusão/genética , Animais , Sítios de Ligação/genética , Células Cultivadas , DNA/genética , Dopamina/genética , Dopamina/metabolismo , Regulação da Expressão Gênica/genética , Substâncias Macromoleculares , Masculino , Neurônios/citologia , Neurônios/metabolismo , Plasmídeos/genética , Ratos , Ratos Wistar , Substância Negra/citologia , Substância Negra/metabolismoRESUMO
Background. Genotoxicity of antibiotic has not been well evaluted, and there is not much information on the genetic risk of quinolone drugs, even though they are widely used as alternative choice drugs in urinary infections. Methods. Pipemidic acid and norfloxacin were tested for their capacity to induce point mutations using the Ames test and DNA damage on Escherichia coli PolA-/PolA+. Results. At non-toxic doses, all of the drugs studied were negative on the E. coli PolA-/PolA+ test with or without in vitro metabolic activation with induced arochlor 1254 rat liver (S9). They did not procedure frameshift mutations in TA98, or base-air substitutions in S. typhimurium hisG46 strains TA100, or UTH8414. Norfloxacin and its induced metabolites in vitro with S9 rat liver were mutagenic to hisG48 strains TA102 and TA104, both of which detect oxidative chemicals. Pipemidic acid induced mutations in S. typhimurium hisG48 strains only when they had an efficient DNA excision repair system. Conclusions. These results suggest that the risk of oxygen-free radical generation from quinolones should be considered