RESUMEN
Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P<0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system.
Asunto(s)
Gastrópodos , Hepatopáncreas/metabolismo , Muramidasa/genética , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/inmunología , Secuencia de Aminoácidos , Animales , Bacterias/metabolismo , Evolución Biológica , Clonación Molecular , Secuencia Conservada/genética , ADN Complementario/análisis , Hepatopáncreas/inmunología , Hepatopáncreas/parasitología , Interacciones Huésped-Parásitos/genética , Datos de Secuencia Molecular , Muramidasa/inmunología , Muramidasa/metabolismo , Schistosoma japonicum/patogenicidad , Transgenes/genéticaRESUMEN
The retrograde axonal transport mechanism of motor neurons has been exploited to deliver the gene encoding Glial cell line-derived neurotrophic factor (GDNF) into the central nervous system to provide trophic support following injury. A nonviral gene delivery system, consisting of a monoclonal antibody (MC192) that binds the neurotrophic receptor, p75(NTR), coupled to poly-L-lysine, was constructed and used to deliver the gene via a receptor-mediated mechanism. The MC192-poly-l-lysine/pGDNF complex was injected into the hind limb of newborn rats to allow gene expression within motor neurons prior to sciatic nerve transection. In adult rats, the gene delivery complex was administrated in gel foam placed on a transected hypoglossal nerve. We show that the delivered construct is internalized following binding to p75(NTR) and is transported into the brain and spinal cord, bypassing the blood-brain barrier. The presence of the GDNF transgene and its transcript could be detected for up to 8 weeks in spinal cord and brain stem. Expression of the GDNF protein rescued 38% of the targeted motor neurons 1 week postinjury in newborn rats while the survival rate in control group was below 12%. In adult rats, neuronal death induced by axotomy was almost completely reversed by the introduction of the transgene (95 +/- 3%). Thus, the significant functional outcomes of this novel gene delivery system are demonstrated both in postnatal and adult motor neurons.