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1.
BMC Biotechnol ; 8: 50, 2008 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-18485214

RESUMO

BACKGROUND: Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein. RESULTS: Secretion level of the fusion protein produced in vitro in BHK cells was approximately 30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04-1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of approximately 150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. CONCLUSION: Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.


Assuntos
Butirilcolinesterase/farmacocinética , Rim/enzimologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/farmacocinética , Animais , Butirilcolinesterase/sangue , Butirilcolinesterase/genética , Linhagem Celular , Cricetinae , Cabras , Humanos , Taxa de Depuração Metabólica , Camundongos , Camundongos Transgênicos , Albumina Sérica/genética , Suínos
2.
Proc Natl Acad Sci U S A ; 104(34): 13603-8, 2007 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-17660298

RESUMO

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat beta-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.


Assuntos
Butirilcolinesterase/metabolismo , Leite/efeitos dos fármacos , Leite/enzimologia , Intoxicação por Organofosfatos , Animais , Animais Geneticamente Modificados , Butirilcolinesterase/genética , Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/farmacocinética , Metabolismo dos Carboidratos , Carboidratos/análise , Regulação Enzimológica da Expressão Gênica , Cabras , Cobaias , Humanos , Camundongos , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética
3.
Theriogenology ; 60(3): 397-406, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12763154

RESUMO

This study was conducted to determine whether or not exogenous gonadotropin releasing hormone (GnRH) alters the timing or improves the synchrony of estrus, the LH surge, and ovulation following estrous synchronization in dwarf goats, and to assess the effects of season on these parameters. In January and June, estrus was synchronized in 12 Pygmy and Nigerian Dwarf goats with a 10-day progestagen sponge, 125 microg cloprostenol i.m. 48 h before sponge removal, and 300 IU equine chorionic gonadotrophin (eCG) i.m. at sponge removal. Six of the 12 goats were given 50 microg GnRH i.m. 24h after sponge removal. Onset of estrus was monitored using two males. Samples for plasma LH were collected at 2 h intervals beginning 22 h after sponge removal and ending at 48 h in January and at 58 h in June. Time of ovulation time was confirmed by laparoscopy at 36, 50, 60, and 74 h in January and at 50, 60, and 74 h in June. Administration of GnRH had no significant effect on the onset of estrus; however, it reduced the interval from sponge removal to the LH surge and improved the synchrony of the LH surge (P<0.05). Treatment with GnRH also reduced the interval from sponge removal to ovulation and improved the synchrony of ovulation (P<0.05). Season had a significant effect on the timing and the synchrony of estrus with and without GnRH treatment (P<0.05). A seasonal shift was also observed in the timing of the LH surge in the absence of GnRH treatment (P<0.05). Further research is required to determine the optimum time for GnRH administration and the minimum effective dose in dwarf goats.


Assuntos
Cabras/fisiologia , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Luteinizante/metabolismo , Ovulação/efeitos dos fármacos , Administração Intravaginal , Animais , Gonadotropina Coriônica/administração & dosagem , Cloprostenol/administração & dosagem , Detecção do Estro , Sincronização do Estro , Feminino , Cavalos , Cinética , Hormônio Luteinizante/sangue , Acetato de Medroxiprogesterona/administração & dosagem , Estações do Ano , Fatores de Tempo
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