RESUMO
BACKGROUND: Adipogenesis is a complex process that involves many genes/proteins at different stages of differentiation. In order to identify genes critical for adipogenesis, we took a novel approach based on phenotype change of individual cell, to search for genes with regulatory roles in adipogenesis genome-wide in 3T3-L1 cells. METHODS: Lentivirus-based inducible random homologous knockdown was used for the screening of functional gene that altered lipid formation in the adipocyte during differentiation. RESULTS: In the present study, we reported the identification of an alternatively spliced mitochondrial oxodicarboxylate carrier (ODC), so named ODC-AS. ODC-AS is different from ODC by replacing 22 amino acids with 29 amino acids at the N-terminal. ODC was widely expressed in most tissues in mouse as determined by multi-tissue cDNA panel polymerase chain reaction. However, ODC-AS was only detected in adipose tissue and in iris and sclera-choroid complex of the eye. The expression of ODC-AS in 3T3-L1 was detected after the induction of differentiation, and reached a peak at day 4 and then reduced thereafter, whereas no ODC transcript detected in the cells neither before nor after differentiation. Knocking down of ODC-AS expression by RNA interference led to significant reduction in lipid accumulation as determined by triglyceride measurement and Nile Red staining, as well as adipogenic marker CEBPalpha, PPARgamma, aP2 and CD36. Although both ODC and ODC-AS are expressed in white and brown adipose tissues, only the expression of ODC-AS was down-regulated in brown adipose tissue by cold exposure. CONCLUSION: These results implicate that ODC-AS may promote lipid accumulation during adipocyte differentiation and play an important role in the regulation of lipid metabolism in adipose tissues.
Assuntos
Adipogenia/genética , Processamento Alternativo , Ácidos Carboxílicos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genoma , Lentivirus/genética , Lipídeos/química , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Especificidade de Órgãos , Transportadores de Ânions Orgânicos/genética , FenótipoRESUMO
During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.
Assuntos
Colágeno/farmacologia , Proteínas de Insetos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Proteínas e Peptídeos Salivares/farmacologia , Animais , Anopheles , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Proteínas de Insetos/genética , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares , Proteínas e Peptídeos Salivares/genéticaAssuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Centrifugação/métodos , Embrião de Mamíferos/fisiologia , Estudos de Viabilidade , Camundongos , Técnicas de Cultura de Tecidos/métodosRESUMO
Mint-1, which is also called as X11 or mammalian Lin10, protein has been implicated in the synaptic vesicle exocytosis and the targeting and localization of synaptic membrane proteins. Here, we established mint-1 gene knockout (mint-1 KO) mice and investigated vesicular and transporter-mediated dopamine (DA) release evoked by high K(+) and methamphetamine (METH), respectively. Compared with wild-type control, high K(+)-evoked striatal DA release was attenuated, but not significantly, in the KO mice as measured by microdialysis method. The METH-induced DA release was significantly attenuated in the KO mice. In addition, METH-induced stereotypy was also significantly attenuated in the KO mice. Mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object as compared with wild-type mice. These results suggest that mint-1 protein is involved in transporter-mediated DA release induced by METH.
