RESUMEN
OBJECTIVE: In rodents scavenger receptor class B type I (SR-BI) is a key molecule for selective uptake of cholesteryl ester from high-density lipoprotein (HDL). This study was aimed to clarify the role of the human SR-BI/CD36 and LIMP-II Analogues-1 (CLA-1) as a molecular target of selective uptake of cholesteryl ester from HDL in vivo. METHODS AND RESULTS: To clarify the function and regulation of CLA-1 in vivo we produced CLA-1 BAC transgenic mice. In spite of abundant hepatic RNA expression of CLA-1, CLA-1 BAC transgenic mice had no significant effect on mouse HDL cholesterol. Although coexpression of a human scaffolding protein PDZK1 along with CLA-1 enhanced hepatic CLA-1 expression, it did not affect mouse HDL cholesterol levels, either. However, in the presence of human apoA-1, HDL cholesterol level and size were significantly reduced in CLA-1 transgenic mice, and its reduction was more pronounced in CLA-1/human PDZK1 double transgenic mouse. CONCLUSIONS: We established a mouse model to study human reverse cholesterol transport by expressing CLA-1, human PDZK1, and human apoA-I gene. Our results imply that enhancing CLA-1 expression by human PDZK1 in the liver can modulate HDL cholesterol metabolism and possibly enhance reverse cholesterol transport to prevent the progression of atherosclerosis in human.
Asunto(s)
Proteínas Portadoras/metabolismo , HDL-Colesterol/metabolismo , Hígado/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Proteínas Portadoras/genética , Ésteres del Colesterol/metabolismo , HDL-Colesterol/sangre , Cromosomas Artificiales Bacterianos , Humanos , Proteínas de la Membrana , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptores Depuradores de Clase B/genéticaRESUMEN
We established a real-time quantitative PCR (RQ-PCR) with which to measure abundance of the asparagine synthetase (AS) mRNA. The level of AS mRNA paralleled AS enzyme activity, as well as the AS protein level detected by Western blotting and by in situ immunostaining. Cytotoxicity tests in vitro showed that the AS mRNA level also synchronized with cellular resistance to L-asparaginase in cell lines. Cellular levels of AS enzyme activity correlated with resistance to L-asparaginase. These results indicate that the AS mRNA level is an index of resistance to L-asparaginase. RQ-PCR is superior to enzyme assays, Western blotting, and immunostaining in the following ways: less labor and time, accurate and reproducible quantitativity, and broad dynamic range. In addition, RQ-PCR could evaluate differences in L-asparaginase sensitivity although immunostaining could not. And in clinical samples, we analyzed eight pediatric leukemia cases by this RQ-PCR to evaluate whether this method was applicable to clinical laboratories and the expression level of AS mRNA in each case were predictable for the effectiveness of L-asparaginase treatment. Consequently, this method was useful enough in defining candidates for selective therapy that targets an AS deficiency.