Garige Mamatha, Azuine Magnus A, Lakshman M Raj
Department of Biochemistry and Molecular Biology, The George Washington University, Washington, DC 20037, USA.
Metabolism. 2006 Jun;55(6):803-10. doi: 10.1016/j.metabol.2006.01.018.
We have previously shown that chronic ethanol feeding stimulates liver cytosolic sialidase (CS) and plasma membrane sialidase (PMS), whereas it decreases lysosomal membrane sialidase (LMS) activities with concomitant alterations in their relative synthetic rate in rat. To understand the molecular mechanism(s) for these changes, we have evaluated the effect of ethanol administration in male Wistar rats as a function of increasing dietary ethanol concentration after 8 weeks of pair-feeding on (i) the expression of CS, PMS, and LMS genes by real-time quantitative polymerase chain reaction method; (ii) their relative transcription rates by nuclear run-on assay; and (iii) the actual amount of these sialidase proteins in the liver fractions of the respective groups by Western blot method. We have demonstrated that the animals fed with 10.6%, 20.8%, and 36% of total calories as ethanol showed a 20% (P<.05), 34% (P<.01), and 69% (P<.01) increase in CS mRNA level, and 22% (P<.05), 26% (P<.01), and 47% (P<.01) increase in PMS mRNA level, but a decrease in LMS mRNA level by 35% (P<.05), 50% (P<.01), and 80% (P<.01), respectively, as compared to controls. Western blot analyses of CS, PMS, and LMS in the liver subfractions showed that changes in protein levels of CS, LMS, and PMS were consistent with the corresponding changes in the respective mRNA levels. Thus, the upregulation of CS and PMS, but not LMS which is down regulated by chronic ethanol, may account for the appearance of asialoconjugates in alcoholics.
我们之前已经表明，长期给予乙醇会刺激肝脏胞质唾液酸酶（CS）和质膜唾液酸酶（PMS），而会降低溶酶体膜唾液酸酶（LMS）的活性，同时大鼠体内它们的相对合成速率也会发生改变。为了理解这些变化的分子机制，我们在雄性Wistar大鼠中进行了配对喂养8周后，评估了随着饮食中乙醇浓度增加给予乙醇的作用，具体如下：（i）通过实时定量聚合酶链反应法检测CS、PMS和LMS基因的表达；（ii）通过核转录分析检测它们的相对转录速率；（iii）通过蛋白质印迹法检测各组大鼠肝脏各部分中这些唾液酸酶蛋白的实际含量。我们已经证明，以乙醇形式摄入总热量10.6%、20.8%和36%的动物，其CS mRNA水平分别升高了20%（P<0.05）、34%（P<0.01）和69%（P<0.01），PMS mRNA水平分别升高了22%（P<0.05）、26%（P<0.01）和47%（P<0.01），但与对照组相比，LMS mRNA水平分别降低了35%（P<0.05）、50%（P<0.01）和80%（P<0.01）。肝脏亚组分中CS、PMS和LMS的蛋白质印迹分析表明，CS、LMS和PMS蛋白水平的变化与各自mRNA水平的相应变化一致。因此，CS和PMS的上调，而非长期乙醇摄入下调的LMS，可能是酗酒者中去唾液酸糖缀合物出现的原因。
