A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

阜豆GmABC基因的克隆及表达分析

利用RACE技术从大豆品种‘阜豆11号’中克隆到1个ABC转运蛋白基因,该基因cDNA全长为4 693bp,其中开放读码框4 341bp,编码1 447个氨基酸,分子量162.5kD,具有高度保守的ATP结合位点,命名为GmABC。蛋白序列比对结果显示,该基因编码蛋白属于PDR(pleiotropic drug resistance)多向耐药性蛋白家族成员。系统进化树分析显示,GmABC与苜蓿PDR亲缘关系最近,相似性达84%。基因表达分析显示,GmABC受镉诱导表达,当Cd2+浓度达到100mg.L-1时,其表达量最高。研究表明,GmABC可能对阜豆镉胁迫抗性发挥重要作用。     

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.     

肝X受体α在泡沫细胞胆固醇流出中的调控作用

以THP-1巨噬细胞源性泡沫细胞为研究对象,观察肝X受体α(LXRα)在THP-1巨噬细胞源性泡沫细胞胆固醇流出中的调控作用.结果发现,22(R)-羟基胆固醇剂量依赖性增加THP-1巨噬细胞源性泡沫细胞胆固醇流出, 而DIDS剂量依赖性减少THP-1巨噬细胞源性泡沫细胞胆固醇流出.逆转录聚合酶链反应显示, 22(R)-羟基胆固醇可增加THP-1巨噬细胞源性泡沫细胞LXRα mRNA的表达, DIDS可抑制THP-1巨噬细胞源性泡沫细胞LXRα mRNA的表达.结果提示,LXRα在巨噬细胞源性泡沫细胞胆固醇流出中起着重要的调控作用,这为开发和寻找抗动脉粥样硬化药物提供了新的思路.     

禾谷缢管蚜ABC转运蛋白基因的克隆、分子特性及表达分析

【目的】ABC（ATP-binding cassette）转运蛋白是一类重要的跨膜蛋白超家族,某些ABC转运蛋白基因在一些害虫的抗药性品系中表达显著提高。本研究旨在克隆禾谷缢管蚜 Rhopalosiphum padi ABC转运蛋白基因RhpaABCG9,RhpaABCG20和RhpaABCG23 cDNA全序列,分析这3个基因在该虫不同发育阶段和不同抗药性品系中的表达模式,为阐明ABC转运蛋白在禾谷缢管蚜抗药性中的作用和其他生理功能,以及深入分析该虫抗药性机理奠定基础。【方法】采用RT-PCR与RACE技术,克隆了基因cDNA全序列;利用实时荧光定量PCR技术,研究这3个基因在禾谷缢管蚜不同生长发育阶段和不同抗药性品系中的表达变化。【结果】获得了RhpaABCG9,RhpaABCG20和RhpaABCG23 3个基因cDNA全序列,其开放阅读框长度分别为2 103,2 436 和2 082 bp,分别编码700, 811和693个氨基酸。结构分析表明,3个蛋白均具有ABC转运蛋白家族典型的结构特征;系统进化分析结果显示,3个蛋白氨基酸序列分别与豌豆蚜Acyrthosiphon pisum各对应氨基酸的序列一致性最高。实时荧光定量PCR分析结果表明,这3个基因在禾谷缢管蚜不同发育时期均不同程度表达。RhpaABCG20在各个发育时期的表达变化差异不显著;RhpaABCG9表达量在4龄若蚜最高,在1龄若蚜最低;RhpaABCG23 表达量在3龄若蚜最高,1龄若蚜最低,其他阶段差异不显著。禾谷缢管蚜异丙威抗性品系中,RhpaABCG20表达量显著高于敏感品系,而RhpaABCG9和RhpaABCG23表达量均低于敏感品系,但差异不显著;禾谷缢管蚜吡虫啉抗性品系中,RhpaABCG20和RhpaABCG23表达量显著高于敏感品系,而RhpaABCG9表达上调不显著。【结论】RhpaABCG9,RhpaABCG20和RhpaABCG23基因可能参与禾谷缢管蚜体内农药的运输,并与禾谷缢管蚜的抗药性具有一定的关系。本研究结果为进一步深入分析禾谷缢管蚜的抗药性机制,以及该虫的抗药性治理与综合防治奠定了基础。     

On the regulatory role of side-chain hydroxylated oxysterols in the brain. Lessons from CYP27A1 transgenic and Cyp27a1−/− mice

The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27^−/− mice, cholesterol synthesis was slightly increased, with increased levels of cholesterol precursors but normal mRNA levels of HMG-CoA reductase and HMG-CoA synthase. The mRNA levels corresponding to LXR target genes were not affected. The results are consistent with the possibility that both 24OH and 27OH are physiological suppressors of cholesterol synthesis in the brain. The results do not support the contention that 27OH is a general activator of LXR target genes in this organ.     

核受体孕烷X 受体在药物诱导性肝损伤中的作用

肝脏在转化、清除异源物的过程中发挥重要作用。持续暴露于高浓度的异源物会导致肝功能受损,称为药物诱导性肝损伤(drug-induced liver injury, DILI)。核受体超家族成员孕烷X受体(pregnane X receptor, PXR)作为肝脏药物清除系统的重要调控因子,通过调节药物代谢酶(drug metabolizing enzymes, DMEs)和转运体的表达,在肝脏药物转化、代谢与清除中发挥重要作用。研究证明PXR参与DILI的发生发展。本文旨在阐述PXR在DILI中的作用及其机制研究进展。     

A Simple Gasometric Method for Determination of Free Amino Acid Contents in Biological Fluids

A simple method for the determination of total amino acid contents in biological fluids which contain various amino acids, peptides and proteins is described. This method is based on the determination in a Gilson differential respirometer of CO₂ gas evolved by the reaction of chloramin-T with α-amino-carboxylic acids in 5% trichloroacetic acid. The use of the trichloroacetic acid greatly suppressed interference by amines and ammonia, thus making this method appreciable to the determination of free amino acids in biological fluids without prior separation. Samples containing 1~20 μmoles of free amino acids can be assayed with accuracy of ±3%. This method was applied to the study of proteolytic digestion of casein.