动态分析沙鼠非酒精性脂肪肝病形成及生化影响

目的分析高脂饮食导致的沙鼠NAFLD疾病进展中脂质代谢、肝功能、抗氧化等方面的变化,探讨沙鼠NAFLD的形成机理。方法雄性沙鼠120只,随机分为对照组和模型组。对照组给予普通饲料,模型组高脂饮食建立NAFLD模型。分别于1、2、4、6、8、16周每组各处死10只动物,观察肝脏病理变化,计算肝指数,检测血清CHO、TG、LDL-C、HDL-C、GOP、GPT及肝组织的SOD、GSH-PX、CAT活性和FFA含量。结果模型组病理观察2周形成单纯性脂肪肝,6周动物门管区有轻度炎症,8周出现腺泡Ⅲ带局灶性窦周/细胞周纤维化,16周肝脏中度纤维化;模型组第1、2、4、6、8、16周时CHO、HDL-C、LDL-C及FAA波动性升高,但均高于同期对照组（P〈0.05,P〈0.01）,TG在1、2、4周高于同期对照组（P〈0.05,P〈0.01）;16周末GOT、GPT出现了显著性升高（P〈0.01）。抗氧化酶GSH-PX、CAT、SOD活性模型组在第8～16周显著性降低（P〈0.05,P〈0.01）。结论高脂饮食使沙鼠2周形成单纯性脂肪肝模型后,随饲喂时间延长,16周末可发展为中度肝纤维化。脂质代谢紊乱与氧化应激在沙鼠NAFLD进展中的发挥了不同的作用。     

长爪沙鼠的遗传多样性分析

利用17个微卫星DNA标记对Z:ZCLA长爪沙鼠封闭群、野生群和近交系进行遗传多样性分析, 评估群体内的遗传变异和群体间的遗传分化。结果表明：在Z:ZCLA封闭群和野生群中共有9个微卫星DNA标记获得稳定的结果, 分别为AF200940、AF200941、AF200942、AF200945、AF200946、AF200947、D11Mit128、PKC和 SCN, 共检测到41个等位基因, 每个基因的等位基因数从1～7不等, 片段大小在120～283 bp之间, 所有位点的平均期望杂合度(He)和多态信息含量(PIC)值分别为0.5032和0.4656, Z:ZCLA封闭群和野生群9个微卫星位点平均有效等位基因数分别为2.78和2.89, 平均基因杂合度分别为0.3704和 0.3893, 平均多态信息含量分别为0.3256和0.3344, 两个群体都表现为中度多态, Z:ZCLA封闭群较野生群稍低; 在3个近交系中共有8个位点获得稳定的扩增结果, 分别为AF200941、AF200942、AF200945、AF200946、AF200947、D11Mit128、PKC和 SCN, 共检测到11个等位基因, 片段大小在140~241 bp之间, 其中5个位点在群体内表现为单态纯合, 3个位点在群体内表现为单态杂合, 所有位点在群体内和群体间均呈单态性, 表明这3个长爪沙鼠品系基本符合近交系的要求, 微卫星标记技术适用于近交系长爪沙鼠的遗传检测。     

人TRAIL基因cDNA的克隆及其在COS-7细胞中的表达

TRAIL(TNF-related apoptosis inducing ligand)是最近克隆的肿瘤坏死因子(TNF)家族的新成员,由于它的蛋白质结构和生物学效应类似于FAS/APO-1L,因此,也被称为APO-2L.     

天麻抗真菌蛋白基因的克隆及其启动子活性

通过构建和筛选天麻(Gastrodia elata Bl.)基因组文库,克隆了一个天麻抗真菌蛋白基因组DNA.该基因组DNA含有一个516碱基组成的编码区,没有内含子结构.其启动子区含有保守的TATA盒及CAAT盒.为研究启动子活性,构建了-1 157 bp启动子区与GUS基因的融合表达载体.并将其用农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法导入烟草(Nicotiana tabacum)中,获得了稳定转化的烟草.利用荧光检测及组织化学染色法对GUS表达进行了分析.结果表明,该启动子能够启动GUS基因在转基因烟草中组织特异性地表达.GUS基因在根中的表达水平最高,茎中次之,叶中只有低水平表达.而且该启动子具有诱导表达活性,可被真菌及水杨酸、茉莉酸强烈诱导表达.     

非酒精性脂肪肝大鼠脂质代谢及病理变化的动态观察

目的通过高脂饮食建立NAFLD大鼠模型,连续监测4～16周模型动物肝功能、脂质代谢、胰岛素抵抗及肝细胞凋亡在NAFLD进展过程中的变化情况及相互关系,为该模型在脂肪肝发病机制、脂肪肝治疗药物评价等方面的应用提供参考依据。方法 SD大鼠50只,除正常对照组外,其余动物饲喂高脂饲料,分别检测4,8,12,16周大鼠血清GLU、CHO、TG、HDL、LDL、GPT、GOT及胰岛素水平,肝脏组织切片进行病理学及细胞凋亡观察,进一步分析大鼠肝功能、脂质代谢、胰岛素抵抗及肝细胞凋亡对肝组织病理改变的影响。结果模型组大鼠4周后就出现肝功能损伤,脂质代谢紊乱、胰岛素抵抗,肝细胞凋亡8 W后明显增加,肝细胞脂变及炎症为肝组织病理变化的主要特征,且造模时间越长,病变程度越严重。结论经过高脂饲料的喂养,SD大鼠在4～16周内可形成病变程度逐步加重的NAFLD模型,肝功能损伤,脂质代谢紊乱及肝细胞凋亡是引起非酒精性脂肪肝中脂肪变性和炎症的重要因素,该模型可应用于脂肪肝治疗药物评价等方面。     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

海洋浮游藻类细胞质膜氧化还原活性与细胞外CA活性的关系

海洋浮游藻类除通过吸收和释放分子与离子来改变其环境的化学成分外,还可通过细胞外表面一些酶的作用引起质膜外化学物质变化。在这方面,海洋浮游藻类一个主要的细胞外表面酶-碳酸酐酶(CA),在经胰蛋白酶处理从细胞质膜上释放出来后,仍保留其催化活性。当细胞外表面CA(简称细胞外CA)具活性时,可催化质膜外HCO_3~-与CO_2的相互转化,为Rubisco(磷酸核酮糖羧化酶)提供一稳定的CO_2流量环境,以维持正常的光合作用。     

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.