酿酒酵母胞内无机焦磷酸酶的分离纯化及性质

An inorganic pyrophosphatase (EC3.6.1.1) from Saccharomyces cerevisiae was purified to PAGE homogeneity by sonication disruption. (NH4)2SO4 fractionation and DEAE-cellulose colunm chromatography. The optimum pH and temperature of the enzyme were 7.4～7. 8 and 60℃, respectively. The Km was 19.3 mmol / L. The enzyme required Mg2+ as a cofactor for hydrolysis of pyrophosphate and was inhibited by Ca2+, Hg2+, Pb2+, Mn2+.     

马槟榔甜蛋白基因(MBLⅡ)的拼接与表达载体的构建

从云南植物Capparismasaikai中提取总DNA,设计引物克隆马槟榔甜蛋白基因MBLⅡ,序列测定后经同源性分析表明与GeneBank中登陆的马槟榔甜蛋白基因MBLⅡ(cDNA)序列一致,表明MBLⅡ不存在内含子序列。利用重叠区扩增基因拼接法(genesplicingbyoverlapextension,geneSOEing)进行体外基因剪接,剪接片段与植物表达载体pVKH相连,构建pVKH-35S-MBLⅡ-Pa、pVKH-35S-S-A B-pA、pVKH-35S-A B-pA、pVKH-35S-S-A-pA、pVKH-35S-S-B-pA,为寻找mabinlin的活性表达形式和翻译后剪接机制打下基础。     

中国汉族人群间-α-胰蛋白酶抑制因子(ITI)的遗传多态性

我们建立了测定间-α-胰蛋白酶抑制因子遗传表型的聚丙烯酰胺凝胶等电聚焦免疫固定技术,应用这种方法对居住在成都地区的汉族群体进行了群体研究和家系调查,结果表明中国汉族人群间-α-胰蛋白酶抑制因子具有遗传多态性,它有希望成为法医血液遗传学新的遗传标记。     

烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响

大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶 (NAPRTase) 的编码基因,通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+,从而恢复了厌氧条件下重组菌E. coli BA014 (BA002/pTrc99a-pncB) 的生长和产丁二酸的性能。然而,BA014在厌氧发酵过程中有大量丙酮酸积累,为进一步提高菌株的丁二酸生产能力,减少副产物丙酮酸的生成,共表达NAPRTase和来自于乳酸乳球菌 NZ9000中丙酮酸羧化酶 (PYC) 的编码基因pyc,构建了重组菌E. coli BA016 (BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明,BA016发酵112 h后,共消耗了35.00 g/L的葡萄糖。发酵结束时,菌体OD600为4.64,产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因,使BA016的丙酮酸积累进一步降低,丁二酸产量进一步提高。     

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.     

作物抗除草剂转基因研究进展

综述了抗除草剂基因研究的意义、研究机理及国内外研究现状,分析了各种转基因方法的优缺点,展望了除草剂抗性育种的发展方向。     

神经元限制性沉默因子对人胰岛素基因转录调控作用的研究

为了研究神经元限制性沉默因子(NRSF)调控神经元及胰岛细胞中神经特异性基因的表达,进一步寻找胰岛细胞中可能存在的其他NRSF调控基因.先用生物信息学手段对相关基因进行了分析.筛选及序列比对发现,人胰岛素核心启动子区有一段与NRSE相似的序列,提示,它可能受NRSF调控.构建了含NRSF基因的慢病毒载体,将其稳定转染于INS-1细胞.构建了3种荧光素酶报告载体:含有人胰岛素启动子-荧光素酶(hInsP-LUC)的慢病毒载体,pGL3-Basic载体和含有2拷贝NRSE样基序-荧光素酶(NRSE-LUC)的报告载体.利用稳定转染及瞬时转染实验观察NRSF对报告载体中荧光素酶活性的影响.利用电泳迁移率变动分析实验观察NRSE样基序与NRSF蛋白的结合情况,并通过竞争结合实验、引入特异性抗体实验证实探针与蛋白质结合的特异性.RT-PCR检测证实,感染空病毒的INS-1细胞不表达NRSF,感染含目的基因慢病毒的INS-1细胞能表达NRSF.将含有hInsP-LUC的慢病毒载体稳定转染于上述2种细胞,荧光素酶活性分析结果显示,NRSF的过表达能明显降低胰岛素启动子的活性.瞬时转染hInsP-LUC报告系统于上述2种细胞,结果也显示NRSF能明显抑制胰岛素启动子-荧光素酶的活性.将含有NRSE-LUC的报告载体瞬时转染于上述2种细胞,结果表明过表达NRSF的INS-1细胞组的荧光素酶相对值比对照组有明显下降.电泳迁移率变动分析实验进一步证实,此NRSE样序列可以与NRSF蛋白特异结合,这种特异结合可以被标准的NRSE序列所竞争.结果表明,人胰岛素启动子中含有NRSE样序列,该序列通过与NRSF蛋白结合从而抑制人胰岛素启动子的转录活性.这一研究工作有助于进一步了解NRSF在胰岛细胞中的调控作用.     

Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux

Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis.     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞