人松弛素原样蛋白H2在大肠杆菌中的表达、分离纯化及鉴定

利用聚合酶链式反应方法 ,从含有人前松弛素原cDNA基因的质粒 pMALp2X-hRLXH2上得到人松弛素原H2的编码基因 ,亚克隆到温度诱导型原核表达载体pBV2 2 0上 ,转化大肠杆菌DH5α细胞。经 4 2℃温度诱导 ,获得了目的基因的高效表达。目的蛋白质以包含体的形式存在于大肠杆菌细胞中。菌体经过超声波破碎 ,包含体裂解 ,蛋白质的体外变性还原 ,复性 ,SephadexG 75凝胶过滤层析 ,反相快速蛋白质液相层析等一系列分离纯化过程 ,得到了高纯度的重组Met 人松弛素原样蛋白H2 ,得率约为 2～ 3mg/L。对纯化的目的蛋白质进行了SDS PAGE电泳纯度分析 ,氨基酸组成分析 ,通过基质辅助激光解吸电离飞行时间质谱法测得目的蛋白质的分子量为 183 90 .4 (理论计算值为 183 92 .3 )。     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

Comparative analysis of vascular endothelial cell activation by TNF-α and LPS in humans and baboons

As an Old World nonhuman primate, baboons have been extensively used for research on dyslipidemia and atherogenesis. With increasing knowledge about the endothelium's role in the initiation and progression of atherosclerosis, the value of the baboon model can be increased by developing it for research on the role of dysfunctional endothelium in atherogenesis. Toward that goal, we have established and validated methods of isolating and culturing baboon femoral artery endothelial cells (BFAECs) and compared baboon endothelial cellular characteristics with those of humans. Our results indicated that baboon and human endothelial cells share similar growth and culture behaviors. As was the case for human endothelial cells, BFAECs responded to tumor necrosis factor (TNF)-α stimulation with increased expression of adhesion molecules (maximum increase for intracellular adhesion molecule (ICAM): 1.76±0.26-fold; vascular cell adhesion molecule (VCAM): 1.65±0.25-fold; E-selectin: 2.86±0.57-fold). However, BFAECs were hyporesponsive to lipopolysaccharide (LPS) (range, 0.25–20 μg/mL) in adhesion molecule expression, whereas 1 μg/mL LPS induced 2.14- to 3.71-fold increases in human endothelial cells. The differential responses to LPS were not related to TLR-2 and toll-like receptor (TLR)-4 expression on the cell surface. And baboon microvascular endothelial cells had similar features as BFAECs. We observed constitutive expression of interleukin (IL)-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)-1 in both human and baboon endothelial cells, and these cytokines were further induced by TNF-α and LPS. We also demonstrated that the responses to TNF-α or LPS varied among baboons maintained under the same dietary and environmental conditions, suggesting that response may be controlled by genetic factors.     

马尾松雌球果的发生和早期发育研究

采用常规石蜡制片技术对马尾松雌球果的发生和早期发育进行了研究。结果表明：雌球果原基发生时间为10月中旬,不同的树龄和着生部位,其发生时间不同。雌球果原基与营养茎端在外部形态及内部细胞组织学分区结构有明显差异。营养茎端外形扁平,内部顶端分生组织结构有顶端原始细胞区、中央母细胞区、形成层状过渡区、周围分生组织区及肋状分生组织区5个明显的分区;而雌球果原基外形呈圆锥状,内部结构只有套层和髓区。12月初,最初的苞片原基在雌球果原基的鳞片的叶腋处产生,之后其由基部向顶部连续发生。翌年1月初,在苞片原基的叶腋处,珠鳞原基发生,发生方向亦为向顶发育。2月底,苞片体积不再发生变化,珠鳞膨大端的基部的近轴面分化出2个倒生胚珠。从雌球果原基发生到胚珠分化历时4个多月。亚热带的冬季气候对马尾松雌球果的生长发育没有明显的抑制作用。     

Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen

The mechanisms underlying better immune protection by mucosal vaccination have remained poorly understood. In our current study we have investigated the mechanisms by which respiratory virus-mediated mucosal vaccination provides remarkably better immune protection against pulmonary tuberculosis than parenteral vaccination. A recombinant adenovirus-based tuberculosis (TB) vaccine expressing Mycobacterium tuberculosis Ag85A (AdAg85A) was administered either intranasally (i.n.) or i.m. to mice, and Ag-specific CD4 and CD8 T cell responses, including frequency, IFN-gamma production, and CTL, were examined in the spleen, lung interstitium, and airway lumen. Although i.m. immunization with AdAg85A led to activation of T cells, particularly CD8 T cells, in the spleen and, to a lesser extent, in the lung interstitium, it failed to elicit any T cell response in the airway lumen. In contrast, although i.n. immunization failed to effectively activate T cells in the spleen, it uniquely elicited higher numbers of Ag-specific CD4 and CD8 T cells in the airway lumen that were capable of IFN-gamma production and cytolytic activities, as assessed by an intratracheal in vivo CTL assay. These airway luminal T cells of i.n. immunized mice or splenic T cells of i.m. immunized mice, upon transfer locally to the lungs of naive SCID mice, conferred immune protection against M. tuberculosis challenge. Our study has demonstrated that the airway luminal T cell population plays an important role in immune protection against pulmonary TB, thus providing mechanistic insights into the superior immune protection conferred by respiratory mucosal TB vaccination.     

局灶性脑缺血再灌损伤时神经细胞内Ca~(2+)时空动态变化的实验研究

本文用插线法制作局灶性脑缺血／再灌损伤模型,利用激光共聚焦扫描显微镜观察活体脑片细胞内Ｃａ２＋的分布及动态变化,结果表明：（１）缺血／再灌时间不同,梗塞面积不同,缺血４小时梗塞面积占同侧半球的１６．３％,缺血４小时再灌２０小时梗塞面积增加到２５．９％,缺血２４小时梗塞面积占同侧半球的６０．４％。（２）本文首次观察到在缺血４小时纹状体区域的Ｃａ２＋变化明显高于皮层,并且再灌后皮层及纹状体区域Ｃａ２＋的含量明显增加     

三峡库区的渔业资源及保护

分析了三峡库区的水域特点,比较了1997-2004年鱼类的捕捞情况,说明三峡库区有着丰富的渔业资源。介绍了三峡库区的主要珍稀特产鱼类。三峡工程的修建,生态环境的破坏,对库区渔业资源造成了严重的威胁。加强对渔业资源的保护和科学合理的开发利用势在必行,有利于保护鱼类多样性,促进渔业资源的发展,维持库区生态平衡。     

深黄被孢霉△^6—脂肪酸脱氢酶基因在转基因烟草中的表达

γ—亚麻酸(GLA)是人体和动物饮食中具有营养作用的重要的多烯不饱和脂肪酸,在大多数油料作物种子中不含有GLA,而只含有其前体物亚油酸,只有少数油料植物种子中含有GLA,如夜来香(Oenothera spp),琉璃苣(Borago officinalis)等。△^6—脂肪酸脱氢酶可将亚油酸转化为γ—亚麻酸,为了能够在传统的油料作物种子中产生GLA,我们将从深黄被孢霉中克隆的△^6—脂肪酸脱氢酶基因,与植物表达载体pGA643连接,构建了重组质粒pGAM—ICL6,将其通过农杆菌介导法,导入模式植物烟草中。经PCR和Southern杂交分析表明该基因已导入并整合到烟草的基因组中,Northern杂交结果表明该基因在转基因烟草的mRNA水平上获得表达。对转基因植株进行脂肪酸分析,结果显示,GLA和十八碳四烯酸(OTA)分别占总脂肪酸含量的19．7％和3．5％。     

含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白