计算机辅助设计可溶性BLyS受体, eBCMA-Fc 融合蛋白及其在大肠杆菌中的表达

B 细胞成熟抗原 (BCMA)是 B淋巴细胞刺激因子(BLyS)的受体之一．它的胞外区与人IgG1 Fc的融合蛋白eBCMA-Fc,又称为诱饵受体,具有拮抗BLyS的活性．为了设计新的拮抗肽,基于BCMA和Fc的晶体结构,通过计算机图形学技术、分子模拟方法,建立了eBCMA-Fc融合蛋白的三维理论结构．利用均方根位移（root mean square distance, RMSD）对eBCMA-Fc融合蛋白与单体eBCMA、Fc构象差异进行分析．融合蛋白eBCMA-Fc中的eBCMA段与单体eBCMA的主链碳原子间RMSD值为0.036 nm,Fc段与单体Fc的主链碳原子间RMSD值为0.064 nm．结果表明,对比单体,融合蛋白eBCMA-Fc并未因eBCMA与Fc直接连接而发生构象的变化．分子对接方法显示,融合蛋白eBCMA-Fc中的BCMA与BLyS作用,而Fc扮演着稳定BCMA构象的支架作用．为进一步验证上述理论分析,构建eBCMA-Fc融合基因,并将载有eBCMA-Fc融合基因的原核表达质粒转化BL21 (DE3)菌、在细菌中表达．目的蛋白经蛋白A亲和柱纯化大约为36 kD,与理论预测值34 kD相近．免疫印迹表明抗人IgG抗体能够识别eBCMA-Fc融合蛋白．ELISA证实,eBCMA-Fc融合蛋白能够结合BLyS．随着eBCMA-Fc融合蛋白增加,结合BLyS的融合蛋白也相应增加．而对照人IgG,即使在高浓度条件下,也不结合BLyS．此外,eBCMA-Fc 融合蛋白能够抑制BLyS对B细胞肿瘤Daudi细胞的作用．这些研究为下一步设计和筛选BLyS拮抗肽提供了实验基础．     

Nerve growth factor promotes formation of lumen-like structures in vitro through inducing apoptosis in human umbilical vein endothelial cells

Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.     

Contrasting effects of plant inter‐ and intraspecific variation on community trait responses to restoration of a sandy grassland ecosystem

Changes in plant community traits along an environmental gradient are caused by interspecific and intraspecific trait variation. However, little is known about the role of interspecific and intraspecific trait variation in plant community responses to the restoration of a sandy grassland ecosystem. We measured five functional traits of 34 species along a restoration gradient of sandy grassland (mobile dune, semi‐fixed dune, fixed dune, and grassland) in Horqin Sand Land, northern China. We examined how community‐level traits varied with habitat changes and soil gradients using both abundance‐weighted and non‐weighted averages of trait values. We quantified the relative contribution of inter‐ and intraspecific trait variation in specific leaf area (SLA), leaf dry matter content (LDMC), leaf carbon content (LCC), leaf nitrogen content (LNC), and plant height to the community response to habitat changes in the restoration of sandy grassland. We found that five weighted community‐average traits varied significantly with habitat changes. Along the soil gradient in the restoration of sandy grassland, plant height, SLA, LDMC, and LCC increased, while LNC decreased. For all traits, there was a greater contribution of interspecific variation to community response in regard to habitat changes relative to that of intraspecific variation. The relative contribution of the interspecific variation effect of an abundance‐weighted trait was greater than that of a non‐weighted trait with regard to all traits except LDMC. A community‐level trait response to habitat changes was due largely to species turnover. Though the intraspecific shift plays a small role in community trait response to habitat changes, it has an effect on plant coexistence and the maintenance of herbaceous plants in sandy grassland habitats. The context dependency of positive and negative covariation between inter‐ and intraspecific variation further suggests that both effects of inter‐ and intraspecific variation on a community trait should be considered when understanding a plant community response to environmental changes in sandy grassland ecosystems.     

荧光定量RT-PCR检测血管内皮细胞PAI-1 mRNA方法的建立

为了建立用荧光定量PCR检测人微血管内皮细胞纤溶酶原激活剂抑制物-1（PAI-1）mRNA表达的方法。提取人微血管内皮细总RNA,经RT-PCR获得靶基因（PAI-1）及管家基因（β-actin）的PCR产物。纯化后,作为标准品梯度稀释,采用SYBR Green I定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、灵敏性和重复性。分析全反式维甲酸对内皮细胞表达PAI-1 mRNA的干预效果。这种定量方法特异性好,检测的灵敏度达10^3拷贝,线性范围为10^4-10^10拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系（r^2〉0.990）,批内变异≤4.93%,批间变异≤9.12%。全反式维甲酸能上调内皮细胞PAI-1 mRNA的表达,且呈剂量依赖性。因此,荧光定量RT-PCR检测血管内皮细胞PAI-1基因表达的方法有助于溶栓药物药理学的研究和新药筛选。     

越冬代棉大卷叶螟的种群动态

对棉大卷叶螟Sylepta derogata Fabricius第5代、第6代室内种群饲养结果显示,该虫的有效越冬虫源为第5代滞育的老熟幼虫,滞育率24.4%～33.1%,第6代棉大卷叶螟的发生对第2年的种群基数影响不大。滞育老熟幼虫的越冬存活率为60.0%～71.9%。次年越冬代羽化的成虫雌虫少,雄虫多,单雌产卵量平均为163.4～198.8粒。     

Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.     

金钱鱼毒腺cDNA表达文库的构建及EST序列分析

以金钱鱼 (Scatophagusargus)毒腺为出发材料 ,构建以真核表达载体pcDNA3 0为基础的金钱鱼毒腺cDNA表达文库 .应用SMARTTM cDNALibraryConstruction技术和生物信息学分析等方法 ,通过对文库克隆的序列测定和初步生物信息学分析 ,获得了 2 0 1个金钱鱼新表达序列标签 (ESTs) ,其中已确定全长cDNA的克隆有 2 7个 ,包括多个核糖体大小亚基蛋白 (ribosomalprotein)、细胞凋亡相关蛋白 (programmedcelldeath 10 ) ,G蛋白信号调控子 (Gproteinsignalingregulator)、胸腺素(thymosinbeta 4 )、延伸因子 (translationelongationfactor 1 alpha)和泛素 (ubiquitin)等 .金钱鱼毒腺cDNA表达文库的成功构建 ,为研究金钱鱼毒腺的活性组分及其作用机制奠定了基础 ,也是分离新基因不可多得的重要资源     

Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1

Heparan sulfate (HS) is a highly sulfated polysaccharide and is present in large quantities on the cell surface and in the extracellular matrix. Herpes simplex virus type 1 (HSV-1) utilizes a specialized cell surface HS, known as 3-O-sulfated HS, as an entry receptor to establish infection. Here, we exploit an approach to inhibiting HSV-1 infection by using a 3-O-sulfated octasaccharide, mimicking the active domain of the entry receptor. The 3-O-sulfated octasaccharide was synthesized by incubating a heparin octasaccharide (3-OH octasaccharide) with HS 3-O-sulfotransferase isoform 3. The resultant 3-O-sulfated octasaccharide has a structure of Delta UA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS3S6S-IdoUA2S-GlcNS6S (where Delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is D-glucosamine, and IdoUA is L-iduronic acid). Results from cell-based assays revealed that the 3-O-sulfated octasaccharide has stronger activity in blocking HSV-1 infection than that of the 3-OH octasaccharide, suggesting that the inhibition of HSV-1 infection requires a unique sulfation moiety. Our results suggest the feasibility of inhibiting HSV-1 infection by blocking viral entry with a specific oligosaccharide.     

WSF-P-1, a novel AMPK activator,promotes adiponectin multimerization in 3T3-L1 adipocytes

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). The HMW adiponectin oligomers possess the most potent insulin-sensitizing activity. WSF-P-1(N-methyl-1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-7H-2,4α-methanonaphthalen-7-amine) is derived from natural sesquiterpene longifolene by chemical modifications. We found that WSF-P-1 activates AMPK in both 3T3-L1 adipocytes and 293T cells in this study. Activation of AMPK by WSF-P-1 promotes the assembly of HMW adiponectin and increases the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. We demonstrated that the Ca²⁺-dependent CaMKK signaling pathway is involved in WSF-P-1-induced AMPK activation and adiponectin multimerization. WSF-P-1 also activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes, making it a potential drug candidate for the treatment of type 2 diabetes, obesity, and other obesity-related metabolic diseases.     

Hierarchically Structured 3D Integrated Electrodes by Galvanic Replacement Reaction for Highly Efficient Water Splitting

A NiFe‐based integrated electrode is fabricated by the spontaneous galvanic replacement reaction on an iron foam. Driven by the different electrochemical potentials between Ni and Fe, the dissolution of surface Fe occurs with electroless plating of Ni on iron foam with no need to access instrumentation and input energy. A facile cyclic voltammetry treatment is subsequently applied to convert the metallic NiFe to NiFeO_x . A series of analytical methods indicates formation of a NiFeO_x film of nanosheets on the iron foam surface. This hierarchically structured three dimensional electrode displays high activity and durability against water oxidation. In 1 m KOH, a current density of 1000 mA cm^?2 is achieved at an overpotential of only 300 mV. This method is readily extended to fabricate CoFe or NiCoFe‐based integrated electrodes for water oxidation. Phosphorization of the bimetallic oxide (NiFeO_x ) generates the bimetallic phosphide (NiFe‐P), which can act as an excellent electrocatalyst for hydrogen production in 1 m KOH. An alkaline electrolyzer is constructed using NiFeO_x and NiFe‐P coated iron foams as anode and cathode, which can realize overall water splitting with a current density of 100 mA cm^?2 at an overpotential of 630 mV.