COPII mitigates ER stress by promoting formation of ER whorls

Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.Subject terms: Endoplasmic reticulum, Collective cell migration     

The influence of RNA integrity,purity and cDNA labelling on glass slide cDNA microarray image quality

The accuracy of gene expression measurements generated using cDNA microarrays is dependent on the quality of the image generated following hybridization of fluorescently labelled cDNA. It is not known how this image is influenced by sample preparation factors which such as RNA quality, cDNA synthesis and labelling efficiency. In this study we used a simple metric based on the ratio of the total feature (F) and background (B) fluorescence, which correlates with the visual assessment of 60 microarray images, to determine the influence of sample preparation on image quality. Results indicate that RNA purity (A260/A280) and integrity (18S:28S ratio) do not strongly influence microarray image quality. cDNA having an nucleotide to dye ratio greater than 100 produced poor microarray images, however, cDNA labelled more efficiently was not a guarantee of a better image. The data also indicate that the array image quality is not improved by loading more cDNA into the hybridization mixture however poor image quality did result from a disproportionate amounts of Cy5 and Cy3 labelled cDNA. This study provides insight into the source of variation in microarray image analysis introduced during sample preparation and will assist in the standardisation of cDNA glass slide microarray protocols.     

蛋白G IgG Fc段结合域的克隆表达及功能研究

旨在研究蛋白G IgG Fc段结合域(PGFB)的克隆、表达及其抗体结合功能,用于抗体的纯化.根据PGFB的氨基酸序列,选择大肠杆菌偏爱的密码子,设计并合成了4个寡核苷酸片段.通过重叠延伸PCR方法合成了PGFB DNA片段,测序鉴定后克隆至原核表达系统pET-28a-c(+)上,转化大肠杆菌,获得表达菌株;IPTG诱导表达PGFB,经Ni+-NTA琼脂糖凝胶层析纯化后偶联到琼脂糖凝胶6B上,用其纯化多克隆抗体.结果显示,PGFB在大肠杆菌BL21(DE3)中获得高效表达,纯化后纯度达到90%以上,相对分子量为12.25 kD,与预期值相符.此外,偶联产物纯化多克隆抗体达到了良好的效果,每毫升基质可结合20 mg抗体.本研究克隆构建并高效表达了具有较好抗体亲和能力的PGFB,为多克隆抗体的快速纯化提供了方便.     

Spinal cord injury increases iron levels: catalytic production of hydroxyl radicals

This study used a weight drop impact injury model to explore the role of iron and the reality of iron-catalyzed hydroxyl radical ((*)OH) formation in secondary spinal cord injury (SCI). The time course of total extracellular iron was measured following SCI by microcannula sampling and atomic absorption spectrophotometry analysis. Immediately following SCI, the total iron concentration increased from an undetectable level to an average of 1.32 microM. The time course of SCI-induced (*)OH-generating catalytic activity in the cord was obtained by determining the ability of tissue homogenate to convert hydrogen peroxide to (*)OH and then measuring 2,3-dihydroxybenzoic acid, a hydroxylation product of salicylate. The concentration of 2,3-DHBA quickly and significantly increased (p <.001) and returned to sham level (p = 1) by 30 min post-SCI. Desferrioxamine (80 and 800 mg/kg body weight) significantly (p <.001) reduced the catalytic activity, suggesting that iron is the major contributor of the activity. Administering FeCl(3) (100 microM)/EDTA (0.5 mM) in ACSF into the cord through a dialysis fiber significantly increased SCI-induced (*)OH production in the extracellular space, demonstrating that Fe(3+) can catalyze (*)OH production in vivo. Our results support that iron-catalyzed (*)OH formation plays a role in the early stage of secondary SCI.     

Preorientation of protein and RNA just before contacting

Protein and RNA molecules interact and form complexes in many biological processes. However, it is still unclear how they can find the correct docking direction before forming complex. In this paper, we study preorientation of RNA and protein separated at a distance of 5–7?Å just before they form contacts and interact with each other only through pure electrostatic interaction when neglecting the influence of other molecules and complicated environment. Since geometric complementary has no meaning at such a distance, this is not a docking problem and so the conventional docking methods, like FTDock, are inapplicable. However, like the usual docking problem, we need to sample all the positions and orientations of RNA surrounding the protein to find the lowest energy orientations between RNA and protein. Therefore, we propose a long-range electrostatic docking-like method using Fast Fourier Transform-based sampling, LEDock, to study this problem. Our results show that the electrostatically induced orientations between RNA and protein at a distance of 5–7?Å are very different from the random ones and are much closer to those in their native complexes. Meanwhile, electrostatic funnels are found around the RNA-binding sites of the proteins in 62 out of 78 bound protein–RNA complexes. We also tried to use LEDock to find RNA-binding residues and it seems to perform slightly better than BindN Server for 23 unbound protein–RNA complexes.     

Pattern and drivers of speciesgenetic diversity correlation in natural forest tree communities across a biodiversity hotspot

Aims Exploring species-genetic diversity correlation (SGDC) is essential for understanding spatial patterns of diversity and the underlying mechanisms. Until now, latitudinal patterns of species diversity (SD) and genetic diversity (GD) were rarely studied simultaneously. As the freezing-tolerance hypothesis predicts a decrease of SD from low to high latitudes and the central-marginal hypothesis predicts a unimodal pattern of GD along latitude, we hypothesized that SD and GD are uncorrelated. We also tested how climatic and edaphic factors affect the correlation between the two levels of biodiversity.     

海洋生态资本理论框架下的生态系统服务评估

海洋生态资本指能够直接或间接作用于人类社会经济生产、提供有用的产品流或服务流的海洋生态资源。海洋生态资本价值由海洋生态资源存量价值和海洋生态系统服务价值组成。海洋生态资本评估包括海洋生态资源存量评估和海洋生态系统服务评估。在海洋生态资本理论框架体系下,针对我国近海生态系统服务的开发与利用情况,建立了评估海洋生态系统服务的物质量和价值量的技术框架。基于物质量可量化、价值量可货币化、数据可获得性三条评估原则,筛选出9个指标定量评估海洋生态系统服务的物质量和价值量,并给出了对应的评估方法、计算公式、参数和数据来源。海洋供给服务采用养殖生产、捕捞生产和氧气生产3个指标评估;海洋供给服务采用气候调节、废弃物处理2个指标评估;海洋文化服务采用休闲娱乐、科研服务2个指标评估;海洋支持服务采用物种多样性维持、生态系统多样性维持2个指标评估。养殖生产、捕捞生产等指标采用市场价格法进行评估;氧气生产、废弃物处理、科研服务等指标采用替代成本法进行评估;气候调节指标采用替代市场价格法进行评估;休闲娱乐指标采用旅行费用法或收入替代法进行评估;物种多样性维持、生态系统多样性维持等指标采用条件价值法进行评估。该套方法体系已经应用于山东省7个沿海地级市和福建省东山湾、罗源湾的近海生态系统服务价值评估,已经得到学术界和海洋管理部门的认可,被国家标准《海洋生态资本评估技术导则》吸收采用。该套方法紧密切合国家生态文明建设需求,可为海洋主管部门的生态资本核算、生态补偿业务、环评审批提供关键技术手段,也为海洋生态系统服务的精确评估提供了科学基础。