Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

利用简便微量法定量检测肠道正常菌群

利用简便微量法定量检测肠道正常菌群中的双歧杆菌和肠杆菌,10次检测结果与常规法无显著性差异。简便微量法主要优点是简便易行,节省人力物力和时间。     

Systems Mapping for Hematopoietic Progenitor Cell Heterogeneity

Cells with the same genotype growing under the same conditions can show different phenotypes, which is known as “population heterogeneity”. The heterogeneity of hematopoietic progenitor cells has an effect on their differentiation potential and lineage choices. However, the genetic mechanisms governing population heterogeneity remain unclear. Here, we present a statistical model for mapping the quantitative trait locus (QTL) that affects hematopoietic cell heterogeneity. This strategy, termed systems mapping, integrates a system of differential equations into the framework for systems mapping, allowing hypotheses regarding the interplay between genetic actions and cell heterogeneity to be tested. A simulation approach based on cell heterogeneity dynamics has been designed to test the statistical properties of the model. This model not only considers the traditional QTLs, but also indicates the methylated QTLs that can illustrate non-genetic individual differences. It has significant implications for probing the molecular, genetic and epigenetic mechanisms of hematopoietic progenitor cell heterogeneity.     

贺兰山两栖爬行动物的物种多样性及区系特征

为掌握贺兰山两栖爬行动物物种多样性及区系特征,于2007-2008年,采用样带调查法对贺兰山两栖爬行动物进行了系统调查。结果表明:贺兰山两栖爬行动物共计2目8科12属19种,其中王锦蛇(Elaphe carinata)和玉斑锦蛇(E.mandarina)为宁夏爬行动物新纪录种;贺兰山两栖爬行动物Shannon多样性指数为2.250,均匀性指数为0.563,其两栖爬行动物科数和种数分别占宁夏两栖爬行动物总科数和种数的72.7%和67.9%,分别占内蒙古两栖爬行动物总科数和种数的66.7%和50.0%;花背蟾蜍(Bufo raddei)、中国林蛙(Rana chensinensis)、荒漠沙蜥(Phrynocephalus przewalskii)、草原沙蜥(P.frontalis)、丽斑麻蜥(Eremias argus)和密点麻蜥(E.multiocellata)是贺兰山优势种,花条蛇(Psammophis lineo-latus)、黄脊游蛇(Coluber spinalis)和虎斑颈槽蛇(Rhabdophis tigrinus)为常见种,其余种类为偶见种;贺兰山两栖爬行动物物种中,14种为古北界物种,5种为广布种,且蒙新区物种成分优势明显,占42.6%,反映了贺兰山两栖爬行动物具有典型的蒙新区西部荒漠亚区的物种组成和区系特征。     

多肽融合标签的移除策略

多肽融合标签能够赋予目标蛋白新的特性,便于目标蛋白的定位、追踪、纯化以及结构和相互作用研究．但在很多情况下,尤其是研究蛋白质结构或分离纯化药用蛋白时,需要将多肽融合标签从融合蛋白上切除,以降低和消除多肽融合标签对目标蛋白结构和功能的影响．可用来切除多肽融合标签的方法主要有4种,即化学法、内切蛋白酶法、外切蛋白酶法以及自我剪切法．介绍和比较了每种方法的基本原理、应用以及研究进展．     

水牛MyD88cDNA的克隆与原核表达

采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88 (mydoid differentiation factor 88,MyD88) cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21 (DE3),经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析.结果显示,克隆到的水牛MyD88 cDNA全长为1 189 bp,含有1个891 bp的开放阅读框,编码296个氨基酸,理论等电点为5.65.经IPTG诱导表达后,得到一个带His·Tag的约39 kD的重组融合蛋白.用抗His单克隆抗体进行Western blotting,得到1条约39 kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达.本研究为进一步开展水牛MyD88的结构功能分析奠定了基础.     

A systems-biology analysis of feedback inhibition in the Sho1 osmotic-stress-response pathway

BACKGROUND: A common property of signal transduction systems is that they rapidly lose their ability to respond to a given stimulus. For instance in yeast, the mitogen-activated protein (MAP) kinase Hog1 is activated and inactivated within minutes, even when the osmotic-stress stimulus is sustained. RESULTS: Here, we used a combination of experimental and computational analyses to investigate the dynamic behavior of Hog1 activation in vivo. Computational modeling suggested that a negative-feedback loop operates early in the pathway and leads to rapid attenuation of Hog1 signaling. Experimental analysis revealed that the membrane-bound osmosensor Sho1 is phosphorylated by Hog1 and that phosphorylation occurs on Ser-166. Moreover, Sho1 exists in a homo-oligomeric complex, and phosphorylation by Hog1 promotes a transition from the oligomeric to monomeric state. A phosphorylation-site mutation (Sho1(S166E)) diminishes the formation of Sho1-oligomers, dampens activation of the Hog1 kinase, and impairs growth in high-salt or sorbitol conditions. CONCLUSIONS: These findings reveal a novel phosphorylation-dependent feedback loop leading to diminished cellular responses to an osmotic-stress stimulus.     

中华山蝠牙齿的脱换模式

笔者于1997年对中华山蝠(Nyctalus velutinus)牙齿的脱换模式进行了研究。共观察幼蝠200只次。中华山蝠初生仔共有22枚乳齿,齿式为2.1.2.0/3.1.2.0,各乳齿略向舌侧倾斜,除乳前臼齿齿冠不分叉外,其余乳齿齿冠均分为三叶。4d龄开始换齿,31d龄左右脱换完毕。乳齿的脱落顺序是:上颌,PM2→PM1→C1→I1→I2;下颌,I1→PM2.I2→I3→PM1→C1;恒齿萌生的顺序为:上颌,M1→PM2.C1→M2→PM1.M3→I1→I2,下颌,M1→I1.PM2→I2.M2→I3.PM1→C1→M3。     

Genetic analysis of IREB2, FAM13A and XRCC5 variants in Chinese Han patients with chronic obstructive pulmonary disease

Recently, variants (rs2568494, rs2869967 and rs3821104) in the IREB2, FAM13A and XRCC5 genes were found to be associated with chronic obstructive pulmonary disease (COPD) in non-Asian populations by genome-wide association study (GWAS) analysis. To evaluate whether variants in these genes are related to COPD in Chinese Han population, we investigated COPD patients of Chinese Han ethnicity from Mainland China. Significant differences in genotypic distributions (χ² = 6.319, p = 0.042 for rs2869967; χ² = 6.062, p = 0.048 for rs3821104) and allele distributions (χ² = 4.014, p = 0.045 for rs2869967; χ² = 5.607, p = 0.018 for rs3821104) were observed between patients and control subjects for variants rs2869967 and rs3821104, whereas no statistically significant associations for genotypic and allelic distribution between IREB2 rs2568494 and COPD phenotype (p > 0.05) were identified. Our results support that FAM13A rs2869967 and XRCC5 rs3821104 are associated with COPD in Chinese Han population.