NOX4-Derived Reactive Oxygen Species Drive Apelin-13-Induced Vascular Smooth Muscle Cell Proliferation via the ERK Pathway

Apelin is the endogenous ligand of the G-protein-coupled receptor, apelin–angiotensin receptor-like 1 (APJ). Vascular smooth muscle cells express both apelin and APJ, which are important regulatory factors in the cardiovascular system. Apelin-13 significantly stimulated vascular smooth muscle cell proliferation. However, little is known about the precise cellular mechanisms responsible for vascular smooth muscle cell proliferation induced by apelin-13. Here, we present novel data that indicate the key role of NADPH oxidase 4-derived reactive oxygen species in proliferation of vascular smooth muscle cells treated with apelin-13. Apelin-13 stimulated reactive oxygen species production in a concentration- and time-dependent manner. Furthermore, DPI impaired apelin-13-induced reactive oxygen species generation and vascular smooth muscle cell proliferation. Apelin-13-treatment increased the expression of NADPH oxidase 4 in a dose-dependent manner. Down-regulation of NADPH oxidase 4 using siRNA prevented apelin-13-induced reactive oxygen species generation and vascular smooth muscle cell proliferation. An increase in reactive molecules can trigger the activation of ERK stress-sensitive signaling pathways. Additionally, siRNA-NOX4 and DPI reversed the phosphorylation of ERK induced by apelin-13. Apelin-13 induced vascular smooth muscle cell proliferation by NOX4-derived ROS via the ERK signaling pathway.     

球囊菌胁迫中华蜜蜂幼虫肠道过程中病原的转录组学研究

【目的】本研究利用RNA-seq技术对球囊菌胁迫的中华蜜蜂(中蜂)幼虫肠道进行深度测序,经趋势分析得到差异表达基因(DEGs)的显著表达模式,进而对胁迫过程中的球囊菌进行转录组学分析。【方法】利用Illumina HiSeq 2500平台对球囊菌胁迫的中蜂幼虫肠道进行深度测序,并利用相关软件进行了深入分析。最后,通过RT-qPCR对RNA-seq数据进行了验证。【结果】本研究共得到球囊菌的41133932条高质量clean reads。22865个DEGs共聚类为8个基因表达模式,其中,16769个DEGs聚类为2个显著上调趋势与2个显著下调趋势。GO富集分析结果显示,显著上调与显著下调趋势中的DEGs分别富集于40与37个GO term,基因富集数最多的为细胞进程(2486 unigenes)。KEGG代谢通路(pathway)富集分析结果显示,显著上调与显著下调趋势中的DEGs分别富集于119和112个pathway,基因富集数最多的分别是氨基酸生物合成(127 unigenes)与核糖体(98 unigenes)。进一步分析表明球囊菌在胁迫中蜂幼虫肠道的过程中通过提高物质合成促进其增殖,而宿主通过抑制球囊菌的蛋白合成抵御病原入侵。富集在MAPK信号通路的11个DEGs的表达水平随着胁迫时间的延长而逐渐下降,推测中蜂幼虫通过抑制该通路而阻遏球囊菌增殖。【结论】本研究不仅为揭示白垩病过程中的球囊菌-中蜂幼虫互作提供了重要信息,也为阐明不同抗性蜂种的球囊菌抗性差异奠定了基础。     

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228

Metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of β-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine β-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of β-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).     

异羟基洋地黄毒甙元(Digoxigenin)标记的RNA探针在丁型肝炎病毒核酸杂交中的应用

应用异羟基洋地黄毒甙元(Digoxigenin)标记的RNA探针,检测了人和黑猩猩血清及肝脏中丁型肝炎病毒核酸,并与~(32)P标记同一探针做了比较。结果表明,异羟基洋地黄毒甙元标记的RNA探针的杂交效果与同位索探针一致(同源cDNA0.2pg),可用于人和动物血清及肝脏标本内HDV核酸的检测。     

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana.However,the leaf development that is specific to plants has been little studies.With the aim of contributing to the genetic dissection of leaf development,we have performed a large-scare screening for mutants with abnormal leaves.Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis,four independent mutant lines have been identified and studied genetically.Phenotypes of these mutant lines represent the defects of four novel muclear genes designated LL1(LOTUS LEAF 1),LL2(LOTUS LEAF2),URO(UPRIGHT ROSETTE),and EIL(ENVIRONMENT CONDITION INDUCED LESION).The phenotypic analysis indicates that these genes play important roles during leaf development.For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2,we have mapped these genes to chromosome regions with an efficient and rapid mapping method.     

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.     

Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

应用结构方程模型解析影响黄连木果实产量 和种子命运的因素

果实(种子)产量和质量是影响植物种群更新的重要因素。为了探明影响黄连木果实产量和种子命运的因素以及这些影响因素之间的相互作用, 作者于2009年对河南省济源市45株黄连木(Pistacia chinensis)结果样树的植株特征、果实特征、果实产量和种子命运等进行了测定, 并用结构方程模型进行综合分析。结果表明: (1)黄连木果实产量与树高、树冠面积和果序大小等特征成正相关, 但与胸径、果实大小相关性不显著; (2)与捕食者饱和假说的预测不一致, 单株果实产量对黄连木广肩小蜂(Eurytoma plotnikovi)的种子捕食率(即虫蛀率)无显著直接负向效应; (3)树高和果实大小对虫蛀率为显著直接正向效应, 胸径对虫蛀率为显著直接负向效应, 显示黄连木广肩小蜂对植株特征和果实特征有一定的选择能力; (4)空壳率与虫蛀率成显著负相关, 空壳果实越多, 越易逃避黄连木广肩小蜂的寄生, 空壳果实的存在对完好种子起到了一定保护作用, 可能是黄连木防御昆虫寄生的重要机制; (5)空壳率和虫蛀率对种子完好率有显著直接负向效应, 而胸径、果序大小和果实产量对完好率为间接正向效应, 树高和果实大小为间接负向效应。可见, 黄连木植株特征和果实特征均不同程度地影响其果实产量和昆虫寄生, 从而影响黄连木的种子质量和种群更新。     

肿瘤干细胞及其耐药机制

肿瘤干细胞是存在于造血系统肿瘤和一些实体瘤中具有干细胞特性的细胞。肿瘤干细胞假说认为,经药物治疗后肿瘤复发和转移与肿瘤干细胞残存有密切关系。其原因可能是肿瘤干细胞高表达ABC转运蛋白和Bcl-2抗凋亡蛋白,同时其本身又具有一些干细胞特性。对肿瘤干细胞耐药机制的研究,将有助于发现新的肿瘤治疗靶点和更好的抗癌策略。     

茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

[目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白（AZC_3085、AZC_3226和AZC_4658）进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。