马铃薯StPSKRs基因的克隆及其亚细胞定位分析

该研究采用同源克隆与PCR扩增方法,从马铃薯品种‘Desiree’中克隆植物磺肽素受体基因StPSKR1和StPSKR2的全长cDNA,并对其进行生物信息学分析及亚细胞定位分析,为深入研究StPSKR1和StPSKR2基因在马铃薯生长发育和生物胁迫中的作用提供理论依据。结果发现:(1)通过同源克隆与PCR扩增获得StPSKR1和StPSKR2的全长cDNA片段,并将其克隆到pGWB5-GFP载体;测序结果显示这2个基因编码的蛋白质与数据库给定的蛋白质序列保持一致,表明成功克隆到StPSKR1和StPSKR2基因。(2)StPSKR1位于马铃薯1号染色体上,cDNA全长3 042 bp,编码1 013个氨基酸,预测蛋白相对分子质量为112.16 kD,理论等电点6.27;StPSKR2位于7号染色体,cDNA全长3 135 bp,编码1 044个氨基酸,相对分子量为114.99 kD,理论等电点6.19。(3)生物信息学分析显示,StPSKR1和StPSKR2都属于跨膜蛋白。(4)亚细胞定位结果显示,StPSKR1和StPSKR2均定位于细胞膜上。     

Nitrogen removal from secondary effluent by using integrated constructed wetland system

The treatment capacity of an integrated constructed wetland system (CWS) that was designed to reduce nitrogen (N) from secondary effluent was explored. The integrated CWS consisted of vertical-flow constructed wetland, floating bed and sand filter. The vertical-flow wetland was filled with gravel, steel slag and peat from the bottom to the top. Vetiver zizanioides was selected to grow in the vertical-flow constructed wetland and Coix lacrymajobi L. was grown in the floating bed. The results showed that the integrated CWS displayed superior removal efficiency for nitrate nitrogen (NO₃⁻-N), ammonia nitrogen (NH₄⁺-N), nitrite nitrogen (NO₂⁻-N), and total nitrogen (TN). The average NO₃⁻-N, NO₂⁻-N, NH₄⁺-N and TN removal efficiencies of the integrated CWS were 98.83%, 95.60%, 98.05% and 92.41%, respectively, during the whole experimental operation. The integrated CWS may have a good potential for removing N from secondary effluent.     

中药有效成分治疗类风湿关节炎研究进展

中药治疗类风湿性关节炎(RA)有几千年的临床应用历史,积累了丰富的经验.近年来,国内外针对中药复方和单味药的抗RA作用开展了大量的实验研究工作,很多中药的活性成分(组分)和作用机制得到初步阐明,开发出青藤碱注射液、雷公藤多苷片和白芍总苷片等新型RA治疗药物.本文对中药有效成分在治疗RA方面的研究进展进行综述.     

Peripheral Effects of Nesfatin-1 on Glucose Homeostasis

Aims/hypothesis

The actions of peripherally administered nesfatin-1 on glucose homeostasis remain controversial. The aim of this study was to characterize the mechanisms by which peripheral nesfatin-1 regulates glucose metabolism.

Methods

The effects of nesfatin-1 on glucose metabolism were examined in mice by continuous infusion of the peptide via osmotic pumps. Changes in AKT phosphorylation and Glut4 were investigated by Western blotting and immnuofluorescent staining. Primary myocytes, adipocytes and hepatocytes were isolated from male mice.

Results

Continuous peripheral infusion of nesfatin-1 altered glucose tolerance and insulin sensitivity in mice fed either normal or high fat diet, while central administration of nesfatin-1 demonstrated no effect. Nesfatin-1 increases insulin secretion in vivo, and in vitro in cultured min6 cells. In addition, nesfatin-1 up-regulates the phosphorylation of AKT in pancreas and min6 islet cells. In mice fed normal diet, peripheral nesfatin-1 significantly increased insulin-stimulated phosphorylation of AKT in skeletal muscle, adipose tissue and liver; similar effects were observed in skeletal muscle and adipose tissue in mice fed high fat diet. At basal conditions and after insulin stimulation, peripheral nesfatin-1 markedly increased GLUT4 membrane translocation in skeletal muscle and adipose tissue in mice fed either diet. In vitro studies showed that nesfatin-1 increased both basal and insulin-stimulated levels of AKT phosphorylation in cells derived from skeletal muscle, adipose tissue and liver.

Conclusions

Our studies demonstrate that nesfatin-1 alters glucose metabolism by mechanisms which increase insulin secretion and insulin sensitivity via altering AKT phosphorylation and GLUT 4 membrane translocation in the skeletal muscle, adipose tissue and liver.     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

基于λ-Red重组酶系统的Ⅳa2基因缺失及重组腺病毒的包装

本研究用λ-Red重组酶介导的PCR打靶方法缺失腺病毒基因组上Ⅳa2基因的大部分编码序列(1104 bp),并获得一种Ⅳa2基因缺失的重组腺病毒。首先构建PCR打靶的含有卡那霉素抗性表达盒的模板质粒pAK,再以pAK为模板扩增出两端含39 bp同源臂的线性DNA片段;将pFG140质粒及线性DNA片段依次转化到宿主菌BW25113/pIJ790中,通过λ-Red重组酶介导的同源重组获得了缺失Ⅳa2基因的腺病毒基因组质粒pFG140-ΔⅣa2(1104)。酶切及DNA测序结果显示,用λ-Red重组酶介导的PCR打靶方法在pFG140上精确地缺失了预计的Ⅳa2基因3'端的1104bp片段。用PCR方法扩增获得Ⅳa2基因全长ORF,插入表达载体pAAV2neo中,构建成Ⅳa2基因表达质粒pAAV2neo-Ⅳa2。将pFG140-ΔⅣa2(1104)与pAAV2neo-Ⅳa2共转染HEK293细胞,成功地获得了重组腺病毒Ad5ΔⅣa2(1104)。Western Blot的结果表明,Ad5ΔⅣa2(1104)病毒感染的HEK293细胞检测不到Ⅳa2蛋白的表达。本研究建立了一种对腺病毒基因组直接进行缺失操作的λ-Red...     

逆转肾小管上皮细胞转分化的Smad信号转导机制

肾小管上皮细胞转分化(tubular epithelial to menchymal transdifferentiation,EMT)是肾小管间质纤维化的重要病理机制之一。致纤维化细胞因子TGF-β通过几种信号转导途径调节EMT,其中TGF-β/Smads信号通路发挥核心作用。目前研究表明,Smad7、HGF、BMP-7等可通过调控Smads信号通路而逆转EMT,这为肾间质纤维化的防治提供了新的思路。该文主要介绍TGF-β/Smads信号通路在EMT发生的作用,以及Smad7、SnoN、HGF、BMP-7等分子是如何通过抑制Smads信号通路而发挥逆转EMT作用的。     

Secretory delivery of heterologous proteins in attenuated <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.     

6-羟多巴胺纹状体内注射制作大鼠帕金森病模型的研究

目的 为拓宽6-OHDA损毁多巴胺能神经元所制备大鼠帕金森病模型的应用范围,采用多位点纹状体内注入6-OHDA的途径来制备模型。方法 研究用SD大鼠,两个针道内四点定位注射,每点注射3μg／μ16-OHDA3μl。结果 术后两周出现缓慢旋转,4周旋转行为达到7转／分并保持稳定;形态学染色可见损毁1周后注射侧黑质酪氨酸羟化酶免疫组化阳性细胞减少20％,2周后减少38％,3～4周减少70％以上,6周后损伤趋缓。高效液相-电化学法活体检测纹状体内多巴胺的代谢产物3、4-二羟基苯乙酸(DOPAC)和高香草酸(HVA),发现注射侧和非注射侧相比含量分别下降98．33％和96．05％;组织匀浆检测损毁侧黑质多巴胺含量下降了73％以上,3、4-二羟基苯乙酸(DOPAC)含量下降60％。结论 纹状体内注射6-OHDA能够制备帕金森病大鼠模型。