新生大鼠下丘脑CRF和AVP神经元对急性低氧的应答

目的:观察肾上腺摘除新生大鼠下丘脑促肾上腺皮质激素释放激素(CRF)和精氨酸加压素(AVP)神经元对急性低氧的应答.方法:在低压氧舱中模拟高海拔低氧,用放免法测定AVP和CRP含量.结果:新生大鼠暴露于急性低氧环境下(模拟5 000 m和7 000 m海拔高度,24 h),其下丘脑CRP在3 d和7 d龄大鼠中无明显变化,但14d、21 d和28 d时低于对照;下丘脑AVP在3 d大鼠中亦无变化,但14 d时低于对照,7 d、21 d及28 d时高于对照.两者对低氧的应答模式随日龄而变化.摘除肾上腺后,14 d、21 d及28 d大鼠下丘脑CRF和AVP含量均显著低于同龄完整大鼠,此时暴露于急性低氧环境下,CRF和AVP无进一步的变化.结论:摘除肾上腺抑制下丘脑CRF和AVP的发育,影响它们对低氧应激的正常应答.     

Decoy engineering of the receptor-like cytoplasmic kinase StPBS1 to defend against virus infection in potato

Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1–RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1^NIa. StPBS1^NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1^NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1^NIa.     

灰色链霉菌胰蛋白酶在变铅青链霉菌中的异源表达及酶学性质分析

胰蛋白酶作为一种重要的丝氨酸蛋白酶被广泛应用于食品、医药和皮革等工业领域.本文成功实现了灰色链霉菌来源的胰蛋白编码基因在变铅青链霉菌中的高效活性表达,并对其酶学性质进行分析比较.以灰色链霉菌ATCC10137基因组为模板,获得胰蛋白酶编码基因sprT并克隆至表达质粒pIJ86,成功构建了重组链霉菌工程菌TK24/pIJ86-sprT.以R2YE和SELF为发酵培养基,最高酶活分别达9.21 U/mL和8.61 U/mL.酶学性质分析表明,和牛胰蛋白酶(BT)相比,重组链霉菌胰蛋白酶(rSGT)的耐酸能力强,具有较广的pH;且rSGT对酰胺键具有更高的特异性;此外,Zn2+和有机溶剂分别对rSGT的酯酶活力和酰胺酶活力具有促进作用;本研究结果为rSGT的性质改造以及工业应用提供了依据.     

重组小麦静息巯基氧化酶的表达、酶学特性及其对面包品质的影响

为发展新型面粉改良酶制剂,利用大肠杆菌Escherichia coli原核表达了小麦静息巯基氧化酶(Wheat quiescin sulfhydryl oxidase,wQSOX)。将合成的wqsox基因构建至pMAL-c5x载体,并在大肠杆菌中进行表达,优化蛋白表达条件后对重组蛋白进行分离纯化及融合标签切除,获得的重组wQSOX蛋白用于酶学性质探究以及面包品质改良。结果表明,合成的截短wqsox基因包含1359 bp,编码453个氨基酸,理论蛋白分子量51 kDa;构建的pMAL-c5x-wqsox重组质粒在E.coli Rosetta gamiB(DE3)中可溶表达了重组蛋白MBP-wQSOX,其最佳表达条件为:诱导温度25℃,诱导剂IPTG浓度0.3 mmol/L,诱导时间6 h;利用Xa因子蛋白酶切除了MBP融合标签,亲和层析纯化得到了wQSOX;wQSOX可催化DTT、GSH和Cys氧化,并伴随着H2O2的生成,其中对DTT表现出最高的底物特异性;酶学性质研究发现,wQSOX最适反应温度和pH分别为50℃和10.0,在高温和碱性环境条件下表现出较好的稳定性;每克面粉中添加1.1 U wQSOX能够显著(P<0.05)提高26.4%的面包比容,降低20.5%的面包芯硬度和24.8%的咀嚼性,表现出了较好的改良面包加工品质能力。研究结果对丰富新型面粉改良酶制剂种类以及推动wQSOX在焙烤行业的应用奠定了理论基础。     

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.     

高粱抗蚜研究进展

高粱(Sorghum bicolour)是世界上最重要的粮食、饲料、酿造和能源作物之一, 也是C₄植物研究的模式植物。蚜虫是农业生产上的重要害虫, 几乎危害所有的栽培作物。危害高粱的蚜虫主要包括高粱蚜(Melanaphis sacchari)、麦二叉蚜(Schizaphis graminum)和玉米蚜(Rhopalosiphum maidis)。高粱的抗蚜资源尚不丰富且缺乏深入系统的研究。目前研究较多的是麦二叉蚜的抗性遗传方面, 已定位20个抗性QTLs, 单一QTL对抗性差异贡献率最高可达80.3%, 对高粱蚜和玉米蚜的研究尚需进一步加强。高粱的理化特性与其抗蚜性能相关, 故可与育种实践相结合。高粱和蚜虫(Acyrthosiphon pisum)的全基因组测序工作已经完成, 这将有助于蚜虫-植物间的相互作用关系及植物对蚜虫的抗性机制研究。目前已克隆到2个抗蚜基因, 且多个抗蚜基因(位点)已被定位在染色体上。该文重点综述了上述研究成果并对高粱抗蚜的研究前景进行了展望。     

乙型脑炎病毒DNA初免-蛋白加强策略在小鼠模型上的免疫效应评价

为了评价基因Ⅰ型乙型脑炎病毒prM-E DNA疫苗与prM和EⅢ融合抗原亚单位疫苗采用DNA初免-蛋白加强免疫策略对小鼠的免疫效果,本研究将prM-E融合基因插入到pVAX1真核表达载体中,构建重组表达载体prM-E-pVAX1作为DNA疫苗进行初免,利用原核表达系统获得的prM和EⅢ融合抗原作为亚单位疫苗进行加强免疫。将32只4−6周龄雌性BALB/c小鼠随机分成4组,设置prM-E-pVAX1 DNA疫苗组、DNA初免-蛋白加强免疫组、prM和EⅢ融合抗原亚单位疫苗组及pVAX1载体对照组,通过ELISA检测血清中特异性抗体水平;通过噬斑减少中和试验滴定中和抗体滴度;通过细胞因子表达丰度和淋巴细胞增殖试验分析不同疫苗免疫组诱导产生的细胞免疫反应。结果表明,用DNA初免-蛋白加强策略免疫的小鼠诱导产生的中和抗体滴度略高于prM和EⅢ融合抗原亚单位疫苗免疫组,显著高于prM-E-pVAX1 DNA疫苗免疫组。DNA初免-蛋白加强策略在小鼠模型中诱导产生了有效的Th1/Th2型免疫反应,特别是显著诱导了Th1型细胞免疫反应。本研究为预防流行性乙型脑炎提供了新的免疫策略和理论参考依据。     

Adhesion of lymphoid cell lines to fibronectin-coated substratum: Biochemical and physiological characterization and the identification of a 140-kDa fibronectin receptor

Little information is available on the interaction between lymphocytes and fibronectin (fn). To gain a better understanding on this issue we examined the adhesion of 12 lymphoid cell lines, each exhibiting different phenotypic characteristics, to fn-coated substratum. Of the cell lines tested, five that adhered to fn possessed B-cell characteristics, while neither the T-cell lines nor the pre-B-cell line adhered. The physiology and biochemistry of adhesion of a B-cell line, MOPC 315, were examined in detail. Our results indicated that (1) the adhesion was a specific and time-dependent process, (2) the adhesion was temperature-dependent and inhibited by metabolic inhibitors, such as KCN and 2-deoxyglucose, (3) the presence of cycloheximide and pretreatment of cells with trypsin inhibited adhesion, (4) a 140-kDa surface protein was immunoprecipitated by anti-fn receptor antibodies, (5) the presence of divalent cations was essential for adhesion, (6) the presence of colchicine had no effect on adhesion, while cytochalasin B partially inhibited adhesion, and (7) the treatment of cells by both phorbol 12-myristate 13-acetate and calcium ionophore A23187 enhanced adhesion. In this study, we have established the interaction between lymphoid cell lines and fn. Such an interaction might play an important role in the behavior of lymphocytes in tissues.