人工锌指核酸酶突变EGFP基因的功能分析

锌指核酸酶技术在基因定点修饰中具有效率高和特异性好等特点,并成功应用于数十种生物。目前,该技术是否能应用羊上尚未报道。为了敲除转基因山羊标记基因 (EGFP),构建了一对针对EGFP外显子上的锌指核酸酶表达载体,将其电转染至转EGFP基因胎儿成纤维细胞中,研究了锌指核酸酶突变EGFP基因的效率和方式,利用基因显微注射单细胞获得获得的转基因 (EGFP) 细胞系作为锌指核酸酶的靶细胞。结果显示,通过锌指核酸酶的突变作用,转染后的细胞发绿色荧光比例下降,测序结果显示在EGFP外显子中插入1个碱基G,导致编码EGFP基因的阅读框改变,从而起到基因突变的作用。结果表明,文中构建的锌指核酸酶对EGFP基因有突变作用,可以为以后获得无标记基因供核细胞进行体细胞核移植生产克隆羊奠定基础。     

Raf激酶抑制蛋白的研究进展

Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的成员。RKIP通过与Raf-1结合,抑制了Ras/Raf-1/MEK/ERK信号转导通路,并在NF-κB及G蛋白偶联受体(GPCR)信号转导通路中也起重要调节作用。RKIP参与细胞凋亡、肿瘤转移、神经发育以及精子发生等病理生理过程,通过研究RKIP能为治疗相关疾病提供新思路新靶点。本文主要介绍RKIP的生物功能,着重于其在神经系统、肿瘤和生殖系统中的研究进展。     

Antibacterial Agents that Inhibit Histidine Protein Kinase YycG of Bacillus subtilis

We demonstrated in vitro that YycG-YycF of Bacillus subtillis constitutes a two-component system and shows a specificity of the sensor protein for the cognate phosphorylation partner. Based on inhibition of such an autophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial agents such as NH125, NH126, NH127, and NH0891.     

Characterization of the RcsC→YojN→RcsB Phosphorelay Signaling Pathway Involved in Capsular Synthesis in Escherichia coli

Escherichia coli and other enteric microorganisms produce an extracellular polysaccharide capsule, called colanic acid, under certain environmental conditions. This capsular synthesis is regulated by the RcsC (sensor kinase)→YojN (phosphotransfer intermediate)→RcsB (response regulator) phosphorelay signal transduction under certain growth conditions. Nonetheless, little is known about signals that exaggerate the Rcs-system. To gain insight into signals that activate the Rcs-system, here we searched for genes that activate the Rcs-system, provided that those on a multicopy plasmid were introduced into E. coli. We identified several such genes, namely, rcsB, rcsA, djlA, lolA, and ompG. The DjlA, LolA, and OmpG proteins are particularly interesting in that they are all located on the cell surface, where the primary sensor RcsC histidine-kinase is localized. Implications of these findings are discussed with special reference to the mechanism by which RcsC perceives external signals.     

低分子肝素的绿色荧光蛋白荧光标记及其活性检测

增强型绿色荧光蛋白（EGFP）是生物领域常用的标记物. 本实验首先利用高碘酸法活化低分子肝素（LMWH）,与EGFP连接得到LMWH EGFP.然后用Sephacryl S-200 HR对其进行初步分离,再用Sephadex G-10 HR进行脱盐纯化. 采用Sephacryl S 200 HR检测LMWH EGFP的纯度,为单一对称峰. 经检测,LMWH-EGFP具有良好的热稳定性和耐碱性. 通过荧光分光检测器检测LMWH-EGFP的λEx为488 nm,λEm为509 nm. 通过抗凝实验发现LMWH EGFP仍具有抗凝活性. 本实验建立了LWMH荧光标记的方法,为多糖的荧光标记提供了新的选择.     

植物激素乙烯作用机制的最新进展

气体植物激素乙烯在植物生长发育及应对胁迫的防御反应中起重要调控作用．通过20多年的研究,利用模式植物拟南芥,勾画出一条自内质网膜受体至细胞核内转录因子的线性乙烯信号转导通路．本文概述了研究乙烯信号转导的方法及乙烯信号转导的基本过程;阐述了最新发现的乙烯信号从内质网膜传递到细胞核的分子机制,即原本定位于内质网膜上的EIN2蛋白其C端被剪切之后进入细胞核,然后通过抑制EBF1／2而稳定转录因子EIN3／EIL1;根据最近多个小组报道EIN3／EIL1直接调控除乙烯响应基因之外的其他生物学过程相关基因,提出了EIN3／EIL1可以作为网络节点整合多条信号通路的新观点;通过分析不同信号通路调控EIN3／EIL1的方式,发现不仅EIN3／EIL1的蛋白稳定性受到调控,而且其转录活性还受到诸如JAZ,DELLA等转录调节因子的调控．本文展望了未来乙烯信号转导通路的研究方向与研究热点．     

Membrane dynamics in mammalian embryogenesis: Implication in signal regulation

Eukaryotes have evolved an array of membrane compartments constituting secretory and endocytic pathways that allow the flow of materials. Both pathways perform important regulatory roles. The secretory pathway is essential for the production of extracellular, secreted signal molecules, but its function is not restricted to a mere route connecting intra‐ and extracellular compartments. Post‐translational modifications also play an integral function in the secretory pathway and are implicated in developmental regulation. The endocytic pathway serves as a platform for relaying signals from the extracellular stimuli to intracellular mediators, and then ultimately inducing signal termination. Here, we discuss recent studies showing that dysfunction in membrane dynamics causes patterning defects in embryogenesis and tissue morphogenesis in mammals. Birth Defects Research (Part C) 108:33–44, 2016. © 2016 Wiley Periodicals, Inc.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.