牛脑液泡型质子泵70kD和33kD亚基基因的表达

已分离了编码牛脑液泡型质子泵的70kD亚基的cDNA,利用聚合酶链反应(PCR)扩增了70kD亚基的编码片段,同时直接从牛脑cDNA库中得到了33kD亚基的编码片段.分别将相应片段连接到PET载体上完成70kD和33kD亚基基因在大肠杆菌中的表达.SDS聚丙烯酰胺凝胶电泳和蛋白质印迹分析表明70kD和33kD亚基基因均得到明显表达.     

PP-1催化亚基（PP-1c）在成体小白鼠不同组织器官中的分化表达与定位

为了确定蛋白磷酸酶-1(protein phosphatase-1)的催化亚基(PP 1c)在小白鼠不同器官组织(肌肉、卵巢、肾、胃、 脾、大脑、心、肝、肺及乳腺)中的表达模式,运用RT-PCR、Western 印迹及荧光免疫组织化学技术等实验手段进行了检测 和分析.结果表明,在mRNA水平, PP-1c在大脑中表达最高,卵巢及肺中表达次之,在肌肉、肾、心、肝中表达较低,在胃 和乳腺中表达最低;在蛋白质水平,肝中表达最高,肾、大脑、肺和乳腺中表达较高,而肌肉、卵巢、心和脾中表达相对较 低,胃中表达最低.免疫荧光组织化学实验结果显示,PP 1c的表达也具有明显的组织特异性和细胞特异性.这些结果为进一 步探讨PP 1在哺乳动物不同组织器官中的功能提供了重要的实验依据.     

Determinants of Voltage-Gated Potassium Channel Surface Expression and Localization in Mammalian Neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K⁺ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca²⁺ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Differential Expression of the Peripheral Benzodiazepine Receptor and Gremlin During Adipogenesis

This study used the mRNA differential display technique to identify differentially expressed genes during the process of adipogenesis in the preadipocyte cell line, 3T3‐L1. 3T3‐L1 cells were treated with dexamethasone, isobutyl‐1‐methylxanthine, and insulin to induce differentiation into mature adipocytes. Cells were collected at three time‐points during differentiation: Day 0 (d0), or nondifferentiated; Day 3 (d3), during differentiation; and Day 10 (d10), >90% of the cells had differentiated into mature adipocytes. Initial studies yielded 18 potentially differentially regulated cDNA candidates (8 down‐regulated and 10 up‐regulated). Reverse Northern and Northern blots confirmed differential expression of six of the candidates. Four of the candidates up‐regulated on d3 and d10 were identified by sequence analysis to be lipoprotein lipase, a well‐known marker of adipocyte differentiation. A fifth candidate that was expressed in d0, but not d3 or d10, was identified as DRM/gremlin, a bone morphogenetic protein antagonist. Finally, a sixth candidate that was increased at d3 and d10 was identified as the peripheral benzodiazepine receptor, which has been implicated in proliferation, differentiation, and cholesterol transport in cells. This study is the first to show that peripheral benzodiazepine receptor and DRM/gremlin are expressed in preadipocyte cell lines and that they are differentially regulated during adipogenesis.     

糖尿病小鼠胚胎早期发育的差异表达基因

目的利用小鼠糖尿病模型,探讨母体糖尿病环境对早期胚胎基因表达的影响。方法ICR雌性小鼠腹腔注射150mg／kg剂量STZ诱发糖尿病,与正常雄鼠交配受孕,取14d胎龄的胚胎,提取胚胎的总RNA。将Cy3和Cy52种荧光分别标记到实验组和对照组的RNA上,制成RNA探针,并与包含24859个基因的表达谱芯片进行杂交及扫描,重复3次实验,采用Agilent扫描仪进行扫描软件读取数据。结果筛选出差异表达基因397个,其中有328个基因在实验组表达量比对照组大2倍,69个基因在实验组表达量比对照组小2倍。结论母体糖尿病环境能影响早期胎儿的基因表达,通过上调代谢相关基因和下调发育相关基因影响小鼠胚胎的早期发育。为深入探讨糖尿病胚胎病理和代谢疾病的分子机理提供了基本数据和研究的方向。     

Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder. We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.     

利用K562红细胞分化模型和cDNA芯片技术筛选参与红细胞分化的新基因(英文)

以氯高铁血红素 (hemin)诱导K5 6 2分化作为体外红细胞分化模型 ,结合cDNA大规模测序、生物信息学分析、基因芯片杂交和NorthernBlot分析等技术 ,筛选红细胞分化相关的新基因 .首先利用大规模测序技术从人胚肾cDNA文库中随机挑选克隆测得 192个EST(expressedsequencetags)片段 ,经在线生物信息学分析 ,得到 79个代表新基因的未知EST片段 ,并在NCBI(NationalCenterofBiotechnologyInformation)dbEST库中登录 .利用 79个ESTcDNA片段制备了基因芯片 .提取分化前后的K5 6 2细胞的mRNA作为荧光标记反转录的模板 ,反转录后的探针用于DNA芯片杂交 .分析杂交后的结果 ,得到了 2个差异表达较明显的基因 ,GenBank登录号分别为AF147772 (187bp)和AF4 776 2(6 30bp) ,并分别命名为EDRG1和EDRG2 (erythroiddifferentiationrelatedgene 1and 2 ) ,相似性检索表明它们属全新基因 ,基因组草图测序数据库检索表明了两个基因的染色体定位 .随后的Northern印迹用于验证了在分化前后的K5 6 2细胞中差异表达 .提示这两个基因参与了红细胞分化过程 .RT PCR检测了EDRG1和EDRG2在人胚胎多组织中的表达 .结果提示 ,EDRG1可能与多种胚组织的正常发育相关 ,尤其在胚脑中高丰度表达 ,而EDRG2则可能参与了胚心和胚肾的组织生成 .生物     

An overview of trafficking and assembly of neurotransmitter receptors and ion channels (Review)

Ionotropic neurotransmitter receptors and voltage-gated ion channels assemble from several homologous and non-homologous subunits. Assembly of these multimeric membrane proteins is a tightly controlled process subject to primary and secondary quality control mechanisms. An assembly pathway involving a dimerization of dimers has been demonstrated for a voltage-gated potassium channel and for different types of glutamate receptors. While many novel C-terminal assembly domains have been identified in various members of the voltage-gated cation channel superfamily, the assembly pathways followed by these proteins remain largely elusive. Recent progress on the recognition of polar residues in the transmembrane segments of membrane proteins by the retrieval factor Rer1 is likely to be relevant for the further investigation of trafficking defects in channelopathies. This mechanism might also contribute to controlling the assembly of ion channels by retrieving unassembled subunits to the endoplasmic reticulum. The endoplasmic reticulum is a metabolic compartment studded with small molecule transporters. This environment provides ligands that have recently been shown to act as pharmacological chaperones in the biogenesis of ligand-gated ion channels. Future progress depends on the improvement of tools, in particular the antibodies used by the field, and the continued exploitation of genetically tractable model organisms in screens and physiological experiments.     

人类单纯性先天性心脏病中TBX5基因的突变及表达研究

本文首次较为完整地报道了藏汉通婚子代群体的14项肤纹参数（其中藏父汉思及汉父藏母各100 例）,并将这些肤纹参数分别与其藏汉父母样本的有关肤纹参数进行比较,再与1000例藏族及1040例 汉族两个大样本的有关肤纹参数进行比较。结果表明：藏汉后代的肤纹特征介于藏族和汉族之间,提示 肤纹参数的多因子遗传本质。     

EF—Tumt和EF—Tsmt在不同发育阶段小鼠各组织中的表达分析

线粒体蛋白质翻译延长因子Ｔｕ和Ｔｓ（ｍｉｔｏｃｈｏｎｄｒｉａｌｅｌｏｎｇａｔｉｏｎｆａｃｔｏｒＴｕａｎｄＴｓ,ＥＦＴｕｍｔａｎｄＥＦＴｓｍｔ）是由核基因编码的两个蛋白质,它们的功能和调控对细胞的生长发育有重要意义。采用ＥＦＴｕｍｔ和ＥＦＴｓｍｔ重组蛋白分别制备了抗ＥＦＴｕｍｔ和抗ＥＦＴｓｍｔ特异抗体并以此检测了它们在小鼠不同发育时期心肌、骨骼肌、肝、脑、脾等组织中的表达。蛋白质印迹结果表明ＥＦＴｕｍｔ和ＥＦＴｓｍｔ在各组织中的表达水平不同、有明显的组织差异性,并都受发育的调节。ＥＦＴｕｍｔ在同一发育时期各组织中的表达及随发育的变化趋势与ＥＦＴｓｍｔ基本一致。结果提示ＥＦＴｕｍｔ和ＥＦＴｓｍｔ的表达水平与组织细胞能量代谢水平密切相关,它们不仅在体内以复合体形式发挥作用,其基因表达可能受同一机制的调控。     

Gm2052基因在小鼠胚胎发育中表达的初步研究

目的:研究预测的编码蛋白基因Gm2052在小鼠胚胎发育阶段的表达模式,为进一步了解该基因的功能奠定基础。方法:通过全胚胎原位杂交技术、组织切片原位杂交技术及半定量RT-PCR方法,对预测的Gm2052基因在小鼠胚胎发育中后期及在新生小鼠中的表达情况进行初步分析。结果:全胚胎原位杂交显示,在E10.5小鼠胚胎中,Gm2052仅在脑中表达;当小鼠胚胎发育至E13.5时,Gm2052在脑、舌、肺、肝脏、胰腺等组织中均有表达。半定量RT-PCR结果显示,在小鼠胚胎中后期(E15.5和E18.5)及新生小鼠(出生后第9 d)中,Gm2052呈动态表达模式。结论:预测基因Gm2052与小鼠脑的发育密切相关,并可能参与小鼠肺、肝脏及胰腺等主要脏器胚期的发育。     

Subunits of Xanthine Dehydrogenase and Their Differential Appearance in Chick Tissues During Development

Xanthine dehydrogenase (XDH) from adult chick liver comprises two polypeptide chains of different size in a molar ratio of 1: 1. The molecular weights of these subunits were estimated to be 155K (α) and 135K (β) daltons (1). However, XDH isolated from the liver of newly hatched chick was not found to represent the equimolar ratio of these two subunits; that is, the amount of subunit β was lower than that of subunit α. While examamining electrophoretically the change in the amounts of these subunits in the liver, the subunit α was found to appear earlier in the embryonic stage, but β only after hatching. In the kidney, however, both subunits were detected before hatching, being consistent with the fact that XDH exists before hatching in the kidney. The two subunits also appeared differentially in the kidney; i.e., subunit α appeared earlier than subunit β. In either tissue, the rate of increase in XDH activity corresponded to that of subunit β. Thus, the synthesis of two subunits of XDH are separately regulated at least until just after hatching.     

Effects of Chronic Ethanol Treatment on the Expression of Calcium Transport Carriers and NMDA/Glutamate Receptor Proteins in Brain Synaptic Membranes

Abstract: Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca-exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca²⁺. Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair-fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 120-kDa exchanger proteins was increased by 14% and that of a 36-kDa exchanger-associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36–72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca²⁺, Mg²⁺-ATPases. The enhanced expression of receptor and exchanger-associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment.     

小麦高分子量麦谷蛋白亚基1Dx5启动子驱动外源基因的表达

将小麦高分子量麦谷蛋白亚基(HMW-GS)基因的胚乳组织特异性表达启动子驱动的外源突变型1Dx5基因和gus基因导入小麦中.对其转基因植株连续3代的跟踪研究表明,突变型1Dx5基因的重复序列导致其表达蛋白分子量增大,并影响其它1Bx17 1By18亚基基因的表达.组织化学分析观察到gus基因在1Dx5基因启动子驱动下的表达表现出胚乳组织特异性,在开花2周后开始表达,表达量呈持续上升,至腊熟期达到最高,其次为籽粒成熟期.     

小鼠早期胚胎发育过程中细胞凋亡及凋亡基因表达的检测

小鼠早期胚胎发育过程中凋亡现象大量存在,细胞凋亡与凋亡基因表达有关。应用彗星电泳法检测小鼠早期胚胎凋亡情况;应用巢式RT-PCR、免疫组化的方法检测了Bcl-2家族成员(Bax、Bcl-2、Bak、Bcl-xl)的表达变化情况。结果显示:随着胚胎细胞数目的增加,凋亡比率逐渐增大;Bax表达量在整个过程中基本不变,Bcl-2表达量逐渐上调,Bak、Bcl-xl的表达量逐渐降低。对小鼠早期胚胎发育过程中的基因表达研究对于揭示早期胚胎发育的机制有重大的意义。     

Expression of individual HMW glutenin subunit genes of wheat (Triticum aestivum L.) in relation to differences in the number and type of homoeologous subunits and differences in genetic background

The amount of individual high-molecular-weight (HMW) glutenin subunits of bread-wheat has been studied in relation to variation at homoeologous loci and in the general genetic background. The relationships between Glu-1 loci have been studied using nearisogenic lines (NILs) of the variety Sicco and in the progenies of two crosses. Substitution of the Sicco Glu-D1 allele by a null-allele resulted in higher amounts of the homoeologous subunits. The presence of a Glu-A1 nullallele did not have a noticeable effect on the amounts of homoeologous subunits. In three out of four NILs and in the sister-lines of two crosses, the amounts of HMW-subunits did not depend on the allele make-up at homoeologous loci. Only in the NIL which contains the Glu-D1 allele, encoding subunits 1Dx2.2 and 1Dy12, was the amount of homoeologous subunits lower than the amount of these subunits in Sicco. This study suggests a relation between the amount of HMW-subunits encoded by an allele and its contribution to bread-making quality. The effect of genetic background has been studied using F4 and F5 lines of two crosses. The total amounts of subunits, relative to the total amount of kernel proteins, showed a considerable variation between lines. The ratio between individual subunits did not differ between genetic backgrounds. Because this ratio is also largely independent of differences in environmental conditions, it is concluded that the relative amount of a subunit is a valuable measure for the detection of genetically-determined differences in the expression of HMW-subunit genes.