大肠杆菌P蛋白和T蛋白的调节结构域互换研究

在细菌、真菌及植物中,分支酸是一种位于关键分叉点上的中间代谢物,是所有芳香族氨基酸合成的共同前体.它可在双功能酶分支酸变位酶(CM)和预苯酸脱水酶(PDT)的催化下合成苯丙氨酸,在另一个双功能酶分支酸变位酶和预苯酸脱氢酶(PDH)的催化下合成酪氨酸.前者被称为P蛋白,后者被称为T蛋白.大肠杆菌P蛋白和T蛋白有着类似的结构,P蛋白由CMp、PDT和调节结构域３个独立结构域组成,其变构调节因子是苯丙氨酸.T蛋白只有CMt和PDH两个独立结构域组成,起变构调节作用的调节结构域与PDH密不可分,其变构调节因子是酪氨酸.为了研究P蛋白和T蛋白的调节结构域的变构调节作用,应用融合蛋白技术将P蛋白和T蛋白的调节结构域进行了互换.结果发现,互换了的调节结构域仍然具有变构调节作用,而且调节结构域的互换导致了变构调节因子的互换,说明调节结构域对酶活性的调节作用是非专一的,而其R结构域与调节因子的结合却是专一的.     

蛋白分子的变构作用是学习过程的基本机制

本文综述了近年在学习记忆神经机制研究中的新进展,分析了腺苷酸环化酶、NMDA受体蛋白和S₁₀₀酸性蛋白等分子发生变构作用的条件及其与学习记忆过程的关系。这些蛋白分子的变构作用既制约于条件刺激又决定于非条件刺激,两种刺激的结合就会实现较大的生物效应。这一规律表明,这些蛋白分子的变构作用是学习过程的基本机制。     

Maf1:一种RNA聚合酶Ⅲ的负调节因子

Maf1是一种RNA聚合酶Ⅲ的负调节因子,在酵母雷帕霉素诱导的营养限制、DNA损伤和分泌通路缺陷反应过程中发挥重要作用。在不利生长条件下,Maf1被蛋白磷酸酶2A脱磷酸化而激活,被运输到细胞核发挥对RNA聚合酶Ⅲ的抑制作用。Maf1还可以被蛋白激酶A磷酸化并在Msn5载体蛋白的协助下运出细胞核而解除抑制。Maf1在真核生物中高度保守,因此相关研究在理解哺乳动物RNA聚合酶Ⅲ转录抑制机理方面可能有重要意义。     

微囊藻毒素对鱼组织匀浆液蛋白磷酸酶活性抑制作用的研究

蛋白磷酸酶是在调节细胞内蛋白磷酸化水平方面具有重要作用的酶。研究表明，蛋白磷酸化水平与肿瘤的促进作用密切相关，激活蛋白激酶C和抑制丝氨酸／苏氨酸蛋白磷酸酶（Ser／ThrProtein…。s…atase，简称PP，根据对抑制剂的敏感性和对二价阳离子的依赖性，分为1，ZA，ZB，ZC四类）的物质都对肿瘤形成起促进作用。近期研究发现，天然有毒物微囊藻毒素对PPI和PPZA活性具有极强的抑制作用l’］。对PPI和PPZA具有极强抑制作用的这一物质的发现有重要意义，从酶学研究来看，由于这类物质对酶抑制作用专一和灵敏，因而可作为理想的分…     

基于弹性网络模型的蛋白质变构路径与关键残基识别研究

目的 变构效应在蛋白质生物学功能执行过程中发挥着重要的调控作用，如何基于蛋白质空间结构，有效识别变构信号的传播路径和关键的残基位点是蛋白质结构-功能关系研究领域的热点科学问题。方法 本研究利用基于弹性网络模型（elastic network model，ENM）的力分布计算方法，通过分析蛋白质对外力的响应过程，来识别体系的变构路径以及变构过程中的关键残基。在该方法中，对蛋白质的关键变构位点施加外力，通过对体系形变以及内力分布情况的分析，有效识别与外力承载区域形变相耦合的关键残基，从而得到力信号在蛋白质结构内的传播路径。结果 利用该方法研究了人类磷酸甘油酸激酶（human phosphoglycerate kinase，hPGK）和蛋白质酪氨酸磷酸酶（protein tyrosine phosphatase，PTP）PDZ2结构域的变构调控路径和关键残基。对于hPGK，识别出从底物结合位点到铰链区的两条变构信号传导路径。对于PTP PDZ2，也成功识别出从配体结合位点传递到蛋白质远端的两条长程变构调控路径。计算结果与实验和分子动力学（molecular dynamics，MD）模拟得到的结果一致。结论 本研究为蛋白质体系关键残基识别及变构路径研究提供了有效的分析方法。     

微囊藻毒素对鱼肝的毒性效应

研究已发现离体条件下微囊藻毒素时鱼的蛋白磷酸酶有极强的抑制作用’‘,”。有些研究证明活体致毒后细胞内蛋白的磷酸化水平增加,而毒素对生物活体致毒时蛋白磷酸酶的情况尚未见报道。本研究用鱼作为实验材料,活体致责时微囊藻毒素对鱼的蛋白磷酸酶及几种其它分子生态毒理学指标的影响。回材料与方法1．l微囊藻毒素L民根据HSYddd的方法卜‘,由日本SllWS湖水华样品中提取。l．2活体实验,对体重约259的鲫鱼腹腔注射LR,剂量分别为IO,50,100和400pg／kg。在lh和3h时取样,分别测定蛋白磷酸酶（Proteiphosphatase,简称PP）活性和…     

乙型肝炎病毒核心蛋白变构调节剂的研究进展

由于乙型肝炎病毒(hepatitis B virus,HBV)的共价闭合环状DNA的持续存在和病毒介导的宿主免疫反应钝化,导致慢性乙型肝炎病毒感染很难治愈。现有的核苷(酸)类似物或聚乙二醇干扰素疗法难以实现高比率的HBV表面抗原清除。目前正在研发的核心蛋白变构调节剂有望大幅度降低血清表面抗原。本文就HBV核心蛋白的结构、功能以及核心蛋白变构调节剂的分类、应用前景等方面进行综述。     

对疯牛病诊断及治疗的新设想

过去 15年来想对疯牛病进行免疫接种是科学家的梦想 ,但都失败了。疯牛病的发病原因早已深知 ,由朊蛋白的变构引起的 ,而正常的朊蛋白 (PrPc)分布在各种细胞 ,最多在脑细胞 ,当一旦变构 (PrPsc)即会发病 ,但困难的是抗体不识别变构的PrPsc。2 0 0 4年 1月 13日 ,加拿大多伦多大学JanetWong报道在他们学校神经退化性疾病研究中心Dr.NeilCashman等有一个新的发现。Cashman研究组发现有三个氨基酸 Try Try Arg是变构后才出现 ,他们认为这三个氨基酸PrPc 的侧链 ,正常时是隐藏在结构里 ,变构时才暴露 ,所以这是PrPsc的特征 ,而且种族有…     

变构菌素生物合成基因簇的研究进展

变构菌素是从Streptomyces griseochromogenes菌株中分离得到的第一个高选择性的蛋白磷酸化酶-1（PP-1）抑制剂。变构菌素及其衍生物在神经系统紊乱、代谢综合症、呼吸系统及相关疾病、免疫抑制、肿瘤治疗等诸多领域都有着广泛的应用前景,因而引起了人们对其生物合成途径的研究兴趣。介绍了近年来变构菌素生物合成途径的研究进展。     

26肽蜂毒素与基因变构IL-2嵌合蛋白的原核表达、纯化及活性测定

在E .coliDH5α中表达26肽蜂毒素与基因变构人白细胞介素2的嵌合体蛋白 ,并进行初步纯化和抗原性检测。利用剪接式重叠延伸 (splicedoverlapextension ,SOE)技术在26肽蜂毒素与基因变构IL-2的基因之间导入四个易形成空间结构的氨基酸残基组成的连接肽 (Gly4Ser)₃,构建嵌合体蛋白的基因,克隆到原核表达载体pGEX4T-2中,重组质粒转化宿主菌DH5α,经IPTG诱导表达目的融合蛋白,纯化蛋白经Thrombin酶切后进行SDS-PAGE与Western-blot鉴定。结果表明重组蛋白有良好的抗原性 。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Areography of the Gymnosperms of China (1) Distribution of the Pinaceae of China

Nine of 10 genera and 119 of approximately 240 species of the Pinaceae occur in China, including 67 endemic species and two endemic genera. In this paper, the distributional maps of all the genera of the Pinaceae are presented (fig. 1-8). The horizontal and vertical distributions of species in each genus are discussed. The analysis of the distribution patterns of the genera indicates that some genera, such as Keteleeria, Tsuga, Pseudotsuga, Cathaya and Pseudolarix, are restricted to the area south of the Qinling Mountains and the Huaihe River, and the others, i. e. Picea, Abies, Larix and Pinus, extend northward to northeastern China. However, all of the genera except Keteleeria and Pinus are not found in very dry areas and tropical mountainous regions of China. The monotypic genera, Cathaya and Pseudolarix, are distributed in eastern and central China. The genus Keteleeria consists of 10 species, 7 of which are concentrated in southern Guizhou, northern Guangxi, southwestern Hunan and easternmost Yunnan. The distribution of the remaining 6 genera shows the maximum concentration in western Sichuan and northwestern Yunnan. (Figs. 2-8). Furthermore, more than third of species of the Pinaceae (37.8%) are also concentrated in western Sichuan and northwestern Yunnan. where a great variety of habitats and different topographic features occur. It is apparent that to conduct our systematic and evolutionary studies on this family in these region is especially needed. The relations between the areal size and the tolerance of species are discussed. The distributions of macrofossils and microfossils of the genera of the Pinaceae ia China are given, and it has been proved that areas of most genera of the family were considerably larger in the past. than at present.     

激光联合注射曲安奈德治疗婴幼儿血管瘤的疗效分析

目的：分析激光联合注射曲安奈德治疗婴幼儿血管瘤的疗效。方法：将120例处于增殖期血管瘤患儿分成观察组和对照组各60例,观察组给予激光联合注射曲安奈德治疗,对照组采用激光治疗,比较两组患者的治疗疗效。结果：观察组和对照组的总有效率分别为96．7％和66．7％,两组比较具有显著差异,P〈0．05;对于表浅病灶,观察组治疗的有效率为96．8％,对照组为84．8％,两组之间无差异,P〉0．05;对于深部病灶,观察组和对照组的总有效率分别为96．6％和44．4％,具有显著差异（P〈0．05）。结论：激光联合注射曲安奈德治疗婴幼儿血管瘤具有较好的疗效,尤其对深部增殖期的血管瘤具有较好的效果,适合在临床推广应用。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

坏疽病原体快速检测方法的临床应用

目的:利用荧光定量PCR技术,建立一种快速、敏感、特异的检测产气荚膜梭菌的方法,及时用于指导临床治疗。方法:以产气荚膜梭菌16srRNA基因作为靶序列,设计一对特异引物和探针,以伤口分泌物和脓液提取的核酸作为模板,利用已经优化的引物和探针进行PCR反应;同时与细菌培养作比较,验证此方法的快速性、敏感性及特异性。结果:建立的反应体系在上游引物浓度为0.45μmol/L,下游引物浓度为0.15μmol/L,探针浓度为0.3μmol/L时,具有很好的敏感性,与21种其他细菌均无交叉反应,其敏感性为9cfu/反应体系。荧光定量PCR检测结果与细菌培养结果完全一致。结论:所建立的荧光定量PCR方法特异、灵敏、快速,能对产气荚膜梭菌感染做出准确的检测报告,具有对战时高发疾病气性坏疽进行快速和定量检测潜质。     

Regulation of formation of the intracellular beta-galactosidase activity of Aspergillus nidulans

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.