家蚕磷酸吡哆醛激酶cDNA的克隆和酶活表征

吡哆醛激酶 (EC 2.7.1.35) 在 ATP 和 Zn2 的存在下,催化吡哆醛的磷酸化反应生成磷酸吡哆醛 (PLP)。在生物体内许多酶促反应中,PLP 是一种重要的辅酶因子。家蚕和哺乳动物一样,需依赖食物中的维生素 B6前体来合成 PLP。文章描述了利用家蚕基因组数据库序列信息及使用 PCR 方法,克隆出编码家蚕吡哆醛激酶的 cDNA (GenBank 登录号:DQ452397)。克隆到的 cDNA 含有一个 894 bp 的完整可读框,编码一条分子量为 33.1 kDa,含 298 个氨基酸残基的蛋白质。序列比对显示此蛋白质序列与人类吡哆醛激酶蛋白序列具有 48.6%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但其氨基酸残基数比哺乳动物和植物克隆到的吡哆醛激酶残基数均少 10 多个残基。多序列比对结果显示,吡哆醛激酶中几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换为其他种类氨基酸残基。采用 T7 启动子和 T7 聚合酶表达系统对克隆到的 cDNA 进行了原核表达并对表达粗提产物进行了酶活检测。实验结果显示表达得到的可溶性蛋白产物占其总蛋白量为 10%,细胞粗提物具有活力为 30 nmol/min/mg 的吡哆醛激酶活性,结果证实了克隆到的 cDNA 编码家蚕中的吡哆醛激酶。     

基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征北大核心CSCD

融合了跨膜肽的抗氧化酶可进入细胞,保护细胞免受放射损伤。然而跨膜肽的跨膜能力没有靶向性,其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞,降低放疗的效果。为此,根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点,在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合MMP-2/9的底物肽X,设计了融合蛋白GST-SOD1-X-R9。该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨膜肽,从而无法进入肿瘤细胞,进而只能进入正常细胞。全基因合成SOD1-X-R9序列,并将其插入原核表达载体pGEX-4T-1中,得到表达质粒,并实现了GST-SOD1-X-R9融合蛋白的可溶表达。GST-SOD1-X-R9经硫酸铵沉淀和GST亲和层析纯化,分子量约为47 kDa,与理论值一致。纯化的融合蛋白的SOD活性和GST活性分别为2954 U/mg和328 U/mg。GST-SOD1-X-R9的SOD活性或GST活性在生理条件下几乎没有变化。该融合蛋白在溶液中可被胶原酶Ⅳ部分水解。分别建立了2D和3D培养的HepG2细胞模型来检验肿瘤微环境中的MMP-2活力对该蛋白跨膜能力的影响。在2D培养模型中,HepG2的MMP-2活力极低,但在3D培养模型中,随着培养时间的增加,HepG2肿瘤球的体积变大,其胞外MMP-2活力也随之增强。GST-SOD1-X-R9在2D培养的HepG2细胞中具有和GST-SOD1-R9蛋白一样的跨膜效率,但在3D培养的HepG2细胞球中的跨膜能力大大降低。本研究为后续深入研究GST-SOD1-X-R9靶向防护正常细胞的氧化损伤效应奠定了基础。     

不同地点51个长白落叶松无性系苗期生长变异和遗传稳定性分析

长白落叶松是我国东北林区非常重要的速生用材树种之一。以不同地点51个长白落叶松无性系为材料,对其2年生苗高和地径进行调查分析,探讨其苗期生长变异和遗传稳定性。结果表明:长白落叶松无性系苗高和地径在地点间、无性系间、地点×无性系间差异均达到极显著水平(P<0.01);不同地点苗高和地径的表型变异系数处于23.89%~36.82%,除四平试验点的地径重复力为0.172外,不同地点苗高和地径的重复力均处于0.573~0.891;遗传稳定性分析结果表明,无性系LO 13等28个无性系的遗传稳定性较高;相关分析结果表明苗高与地径呈极显著正相关关系;利用多性状综合评价法,以20%的入选率对无性系进行选择,不同地点均选出10个综合表现较好的无性系,其中无性系LO 10和LO 26在不同地点均被入选优良无性系,且其遗传稳定性较强,可将其初选为优良无性系。     

Comparative study of symmetric and asymmetric somatic hybridization between common wheat and Haynaldia villosa

Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts of Triticum aestivum (cv. Jinan 177) and protoplasts of Haynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose γ-fusion combinations. GISH (genomic in situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion     

壳聚糖固定化AS1．398中性蛋白酶稳定性的研究

对以壳聚糖为载体,由戊二醛作交联剂。制备的固定化ＡＳ１．３９８中性蛋白酶的稳定性进行了研究。实验结果表明,固定化ＡＳ１．３９８中性蛋白酶对热、乙醇、尿素以及ｐＨ值的稳定性均有明显的提高。     

基于物种多样性与生物量关系的草地群落稳定性对全球变暖的响应研究进展

全球变暖背景下物种多样性和生物量的空间变化格局及其对温度升高的响应是生态学研究的热点内容,是探究群落稳定性响应全球变暖的关键.物种分布是多个生态过程相互作用的产物,温度升高通过改变群落关键种的作用影响物种多样性,从而改变群落稳定性.关于草地群落稳定性对全球变暖的响应过程,国内外学者的相关研究存在很大差异.该文作者结合自...     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.     

RNA结合蛋白调节mRNA稳定性的作用机制

免疫稳态的维持涉及多种细胞因子的基因表达调控,其中在转录后水平对mRNA稳定性的调控起重要作用。ARE(AU-rich element)位于mRNA 3'UTR(非编码区),富含AU碱基,某些RNA结合蛋白通过识别结合ARE影响mRNA的稳定性。本文综合最新研究,概述了TTP、HUR等RNA结合蛋白对mRNA稳定性的调节机制及其在信号通路中的作用。     

食管鳞癌染色体及基因组DNA畸变研究进展

食管鳞癌是我国常见的消化道恶性肿瘤,进展快且预后差。由于早期一般无明显症状,临床确诊的食管鳞癌大多已发展到了中晚期,治愈难度较大。越来越多的证据表明,在食管鳞癌发生发展过程中,染色体及基因组DNA畸变均是最常见的遗传学改变。文章就食管鳞癌染色体及基因组水平异常的研究进展作一综述。     

Principles of protein folding--a perspective from simple exact models.

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.