人泛素结合酶UbcH5c活性突变体的构建·纯化以及催化活性研究

[目的]构建人泛素结合酶UbcH5c的活性位点突变体S22R和F62A,同时分析这2种突变体蛋白与野生型蛋白在泛素化反应中的活性差异。[方法]通过点突变(Site-Directed Mutagenesis)的方法构建2种突变体质粒,利用0.5 mmol/L IPTG分别诱导2种突变体蛋白在大肠杆菌中进行表达;将菌体超声破碎后,依次利用阳离子交换层析法对UbcH5c突变蛋白进行分离纯化,最后利用体外泛素化酶促反应结合蛋白免疫印迹杂交的方法分析野生型UbcH5c与其活性突变体UbcH5c S22R和UbcH5c F62A在泛素化修饰上的差异。[结果]人泛素结合酶UbcH5c的2个突变体蛋白S22R、F62A的泛素化修饰程度明显弱于野生型蛋白。[结论]突变体S22R和F62A突变均不同程度的改变了人泛素结合酶UbcH5c与泛素分子之间的结合活性,即2个突变的位点中第62位苯丙氨酸残基及第22位丝氨酸残基均是UbcH5c结合泛素的关键位点,而且后者对于UbcH5c结合泛素活性的影响更大。     

Characterization of cytoplasmic male sterility in Petunia hybrida and Zea mays. Localization and activity of cytochrome c oxidase

Summary Anthers of male fertile, cytoplasmic male sterile (cms), and restored male fertile Petunia hybrida, are analyzed for cytochrome c oxidase (cox) activity in subsequent stages of microsporogenesis, and compared with anthers of male fertile, cms-S and cms-C Zea mays. The cox activity is determined in anther extracts and cytochemically. In petunia anthers, the first differences in cox activity occur from meiosis onward. However, at these stages, the initial symptoms of degeneration are already apparent. It is suggested that the decline in enzyme activity of the cms petunia anthers is the result rather than the cause of the non-formation of functional pollen.In maize anthers, the cox activity of sterile-type anthers is reduced in comparison with fertile-type anthers from premeiosis onward. There are also consistent cytochemical differences in the mitochondrial organization of cox activity between pollen of cms-S and male ferile maize anthers. In fertile-type mitochondria, the DAB reaction product indicating cox activity is localized in the cristae and within the space between the outer and inner limiting membranes of the organelles. In mitochondria of pollen of cms-S maize, cox activity is only observed between the outer and inner membranes of the mitochondria. The biochemical and cytochemical differences are observed at stages of development at which no structural signs of degeneration are apparent. The results suggest that cms in maize correlates with deviations in cytochrome c oxidase activity.Abbreviations cox cytochrome c oxidase - DAB 3,3-diaminobenzidine     

Cloning of vacuolar H<Superscript>+</Superscript>-ATPase subunit c genes from Japanese iris,and functional characterization in yeast

Five different isoforms (IrlVHA-c1-c5) of V-ATPase subunit c (VHA-c) were cloned from a Japanese iris (Iris lactea Pall. var. chinensis Fisch. Koidz) cDNA library using degenerate primers PCR and the 5'-RACE technique. The sequence analysis showed the open reading frame (ORF) of the IrlVHA-c1 c5 to be 495 bp, corresponding to a protein of 164 amino acids. Among the five isoforms, IrlVHA-c1 and IrlVHA-c2 are completely homologous. The IrlVHA-c protein is localized at the vacuolar membrane as indicated by a g...     

Little effect of forest age on oribatid mites on the bark of trees

This study investigates the effect of forest age (20-, 50-, 160-year-old, and primeval forest) on oribatid mite communities on the bark of oak trees in the National Park Pusza Białowieża in eastern Poland. We hypothesized that oribatid mite diversity on bark peaks at forests of intermediate age and that the number of parthenogenetic species of oribatid mites is highest in young stands. In contrast to these hypotheses, the diversity, density, number of juveniles, community structure and the mode of reproduction of oribatid mite species were not significantly affected by forest age. None of the oribatid mite species occurred exclusively on trees of a specific age. The results suggest that oribatid mite communities on the bark of trees are minimally affected by tree harvesting regimes. In contrast to oribatid mites in soil, communities on bark appear to be less sensitive to disturbances.     

布氏田鼠肝脏内线粒体蛋白含量及细胞色素c氧化酶活性的变化

为探讨线粒体能量代谢中细胞色素c氧化酶(cytochromecoxidase,CcO)在布氏田鼠(Microtusbrandti)生长发育过程中的作用,应用紫外分光光度法测定了不同日龄的幼鼠肝脏内线粒体蛋白含量及CcO活性。结果发现根据幼鼠肝脏内线粒体蛋白的含量可将生长发育大致分为3个阶段:1～3日龄,4～19日龄,20日龄至成体。CcO活性变化趋势则呈倒钟形,以17日龄的CcO活性为最高。据此推测布氏田鼠肝脏内CcO的活性可能与恒温机制的建立有关系。     

北京植物新资料

本文收录了<北京植物志>未收录的植物计4种,2变型.它们是白花大叶铁线莲、掌叶大黄、小叶羊角芹、丁香叶忍冬、多头苦荬菜.白花大叶铁线莲为新变型,羊角芹属与岩风属为北京新记录属.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

基于RS和GIS的林型结构与森林涵养水源关系的研究

在运用 RS和 GIS技术划分出水库流域土地利用类型的基础上 ,采用灰色关联分析法 ,结合水文数据 ,分析流域内不同林型和森林覆被率 (共 8个因子 )与年降雨量、年径流量之间的关系。分析得出了流域内涵养水源功能较强的优势林型 ,为合理调整流域内森林结构布局 ,充分发挥森林的水源涵养功能提供参考。研究表明 ,流域内涵养水源功能最强的林型为柞树纯林 ,其次是落叶松和白桦构成的混交林。     

土壤中碳酸盐的碳氧同位素分析

本文采用磷酸法分析土壤中碳酸盐的碳、氧同位素组成,对分析方法和制样装置作了叙述,比较了经不同预处理土壤的碳、氧同位素值,指出土壤有机碳的存在不影响碳酸盐同位素组成的精密测定,有氧灼烧预处理土壤样品对分析结果有明显影响,使δ~13C和δ~18O值降低。测定多种标准样品表明,此方法准确、可靠,重复制样测定的标准偏差在0.1‰以内。     

甲状腺肿瘤中p53、bcl-2及c-myc蛋白的表达

目的 :探讨p5 3、bcl 2和c myc蛋白在甲状腺肿瘤中的表达意义。 方法 :应用免疫组化EnvisionTM法对 73例甲状腺肿瘤中 p5 3、bcl 2和c myc蛋白的表达进行检测。 结果 :3 6例腺瘤中 p5 3、bcl 2和c myc蛋白的阳性表达率分别为 :0 0 %( 0 / 3 6)、88.6%( 3 2 / 3 6)和 5 0 .0 %( 18/ 3 6) ;3 7例腺癌则分别为 :2 1.6%( 8/ 3 7)、64 .9%( 2 4/ 3 7)和 89.2 %( 3 3 / 3 7) ;p5 3、bcl 2、c myc蛋白分别在 3 6例腺瘤与 3 7例腺癌之间比较差异均有显著性 (P <0 .0 5或P <0 .0 1)。p5 3蛋白阳性表达的腺癌 8例 ,其bc1 2及c myc蛋白阳性表达率分别为 75 .0 %( 6/ 8)和 10 0 %( 8/ 8) ,在腺癌中 p5 3蛋白表达与bcl 2蛋白表达仅呈低度正相关 (r =0 .3 5 1,P <0 .0 5 )。 5例淋巴结转移性甲状腺癌中p5 3蛋白阳性表达率为 60 0 %( 3 / 5 ) ,但均无bc1 2蛋白阳性表达。结论 :p5 3、bcl 2及c myc蛋白可能共同参与甲状腺癌的发生发展 ;检测 p5 3蛋白有助于甲状腺良恶性肿瘤的鉴别