栝楼籽核糖体失活蛋白的纯化、性质及晶体生长研究

栝楼（Ｔｒｉｃｈｏｓａｎｔｈｅｓｋｉｒｉｌｏｗｉ）籽经粉碎抽提、硫酸铵沉淀、阳离子交换及凝胶过滤柱层析等步骤,得到一种单链核糖体失活蛋白－Ｔｒｉｃｈｏｋｉｒｉｎ（ＴＣＫ）．ＳＤＳ－ＰＡＧＥ和ＩＥＦ显示为单一条带,其分子量为２９ｋＤ,ｐＩ≥９．３,含糖量约为１．７５％．该蛋白对兔网织红细胞裂解液系统的蛋白质合成具较强的抑制活性,ＩＣ５０为６．７×１０－１０ｍｏｌ／Ｌ．改进了纯化方法,提高了产率,并培养出晶体．     

植物热激蛋白及其功能

植物热激蛋白及其功能＊刘德立（华中师范大学生物系,武汉４３００７０）ＨＥＡＴ－ＳＨＯＣＫＰＲＯＴＥＩＮＳＯＦＰＬＡＮＴＳＡＮＤＴＨＥＩＲＦＵＮＣＴＩＯＮＳＬｉｕＤｅ－ｌｉ（ＢｉｏｌｏｇｙＤｅｐａｒｔｍｅｎｔｏｆＨｕａｚｈｏｎｇＮｏｒｍａｌＵｎｉｕｅｒ...     

热激蛋白的分子生物学研究进展

热激蛋白的分子生物学研究进展费云标,黄涛,舒念红,江勇（中国科学院发育生物研究所,北京１０００８０）ＡＤＶＡＮＣＥＳＯＦＭＯＬＥＣＵＬＡＲＢＩＯＬＯＧＹＯＮＨＥＡＴＳＨＯＣＫＰＲＯＴＥＩＮＳ￥ＦｅｉＹｕｎ－ｂｉａｏ;ＨｕａｎｇＴａｏ;ＳｈｕＮｉａｎ－...     

哺乳类细胞中周期蛋白依赖激酶抑制因子

近两年,人们相继发现了一组能结合性抑制周期蛋白依赖激酶（ＣＤＫｓ）活性的蛋白因子──ＣＤＫ抑制因子（ＣＫＩｓ）。它们分别是：ｐ２１、ｐ１６、ｐ１５、ｐ２７和ＣＤＩｌ。ｐ２１和ｐ２７有一定同源性,能抑制多种ＣＤＫｓ的活性;ｐ１６和ｐ１５则同源性更高,能特异地与ＣＤＫ４、ＣＤＫ６结合;ＣＤ１１的结合特异性还有待进一步的研究。ｐ２１的表达受ｐ５３的正调控;ＴＧＦ－β则上调ｐ１５的表达以及ｐ２７的抑制活性。以上表明ＣＫｌｓ不仅是ＣＤＫｓ活性的调控者,而且还是抑癌因子与细胞周期调控之间的直接联系者。     

人类免疫缺陷病毒1型（HIV—1）核心蛋白（p24）在大肠杆菌中的表达,纯化…

在大肠杆菌中,利用新构建的含Ｔ７噬菌体ｇ－１０核糖体结合位点（ＲＢＳ）,以及λ噬菌体ＰＲ启动子的新型原核表达载体,通过表达ｇａｇ－ｐｏｌ基因片段,获得了具有天然序更的人类免疫缺陷病毒１型（ＨＩＶ－１）核心蛋白Ｐ２４（ＣＡ）的高效表达,克隆的ｇａｇ－ｐｏｌ基因片段 在其阅读框架移位区域插入了４ｂｐ碱基,其的病毒蛋白酶在阅读框架上与ｇａｇ一致,从而实现了对ｇａｇ－ｐｏｌ融合蛋白有有效加工,产生成熟的核     

应用硫氧还蛋白促进外源蛋白在大肠杆菌的可溶性表达

为了观察硫氧还蛋白（ＴｒｘＡ）促进外源蛋白在大肠杆菌中可溶性表达的作用,我们从质粒ｐＥＴ－３２ａ（＋）上克隆了ｔｒｘＡ基因,构建了ＴｒｘＡ表达质粒ｐＴ－ＴｒｘＡ。将该质粒与其它蛋白基因的表达质粒共同转化Ｅ．ｃｏｌｉ并同时获得表达。结果表明,共表达ＴｒｘＡ可以明显促进外源蛋白,如甲状旁腺激素相关蛋白（ＰＴＨｒＰ）、ＰＴＨｒＰ受体（ＰＴＨｒＰ－Ｒ）的血管内皮生长因子（ＶＥＧＦ）的可溶性表达。说明共表达     

金属硫蛋白的生物学特性及生理作用

金属硫蛋白的生物学特性及生理作用朱赓伯（苏州医学院生化教研室,苏州２１５００７）关键词金属硫蛋白金属硫蛋白又名金属硫组氨酸甲基内盐（ｍｅｔａｌｌｏｔｈｉｏｎｅｉｎｓ,ＭＴ）。三十年前美国哈佛大学的Ｂ．Ｌ．Ｖａｌｌｅｅ首次在马的肾脏中发现Ｃｄ－ＭＴ和Ｚ...     

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术,从括楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明,克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％,９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９     

慢性呼吸衰竭病人红细胞膜蛋白和红细胞变形能力的研究

目的：探讨红细胞膜蛋白在红细胞变形性改变中的作用。方法：参照Ｌｅａｍｍｌｉ和Ｐｅａｃｏｃｋ方法,测定了肺心病Ⅰ型呼吸衰竭（Ⅰ组）１８例、Ⅱ型呼吸衰竭（Ⅱ组）１８例和健康对照（ＣＧ）２０例的红细胞膜带３蛋白、膜收缩蛋白二聚体（ＳｐＤ）和四聚体（ＳｐＴ）的相对含量与红细胞变形能力。结果：Ⅰ、Ⅱ组带３蛋白、ＳｐＤ、ＳｐＴ相对含量和红细胞变形指数（ＤＩ）与对照组均有显著差异,且肺心病病人的ＤＩ与带３蛋白相对含量呈显著正相关,与ＳｐＤ／ＳｐＴ比值呈显著负相关。结论：带３蛋白和膜收缩蛋白的异常,可能是导致肺心病人红细胞变形能力降低的重要因素之一。     

结构分枝杆菌分泌蛋白研究进展

结构分枝杆菌（ＭＴＢ）的分泌蛋白是目前发现对ＭＴＢ感染保护性最好的一组蛋白,对结构病的预防和诊断具有重要意义。本文从其组成、生物学功能、免疫性及其在疫苗研制和ＭＴＢ诊断中的潜在价值作一概述。     

Membrane association of flagellar creatine kinase in the sperm phosphocreatine shuttle.

The flagellar creatine kinase (TCK) of Strongylocentrotus purpuratus sperm is both a principal component of sperm tail membrane preparations and a cytosolic enzyme. An improved purification scheme identified three pools of TCK, termed TCK I, TCK II, and TCK III. TCK I and II were essentially homogeneous protein preparations, while TCK III was heavily contaminated with other flagellar proteins, predominantly guanylate cyclase, and alpha- and beta-tubulin. The three TCK species are roughly present in a 1:10:1 ratio as assessed by activity measurements. TCK I and II are similar proteins as shown by two-dimensional gel electrophoresis, partial proteolytic fragmentation, and cellulose polyacetate electrophoresis and have the same pH-dependent specific activity. However, they are functionally distinct with respect to their capacity to associate with lipids. TCK II associated readily with phospholipid liposomes and detergent micelles, while TCK I did not. Association of TCK II was as a protein monomer with an apparent Kd of approximately 1-2 mM at a 10(4):1 lipid or detergent to protein ratio. Whereas the Kd estimates were pH independent, the rate of association increased 2-3-fold between pH 6.5 and 8. The data are consistent with membrane-association of TCK II being a two-step process, involving a pH-dependent, intramolecular, TCK-specific step and a charge-facilitated, but pH-independent, membrane association step. Membrane association of TCK may, together with microtubule association (Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317) represent a mechanism required for specific accumulation of the enzyme within the flagellum.     

Myristoylation of flagellar creatine kinase in the sperm phosphocreatine shuttle is linked to its membrane association properties.

TCK, the flagellar creatine kinase (ATP:creatine N-phosphotransferase) of sperm from the sea urchin Strongylocentrotus purpuratus is a membrane-associated lipophilic protein involved in energy transport. The cDNA derived protein sequence contains a consensus site sufficient for the covalent attachment of myristate. To examine whether TCK was myristoylated, mouse fibroblast Swiss 3T3 and baby hamster kidney cell lines were transfected with a cDNA encoding the entire TCK protein linked to a metallothionein promotor. TCK expression was induced by zinc and paralleled by incorporation of [3H]myristic acid derived label into the protein. 3H Label incorporated into TCK was resistant to hydroxylamine treatment. The 3H-labeled material released from TCK by acid methanolysis eluted from a C18 reverse phase high pressure liquid chromatography column at the positions of myristic acid and methylmyristate. Thus, TCK expressed in transfected mammalian cell lines contains authentic myristic acid, covalently attached through amide linkage. [3H]Myristoyl TCK comigrated on two-dimensional gels with the purified lipophilic isoform TCK II from sea urchins. Furthermore, like TCK II, [3H]myristoyl TCK associated with phospholipid liposomes, suggesting that myristoylation may mediate the observed membrane association of TCK. Myristoylation of sea urchin sperm flagellar creatine kinase may play a role in confining this enzyme to the flagellum during spermatogenesis.     

小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

电子鼻技术在快速检测小麦矮腥黑穗病菌中的应用

由小麦矮腥黑穗病菌(TCK)引起的小麦矮腥黑穗病是我国重要的检疫性病害,为明确电子鼻技术在快速检测小麦矮腥黑穗病菌方面的可行性,利用电子鼻对含有TCK和小麦光腥黑穗病菌(TFL)不同冬孢子数(50g小麦种子中冬孢子数分别为0、100、101、102、103、104、105)的小麦进行了检测,采用主成分分析法(PCA)和线性判别法(LDA)进行数据分析。结果发现,这2种分析方法均可将不含TCK冬孢子的小麦和含TCK冬孢子的小麦区分开来,而且通过LDA分析,可将TCK冬孢子含量为100、101、102及103以上的处理区分开来。另外,通过PCA分析,可将TCK与TFL区分开来。此结果为电子鼻技术在快速检测小麦矮腥黑穗病菌中的应用奠定了基础。     

实时荧光PCR技术对小麦矮腥黑穗病菌的检测

通过对小麦矮腥黑穗病菌(TCK)及其近似种小麦网腥黑穗病菌(TCT)和小麦光腥黑穗病菌(TFL)的rDNA序列ETS区间测序比较分析,找出了TCK相对于TCT和TFL的特异性序列,并根据TCK的特异性序列设计了实时荧光PCR探针,利用实时荧光PCR技术成功实现了对TCK的检测。     

Traditional Climatic Knowledge: Orchardists' perceptions of and adaptation to climate change in the Campania region (Southern Italy)

Climate change is projected to have severe changes in the Mediterranean area, however, few studies have investigated environmental resource managers’ perceptions and adaptations to climatic change in the area. Our research investigates the use of orchardists' observations for bioindicating climate variations and of their experience for defining possible coping and adaptation strategies. Interviews were conducted with orchardists cultivating apple orchards for at least 30 years in the Campania region (southern Italy) to obtain observations on climate, which were then compared with climate data analyses. Orchardists reported a more unpredictable seasonality and shifting climate conditions, perceived as beginning 20–30 years ago. Climate data analysis seems to corroborate orchardists' perceptions. Traditional Ecological Knowledge specifically addressed to climate and weather is here defined as Traditional Climatic Knowledge (TCK). TCK is a key factor in environmental management.     

Development of a SCAR marker for molecular detection and diagnosis of Tilletia controversa Kühn,the causal fungus of wheat dwarf bunt

Tilletia controversa Kühn (TCK) is an important quarantine pathogen that causes wheat dwarf bunt and results in devastating damage to wheat production. The fungus is difficult to be distinguished from T. caries and T. laevis, which cause wheat common bunt, based on morphological, physiological and symptomatological characteristics of the pathogens. The traditional detection of the fungus can be a long and tedious process with poor accuracy. The inter-simple sequence repeat (ISSR) technique has been used for identifying molecular markers for detection of TCK. Of 28 ISSR primers screened, ISSR-859 amplified a specific 678 bp DNA fragment from all TCK isolates but not from any isolates of the common bunt fungi or other pathogenic fungi tested. Based on the fragment sequence, a pair of sequence characterized amplified region (SCAR) primers was designed, which amplified a 372 bp DNA fragment specifically in TCK. The SCAR marker was detected using as low as 1 ng template DNA of TCK, and was also detected using broken teliospores and DNA from asymptomatic wheat samples. We developed the SYBR Green I and TaqMan Green I and TaqMan real-time polymorphism chain reaction methods to detect TCK with the detection limit of 0.1 fg with asymptomatic wheat samples. Further work is needed to develop a rapid test kit for this pathogenic fungus using the designed specific primers.     

A Casein Kinase-Like Kinase Phosphorylates β-Tubulin and May Be a Microtubule-Associated Protein

The hypothesis that casein kinase II (CKII) is a microtubule-associated protein kinase was investigated using a neuronal cell line and bovine brain. Heparin, an inhibitor of CKII, inhibited the phosphorylation of a PC12 cytosolic protein whose molecular weight was similar to that of beta-tubulin. Partially purified PC12 CKII was immunoreactive to an antibody directed against bovine CKII and was able to phosphorylate purified beta-tubulin in a heparin-inhibitable manner when the concentration of tubulin was less than 50 micrograms/ml. To better determine if CKII is a microtubule-associated protein kinase, bovine brain tubulin was chromatographed on FPLC Mono Q and phosphocellulose columns. Several tubulin casein kinase (TCK) activities were apparent. All TCK activities phosphorylated tubulin and casein, but none was able to phosphorylate the CKII-specific synthetic peptide RRREEETEEE. One of these TCK fractions was immunoreactive to the antibody directed against CKII, and this antibody labeled a 50-kDa molecular mass band that had a molecular mass distinctly different from those of the subunits of CKII. Thus, we suggest that a CKII-like protein, but not CKII, might be a microtubule-associated protein.