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用定点诱变技术鉴定蛇毒解联蛋白RGD侧翼氨基酸残基的结构和功能(英文)

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Acta Biochimica et Biophysica Sinica 2001年第2期33卷 153-157页

作者：徐存拴,rahman salman 河南师范大学生命科学学院!新乡453002 中国伦敦大学皇家医学院血友病研究中心!伦敦SE17EH 英国

为了证实蛇毒蛋白RGD侧翼氨基酸残基对ADP诱导的血小板凝聚抑制作用的重要性 ,分析了RGD环上游氨基酸残基对解联蛋白活性的影响。将位于两种解联蛋白蝮蛇毒素 (kistrin)和华丽蛇毒素 (elegantin)之间的氨基酸残基和RGD环内的氨基酸残基... 详细信息

为了证实蛇毒蛋白RGD侧翼氨基酸残基对ADP诱导的血小板凝聚抑制作用的重要性 ,分析了RGD环上游氨基酸残基对解联蛋白活性的影响。将位于两种解联蛋白蝮蛇毒素 (kistrin)和华丽蛇毒素 (elegantin)之间的氨基酸残基和RGD环内的氨基酸残基进行杂交。用定点诱变技术将elegantin一级结构内的KKKR4 4T4 5I4 6/A50 RGDN54 P55分别突变为SKAG4 4T4 5I4 6/P50 RGDM54 P55和SKAG4 4I4 6/P50 RGDM54 P55,这些序列和kistrin中相应的序列S39RAGT4 3/P50 RGDM52 P53 有相似之处 ,序列由KKKR4 4T4 5I4 6/A50 RGDN54 P55突变为SKAG4 4T4 5I4 6/P50 RGDM54 P55,显著地降低了elegantin对血小板凝聚的抑制因子的活性 ,而T4 5的缺失 (KKKR4 4T4 5I4 6/A50 RGDN54 P55突变为SKAG4 4I4 6/P50 RGDM54 P55)却增强了elegantin对血小板凝聚的抑制作用 ,进一步研究证明它们的电泳特性是不同的。表明了RGD模体上游 39～ 45位的氨基酸与RGD侧翼氨基酸残基序列对解联蛋白的结构和功能具有重要作用

关键词：定点诱变整联蛋白解联蛋白 RGD模体(motif) 蛇毒蛋白

Large scale of identification of differentially expressed genes in the regenerating rat liver after SISPH

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Science China(Life Sciences) 2005年第6期48卷 624-635页

作者： rahman salman Homophilia Research Center London University London SE17EH United Kingdom

Extensive gene expression analysis was carried out after a 0, 4, 36, 72, 96 h short interval successive partial hepatectomy (SISPH) was performed. A total of 185 elements were identified as differing by more than two-fold in their expression levels at one or more time points. Of these 185 elements, 103 were up-regulated, 82 were down-regulated and 86 elements were unreported genes. Quite a few genes were previously unknown to be involved in liver regenera-tion (LR). Using cluster and general analysis, we found that the genes at five time points of the SISPH share eight different types of different expression profiles and eight distinct temporal in-duction or suppression patterns. A comparison of the gene expression in SISPH with that after PH found that 41 genes were specifically altered in SISPH, and 144 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but they were present in different amounts at the different time points. The conclusions are that (i) microarrays combined with suppressive subtractive hybridization (SSH) can effectively identify genes involved in LR on a large scale; (ii) more genes were up-regulated than down-regulated; (iii) there are fewer abun-dantly expressed genes than those with increased levels of 2-5 fold.

关键词： partial hepatectomy (PH), short interval successive partial hepatectomy (SISPH), liver regeneration (LR), subtracted cDNA libraries, complementary DNA microarray, cluster analysis.

Correlation analysis of liver tumor-associated genes with liver regeneration

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World Journal of Gastroenterology 2007年第24期13卷 3323-3332页

作者： Cun-Shuan Xu Shou-Bing Zhang Xiao-Guang Chen salman rahman College of Life Science Henan Normal University Xinxiang 453007 Henan Province China Key Laboratory for Cell Differentiation Regulation Xinxiang 453007 Henan Province China Laboratory of Thrombosis and Vascular Remodelling Division of Cardiovascular Medicine King's College London School of Medicine St Thomas' Hospital Lambeth Palace Road London SE1 7EH United Kingdom

AIM: To study at transcriptional level the similarities and differences of the physiological and biochemical activities between liver tumor (LT) and regenerating liver cells. METHODS: LT-associated genes and their expression changes in LT were obtained from databases and scientific articles, and their expression profiles in rat liver regeneration (LR) were detected using Rat Genome 230 2.0 array. Subsequently their expression changes in LT and LR were compared and analyzed. RESULTS: One hundred and twenty one LT-associated genes were found to be LR-associated. Thirty four genes were up-regulated, and 14 genes were down-regulated in both LT and regenerating liver; 20 genes up-regulated in LT were down-regulated in regenerating liver; 21 up-regulated genes and 16 down-regulated genes in LT were up-regulated at some time points and down-regulated at others during LR. CONCLUSION: Results suggested that apoptosis activity suppressed in LT was still active in regenerating liver, and there are lots of similarities and differences between the LT and regenerating liver at the aspects of cell growth, proliferation, differentiation, migration and angiogenesis.

关键词： Partial hepatectomy Rat Genome 230 2.0Array Apoptosis Liver regeneration-associated gene Liver tumor-associated gene

R^(49)A^(50)RGDN^(54)P^(55)突变成I^(49)P^(50)RGDM^(54)P^(55)对华丽蛇毒素活性的影响(英文)

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河南科学 2003年第4期21卷 426-430页

作者：徐存拴 salman rahman 河南师范大学生命科学学院伦敦大学皇家医学院凝血研究室

用基因点突变技术将已知具有强抑制血小板凝集作用的去整合蛋白———kistrin的RGD袢区IPRGDMP替换抑制血小板凝集作用较弱的elegantinRGD袢相应部位的RARGDNP,重构一个新的杂合的去整合蛋白,然后,分析该杂合蛋白对血小板凝集的抑制作... 详细信息

用基因点突变技术将已知具有强抑制血小板凝集作用的去整合蛋白———kistrin的RGD袢区IPRGDMP替换抑制血小板凝集作用较弱的elegantinRGD袢相应部位的RARGDNP,重构一个新的杂合的去整合蛋白,然后,分析该杂合蛋白对血小板凝集的抑制作用。结果表明,重组的杂合去整合蛋白比野生型elegantin具有更强的抑制血小板凝集活性。推测RGD顺序两翼的氨基酸残基在保持去整合蛋白的结构和功能中起重要作用。

关键词：华丽蛇毒素基因点突变去整合蛋白血小板凝集 IPRGDMP RARGDNP

华丽蛇毒素RGD序列上游点突变对结构和功能的影响分析

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华丽蛇毒素RGD序列上游点突变对结构和功能的影响分析

中国细胞生物学学会医学细胞生物学、免疫细胞生物学和发育生物学专业委员会学术研讨会

作者：徐存拴辛泽华 rahman salman 河南师范大学生命科学学院河南省生物工程重点实验室英国皇家医学院血友病研究中心

解联蛋白（disintegrin）是一类存在于蛇毒中的血小板凝集抑制因子。研究表明,Arg-GLT-Asp（RGD）序列与解联蛋白的生物学活性有密切关系,紧靠RGD序列两翼的氨基酸残基有调节解联蛋白抑制ADP-诱导血小板凝集作用,RGD序列侧翼的第45位氨... 详细信息

解联蛋白（disintegrin）是一类存在于蛇毒中的血小板凝集抑制因子。研究表明,Arg-GLT-Asp（RGD）序列与解联蛋白的生物学活性有密切关系,紧靠RGD序列两翼的氨基酸残基有调节解联蛋白抑制ADP-诱导血小板凝集作用,RGD序列侧翼的第45位氨基酸残基位于该类蛋白内部形成的小槽内,这个小槽在RGD袢形成发夹结构方面起重要作用。在此基础上,本文进一步分析了RGD袢上游区域氨基酸残基在调节解联蛋白功能中的作用。具体方法是:用基因点突变技术将华丽蛇毒素的KKKRTI突变成腹蛇毒素的SKAGTI和SKAGI,然后分析突变蛋白的结构、性质和功能变化。结果表明,前一种突变KKKRTI→SKAGTI显著地降低了华丽蛇毒素对血小板凝集的抑制活性,后一种突变SKAGIT→SKAGI有效地恢复了华丽蛇毒素抑制血小板凝集的作用。同时,这两种突变也改变了含点突变基因的pGEX-3X质粒在受体细胞（***）内的表达,例如,重组于pGEX-3X质粒的野生型腹蛇毒基因和华丽蛇毒素基因以可溶性形式表达,而重组于相同载体的点突变基因表达的目的蛋白是不溶性的,为此,本文又通过构建表达难溶蛋白载体的方法获得了突变基因的可溶性表达产物。本文首次证实了RGD上游的39.45位氨基酸残基与解联蛋白的结构和功能有密切关系。

关键词：基因点突变整联蛋白解联蛋白 RGD序列 RGD袢 ADP诱导的血小板凝集腹蛇毒素华丽蛇毒素

大规模鉴定大鼠0,4,36,72,96h短间隔连续部分肝切除中再生肝差异表达的基因

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中国科学（C辑） 2005年第2期35卷 153-163页

作者：徐存拴袁金云韩鸿鹏常翠芳李文强杨柯金赵利峰李玉昌张会勇 salman rahman 河南师范大学生命科学学院新乡453002 河南省部共建细胞分化调控国家重点实验室新乡453007 Homophilia Research Center London UniversityLondon SE17EHUnited Kingdom

用抑制性消减杂交方法(SSH)构建了短间隔连续部分肝切除(SISPH)再生肝的消减cDNA文库,从中筛选出了551个与肝再生相关的基因,把这些基因制成cDNA微阵列(cDNA芯片),分析它们在0h正常肝及4,36,72,96h再生肝中的动态变化发现,185个基因至... 详细信息

用抑制性消减杂交方法(SSH)构建了短间隔连续部分肝切除(SISPH)再生肝的消减cDNA文库,从中筛选出了551个与肝再生相关的基因,把这些基因制成cDNA微阵列(cDNA芯片),分析它们在0h正常肝及4,36,72,96h再生肝中的动态变化发现,185个基因至少在肝再生的一个时间点表达变化达2倍以上;185个基因中的86个属未报道的基因,99个为已报道的基因,但在此之前尚不知道它们与肝再生有关;185个基因中的103个在肝再生中表现上调表达,82个表现下调表达.用GeneMath软件和GeneSpring方法对这些基因在肝再生中的表达轮廓进行聚类分析表明,基因的表达模式可分为8组,即早期诱导、中期诱导、晚期诱导、持续诱导、早期抑制、中期抑制、晚期抑制和持续抑制.与一次性部分肝切除(PH)相比,41个基因在SISPH中特异性表达,其他基因在两个模型中的表达趋势相同,但在各时间点的表达丰度有差异.综合分析可见,抑制性消减杂交技术与基因芯片技术相结合是研究再生肝差异表达基因的有效方法;肝再生中上调表达的基因多于下调表达的基因;早期诱导的基因多于晚期诱导的基因;诱导表达幅度大的基因少于诱导表达幅度小的基因.

关键词：短间隔连续部分肝切除抑制性消减杂交技术 cDNA微阵列大鼠鉴定规模 cDNA文库 cDNA芯片差异表达基因基因芯片技术诱导表达肝再生特异性表达杂交方法变化发现表达变化表达模式综合分析有效方法时间点分析表

大鼠基因组的肝再生相关基因鉴定及表达谱分析

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大鼠基因组的肝再生相关基因鉴定及表达谱分析

中国细胞生物学学会第九次会员代表大会暨青年学术大会

作者：徐存拴常翠芳陈晓光 salman rahman 河南师范大学生命科学学院 The Division of Vascular Surgery University of Washington School of Medicine and Veterons Affairs Puget Sound Health Care SystemSeattleWashington 98108

大鼠部分肝切除(partial hepatectomy,PH)后,残肝细胞经过激活、去分化、增殖、再分化及组织结构功能重建等生理生化过程、持续约一周时间将丢失的肝组织补偿,这一过程称为肝再生(liver regeneration, LR)。到目前为止,人们尚不完全清... 详细信息

大鼠部分肝切除(partial hepatectomy,PH)后,残肝细胞经过激活、去分化、增殖、再分化及组织结构功能重建等生理生化过程、持续约一周时间将丢失的肝组织补偿,这一过程称为肝再生(liver regeneration, LR)。到目前为止,人们尚不完全清楚哪些基因参与肝再生、它们在肝再生中的表达谱及肝再生所涉及生理生化活动进程。为此,本文用含14 620个已知基因的 Rat Genome 230 2．0芯片分析了大鼠 PH 后恢复

关键词：大鼠部分肝切除肝再生相关基因基因芯片生物信息学系统生物学

Analysis of the expressed genes in the regenerating rat liver after partial hepatectomy

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Analysis of the expressed genes in the regenerating rat live...

中国细胞生物学学会第八届会员代表大会暨学术大会

作者：常翠芳袁金云李文强韩鸿鹏杨柯金赵利峰李玉昌张会勇章静波徐存拴 salman rahman College of Life Sciences Henan Normal University Xinxiang 453002 China Key Laboratory for Cell Differentiation Regulation built by Province and Ministry Xinxiang 453002 China Homophilia Research Center London UniversityLondon SE17EHUnited Kingdom

It was confirmed that lots of genes participating in liver regeneration (Fukuhara et al., 2003;Yang et al., 1999). Learning gene expression patterns in liver regeneration may help to reveal how liver regeneration is initiated and controlled as well as how liver disease is leaded. In the present study, 576 elements from 4687 ESTs in the subtracted cDNA libraries were made into an in-house cDNA micro array to analyze gene expression profiles of the regenerating rat liver after PH. Hybridized with RNA from four rat livers at each time point (taken at 0, 2,4, 8,12, 16, 24, 36, 48, 72, 96 and 144h after PH), 300 elements are discovered up- or down-regulated more than two folds at one or more time points after PH. The sequence analysis showed that 152 genes were unreported, and 133 genes were reported, but they were uncertain that which role they play in liver regeneration. Analyzing their expression differences in the regenerating liver at different time points after PH showed that in the priming phase of liver regeneration (LR), the expression of 34 genes was rapidly altered within 6h and then recovered gradually to control levels in 48-72h after PH. In the cell cycle progression phase of 8-36h after PH, the expression of 86 genes was altered markedly. In the terminal phase of LR over 72h after PH, the expression of 13 genes was altered distinctly. Following biochemical actions, the 133 elements were respectively involved in stress response, in glycometabolism, in fatty and stearoyl metabolism, in oxidation and reduction response, in regulation-proteins, in glycoproteins, in lipid-proteins, in nucleolar proteins, in receptors, in factors, in hemoglobins, in immunological proteins, in chaperonins, in cytoskelets, in ribosomal proteins, in marker proteins, in amino acid enzymes, in nuclearase, in proteolytic enzymes, in proteinase inhibitors, in phopshorylases, in phosphatases, in synthase, in transferases. Their action is being *** analysis of gene express

关键词： Partial hepatectomy (PH) Subtracted cDNA libraries Complementary DNA micro array Liver regeneration (LR) Cluster analysis

Identification of the Expressed Genes in the Regenerating Rat Liver in 0-4-8-12h Short Interval Successive Partial Heptectomy

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Identification of the Expressed Genes in the Regenerating Ra...

中国细胞生物学学会第八届会员代表大会暨学术大会

作者：袁金云韩鸿鹏李文强常翠芳杨柯金赵利峰李玉昌张会勇章静波徐存拴 salman rahman College of Life Sciences Henan Normal University Xinxiang 453002 China Key Laboratory for Cell Differentiation Regulation built by Province and Ministry Xinxiang 453002 China Homophilia Research Center London University London UniversityLondon SE17EHUnited Kingdom

The mammalian liver has a very strong regeneration capacity after partial hepatectomy (PH) (Michalopoulos & Defrance, 1997;Fausto, 2000). To further learn the genes participating in the liver regeneration (LR), our laboratory studied the gene expression profiles in regenerating liver of 0-4-8-12h short interval successive partial hepatectomy (SISPH) by microarray (cDNA chip) of 551 cDNA screened from subtracted cDNA libraries of the rat liver, and analyzed these data in virtue of Microsoft Excel and GeneSpring. 183 elements were discovered either up- or down-regulated more than twofold at one or more time points in SISPH, of which 86 genes were unreported, and other 97 were reported, which role they play in LR is still unknown;106 genes were down regulated to less than 0.5;54 genes were up regulated to 2-5 folds, and 23 genes were strongly up regulated to more than 5 folds, of which the maximum was up regulated over 50 folds. Briefly, the genes expressed consistently are less than that immediately, and the genes expressed in high abundance are much less than that increased 2-5 folds;these genes functionally involved in stress response, in glycometabolism, in stearoyl metabolism, in oxidation and reduction response, in regulation-proteins, in glycoproteins, in lipid-proteins, in nucleolar proteins, in receptors, in factors, in hemoglobins, in immunological proteises, in cytoskelets, in marker proteins, in amino acid enzymes, in proteolytic enzymes, in proteinase inhibitors, in phopshorylase, in phosphatases, in synthases and in transferases. Their function is being *** cluster analysis and generalization analysis showed the gene expression profiles had no much similarity in the four time points of 0-4-8-12h SISPH. κ-means analysis generated 7 kind of ramose gene expression clusters and five distinct temporal induction or suppression patterns following immediate induction, late induction, consistent induction, late suppression, consistent suppression. A compar

关键词： Complementary DNA microarray Partial hepatectomy (PH) Short interval successive partial hepatectomy (SISPH) Liver regeneration (LR) Cluster analysis Subtracted cDNA libraries