重组内皮抑素的质谱分析及对内皮细胞周期的影响

采用质谱、圆二色谱、荧光光谱与细胞周期分析技术研究了重组内皮抑素的蛋白结构和对内皮细胞的作用机制.研究发现,以包涵体方式表达的蛋白中会有未被降解的N末端甲硫氨酸产物,重组内皮抑素在G2期阻断内皮细胞的生长.表明内皮抑素引发内皮细胞凋亡与细胞周期相关,为抗血管生成治疗肿瘤研究奠定了实验基础.     

重组内皮抑素的性质表征与生物活性分析

通过SDS—PAGE电泳、基质辅助激光解吸电离飞行时间质谱、高效液相色谱、地高辛方法、蛋白印迹方法、鸡胚尿囊膜血管生成抑制实验和透射电子显微镜法对重组内皮抑素进行纯度分析、鉴定和生物活性研究。重组内皮抑素纯度达到99％，能够抑制鸡胚尿囊膜新血管生成和人脐静脉内皮细胞ECV304增殖，引发内皮细胞的凋亡。     

肿瘤血管生成抑制剂

肿瘤血管生成的药物治疗是当前有关肿瘤的热点研究领域,目前已经有数种肿瘤血管生成抑制剂上市。肿瘤血管生成抑制剂能够抑制肿瘤的生长和转移,甚至使肿瘤消退,此类药物的研究开发可为肿瘤患者提供高效、低毒且抗瘤谱更广的药物。本文综述了近年来血管生成抑制剂的研究进展,首先介绍了间接血管生成抑制剂,此类药物中作用于血管内皮细胞生长因子受体信号通路的药物是目前最成功的一类血管生成抑制剂;其次介绍了直接血管生成抑制剂,使用这类药物更有可能避免间接抑制剂所引起的血管生成援救反应;然后在其他途径肿瘤血管生成抑制剂部分,本文详细介绍了作用机制尚不明确的沙利度胺及其衍生物;最后,本文分析讨论了这类药物开发所遇到的一些问题,如抗血管生成治疗新理论的挑战和耐药性等,并指出了未来发展方向。     

R-or-S-Thalidomide合成新工艺的研究

沙利度胺(thalidom ide)具有抗炎、免疫调节、治疗风湿性疾病和抗肿瘤等作用。近期对沙利度胺的研究热点集中于肿瘤治疗中抗血管生成,其作用机制是通过非白由基介导的DNA氧化损伤直接抑制肿瘤细胞的生长和存活,抑制肿瘤血管新生,抑制促瘤细胞生长因子的分泌,降低其活性;同时,它     

荧光光谱和圆二色谱研究重组内皮抑素与芦荟大黄素的相互作用

利用荧光光谱法和圆二色谱法研究了重组内皮抑素与中药有效成分芦荟大黄素的相互作用机制,求得两者在25℃和37℃的结合常数Ka为6.623×104和3.796×104。研究发现,Zn2+等5种金属离子能够竞争性降低重组内皮抑制与芦荟大黄素的结合常数,下降幅度为35%～55%,并且其影响程度随温度升高而增强。鸡胚尿囊膜血管生成实验结果表明,芦荟大黄素能增强重组内皮抑素的抗血管生成活性,使其对新生血管的抑制作用提高50%。     

熊果酸衍生物的合成及抗肿瘤血管生成活性

对熊果酸C28位与C3位进行结构修饰得到了24个衍生物,并利用1H NMR,13C NMR,MS及HR-MS对这些化合物进行了结构表征.进一步通过MTT法,以内皮细胞HUVEC为主要模型,研究了24个衍生物抗肿瘤血管生成的活性,同时以A549,Bel-7402及MCF-7细胞为模型研究了上述衍生物对肿瘤细胞的抑制活性.研究结果表明,与熊果酸相比,化合物5,9,12e和14e对HUVEC细胞有较好的选择性,化合物12a和13h比熊果酸的抗肿瘤血管生成活性略高,因此通过适当改变熊果酸C28位的结构可以提高其对内皮细胞HUVEC的选择性,增强抗肿瘤血管生成活性.本文结果表明,熊果酸及其衍生物是潜在的具有抗肿瘤血管生成作用的先导化合物,通过有效的结构优化可能得到新型的抗肿瘤血管生成的化合物.     

稀土离子对重组内皮抑素与肝素相互作用的影响

通过荧光光谱法和圆二色谱法研究了重组内皮抑素与稀土离子和肝素的作用.结果表明,稀土离子和肝素都可以与重组内皮抑素结合并引起蛋白的荧光猝灭和二级结构的改变,稀土离子与重组内皮抑素的结合会影响其与肝素的相互作用,并降低重组内皮抑素对人脐静脉内皮细胞的增殖抑制活性.     

基因重组可溶性非融合血管生成抑制剂Kringle 5的色谱分离和纯化

Kringle 5是血纤维蛋白溶酶原中特异抑制内皮细胞增生和迁移活性最高的一种血管生成抑制剂。该实验在前期成功克隆和表达可溶性非融合血管生成抑制剂Kringle 5的基础上,建立了一种两步色谱法分离纯化Kringle 5的方法。首先用SP Sepharose Fast Flow强阳离子交换色谱柱对Kringle 5重组菌体破碎上清液进行初步分离,然后再用丙烯葡聚糖凝胶S-100 HR凝胶排阻色谱柱对其进行进一步的纯化。采用本方法得到的可溶性非融合血管生成抑制剂Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和高效凝胶排阻色谱检测其纯度大于98%,通过鸡胚尿囊膜法确定这种蛋白质具有抑制内皮毛细血管生长的活性。     

环肽类组蛋白去乙酰化酶抑制剂

组蛋白去乙酰化酶抑制剂是一类以组蛋白去乙酰化酶为靶点的新型靶向抗肿瘤药物,在抗增殖、促凋亡、促分化、阻滞细胞生长周期、抗血管生成等方面有很好的作用。组蛋白去乙酰化酶抑制剂以其独特的抗肿瘤作用机理,在研发肿瘤治疗药物中占有重要地位。在组蛋白去乙酰化酶抑制剂分类中,环肽类抑制剂结构最为复杂,对多种类型的实体瘤及血液学癌细胞均具有良好的对抗作用。本文针对天然和化学合成的环肽类组蛋白去乙酰化酶抑制剂中金属结合区、表面识别区以及连接区的结构特点进行了综述,并描述了各类抑制剂对酶的抑制活性和抗肿瘤增殖活性。对抑制剂不同结构区的修饰、改造可以使抑制剂对不同肿瘤细胞具有高效性和特异性作用,通过构效关系研究寻找具有高效低毒、靶向性环肽类抑制剂的结构规律,可为研究开发抗肿瘤药物提供帮助。     

VEGFR2活性腔性质以及与抑制剂的结合模式研究

VEGFR2介导肿瘤诱导的血管生成作用, 是抑制肿瘤生长和转移的新靶点. 为深入探讨VEGFR2活性腔性质以及与抑制剂的结合模式, 采用多拷贝同时搜寻法(MCSS)研究VEGFR2活性腔的性质, 然后用分子对接方法对5个已上临床的VEGFR抑制剂与VEGFR2活性腔进行对接计算, 讨论它们的结合模式, 确定与配体结合相关的关键残基. 研究发现: 疏水腔I, II是配体结合的关键区域, 残基Glu915, Cys917是关键的氢键作用位点, Lys866, Glu883和Asp1044形成的极性区域对提高配体亲合力很重要, 疏水腔III和极性腔IV是额外增强配体结合力的区域, IV区的Arg1030可提供额外的氢键作用位点. 本研究可为全新VEGFR2抑制剂的合理药物设计提供理论依据, 为寻找新的抗肿瘤药物奠定基础.     

Purification and characterization of recombinant murine endostatin in E. coli

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent.     

Determination of the disulfide bond pattern of the endogenous and recombinant angiogenesis inhibitor endostatin by mass spectrometry.

Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed.     

A molecular dynamics study of human endostatin and its synthetic fragments with antiangiogenic properties

Human endostatin is one of the better characterized endogenous angiogenesis inhibitors, and its ability to modulate vascularization of tumours could be of great therapeutic interest. These properties are not exclusive to the full-length protein, but are shared by some of its synthetic fragments. A number of research groups have partitioned human endostatin in different peptides and have investigated their activity, in order to collect a body of experimental data which could be important in shedding new light on their structure-activity relationships. It was also reported that a small active fragment can become inactive when contained in a larger fragment, revealing an apparent discrepancy in the experimental results. Very few studies have been devoted to the computational analysis of these systems and to the rationalization of their properties using molecular modelling. Through molecular dynamics simulations of human endostatin and of four synthetic fragments, we have been able to rationalize the experimental findings. In particular, we have identified a pattern consisting of six amino acids, namely R-R(G)-A-D-R-A, which appears to be an active epitope if it is properly exposed to the solvent. Interestingly, this pattern can be already present in sequential order in the primary structure, or it can be generated by the spatial approach of two groups of residues, far apart in the primary structure, as an effect of the peptide folding. Comparing the structural features and the time evolution of all the simulated peptides we provide a coherent explanation of their activity or inactivity.     

烟草与尿样中的代谢标记物

卷烟烟气进入人体后会对人体的代谢物产生影响,目前研究较多的是稠环芳烃类、烟草特有亚硝胺类和尼古丁的代谢物。文章分别介绍了稠环芳烃类代谢物1-羟基芘,烟草特有亚硝胺类代谢物NNAL和NNAL-Gluc,尼古丁的代谢物可天宁,对这些代谢物的检测方法、体内的含量水平、各种条件的影响进行了综述。     

Capillary electrophoresis of proteins 2005-2007

This review article with 239 references describes recent developments in capillary electrophoresis of proteins, and covers the two years since the previous review (V. Dolník, Electrophoresis 2006, 27, 126-141) through spring 2007. It includes topics related to CE of proteins, such as sample pretreatment, wall coatings, improving separation, various forms of detection, and special electrophoretic techniques including ACE, CIEF, capillary ITP, and CEC. The paper describes applications of CE to analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals and recombinant protein preparations.     

Capillary electrophoresis of proteins 2003-2005

This review article with 304 references describes recent developments in CE of proteins, and covers the two years since the previous review (Hutterer, K., Dolník, V., Electrophoresis 2003, 24, 3998-4012) through Spring 2005. It covers topics related to CE of proteins, including modeling of the electrophoretic migration of proteins, sample pretreatment, wall coatings, improving separation, various forms of detection, special electrophoretic techniques such as affinity CE, CIEF, and applications of CE to the analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals, and recombinant protein preparations.     

Anti-tumor Effects of a Recombinant Fowlpox Virus Expressing Apoptin on Human Cervical Carcinoma in Vivo and in Vitro

Apoptin is a chicken anemia virus-derived,p53-independent,bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis of various human tumor cells,but not of normal diploid cells.To explore the application of apoptin in tumor gene therapy,we used a recombinant fowlpox virus expressing apoptin protein (vFV-Apoptin) to investigate the anti-tumor effectes of vFV-Apoptin on human cervical carcinoma(HeLa) cells in vivo and in vitro through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetraz...