RGD结合型阿克拉霉素A脂质体的抑瘤作用

目的：观察RGD多肽结合型阿克拉霉素A长循环脂质体（RGD—ACM—A—liposome）对动物实验肿瘤的抑制作用。方法：以逆相蒸发法制备ACM—A长循环脂质体,并通过反应与RGD基元多肽进行共价键结合．然后静脉注射于荷瘤小鼠体内,之后处死动物,解剖肿瘤,计算并比较抑瘤率及生命延长率。结果：RGD多肽结合型阿克拉霉素A长循环脂质体对小鼠S180和EAC腹水癌具有较好的抑制作用。结论：RGD多肽结合型阿克拉霉素A长循环脂质体可能通过导致血管凋亡以及局部释放抗癌药杀灭癌细胞,对新生肿瘤具有抑制与治疗作用。     

蛇毒解离素对肿瘤侵袭、转移的影响

解离素是蛇毒中的一类活性蛋白质,因含特异性RGD(精-甘-天冬氨酸,Arg—Gly—Asp)或KGD(精-甘-天冬氨酸,Lys—Gly—Asp)基序,可阻断细胞膜表面的整合素与其配体结合,具有抑制血小板聚集、影响细胞迁移和抑制血管生成等作用,从而在多个环节遏制肿瘤细胞的侵袭和转移。     

含有RGD序列多肽构效关系的研究

目的：探讨含RGD多肽的构效关系。方法：采用分子力学和量子化学方法对某些含RGD序列多肽的化学结构进行分子模拟和量化计算。结果：优化了各化合物的空间结构，得到了优势构象和优势构象的能量，研究了分子的电荷分布，前线轨道能量等。结论：借助于理论计算可解释含RGD（Arg-Gly-Asp)序列多肽的构效关系，确定了多肽与受体结合时的活性位点，对其活性差异给予较好的分析，可望为合成具有更高活性的含RGD序列多肽提供理论指导。     

RGD肽修饰的异长春花碱-粉防己碱脂质体的制备及体外抑瘤作用研究

目的:制备RGD肽修饰的异长春花碱(VRB)-粉防己碱(TET)脂质体,研究其对脑胶质瘤C6细胞的抑制作用。方法:采用薄膜分散法和硫酸铵梯度法制备RGD肽修饰的VRB-TET脂质体,观察其形态和粒径分布,测定其中VRB的含量;以磺酰罗丹明B法分别测定空白靶向脂质体、VRB普通脂质体和RGD肽修饰的VRB-TET脂质体对C6细胞的抑制作用。结果:所制RGD肽修饰的VRB-TET脂质体呈圆球状或类圆球状,表面光滑,粒径为120 nm左右,其中VRB的平均含量为28.27μg/m L(RSD=0.38%,n=3)。空白靶向脂质体对C6细胞生长无显著影响;RGD肽修饰的VRB-TET脂质体能明显抑制C6细胞生长,其作用后细胞存活率明显低于VRB普通脂质体(P<0.05)。结论:成功制得RGD肽修饰的VRB-TET脂质体,其具有明显的抑制C6细胞生长的作用。     

链霉素抗元结构的探讨

纯链霉素、纯双氢链霉素与福氏完全佐剂免疫家兔可以产生抗链霉素及抗双氢链霉素抗体。我们用间接血凝抑制试验方法研究了链霉素和双氢链霉素的抗元结构,以链霉素、双氢链霉素、二链霉胺及链霉素降解物;链霉胍、麦芽酚,链霉二醣胺、链霉素酸、链霉肟等为抑制剂进行间接血凝抑制试验的结果表明二链霉胺抑制量最小,为0.15μg。推测其结构与链霉素抗元决定簇结构相似。链霉素的抗体是链霉素的醛基与蛋白质氨基共价结合形成的抗元产生的。双氢链霉素产生两种抗体,一为相应于链霉素抗体,另一种可能为双氢链霉素的胍基与蛋白质、多肽等物质作用形成抗元产生的。     

链霉素抗元结构的探讨

纯链霉素、纯双氢链霉素与福氏完全佐剂免疫家兔可以产生抗链霉素及抗双氢链霉素抗体。我们用间接血凝抑制试验方法研究了链霉素和双氢链霉素的抗元结构,以链霉素、双氢链霉素、二链霉胺及链霉素降解物;链霉胍、麦芽酚,链霉二醣胺、链霉素酸、链霉肟等为抑制剂进行间接血凝抑制试验的结果表明二链霉胺抑制量最小,为0.15μg。推测其结构与链霉素抗元决定簇结构相似。链霉素的抗体是链霉素的醛基与蛋白质氨基共价结合形成的抗元产生的。双氢链霉素产生两种抗体,一为相应于链霉素抗体,另一种可能为双氢链霉素的胍基与蛋白质、多肽等物质作用形成抗元产生的。     

生长抑制素与生长抑制素瘤综合征

生长抑制素是在丘脑下部的一种能抑制垂体生长激素分泌的多肽样物质,1972年Gui-llemin 在研究生长激素释放因子时,自羊的下丘脑成功地分离提纯出生长激素释放抑制激素(生长抑制素),生长抑制素具有强大的抑制生长激素分泌的能力。由于生长抑制素作用广泛,除抑制生长激素的分泌外,而且还能影响胰腺功能、胃肠道激素的分泌、胃肠道的运动与吸收以及催乳素、TSH、ACTH 等的分泌。兼备了神经内分泌、神经传递物、邻分泌(Pa-rahormone)与传统内分泌激素的多样作用,因而受到学者们越来越多的重视。一、生长抑制素的结构与血浆内生长抑制素样免疫活性物(SLI)生长抑制素由14个氨基酸所构成的多肽,其线型或环状结构具有     

RGD多肽结合型长循环脂质体的肿瘤靶向性研究

目的 :研究RGD(精氨酸、甘氨酸、天门冬氨酸 )多肽结合型长循环脂质体的肿瘤靶向性。方法 :将长循环脂质体 ,通过共价键反应与RGD基元多肽进行结合 ,然后研究其在荷瘤小白鼠体内的肿瘤靶向性。结果 :RGD结合型长循环脂质体在Colon26荷瘤小白鼠体内具有较好的肿瘤靶向性。结论 :含RGD基元多肽结合型长循环脂质体可能成为一种新型的受体介导靶向制剂     

轻链二硫键缺失突变促进链间重组二硫键BDD-FVⅢ变构体分泌

凝血VⅢ因子(coagulation factor VⅢ, FVⅢ)分子内含有8对二硫键,参与维持其结构和功能,轻链A3结构域中Cys1899/Cys1903间的二硫键阻碍分泌,本室曾通过在FVⅢ重链和轻链间引入重组二硫键,证明其可促进消除B结构域的FVⅢ (B domain-deleted FVⅢ, BDD-FVⅢ)重、轻链异源二聚体的组装和分泌。本文在此基础上将BDD-FVⅢ的重链A2区Met662和轻链A3区Asp1828突变为Cys,从而构建可形成链间二硫键的变构体F8C。将F8C轻链A3区Cys1899和Cys1903突变为Gly,从而消除内源性二硫键,得到变构体F8CG,并探索这两种BDD-FVⅢ变构体的分泌促进作用。通过HEK293和COS-7细胞的基因瞬时转染实验,检测细胞表达变构体多肽的二硫键形成情况和培养上清中变构体多肽的分泌量和凝血活性,并检测变构体的血管性血友病因子(von Willebrand factor, vWF)结合力。结果显示,细胞转基因表达产物F8C和F8CG均以二硫键连接的异源二聚体多肽为主,而野生型BDD-FVⅢ以易于解离的异源二聚体多肽为主。F8C分泌量和活性明显高于野生型BDD-FVⅢ, F8CG的分泌量和活性相对于F8C得到进一步提高,显示轻链二硫键的破坏对分泌具有促进作用,而对vWF的结合力无显著性影响。本文为进一步的变构体体内转基因研究奠定了基础。     

抗蛋白酶降解肽类药物的结构修饰研究进展

多肽类药物的蛋白酶降解稳定性是决定该药物是否长效的重要因素。本文综述了近年来用以提高多肽药物蛋白酶降解稳定性的主要策略,尤其对特定位置如N端、C端及氨基酸侧链等的化学结构修饰进行了分类和概述,探讨了这些修饰方法对发展多肽药物的贡献及不足。     

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys189 is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from PII SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu276 of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other PIII SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other PIII members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain.     

Recombinant expression of mutants of the Frankenstein disintegrin, RTS-ocellatusin. Evidence for the independent origin of RGD and KTS/RTS disintegrins

The requirements to transform a short disintegrin of the RGD clade into an RTS disintegrin, were investigated through the generation of recombinant mutants of ocellatusin in which the RGD tripeptide was substituted for RTS in different positions along the integrin-specificity loop. Any attempt to create an active integrin α₁β₁ inhibitory motif within the specificity loop of ocellatusin was unsuccessful. Replacing the whole RGD-loop of ocellatusin by the RTS-loop of jerdostatin was neither sufficient for confering α₁β₁ binding specificity to this ocellatusin-RTS Frankenstein² mutant. Factors other than the integrin-binding loop sequence per se are thus required to transform a disintegrin scaffold from the RGD clade into another scaffold from the RTS/KTS clade. Moreover, our results provide evidences, that the RTS/KTS short disintegrins have potentially been recruited into the venom gland of Eurasian vipers independently from the canonical neofunctionalization pathway of the RGD disintegrins. PCR-amplifications of jerdostatin-like sequences from a number of taxa across reptiles, including snakes (Crotalinae, Viperinae, and Elapidae taxa) and lizards (Lacertidae and Iguanidae) clearly showed that genes coding for RTS/KTS disintegrins existed long before the split of Lacertidae and Iguania, thus predating the recruitment of the SVMP precursors of disintegrins, providing strong support for the view of an independent evolutionary history of the RTS/KTS and the RGD clades of short disintegrins.     

Non-RGD-containing snake venom disintegrins,functional and structural relations

Snake venom disintegrins are present in a variety of species and are functionally divided into three families: RGD, MLD and R/KTS. The RGD family of disintegrins, which bind and inhibit the physiological functions of RGD-dependent integrins, constitute the largest and most investigated family. This review will be focused on characterization of two relatively new families of snake venom disintegrins, expressing in their active site MLD and R/KTS motifs. The MLD motif, present only in heterodimeric disintegrins, mediates binding of these disintegrins to α4β1, α4β7 and α9β1 integrins, whereas the presence of a KTS or RTS sequence in the active site selectively directs activity of disintegrins to the collagen receptor α1β1 integrin. Structurally, KTS-disintegrins are short, monomeric molecules containing 41 amino acids in its polypeptide chain. Biological activities of MLD and KTS-disintegrins were investigated in many systems in vitro and in vivo. Purified disintegrins are non-toxic in therapeutic doses in rodent and avian models. Their modulatory properties were observed in investigations of cancer angiogenesis and metastasis, immunosuppression of IDDM (insulin-dependent diabetes mellitus) and asthma, as well as in neurodegenerative assays and cell apoptosis.     

Molecular diversity of disintegrin-like domains within metalloproteinase precursors of Bothrops jararaca.

Disintegrins are small peptides isolated from the venom of several snake families which act as integrin-antagonists or agonists, interacting with a variety of biological processes mediated by integrins. In this work we describe five new disintegrin-like domains within metalloproteinase precursor sequences, obtained from a Bothrops jararaca venom gland cDNA library. Among the new disintegrin-like domains, four were contained in PIII metalloproteinase precursors, with three of them presenting ECD-motifs and one presenting a new KCD-motif. Moreover, we found three disintegrin-like domains within PII metalloproteinase precursors. Two of them are similar to the already described disintegrins jarastatin and jararacin. The third molecule is unusual, presenting some typical PIII metalloproteinase characteristics but lacking the cysteine-rich domain being, thus, classified as a PII metalloproteinase. Only few reports presented molecules with these characteristics. Sequence analysis suggests that these molecules are intermediate steps between the more ancient PIII and the more recent PII metalloproteinases. We also investigated disintegrin N-terminus diversity in B. jararaca crude venom by purifying jarastatin and jararacin and analyzing them by mass spectrometry.     

Sodium dodecyl sulfate-stable complexes of echistatin and RGD-dependent integrins: a novel approach to study integrins

This study shows that disintegrins, echistatin as a model, can be used as a radiolabeled probe to simultaneously detect the presence of individual RGD-dependent integrins on cardiac fibroblasts. Binding of (125)I-echistatin to fibroblasts was proportional to cell number, time dependent, reversible, saturable, specific, and membrane bound. SDS-polyacrylamide gel electrophoresis and autoradiograms revealed that (125)I-echistatin was associated with three radioactive protein bands of 180, 210, and 220 kDa that were identified by RGD affinity chromatography, immunoblotting, and immunoneutralization as alpha(v)beta(3), alpha(3)beta(1)/alpha(5)beta(1)/alpha(v)beta(1), and alpha(8)beta(1) heterodimeric integrins, respectively. These results suggest that echistatin binds to RGD-dependent integrins, forming SDS-stable complexes in the absence of chemical cross-linkers, reducing conditions and heating. As assessed by radioligand-binding filtration, disintegrins displayed binding characteristics with an IC(50) ranging from 0.044 to 1.1 nM, but with slope factors lower than 1, indicating the presence of several binding sites. Resolved by SDS-polyacrylamide gel electrophoresis to reveal echistatin-integrin complexes, disintegrins and RGD peptides displayed different binding affinities to individual RGD-dependent integrins present on cardiac fibroblasts. Elegantin and flavostatin demonstrated the highest affinity toward integrins, whereas flavoridin and acPenRGDC had a greater specificity toward alpha(v)beta(3)-integrin. In summary, echistatin forms SDS-stable complexes with RGD-dependent integrins. This model offers a novel way to visualize RGD-dependent integrins, to investigate their activation state, and to determine the integrin specificity of RGD peptides.     

cDNA cloning of a novel P-I lebetase isoform Le-4.

In order to better understand the function of fibrinolytic enzyme lebetase isoforms and the synthesis of disintegrins we have isolated a cDNA encoding the most basic isoform (Le-4) from the cDNA library prepared from the poly(A)(+) RNA of the venomous gland of an individual Vipera lebetina snake. The truncated 5'-sequence of 1112 basepairs encodes the mature protein with 203 amino acid residues with calculated isoelectric point and size of 5.6 and 22,930 Da, respectively. Multiple comparison of the deduced amino acid sequence of the metalloprotease part of Le-4 is related to short reprolysins, identities were within the range of 60--87%. The two lebetase isoforms are synthesized in different way: Le-4 is synthesized with metalloprotease domain only; Le-3 is synthesized with metalloprotease and disintegrin-like domain and processed posttranslationally. The sequence of the disintegrin-like part of Le-3 is identical to A-chain of the heterodimeric disintegrin VLO5 from Vipera lebetina obtusa venom (Calvete et al., 2003).     

Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC50s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.