Effects of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation and characterization of a prothrombin activator

The action of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation was examined in vitro and a strong anticoagulant effect was observed. This action was abolished after treatment with a specific inhibitor of phospholipase A(2) activity (p-bromophenacyl bromide), revealing a procoagulant action in low concentrations of treated venom (around 1 mu g/ml). The effect of the venom an haemostasis was further characterized by measuring its ability to activate purified blood coagulation factors. It is concluded that A. halys pallas venom contains prothrombin activation activity. A prothrombin activator (aharin) was purified from the venom by Sephadex G-75 gel filtration and ion-exchange chromatography on a Mono-Q column. It consisted of a single polypeptide chain, with a mol. wt of 63,000. Purified aharin possessed no amidolytic activity on chromogenic substrates. It did not act on other blood coagulation factors, such as factor X and plasminogen, nor did it cleave or clot purified fibrinogen. The prothrombin activation activity of aharin was readily inhibited by ethylenediamine tetracetic acid (a metal chelator), but specific serine protease inhibitors such as diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride had no effect on it. These observations suggest that, like those prothrombin activators from Echis carinatus and Bothrops atrox venoms, the prothrombin activator from A. halys pallas venom is a metalloproteinase. (C) 1998 Elsevier Science Ltd. All rights reserved.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVl

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta(1)) and 22 kDa (beta(2)), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta(1) and beta(2) subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between 1 and 2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the U. and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against UNA inhibited the aggregation response to stejnulxin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha(2)beta(1) as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

A snake venom metalloproteinase that inhibited cell proliferation and induced morphological changes of ECV304 cells

TSV-DM, a basic metalloproteinase with a molecular weight of 110 kDa, was purified from Trimeresurus stejnegeri venom. TSV-DM degraded the A alpha chain of fibrinogen more rapidly than the B beta chain in a dose dependent manner. The cDNA of TSV-DM encode

Isolation and cloning of a metalloproteinase from king cobra snake venom

A 50 kDa fibrinogenolytic protease, ohagin, from the venom of Ophiophagus hannah was isolated by a combination of gel filtration, ion-exchange and heparin affinity chromatography. Ohagin specifically degraded the alpha-chain of human fibrinogen and the pr

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin. was purified by DEAF A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg, The fibrinopeptides released, identified by HPLC consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it. indicating it is venom serine protease. (C) 2000 Elsevier Science Ltd. All rights reserved.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded a-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin. (C) 2004 Elsevier Ltd. All rights reserved.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) lb is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCgamma2. Mucetininduced phosphorylation of the Fcgamma chain of platelet was greatly promoted by inhibition of alpha(llb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream Of alpha(llb)beta(3) activation are involved in dephosphorylation of Fcgamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(llb)beta(3). Inhibition Of alpha(llb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation Of alpha(llb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxaneA(2) are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom

A novel kinin-releasing and fibrin (ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenymethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aalpha-chain of human fibrinogen with lower activity towards Bbeta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Evolutionary implications of multiple SINE insertions in an intronic region from diverse mammals

An analysis of the nuclear beta-fibrinogen intron 7 locus from 30 taxa representing 12 placental orders of mammals reveals the enriched occurrences of short interspersed clement (SINE) insertion events. Mammalian-wide interspersed repeats (MIRs) are present at orthologous sites of all examined species except those in the order Rodentia. The higher substitution rate in mouse and a rare MIR deletion from rat account for the absence of MIR in the rodents. A minimum of five lineage-specific SINE sequences are also found to have independently inserted into this intron in Carnivora, Artiodactyla and Lagomorpha. In the case of Carnivora, the unique amplification pattern of order-specific CAN SINE provides important evidence for the "pan-carnivore" hypothesis of this repeat element and reveals that the CAN SINE family may still be active today. Particularly interesting is the finding that all identified lineage-specific SINE elements show a strong tendency to insert within or in very close proximity to the preexisting MIRs for their efficient integrations, suggesting that the MIR clement is a hot spot for successive insertions of other SINEs. The unexpected MIR excision as a result of a random deletion in the rat intron locus and the non-random site targeting detected by this study indicate that SINEs actually have a greater insertional flexibility and regional specificity than had previously been recognized. Implications for SINE sequence evolution upon and following integration, as well as the fascinating interactions between retroposons and the host genomes are discussed.

核基因在鸟类分子系统发育学研究中的应用

核基因作为一种新的遗传标记,近年来被广泛应用于鸟类分子系统发育研究中.核基因与线粒体基因位于不同的遗传载体上,因此被引入到系统发育学研究中为物种树的重建提供独立的证据.常用的外显子标记为重组激活基因1(RAG-1),重组激活基因2(RAG-2),癌基因c-myc,原癌基因c-mos,它们由于缓慢的进化速率而被用于鸟类高级分类阶元的系统学研究中.常用的内含子标记是β纤维蛋白原基因内含子7(β-fibrinogen intron7,β-fibint7),肌红蛋白基因内含子Ⅱ(myoglobin intionⅡ).内含子标记通常与线粒体序列联合使用,形成具有互补系统发育信号的数据集,应用于各种分类阶元的系统学研究中.

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin. (C) 2009 Elsevier Ltd. All rights reserved.

凤鹛属鸟类（Yuhina, Aves）的系统发育研究

本研究以凤鹛属（Yuhina）鸟类为研究对象，分别使用分子生物学性状和形 态学性状来重建凤鹛属种间的系统发育关系，为东南亚地区鹛类的起源与进化研 究提出新的见解。分子生物学研究的内群不仅包括了凤鹛属（11 种中的10 种）， 而且还包括了菲律宾特有穗鹛属（Stachyris）和绣眼鸟属（Zosterops）等近缘种。 通过测定线粒体上两个蛋白编码基因Cyt b、ND3 的全长序列和两个RNA 编码 基因 12S, 16S 的部分序列，得到长度为2379bp 的联合序列，使用最大简约法、 最大似然法和贝叶斯法进行系统发育分析的结果表明，菲律宾岛特有的穗鹛（金 冠穗鹛S. dennistouni、纹穗鹛S. striata、怀氏穗鹛S. whiteheadi）与绣眼鸟属（暗 绿绣眼Z. japonicus、红胁绣眼Z. erythropleurus、灰腹绣眼Z. palpebrosus）聚为 一支，位于凤鹛属支系内部。通过凤鹛、菲律宾穗鹛、绣眼鸟三个类群羽冠性状 演化趋势的分析，发现羽冠性状在菲律宾特有穗鹛和绣眼鸟支系中发生丢失。与 前人的研究结果相比，新发现纹喉凤鹛Y. gularis 与棕肛凤鹛Y. occipitalis 是姐妹 群，黄颈凤鹛Y. flavicollis 和白项凤鹛Y. bakeri 是姐妹群，这两个姐妹群又聚为 一大支的关系。 在分类问题上，暗绿绣眼、红胁绣眼和灰腹绣眼在本研究中与鹛类表现出近 缘关系，与前人使用绣眼鸟属其它物种进行的相关研究显示出高度一致性，说明 绣眼鸟属的系统学地位存在很大疑问，应该扩大取样进行深入研究。栗冠凤鹛 Y. everetti 与栗耳凤鹛Y. castaniceps 聚为姐妹群的关系，但是二者之间较小的遗 传距离和形态学差别显示二者之间应该为亚种关系，而不是种级关系。依据栗耳 凤鹛特有的尾羽形态提出的Staphiada 属没有得到本分子生物学研究结果的支持 （Harrison, 1986a, b）。在白腹凤鹛Y. zantholeuca 不属于凤鹛属支系这一结果的 提示下，发现白腹凤鹛独有的形态学特征，例如羽冠由冠羽形成、鼻孔半裸露、 飞羽外翈没有异色羽缘，这些独有的特征与其特殊的系统学地位相对应。因此， 我们支持前人提出的将白腹凤鹛从凤鹛属中分出去，单独成Erpornis 属的建议 （Cibois et al., 2002）。 在历史生物地理学方面，根据菲律宾特有穗鹛和绣眼鸟的近缘关系，认为菲 律宾特有穗鹛的祖先具有与现在绣眼鸟一样的跨海迁移的能力，在中新世末期（5.74Mya）由喜马拉雅地区或印度支那起源地经大巽他地区扩散到菲律宾岛屿 上，经过长期独立进化后，形成现在高度特有的穗鹛支系。更新世冰期时，大巽 他地区海平面下降，使栗冠凤鹛得以扩散至婆罗州（1.66Mya）。在上新世初期台 湾岛成陆后，由于台湾岛与中国大陆相连，褐头凤鹛Y. brunneiceps 的祖先扩散 至台湾岛屿上（5.05Mya）。高黎贡山为第四纪冰期时凤鹛属鸟类的避难场所，因 此形成了现在凤鹛属鸟类在高黎贡山地区密集分布的格局。喜马拉雅山和青藏高 原的隆起导致青藏高原上植被带的变化和喜马拉雅山南麓在第四纪冰期中避难 所的作用，是纹喉凤鹛、棕肛凤鹛、黄颈凤鹛、白项凤鹛等种类现今在喜马拉雅 山南麓密集分布的可能原因。 在凤鹛属分子系统学的研究基础上，从形态学角度探讨凤鹛属除白领凤鹛外 其它物种之间的系统发育关系。使用最先分化出来的白领凤鹛为外群以外群比较 法进行51 个形态学特征的性状极化，使用最大简约法分析后，结果不仅支持一 些分子系统学揭示的物种之间的亲缘关系，例如，栗耳凤鹛与栗冠凤鹛姐妹群的 关系，黑颏凤鹛和褐头凤鹛姐妹群的关系，三种绣眼鸟聚为一支，再与怀氏穗鹛 聚为一个大支；而且发现了新的系统关系，黄颈凤鹛与缅甸凤鹛Y. humilis 是姐 妹群，具有浅色下体的凤鹛属物种具有较近的系统关系。分子数据和形态数据对 凤鹛属系统发育树中部节点解决能力的差异，说明凤鹛属物种之间分化间隔时间 很短。 通过综述近年来核基因在鸟类分子系统发育研究中的应用情况，建议未来研 究中增加核基因的内含子序列，例如肌球素基因内含子2（ myoglobin intron Ⅱ） 或β纤维蛋白原基因内含子7（β-fibrinogen intron7, β-fibint 7）来解决本研究 中未确定的末端分枝分歧顺序。