抗蛇毒IgY对尖吻蝮(Deinagkistrodon acutus)蛇毒主要活性的中和作用研究

目的评价抗尖吻蝮蛇(D.acutus)毒Ig Y对该蛇毒主要活性的中和能力。方法通过不同浓度Ig Y与该蛇毒混合孵育后,再用生化方法检测该蛇毒的多种活性。结果体外实验表明,Ig Y能明显地中和该蛇毒的PLA_2、5'-核苷酸酶、透明质酸酶、金属蛋白酶和丝氨酸蛋白酶(纤维蛋白酶)等酶活性;小鼠实验表明,Ig Y对小鼠的半有效保护剂量(ED_(50))为1 131.09μg。结论该抗尖吻蝮蛇毒Ig Y具有很好的中和活性,且Ig Y无明显的毒性。     

尖吻蝮蛇毒和蝮蛇毒凝血酶样酶促凝的协同作用

尖吻蝮蛇毒和蝮蛇毒凝血酶样酶促凝的协同作用魏文利１，孙家钧，陈家树（中山医科大学药理教研室，广州５１００８９，中国）关键词尖吻蝮蛇；蝮蛇；蛇毒；凝血酶类；血液凝固目的：尖吻蝮蛇（ＤＡ）和蝮蛇（ＡＨ）蛇毒凝血酶样酶（ＴＬＥ）对促凝作用的协同效果．方法：...     

三种蛇毒类凝血酶对纤维蛋白原的作用方式

目的以纤维蛋白原为底物,考察巴西矛头蝮蛇(Bothrops atrox)、尖吻蝮蛇(Agkistrodon acutus)、长白白眉蝮蛇(Agkistrodon halys pullas)3种蛇毒类凝血酶对纤维蛋白原的作用方式。方法采用SDS-PAGE及RP-HPLC法对作用结果进行检测。结果 3种蛇毒类凝血酶对纤维蛋白原的作用方式不完全相同,巴西矛头蝮蛇、尖吻蝮蛇2种蛇毒类凝血酶只作用于纤维蛋白原的α链,对β、γ链则无作用,长白白眉蝮蛇毒类凝血酶起初作用于纤维蛋白原的β链,对α链作用较弱,随着时间的延长对α链作用增强,对γ链无作用。结论巴西矛头蝮蛇、尖吻蝮蛇2种蛇毒类凝血酶属于SVTLE-A型,而长白白眉蝮蛇毒类凝血酶则属于SVTLE-AB型。     

一种简易的家兔颈动脉血栓模型形成方法和蕲蛇酶的溶栓作用

<正> 为研究药物对血栓的溶解作用,我们设计了一种简易的实验方法,可在家兔颈动脉形成血小板性动脉血栓模型,并以此模型比较了尿激酶和一种从尖吻蝮蛇毒纯化的抗血栓新药——蕲蛇酶(Acutinase)的溶栓效应。1 实验材料和方法家兔26只,体重1.5～2.5kg,兼用。蕲蛇酶注射液,为一种从尖吻蝮(Agk-istrodon acutus)蛇毒用离子交换柱层析和分子筛过滤方法分离、纯化的凝血酶样酶     

短尾蝮蛇毒中磷脂结合抗凝蛋白的急性毒性实验

近年来,国内外的学者们对蝮蛇毒进行大量的研究,了解到蝮蛇毒中主要含有精氨酸酯酶、类凝血酶、激肽释放酶、纤溶酶和磷脂酶A2,并以富含精氨酸酯酶为特征,而类凝血酶、激肽释放酶和纤溶酶一般都具有精氨酸酯酶活力。我国在70年代后期开始研制蛇毒酶制剂,主要作为抗凝剂治疗血栓性疾病。分别有以下几种:东北白眉蝮蛇毒为原料制成的“蝮蛇清栓酶”;以江浙蝮蛇毒为原料制成的“蝮蛇抗栓酶”和以尖吻蝮蛇毒为原料制成的“蕲蛇毒去纤酶”。这几种酶制剂,虽然商品名称不同但均为非纯化成分,主要有效成分均为精氨酸酯酶,纯度不高[1]。本试验对短尾…     

皖南尖吻蝮蛇毒提取物体外抗肿瘤活性的实验研究

目的：从中国皖南尖吻蝮蛇（Agkistrodon acutus）蛇毒中提取蛇毒金属蛋白酶x（snake venom metallopmteinases,SVMP-x）并观察其体外抗癌活性。方法：尖吻蝮蛇粗毒冻干粉溶解并经过离子交换和凝胶过滤后纯化后得到SVMP-x组分,CCK．8比色法测定该组分对体外培养的肿瘤细胞的细胞毒作用。结果：从中国皖南尖吻蝮蛇毒中分离出一种单一组分（sVMP-x）,sVMP-x呈剂量依赖性抑制体外培养的人胃癌细胞株（SGC7901）、结肠癌细胞株（sw480）、神经母细胞瘤细胞株（SH—SY5Y）、肝癌细胞株（HepG2）及小鼠黑色素瘤细胞株（B16）的增殖,并且对SGC7901及B16的抑制作用强于5．氟脲嘧啶（5-FU）,对SGC7901的抑制作用呈剂量一时间依赖关系。结论：从尖吻蝮蛇粗毒冻干粉中成功提取出抗肿瘤组分SVMP-x,该组分对体外培养的肿瘤细胞有明显的抑制作用。     

尖吻蝮蛇蛇毒中毒镇痛成分的研究

目的 利用国产尖吻蝮蛇蛇毒筛选阿片受体激动剂,从中寻找无成瘾性新型镇痛药物。方法 通过层析分离方法,从国产尖吻蝮蛇蛇毒中获得电泳纯样品,采用微生理测定仪测定样品对CHO－μ受体细胞的活性,同时进行动物热板法镇痛试验,小鼠竖尾、跳跃式成瘾性试验。结果 从尖吻蝮蛇蛇毒中获得一分子量为1．2万的电泳纯蛋白质,且与CHO-μ受体细胞亲和力活性很高,该成分对小鼠具有较高的镇痛作用,无成瘾性,无毒性。结论 该成分具有较好的临床应用价值。     

超声雾化吸入尖吻蝮蛇血凝酶和凝血酶治疗咯血临床观察

咯血是支气管和肺部疾病的常见症状,也是呼吸科常见的急症之一.肺结核血管破损和空洞内血管瘤破裂,支气管扩张、肺癌、肺囊肿均可出现大咯血,特别是支气管动脉因压力高,或患者伴有肺动脉高压的情况下,一旦出血,咯血量大且来势凶猛,及时控制大咯血,是减少死亡的关键[1-2].咯血的治疗以静脉注射止血药和血管扩张剂为主,必要时进行手术治疗[3-4].尖吻蝮蛇血凝酶(HCA)是一种从尖吻蝮蛇毒液中提取分离出的蛇毒类凝血酶,蛇毒类凝血酶作为一种动物来源的蛋白酶类止血药,由于其止血效果确切,且止血作用不受肝素干扰等,近年来在临床中已被广泛应用[5].凝血酶是临床常用的治疗出血的药物[6].本院采用超声雾化吸人尖吻蝮蛇血凝酶和凝血酶治疗咯血,现报告如下.     

《武夷山蛇药》鉴定会在福州召开

《武夷山蛇药》由建宁县医院和福建医大药理教研室等二十七个单位参加研制和临床验证。五年来共治疗各种毒蛇咬伤357例,治愈率为96.6％,其中尖吻蝮蛇咬伤81例,治愈率为92.5％。经药理试验说明,该药的主要成分白芷、徐长卿和半边莲对眼镜蛇毒、眼镜王蛇毒和银环蛇毒中毒的动物均有显著的保护效果。福建省卫生厅五月下旬在福州召开鉴定会。鉴定结果认为:该药对竹叶青蛇、龟壳花蛇咬伤患者具有良好的疗效;对尖吻蝮蛇咬伤的轻型患者有一定疗效;对中、重、危型患者结合中西医结合治疗,也有一定疗效。建议该药散剂可以投产推广应用,针剂可     

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A₂ inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A₂, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.     

Screening of TNFR1 Binding Peptides from Deinagkistrodon acutus Venom through Phage Display

The venomous species Deinagkistrodon acutus has been used as anti-inflammatory medicine in China for a long time. It has been proven to have anti-inflammatory activity, but its specific anti-inflammatory components have not yet been fully elucidated. Tumor necrosis factor receptor-1 (TNFR1), which participates in important intracellular signaling pathways, mediates apoptosis, and functions as a regulator of inflammation, is often used as the target to develop anti-inflammatory drugs. The small peptides of snake venom have the advantages of weak immunogenicity and strong activity. To obtain the specific TNFR1 binding peptides, we constructed a T7 phage library of D. acutus venom glands, and then performed biopanning against TNFR1 on the constructed library. After biopanning three times, several sequences with potential binding capacity were obtained and one 41-amino acid peptide was selected through a series of biological analyses including sequence length, solubility, and simulated affinity, named DAvp-1. After synthesis, the binding capacity of DAvp-1 and TNFR1 was verified using surface plasmon resonance technology (SPR). Conclusively, by applying phage display technology, this work depicts the successful screening of a promising peptide DAvp-1 from D. acutus venom that binds to TNFR1. Additionally, our study emphasizes the usefulness of phage display technology for studies on screening natural product components.     

Rosmarinic acid in <Emphasis Type="Italic">Argusia argentea</Emphasis> inhibits snake venom-induced hemorrhage

A methanolic extract of Argusia (or Messerschmidia or Tournefortia) argentea (Boraginaceae) significantly inhibited hemorrhage induced by crude venom of Trimeresurus flavoviridis. The extract was then separated according to antivenom activity by using silica gel column chromatography and HPLC equipped with an octadecylsilanized silica gel (ODS) column to afford rosmarinic acid (RA) (1) as an active principle. RA (1) significantly inhibited the hemorrhagic effect of crude venoms of T. flavoviridis, Crotalus atrox, Gloydius blomhoffii, Bitis arietans as well as snake venom metalloproteinases, HT-b (C. atrox), bilitoxin 2 (Agkistrodon bilineatus), HF (B. arietans), and Ac1-proteinase (Deinagkistrodon acutus). This is the first report of the antihemorrhage activity of RA (1), and RA (1) greatly contributes to the antihemorrhagic efficiency of A. argentea against crude snake venoms and hemorrhagic toxins.     

Toxoids and Antivenoms of Venomous Snakes in Taiwan

Abstract

There are in Taiwan six major venomous snakes which can inflict severe bite on human victims. They are three hemorrhagic species i.e. the Taiwan habu (Trimeresurus mucrosquamatus), the green tree viper (Trimeresurus stejnegeri), and the hundred-pace snake (Deinagkistrodon acutus); two neurotoxic species, i.e. the cobra (Naja naja atra) and the krait (Bungarus multicinctus); and the Russell's viper (Daboia r. formosensis) whose venom is both coagulopathy and neurotoxic. Our aim has been the production of highly potent antivenoms for snake-bite treatment in this country. Among individual antivenoms for Taiwan venomous snakes, only those from the pitvipers show partial cross-neutralizing capacity with venoms of other pit vipers.

As all snake venoms are quite lethal to animals, it is important to tame or detoxify the crude venom before using it on the animal to obtain antivenoms. We have demonstrated that glutaraldehyde can be used successfully not only in the detoxification of snake venoms but also improving their immunogenecity. Protocols for toxoid preparation from the crude venoms in the process of manufacturing highly potent antisera have been improved in our institute. Two bivalent equine antivenoms specific for either the combined glutaraldehyde-treated venoms of N. n. atra and B. multicinctus or those of T. mucrosquamatus and T. stejnegeri were successfully produced and proven to be effective and useful. A tetravalent equine antivenom has been prepared likewise against the four major viperid venoms in Taiwan. Recently, we also developed a process to prepare an efficient hexavalent antivenom against all the six major venomous snakes.     

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom.

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom. Toxicon15, 161–167, 1977.—In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon acutus venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did ε-aminocaproic acid. Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due-to a direct action on fibrin. Both EDTA (5 × 10^-4 M) and cysteine (5 × 10^-3 M) completely inhibited the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the fibrinolytic principle.     

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.     

Inflammation induced by Bothrops asper venom

Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by B. asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by B. asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A₂, a metalloproteinase and serine proteinases isolated from B. asper venom are also described.     

Studies on the specificity of CNF, a phospholipase A2 inhibitor isolated from the blood plasma of the South American rattlesnake (Crotalus durissus terrificus). I. Interaction with PLA2 from Lachesis muta muta snake venom

A phospholipase A₂ inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A₂ activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A₂ (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA–CB of crotoxin.In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA₂ activity of the whole venom from the bushmaster snake (Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA₂ active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA₂, but extended to the three acidic forms as well.     

Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom

J. J. Zhang, Z. X. Chen, Y. Z. He and X. Xu. Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom. Toxicon22, 931–935, 1984. — AaHI, a proteolytic hemorrhagic toxin from A. acutus venom, contains one g-atom Zn per mole protein and 2 g-atoms Ca per mole protein. AaHI is activated by calcium and slightly inhibited by zinc. When treated with EDTA, AaHI is completely inactivated. When dialyzed against 1,10-O-phenanthroline, 50–80% of the metal content and activities are lost, while 90% of the hemorrhagic and proteolytic activities are retained when dialyzed against 1,10-O-phenanthroline containing 5mM Ca²⁺. The results suggest that calcium is essential for the hemorrhagic and proteolytic activities. The circular dichroism spectra show that calcium may play an important role in maintaining a proper structure for AaHI. The function of zinc is not clear.     

A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venom

Phospholipase A₂ (PLA₂, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A₂ from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA₂s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA₂s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.