蛇毒的研究与利用——Ⅴ.福建产圆斑蝰蛇(Vipera russelli siamensis)蛇毒的柱层析分离及酶活力和生理效应的测定

用羧甲基葡聚糖凝胶C-50对福建产圆斑蝰蛇(Vipera russelli siamensis Smith)蛇毒进行了柱层析分离,获得12个蛋白峰。其中有2个组份具有促凝作用,4个组份具有抗凝作用。对各蛋白峰进行7种酶活力测定的结果表明,福建产圆斑蝰蛇毒的促凝作用与精氨酸酯酶无明显关系。而致死毒性与磷脂酶A 活力相重迭。粗毒具有较高的精氨酸酯酶、磷脂酶A 和磷酸二酯酶活力,而无出血和纤维蛋白溶解作用。     

圆斑蝰蛇咬伤中毒35例临床分析

目的分析圆斑蝰蛇咬伤中毒的临床特点,总结圆斑蝰蛇咬伤中毒合理有效的治疗方法。方法对2006年6月~2017年12月广西中医药大学第一附属医院与广西医科大学第一附属医院收治确诊为圆斑蝰蛇咬伤中毒患者的临床资料进行回顾性分析。结果 (1)一般情况:本组35例患者中,男21例(60.00%),女14例(40.00%),男女比例为3∶2;年龄8~63岁,平均(44.73±5.84)岁。(2)临床特征与转归:35例患者均出现凝血障碍的症状,其中并发非典型DIC 8例(22.85%),并发急性肾功能衰竭8例(22.85%),并发内脏出血10例(28.57%),并发脑出血5例(14.29%)。全部患者均治愈,治愈率100%。(3)应用抗蝮蛇毒血清治疗情况:应用抗蝮蛇毒血清治疗(应用抗蝮蛇毒血清组)17例,未用抗蝮蛇毒血清治疗(未应用抗蝮蛇毒血清组)18例,两组的疗效和并发症比较差异无统计学意义(P0.05)。结论 (1)圆斑蝰蛇咬伤中毒以青壮年男性多见,咬伤部位多见于下肢,好发于夏秋两季。(2)圆斑蝰蛇咬伤中毒出现凝血功能障碍、非典型DIC综合征表现,可能是消耗性凝血功能障碍导致。(3)应用抗蝮蛇毒血清治疗圆斑蝰蛇咬伤中毒是否有效还需更多的循证医学资料支持。     

蛇毒的研究与利用——Ⅴ.福建产圆斑蝰蛇(Vipera russelli siamensis)蛇毒的柱层析分离及酶活力和生理效应的测定

用羧甲基葡聚糖凝胶C-50对福建产圆斑蝰蛇(Vipera russelli siamensis Smith)蛇毒进行了柱层析分离,获得12个蛋白峰。其中有2个组份具有促凝作用,4个组份具有抗凝作用。对各蛋白峰进行7种酶活力测定的结果表明,福建产圆斑蝰蛇毒的促凝作用与精氨酸酯酶无明显关系。而致死毒性与磷脂酶A活力相重迭。粗毒具有较高的精氨酸酯酶、磷脂酶A和磷酸二酯酶活力,而无出血和纤维蛋白溶解作用。     

新型抗蝰蛇毒血清对不同蝰蛇毒作用的实验研究

新型抗蝰蛇毒血清是采用国产圆斑蝰蛇毒与缅甸蝰蛇毒按一定比例免疫马匹后 ,从马血浆中精制而成。作者通过免疫电泳及动物实验 ,观察新型抗蝰蛇毒血清对国产圆斑蝰蛇毒与缅甸蝰蛇毒的免疫沉淀反应、体外中和作用、体内保护作用等方面的作用。结果 :急性毒性实验 :国产圆斑蝰蛇毒和缅甸蝰蛇毒的 LD50 (腹腔注射 )分别为 (0 .30 6± 0 .0 0 3) mg/ kg、 (0 .2 90± 0 .0 0 3) mgkg。体外中和实验表明新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒均有中和作用 ,其中和抗体效价依次为 2 750μg/ ml (约 450 LD50 )、 2 4 90μg/ml(约 430 L…     

尖吻蝮(Agkistrodon acutus)蛇毒凝血酶样成分的研究——Ⅲ.蛇毒凝血酶止血效应的研究

将纯化得到的蛇毒凝血酶(TLE_3、TLE_4),经家兔体内实验表明,2—3凝血酶单位/kg体重剂量能显著地使家兔全血凝血时间缩短1/3—1/2。药后1小时即有促凝作用,以2—4小时凝血(止血)效应最强,12小时已消失。与Holleman, W. H.等自美洲矛头蝮蛇毒中得到的蛇毒凝血酶(Hemocoagulase)相似。经家兔及家犬实验性创伤止血实验表明,对创伤出血有止血作用。     

圆斑蝰蛇咬伤致脑出血后脑梗死1例报告（英文）

圆斑蝰蛇(Daboia russelii siamensis),又称百步金钱豹、卢氏蝰蛇、锁蛇,为蛇亚目蝰蛇科蝰亚科山蝰属的一种血循毒毒蛇,为我国南方常见毒蛇之一,主要集中于广西、广东、台湾等人口稠密地区,亦分布在缅甸、泰国、柬埔寨、印度尼西亚等地。圆斑蝰蛇活跃于平原或丘陵地带,具有高攻击性,其在攻击时常采用突袭,所以患者被咬伤时多在毫无心理准备的的情况下发生。因此,圆斑蝰蛇咬伤的记录和报道居高不下。在上述地区,平均每年毒蛇咬伤发病率高达5‰,超过20000人因此死亡。根据文献报道,相比脑梗死患者,圆斑蝰蛇咬伤患者更易出现颅内出血症状。本文首次报告圆斑蝰蛇咬伤致脑出血后出血性脑梗死1例。     

蛇志 2006年第18卷 总目次

第1期(2006年3月)抗中华眼镜蛇毒鸡卵黄抗体的制备及其效价测定………………………………………………………………………刘四红,侯力强,孔天翰(1)广西圆斑蝰蛇毒凝血因子X激活物在实验鼠体内的促凝血作用研究……………………………………………王艳妮,左从林,陈明霞,等(6)蝮蛇毒蛋白C激活物对凝血系统的影响…………………………………………………………………………张受涛,文尚武,周志泳,等(11)抗海蛇毒血清用于海蛇咬伤早期急救与监护的实验研究…………………………………………………………厉瑛,张黎明,桂莉,等(15)降纤酶治疗慢性肺…     

抗蝮蛇毒血清治疗圆斑蝰蛇咬伤73例体会

目的总结抗蝮蛇毒血清治疗圆斑蝰蛇咬伤的方法及效果。方法对我院2005年6月~2013年6月收治的73例圆斑蝰蛇咬伤病例资料进行回顾性分析。结果 73例患者均注射了抗蝮蛇毒血清,治愈69例,其中7例住院3天后出现肾功能障碍,转内科血透治疗;6例出血较多,给予输浓缩红细胞2~4u。死亡4例,均为圆斑蝰蛇咬伤并发急性肾功能衰竭患者。结论圆斑蝰蛇咬伤的患者需住院综合治疗观察,注射抗蝮蛇毒血清安慰效果大于实际效果。     

《动物学研究》增刊简介

《动物学研究》第2卷第4期增刊(1981) 本期增刊为“蛇毒研究与蛇伤治疗”。主要内容是:福建产圆斑蝰蛇(Vipera russclli siamensis)蛇毒磷酯酶A的分离纯化及部分性质;毒性磷酯酶A_2的氨基酸组成和N-末端部分氨基酸顺序测定;浙江蝮蛇毒碱性磷脂酶A的分离纯化及性质;我国10种蛇毒磷脂酶A活力比较;广东金环蛇细胞毒素Ⅷ的化学组成和部分一级结构;眼镜蛇膜毒素对菠菜叶绿体光合膜的影响;中华眼镜蛇毒膜毒素对鼠肝线粒体的作用;眼镜王蛇毒中L-氨基酸氧化酶的分离纯     

抗蝰蛇毒血清治疗蝰蛇伤临床疗效考察

圆斑蝰蛇是我国常见剧毒蛇 ,其毒素属血循毒 ,伤者发病快、病情凶险、病死率高。为了考察国产抗蝰蛇毒血清治疗蝰蛇伤的疗效、治疗剂量和不良反应 ,作者将 335例蝰蛇伤患者 (临床分型 :轻型 1 62例 ,重型 95例 ,危重型 78例 )分为用抗蝰蛇毒血清治疗组 32 2例 (其中危重型 69例 )和不用抗蛇毒血清对照组 1 3例 (其中危重型9例 )。治疗组中 1 61例先用 1支 ( 5 0 0 0国际单位 )静脉推注或滴注 ,追加 0 .5支肌注及另外加抗银环蛇毒血清或抗五步蛇毒血清各 1支 ,81例使用 2支 ,42例使用 3支 ,使用 4支以上 35例。并给予辅助治疗 ,如口服或外敷…     

Differential action of proteases from Trimeresurus malabaricus, Naja naja and Daboia russellii venoms on hemostasis

The action of venom proteases and their role in hemostasis has been compared in the venoms of Trimeresurus malabaricus, Daboia russellii and Naja naja from the Southern region of Western Ghats, India. These venoms exhibit varying amounts of proteolytic activity and also influence hemostasis differently. Casein hydrolyzing activity of T. malabaricus venoms was 16 and 24 fold higher than those of N. naja and D. russellii venoms, respectively. With the synthetic substrate TAME, the highest activity was observed in T. malabaricus venom. N. naja venom did not hydrolyze TAME even at higher concentrations. These variations in proteolytic activity also influenced the coagulation process. T. malabaricus and D. russellii venoms are strongly procoagulant and reduce the re-calcification time from 148 to 14 and 12 s, respectively. Similarly, both T. malabaricus and D. russellii venoms reduce the prothrombin time from 12.5 to 6.0 s. On the other hand, N. naja venom is anticoagulant and prolongs re-calcification time to 600 s and prothrombin time to 42 s. In spite of varied effects on hemostasis, all the venoms hydrolyze fibrinogen. T. malabaricus venom hydrolyses both Aalpha and Bbeta subunits. While D. russellii and N. naja venoms hydrolyse only Aalpha. None of these venoms hydrolyze the gamma subunit of fibrinogen. Inhibition studies with specific protease inhibitors revealed that both N. naja and T. malabaricus venoms contain only metalloproteases. D. russellii venom contained both serine and metalloproteases. Only, T. malabaricus venom exhibited thrombin-like activity and induces fibrin clot formation with purified fibrinogen within 58 s. Even though D. russellii venom exhibits procoagulant activity, it did not show thrombin-like activity and may act on other coagulation factors.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom ( Apis mellifera ), three snake venoms ( Naja naja sputatrix, Vipera russellii and Crotalus adamanteus ) and the polypeptide melittin was investigated against Escherichia coli . Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom > melittin > N. naja sputatrix venom ≫ V. russellii venom > C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

New insights into the functions and N-glycan structures of factor X activator from Russell's viper venom

The coagulation factor X activator from Russell's viper venom (RVV-X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C-terminal extension with four additional residues to that published previously. Both light chains showed 77-81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell's viper envenomation. The N-glycans released from each subunit were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex-type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl-Lewis epitopes, as confirmed by western blotting analyses. As these glyco-epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV-X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV-X than by native RVV-X.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom (Apis mellifera), three snake venoms (Naja naja sputatrix, Vipera russellii and Crotalus adamanteus) and the polypeptide melittin was investigated against Escherichia coli. Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom greater than melittin greater than N. naja sputatrix venom much greater than V. russellii venom greater than C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains.

We determined the complete amino acid sequence of RVV-X, the blood coagulation factor X activating enzyme, isolated from Russell's viper venom and studied structure-function relationships. RVV-X (M(r) 79,000) consists of a disulfide-bonded two-chain glycoprotein with a heavy chain of M(r) 59,000 and a light chain of heterogeneous M(r) 18,000 (LC1) and 21,000 (LC2). These chains were separated after reduction and S-pyridylethylation, and the isolated major component LC1 was used for sequence analysis. The heavy chain consists of 427 residues containing four asparagine-linked oligosaccharides, and its entire sequence was similar to that of the high molecular mass hemorrhagic protein, HR1B, isolated from the venom of Trimere-surus flavoviridis. The heavy chain contains three distinct domains, metalloproteinase, disintegrin (platelet aggregation inhibitor)-like and unknown cysteine-rich domains. On the other hand, light chain LC1 consists of 123 amino acid residues containing one asparagine-linked oligosaccharide and shows sequence homology similar to that found in the so-called C-type (Ca(2+)-dependent) lectins. Therefore, RVV-X is a novel metalloproteinase containing a mosaic structure with distintegrin-like, cysteine-rich, and C-type lectin-like domains. RVV-X potently inhibits collagen- and ADP-stimulated platelet aggregations, probably via its distintegrin-like domain, although this domain does not contain the Arg-Gly-Asp sequence which is conserved in various venom distintegrins and which is thought to be one of the interaction sites for platelet integrins. Our findings also indicate that snake venom factor IX/factor X-binding protein with a C-type lectin structure (Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266, 14903-14911) inhibits RVV-X-catalyzed factor X activation; hence, the light chain of RVV-X probably participates in recognizing some portion of the zymogen factor X.     

圆斑蝰蛇毒七个C-型凝集素样蛋白亚基的cDNA克隆与序列分析

按照Promega公司的mRNA提取试剂盒操作手册,从圆斑蝰蛇（Daboia russellii siamensis）的毒腺中提取mRNA;利用RT-PCR的方法进行体外扩增,获得C-型凝集素蛋白的基因,克隆到pMD18-T载体中。随机挑选14个阳性克隆进行核酸测序,获得7个编码不同蛇毒C-型凝集素样蛋白亚基的cDNA,分别命名为DRS-L1、DRS-L2、DRS-L3、DRS-L4、DRS-L5、DRS-L6和DRS-L7。由基因序列推导出的氨基酸序列表明,克隆到的7个蛇毒C-型凝集素样蛋白的亚基中均有糖识别结构域存在。BLAST分析显示,仅有DRS-L1的蛋白序列与目前已知的蛇毒C-型凝集素样蛋白的α亚基相似。序列同源性比较并结合半胱氨酸位点分析,推测DRS-L1和DRS—L2可能分别是圆斑蝰蛇毒X因子激活剂的轻链LC2和LC1。DRS-L3和DRS-L4可能是高分子量的蛇毒C-型凝集素样蛋白的β亚基,而DRS-L5和DRS-L6可能是低分子量的蛇毒C-型凝集素样蛋白的β亚基。DRS—L7可能是类似于血小板膜糖蛋白Ib结合蛋白的β亚基。     

Allosteric aptamers controlling a signal amplification cascade allow visual detection of molecules at picomolar concentrations

A broadly applicable homogeneous detection system has been developed. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition (precipitation) of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF(165)) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF(165), reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus, triggering the cascade and enabling the phase transition. As few as 5 fmol of VEGF(165) could be detected by the naked eye within an hour. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout and can be modified to detect molecules to which aptamers can be obtained.     

Isolation and characterization of nerve growth factor from Vipera berus berus (common viper) venom

Nerve growth factor from Vipera berus berus venom was purified by gel filtration on Sephadex G-100 (superfine), ion-exchange-chromatography on DEAE-Sephadex A-50 and chromatofocusing on PBE 118. The Vipera berus berus venom NGF consists of multiple molecular forms with pls in the interval 9.1-9.7. All isoforms have identical mol. wts approximately 35,000 +/- 3000 (in gel filtration) and 17,000 +/- 2000, 15,000 +/- 2000 (by SDS electrophoresis with beta-mercaptethanol). V. berus berus venom NGF reacted with monoclonal antibodies against Viper lebetina NGF and caused differentiation of pheochromocytoma PC12 cells.     

Alpha-lipoic acid: an inhibitor of secretory phospholipase A2 with anti-inflammatory activity

Alpha-lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) are powerful antioxidants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. The mechanism of anti-inflammatory activity of ALA and DHALA is not known. The present study describes the interaction of ALA and DHALA with pro-inflammatory secretory PLA(2) enzymes from inflammatory fluids and snake venoms. In vitro enzymatic inhibition of sPLA(2) from Vipera russellii, Naja naja and partially purified sPLA(2) enzymes from human ascitic fluid (HAF), human pleural fluid (HPF) and normal human serum (HS) by ALA and DHLA was studied using (14)C-oleate labeled Escherichia coli as the substrate. Biophysical interaction of ALA with sPLA(2) was studied by fluorescent spectral analysis and circular dichroism studies. In vivo anti-inflammatory activity was checked using sPLA(2) induced mouse paw edema model. ALA but not DHLA inhibited purified sPLA(2) enzymes from V. russellii, N. naja and partially purified HAF, HPF and HS in a dose dependent manner. This data indicated that ALA is critical for inhibition. IC(50) value calculated for these enzymes ranges from 0.75 to 3.0 microM. The inhibition is independent of calcium and substrate concentration. Inflammatory sPLA(2) enzymes are more sensitive to inhibition by ALA than snake venom sPLA(2) enzymes. ALA quenched the fluorescence intensity of sPLA(2) enzyme in a dose dependent manner. Apparent shift in the far UV-CD spectra of sPLA(2) with ALA indicated change in its alpha-helical confirmation and these results suggest its direct interaction with the enzyme. ALA inhibits the sPLA(2) induced mouse paw edema in a dose dependent manner and confirms the sPLA(2) inhibitory activity in vivo also. These data suggest that ALA may act as an endogenous regulator of sPLA(2) enzyme activity and suppress inflammatory reactions.     

乌梢蛇血清的抗出血因子:一个有前途的抗蛇毒药物原料

用柱层析和聚丙烯酰胺凝胶盘状电泳法,从乌梢蛇血清中分离纯化了一个抗出血因子。用SDS-聚丙烯酰胺凝胶电泳法测得其分子量大约为65 kD;测定了五种蝮亚科蛇毒(尖吻蝮、竹叶青蛇、原矛头蝮、哈扑和短尾蝮)的最小出血剂量和乌梢蛇血清中抗出血因子对这五种蛇毒的抗出血活性;还测定了七种蛇毒(除上述五种毒蛇外,还包括圆斑蝰和银环蛇)的半数致死量,以及抗出血因子对中毒小鼠的治疗作用。结果显示:从乌梢蛇血清中提纯的抗出血因子的抗蛇毒活性,不仅可以抵抗它的捕食者尖吻蝮的蛇毒,而且还可以抵抗具出血活性的其它蛇毒;但它对不具出血活性的银环蛇毒的致死抑制作用不明显。该抗出血因子不仅在体外实验表现出强的中和出血毒素的活性,而且在体内实验中亦表现出对中毒小鼠良好的治疗作用,因而可能成为新的抗蛇毒药物的有前途的原料。乌梢蛇血清对血循毒的中和能力的获得,可能归因于尖吻蝮与乌梢蛇之间捕食与被捕食相互作用的关系。