《蛇及蛇毒医用研究与应用》第二集出版

1990年11月15日,中国蛇协第七次学术研讨会议在天津举行。蛇及蛇毒医用研究与应用为这次会议的论文集。文集汇集学术论文共244篇,重点突出蛇毒的研究与应用以及血栓病有关的文章。主要内容有:蛇毒的研究与综述、蛇毒在脑血管病中的应用、蛇毒在外科、皮肤科病的应用、蛇毒在其他疾病中的应用,蛇毒及与     

莽山烙铁头蛇毒液蛋白质组学研究(英文)

目的收集一条中国特有品种莽山烙铁头蛇的新鲜毒液,对其蛇毒蛋白作生化分析。方法先以电泳胶片法分析比较莽山烙铁头蛇毒与中国大陆及台湾、日本琉球各地其它品种的烙铁头蛇和各地其他品种的烙铁头蛇的粗蛇毒,在凝胶电泳中展现之异同。然后利用高效液相色谱法(HPLC)将莽山烙铁头蛇毒中的磷脂酶分离,再以质谱仪等鉴定,并与以往相同方法分析莽山烙铁头蛇所得到的磷脂酶分析结果比较。结果我们的研究证实了莽山烙铁头蛇毒含唯一主要的磷脂酶,其含量大约占蛇毒总重量的58%,与中国其他烙铁头蛇品种及日本琉球烙铁头蛇的蛇毒某些碱性磷脂酶之氨基酸序列及蛋白结构特别相似。此碱性磷脂酶主要毒性是水肿、局部炎症及肌肉坏死。结论莽山烙铁头蛇咬伤的临床表现为抗凝与出血,局部炎症坏死等特点,亦可由其蛇毒成分得到印证与解释。     

新型蛇毒制剂──青龙胶囊的临床应用概况

新型蛇毒制剂──青龙胶囊的临床应用概况中国蛇协蛇毒研究所邓禄延,覃公平,胡征林近年来,随着蛇毒的研究与医用的进一步发展,各种蛇毒制剂也应运而生,各国医药学家在开拓蛇毒医用研究领域中,以防治血栓病及攻克癌症为主要方面,已取得了可喜的成果。１９５３年Ｍｏ...     

关于生产多价抗蛇毒血清的初探

我国目前尚无多价抗蛇毒血清产品。本文试验仅是初步摸索,对目前抗蛇毒血清制品进行了分析并提出了制造多价抗蛇毒血清的思路及生产方法。认为可以分为抗血循毒性蛇毒血清及抗神经毒性蛇毒血清二大类产品及几种制造方法。本文采用二种单价免疫马血浆混合后进行精制的方法来制造多价抗血循毒性蛇毒血清,并获得了成功,同时,对具体精制生产上的问题进行了讨论并提出了解决方法。     

八种常见国产蛇毒对白血病细胞杀伤作用研究

用体外细胞培养的方法,观察了我国常见的八种蛇毒（眼镜蛇毒、蝮蛇毒、眼镜王蛇毒、竹叶青蛇毒、蝰蛇毒、烙铁头蛇毒、银环蛇毒及金环蛇毒）对人白血病Ｔ淋巴细胞系ＣＥＭ细胞、人单核细胞白血病Ｕ９３７细胞及人早幼粒细胞白血病ＨＬ６Ｏ细胞的生长曲线、存活率及分裂指数的影响。结果发现八种常见蛇毒中眼镜蛇毒对白血病细胞的杀伤作用最强,蝮蛇毒次之（与空白对照组相比,Ｐ值均小于０．０１）,其余六种蛇毒的作用则很弱。但无论是眼镜蛇毒还是蝮蛇毒,其杀伤淋巴细胞白血病、单核细胞白血病及早幼粒细胞白血病细胞的作用之间无显著差异。     

几种蛇毒与鲎试剂产生凝胶反应的实验研究

目的探讨蛇毒能否与鲎试剂产生凝胶反应．建立中药及有效成分抗蛇毒作用的试验途径。方法采用鲎试剂试管凝胶反应法。结果不同的蛇毒能使鲎试剂产生凝胶反应的浓度不同,眼镜蛇毒与竹叶青蛇毒为5μg／ml．五步蛇毒0．32μg／ml．蝮蛇毒37．5μg／ml,蝰蛇毒2．5pg／ml。结论蛇毒能与鲎试剂产生凝胶反应。     

蛇毒抗肿瘤作用研究进展

蛇毒含有多种酶类和毒性蛋白,具有广泛的生物学活性。对蛇毒各种组分的研究,发现蛇毒具有抗血栓,止血,镇痛及抗肿瘤的作用。本就其抗肿瘤方面的研究进展进行了综述,介绍了蛇毒的抑瘤机制,抑瘤活性成分的分离纯化,理化特性及研究方向和应用前景。     

蛇毒L-氨基酸氧化酶抗肿瘤作用研究进展

L-氨基酸氧化酶（LAAO）广泛地存在于各种蛇毒中。近年来许多文献报道了蛇毒L-氨基酸氧化酶具有与血小板相互作用、细胞毒性及诱导细胞凋亡作用,还报道它们具有出血或溶血活性、引起水肿及抗细菌、抗艾滋病病毒的特性。本文就其抗肿瘤方面的研究进行综述,介绍蛇毒L-氨基酸氧化酶的抑瘤机制、理化性质及国内外研究现状和应用前景。     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

尖吻蝮（Deinagkistrodon acutus）生长过程不同时期排毒量及蛇毒组分的变化

对人工培育的4个年龄段尖吻蝮蛇毒和野生成体尖吻蝮蛇毒进行了聚丙烯酰胺凝胶电泳分析。结果显示：3龄以前尖吻蝮蛇毒蛋白电泳图谱,无论是在分带、泳动率及相应组分的量等方面,均呈现一定的差异,但随着尖吻蝮蛇龄的增长,蛇毒蛋白电泳图谱与野生成蛇蛇毒蛋白电泳图谱越来越接近。3龄尖吻蝮生长发育达到性成熟,其蛇毒蛋白电泳图谱与野生成蛇趋于一致。蛇毒组分的变化,提示了尖吻蝮的生长发育进程。     

Crystal structure of Jararacussin‐I: The highly negatively charged catalytic interface contributes to macromolecular selectivity in snake venom thrombin‐like enzymes

Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin‐like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin‐I) was determined at 2.48 Å resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three‐dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin‐I is highly negatively charged, which contributes to its unique macromolecular selectivity.     

Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca²⁺ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.     

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG‐75, DEAE‐Sephadex A‐50, and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). VLCII as a thrombin‐like enzyme was able to hydrolyze Nα‐CBZ L‐arginine‐p‐nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10‐phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration‐dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.     

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bβ chain and BpirSP41 on both Aα and Bβ chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of ∼3.5 μg versus 20 μg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu²⁺ ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure–function relationship of SVSPs.     

Aqueous Leaf Extract of Jatropha gossypiifolia L. (Euphorbiaceae) Inhibits Enzymatic and Biological Actions of Bothrops jararaca Snake Venom

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A₂ (Lys49 PLA₂). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.     

High resolution analysis of snake venom metalloproteinase (SVMP) peptide bond cleavage specificity using proteome based peptide libraries and mass spectrometry

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1' site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and P' sites suggesting functional differences between these proteinases. The PI SVMPs, leucurolysin-a, atrolysin C, and BaP1, showed preferences across the full P4 to P4' range whereas the PIII SVMP bothropasin showed a narrower range of preferences across the sites. In silico docking experiments with the experimentally derived consensus sequences as well as with comparison of the results to those in the literature regarding peptide bond specificity based on both peptide and protein substrates give rise to a fresh understanding of the specificity of these SVMPS and may serve as a foundation for future experiments to better elucidate their mechanism of action in the complex pathophysiology of snakebite envenomation.     

沙蜇(Stomolophus meleagris)刺丝囊毒素生物活性的初步分析

从沙蜇触手提取刺丝囊细胞毒素,并对该毒素进行溶血活性、致死活性、SOD活性和抗肿瘤活性的研究。结果显示,沙蜇毒素具有明显的溶血活性,其半溶血率(HU50)约为10.5μg/ml;该毒素还对草鱼显示出较强的致死活性,半致死量(LD50)为50μg毒素/g鱼;同时该毒素具有明显的SOD活性和抗肿瘤活性,当毒素浓度为18μg/ml时其总SOD活性为161 U/mg,而毒素浓度为1 mg/ml时,该毒素对肝癌细胞Bel-7402表现出显著的抑制效果,其抑制率达到54.9%。因此,有必要对沙蜇毒素内的生物活性组分进行深入研究,为沙蜇毒素的开发利用提供依据。     

切叶蚁亚科蚂蚁的防御性生物碱

蚂蚁(蚁科)有显著的群居特性,个体间分工明确,并具有复杂的合作策略,以保护巢穴不受捕食者、病原微生物、蚂蚁竞争者的侵害,以及捕食猎物等。切叶蚁亚科是蚁科的最大类群,共有147个现生属,主要通过喷洒富含生物碱的毒液分泌物进行防御和捕猎。本文综述了该亚科防御性生物碱的组分及属种分布特征,其结构类型包括哌啶、吡啶、吡咯、吲哚里西啶、吡咯里西啶和脂肪胺等,并对其功能及应用进行了概述和展望。哌啶类生物碱是火蚁属Solenopsis毒液的典型特征,而吡咯烷、吡咯啉和吡咯里西啶类生物碱是小家蚁属Monomorium毒液的主要成分。吲哚里西啶类生物碱是脊红蚁属Myrmicaria蚂蚁和火蚁属贼蚁毒液的主要成分。除此之外,脂肪胺也是小家蚁属蚂蚁的毒液成分。这些毒液生物碱成分具有显著的生物活性,在农药和生物医药领域具有重要的应用前景。     

Behavioural observations and neurophysiological responses of cockroaches envenomated with Latrodectus katipo venom

1. The behaviour of American cockroaches envenomated with Latrodectus katipo venom was correlated with neurophysiological events recorded from an isolated cockroach CNS preparation to which venom was applied. 2. The injection of a venom extract into cockroaches caused convulsions (3 behavioural categories: quivering, jerking and hyperextension) which eventually led to paralysis and death. 3. Changes in the spontaneous activity of nerve 5 (from the metathoracic ganglion) corresponded to the time course of envenomation behaviours. An initial increase in discharge peaked 5-10 min after application and subsequently decreased to become irreversibly blocked.