尖吻蝮(Agkistrodon acutus)蛇毒凝血酶样成分的研究 Ⅱ.酶学与血液学活性的研究

对四个凝血酶样成分(TLP)的酶学性质的比较研究表明,它们都具有凝结(血)活性和精氨酸酯酶活性。其凝结活力TLP_4>TLP_3>TLP_1>TLP_2,Ca~(++)有激活作用,但不被肝素、EDTA所抑制;精氨酸酯酶活力以TLP_1、TLP_2较高,Ca~(++)、EDTA无明显的激活或抑制作用;TLP_1对热比较稳定,TLP_4对于酸碱变化比较稳定。经动物体内试验表明,TLP_1、TLP_2具有较强的去纤抗凝作用,TLP_3、TLP_4不显示抗凝作用,而显示出较强的促凝血作用。结果表明,尖吻蝮蛇毒与美洲矛头蝮蛇毒一样,同时含有去纤酶(Defibrase)和蛇毒凝血酶(Hemocoagulase)。这一结果对该蛇毒的研究与应用是很有意义的。     

Small‐molecule reductants inhibit multicatalytic activity of AA‐NADase from Agkistrodon acutus venom by reducing the disulfide‐bonds and Cu(II) of enzyme

AA‐NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA‐NADase contains Cu(II) and has disulfide‐bond linkages between two peptide chains. The effects of the reduction of the disulfide‐bonds and Cu(II) in AA‐NADase by small‐molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA‐NADase has six disulfide‐bonds and fifteen free cysteine residues. L‐ascorbate inhibits AA‐NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA‐NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2‐carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide‐bonds in AA‐NADase. Apo‐AA‐NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo‐AA‐NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide‐bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA‐NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L‐ascorbate are endogenous inhibitors to the multicatalytic activity of AA‐NADase. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 141–149, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

蝮蛇毒蛋白C激活物对凝血系统的影响

目的研究蝮蛇毒纯化蛋白C激活物(PCA)的抗凝机制。方法测定PCA对正常人混浆KPTT、PT、TT的影响,对血液凝血活酶生成试验(BTGT)及PA二期法的影响以及对白兔的KPTT和Fgn的影响。结果PCA在最终浓度为0.025mg/L时对正常人混浆KPTT可明显延长,但PT和TT不受影响;当它的浓度增加到50mg/L,PT也可延长,但TT依然无明显变化;最终浓度为0.0125mg/L时,血液凝血活酶生成明显受抑制;当它的浓度增加到2.5mg/L,凝血酶生成显著减少,但抗凝血酶时间始终无明显变化;PCA可明显延长白兔的KPTT。结论PCA在低浓度时首先抑制凝血系统第一阶段内凝途径,在高浓度时也妨碍外凝途径或共同途径,BTGT和PA二期法比KPTT和PT敏感;PCA具有体内抗凝作用。     

长白蝮蛇类凝血酶基因大肠杆菌表达研究

从长白蝮蛇克隆的类凝血酶Ussurin Cdna被克隆进入pET-32α（＋）中与5′端与Thx-Tag和6xHis相连的T7启动子下,构建成大肠杆菌表达载体pET-32α（＋）-Ussurin;再转化感受态E.coli BL（21）DE3细胞,构成表达工程菌株;于IPTG诱导下表达,在细胞质中产生约占细胞质总蛋白10％的具有生物活性的Ussurin。     

Size-based variation in antipredator behavior within a snake (Agkistrodon piscivorus) population

Variation in an animal's response to a predator likely reflectsthe complex interaction of factors that influence predationrisk. Due to their high degree of behavioral variation and simplifiedbauplan, snakes offer a unique model for investigating the influenceof sex and body size on antipredator behavior. We examined variationin antipredator behavior within a cottonmouth (Agkistrodon piscivorusleucostoma) population. Behavioral responses to human-inducedpredation risk were compared across a continuous scale of bodysize. Defensive responses significantly declined with increasingbody size. After controlling for body size, no differences betweenthe sexes were detected. Although this study suggests that variationin antipredator behavior is, in part, related to body size,some studies on snakes have not found this relationship. Likewise,some studies have demonstrated differences between sexes. Suchdisparate patterns of variation indicate a need for future comparativestudies examining the complex interaction of factors that mayinfluence predator–prey relationships.     

PCR方法在蕲蛇中的鉴别应用

目的探讨PCR方法在蕲蛇鉴别中的可行性。方法采用PCR方法对蕲蛇对照品、蕲蛇正品和蕲蛇混淆品目的基因扩增及琼脂糖凝胶电泳分析。结果蕲蛇对照品、蕲蛇正品在291bp处有目的条带,而蕲蛇混淆品未出现目的条带。结论PCR方法鉴别蕲蛇是一种可行、客观且易于操作的有效方法。     

蝮蛇毒介导小鼠急性心肌损伤及心肌组织TNF-α表达的研究

目的探讨蝮蛇毒介导小鼠心肌组织中TNF-α蛋白表达变化与不同蝮蛇毒剂量、中毒时间的关系。方法确定LD50蝮蛇毒注射剂量。将96只小鼠随机分为空白对照组、蛇毒低剂量(0.5mg/kg)组、蛇毒高剂量(1.0mg/kg)组,并按中毒时间设4水平组,即3、6、24、48h组,采集小鼠心肌标本,免疫组化检测心肌组织中TNF-α蛋白的表达情况。结果蛇毒低剂量组及蛇毒高剂量组小鼠心肌TNF-α蛋白表达明显高于空白对照组(均P0.05);而蛇毒高剂量组明显高于低剂量组(P0.05);6h组明显高于3h组(P0.05);24h组高于6h组(P0.05);48h组高于24h组(P0.05)。随着蝮蛇毒剂量的增加、中毒时间的延长,心肌TNF-α蛋白的表达越高。结论蝮蛇毒导致小鼠急性心肌损伤,TNF-α蛋白表达增高,且随着蝮蛇毒剂量的增加、中毒时间的延长,心肌TNF-α蛋白的表达越高,呈一定的时间、剂量效应趋势,提示TNF-α蛋白过度表达可能是蝮蛇毒导致小鼠急性心肌损伤的机制之一。     

Post‐birth separation affects the affiliative behaviour of kin in a pitviper with maternal attendance

The relatedness of individuals can have pronounced effects on behavioural interactions, as engaging in mutually beneficial behaviours with kin can increase inclusive fitness. Parental care can be particularly important for kin discrimination in birds and mammals, but similar studies have not been conducted on species exhibiting more rudimentary forms of care. Maternal attendance of young is ubiquitous among viviparous temperate pitvipers, but the adaptive value of this behaviour has received little attention. We sought to determine if being deprived of a maternal attendance period as neonates altered how cottonmouths (Agkistrodon piscivorus Lacépède), a common North American pitviper, responded to kin vs. non‐kin. We measured the affiliative behaviour of related and unrelated juvenile–juvenile and mother–juvenile pairs that had been allowed a maternal attendance period or had been separated since birth. We found that maternal attendance was not required for sibling or mother–offspring recognition, but did enhance female affiliative behaviour overall, and particularly that of sisters. In contrast, related juveniles that were separated at birth showed a reversal of the sex‐specific affiliative behaviour observed in maternally attended juveniles. Post‐birth separation had only a modest effect on mother–juvenile affiliative behaviour, and no effect on the strong affiliation between mothers and daughters. The patterns of affiliative behaviour observed in maternally attended snakes corresponded to patterns that have emerged from previous captive and field studies of pitvipers; however, the behaviour of juveniles separated at birth was atypical. Thus, it is possible that maternal attendance plays some role in the development of adaptive sex‐specific and kin‐directed affiliative behaviour in pitvipers.     

Purification, gene cloning and expression of an acidic phospholipase A2 from Agkistrodon shedaoensis Zhao

A protein with the activity of phospholipase A2 named asAPLA2 was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column, Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/ L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the N-terminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares. The gene was then cloned into the expression plasmid pET-40b( ) and expressed in E. coli BL21(DE3). Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native     

A novel P-I class metalloproteinase with broad substrate-cleaving activity, agkislysin, from Agkistrodon acutus venom

The venom of Viperdae is a rich source of metalloproteinases, which have potential clinical applications for lowering plasma fibrinogen or dissolving thrombi. Recently, we purified a novel proteinase from Formosan Agkistrodon acutus venom using DEAE-Sephadex A-50 and Mono-Q HR 5/5 column chromatography. The purified getatinolytic component, agkislysin, is a 22kDa-monomeric protein without Asn-linked sugar. Functional characterization showed that agkislysin possessed both fibronectin- and type IV collagen-cleaving activities. In addition, agkislysin preferentially cleaved the Aalpha chain of fibrinogen, followed by the Bbeta chain, while the gamma chain was finally affected. Furthermore, agkislysin was also capable of cleaving prothrombin into various fragments, as well as suppressing ristocetin-induced platelet aggregation by hydrolyzing von Willebrand factor. However, the proteolytic activity of agkislysin was slightly enhanced by divalent metal ions and completely inhibited by chelating agents, but not serine proteinase inhibitor. These findings suggest that agkislysin is a P-I class metalloproteinase with unique biological properties.