蝮蛇毒抗凝血活酶组份及几种蛇毒粗毒对人红细胞和血小板的作用

蝮蛇毒抗凝血活酶组分及蝗蛇毒、圆斑蝰蛇毒和眼镜蛇毒粗毒,不仅能够水解血浆中的磷脂,而且还能水解完整人红细胞膜和完整人血小板膜上的磷脂。但是五步蛇毒和金环蛇毒粗毒却不能水解完整人血小板膜上的磷脂。 扫描电镜观察表明,由于抗凝血活酶组份和几种蛇毒粗毒的作用,人红细胞和人血小板的形态发生了巨大的变化;人红细胞由正常的双圆盘形变成带刺的小球,人血小板的外形变成蜂窝状。     

荧光原位杂交技术和流式细胞仪用于环境样品中产毒及非产毒微囊藻的定量监测

全球范围内,高频次、大范围暴发的蓝藻水华对淡水水体环境造成严重影响.微囊藻因其在生长特别是衰亡过程中向水体释放微囊藻毒素而威胁人类健康.因此,分析其产毒株及非产毒株在环境样品中的组成,建立产毒蓝藻的预报及评价体系显得极为重要.本文采用荧光原位杂交技术结合流式细胞技术实现对环境样品中产毒藻株的鉴别与定量.针对目标基因mcyA设计的、以地高辛标记的双链DNA探针可有效应用于产毒微囊藻FACHB905和PCC7806的鉴别.分别对来自滇池、太湖和关桥的11个样品进行分析显示,该方法与传统的形态学鉴定及PCR方法有较好的匹配.荧光原位杂交技术与流式细胞相结合可有效鉴别产毒与非产毒微囊藻,尤其可以对野外样品中产毒与非产毒藻株进行简便、可视化地鉴别,从而达到对产毒微囊藻水华早期预警的目的.     

(Na~++K~+)—ATP酶与磷脂膜的相互作用

本文研究了不同磷脂对兔肾外髓质(Na~++K~+)-ATP酶活性的影响、结果表明,DOPC、PG重组活性最高,用DMPC重组导致酶失活,酸性磷脂有利于维持该酶活性.DSC及自旋标记ESR实验结果示出(Na~++K~+)-ATP酶有选择地与酸性磷脂相互作用.     

(Na~++K~+)—ATP酶与磷脂膜的相互作用

本文研究了不同磷脂对兔肾外髓质(Na~++K~+)-ATP酶活性的影响、结果表明,DOPC、PG重组活性最高,用DMPC重组导致酶失活,酸性磷脂有利于维持该酶活性.DSC及自旋标记ESR实验结果示出(Na~++K~+)-ATP酶有选择地与酸性磷脂相互作用.     

虎纹捕鸟蛛毒的生物学活性鉴定

本文报道了采集广西产虎纹捕鸟蛛（Selenocosmia huwena)毒液的方法,并对粗毒进行了部分生物学活性的测定,该粗毒对小鼠和蜚蠊的LD50分钟为1．16mg/kg和300ug/g,该粗毒具有透明质酸酶,碱性磷酸酶,蛋白水解酶和脱氧核糖核酸酶活性,未测到磷脂酶A和胆碱脂酶性,通过电生理实验发现该粗毒含有阻断蟾蜍神经肌肉接头传递的毒素,并观察到当小鼠接受腹腔注射致死剂量（5mg/kg)粗毒后便迅速出现呼吸麻痹。     

重组人磷脂酶D2在体内能明显抑制慢性哮喘豚鼠血清中GPI-PLD的表达

 通过建立慢性豚鼠哮喘模型,研究重组人磷脂酶D2(rhPLD2)干预慢性哮喘豚鼠血清中糖基化磷脂酰肌醇特异性磷脂酶D(GPI-PLD)含量的变化,探寻可能的机制. 以rhPLD2干预慢性哮喘豚鼠,用TX-114分相法检测豚鼠血清中GPI-PLD酶活性.与正常状态豚鼠相比较,慢性哮喘状态豚鼠其血清中GPI-PLD酶活性显著升高.但以rhPLD2及地塞米松干预慢性哮喘豚鼠后,该指标显著下降. rhPLD2可通过抑制其血清中GPI-PLD酶活性表达,来抑制哮喘发生过程中的炎症反应.     

食品中椰毒假单胞菌微滴式数字PCR定量检测方法的建立

椰毒假单胞菌可污染包括发酵米面等多种食物,其产生的两种毒素导致严重的食物中毒,威胁人体健康。基于数字PCR技术,建立了针对椰毒假单胞菌的定量测量方法。该方法具有较好的特异性与重复性,人工污染糯米汤样品中的检出限为361 CFU/mL。与平板培养法相比,基于数字PCR技术的测量方法缩短了测量时间,有利于椰毒假单胞菌的快速准确测量,未来可在食品安全监测与中毒分析等领域进行应用。     

白色念珠菌磷脂酶活性与其毒力因子关系的研究

目的 探讨白色念珠菌磷脂酶活性与其毒力因子之间的关系以及与其遗传特征的相关性.方法 蛋黄平板法检测80株白色念珠菌磷脂酶活性,并测定磷脂酶高活性组、低活性组白色念珠菌的溶血活性、细胞表面疏水性、黏附上皮细胞能力及RAPD-PCR分析2组菌株的基因型特征.结果 磷脂酶阳性检出率为97.5%,2组PZ均值分别为0.639±0.043、0.847±0.079(P<0.05);2组的溶血活性、疏水率差异均无显著性(P>0.05),而磷脂酶高活性组菌株对上皮细胞的黏附力显著高于磷脂酶低活性组(P<0.05),磷脂酶活性与黏附力呈正相关(r=0.773,P<0.01).RAPD结果 表明磷脂酶活性与RAPD各电泳条带之间的回归系数不尽相同.结论 磷脂酶活性不能直接反应菌株其他毒力的情况,且RAPD分型也不能全面地反映磷脂酶这一单一的毒力特征,而是对菌株毒力特征的一个综合反映.     

大鼠肝CTP:磷酸胆碱胞苷酰转移酶的部分纯化及其特性

 介绍一种部分纯化CTP:磷酸胆碱胞苷酰转移酶(CT)的方法,并对部分纯化CT的性质进行了研究。经盐析、DEAE-纤维素、磷酸-纤维素及CTP-聚琼脂糖柱层析可将大鼠肝胞液中的CT纯化100倍以上,结果重复性好。CT在胞液中及经初步纯化性质稳定,但进一步纯化则其活性很易丧失。鼠肝总磷脂及油酸可激活CT、使其聚合,磷脂酰絲氨酸是其中起主要作用的CT激活物;CT聚合物可被辛基-葡萄糖苷解聚,且保留80％酶活性。用CT常规底物CTP的类似物观察CT的特异性,发现dCTP是比CTP更好的底物,CDP、dCDP能抑制CT对CTP或dCTP的作用,而CMP、dCMP对酶活性无影响。结果提示分子中糖的2′-OH并非CT底物所必需,而和胞苷相连的三个磷酸则不可缺少。     

雌性和雄性虎纹捕鸟蛛粗毒蛋白质含量比较分析

自然界虎纹捕鸟蛛雌蛛数量远多于雄蛛数量,为了探究雌、雄蛛粗毒的差异,用不同方法比较了单个虎纹捕鸟蛛雌、雄蛛粗毒的特征.雌蛛单次螫毒量为(26.5±2.47)μL,冻干后粗毒质量为(5.01±0.78)mg,明显高于雄蛛单次螫毒量(10.83±1.35)μL,冻干后粗毒质量(2.05±0.17)mg;用Lowry法和Bradford法测定雌蛛和雄蛛粗毒的蛋白质含量,两种方法均表明雄蛛粗毒中蛋白含量高于雌蛛.用反相高效液相色谱分离雌蛛和雄蛛粗毒蛋白,并215 nm检测色谱图,发现大部分洗脱峰重叠,而雄蛛色谱图中多两个主峰.Tricine SDS-PAGE电泳分析表明雌蛛粗毒相对分子质量小于10 kD的蛋白质含量较高;而Tris SDS-PAGE电泳分析表明雌蛛粗毒相对分子质量大于10 kD的蛋白质含量较低,基于雌蛛和雄蛛粗毒蛋白含量的差异,有必要对虎纹捕鸟蛛雌、雄蛛粗毒进行独立研究.     

Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus)

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.     

广西眼镜王蛇毒碱性磷脂酶A_2(PLA_2)的分离纯化及其性质的研究

广西眼镜王蛇毒用羧甲基纤维素CM-52、磷酸纤维素P-11和Sepharose CL-6B柱层析纯化,得到一个在聚丙烯酰胺凝胶电泳上为单一蛋白带,PLA_2的比活性较原蛇毒提高3.6倍,分子量的为13000,由122个氨基酸组成,_pI为8.9,具有良好的热稳定性。从碱性PLA_2对红细胞影响的电镜观察可见,对人的红细胞膜有明显的作用,而对山羊红细胞作用不明显。PLA_2无论对人还是对山羊、兔和豚鼠红细胞电泳速度都有明显的迟缓作用。     

Effect of heparin and antivenom on skeletal muscle damage produced by Bothrops jararacussu venom

We examined the effect of treatment with heparin and polyvalent antivenom on mice muscle Extensor digitorum longus (EDL) regeneration, after damage induced by injection of Bothrops jararacussu crude venom over the muscle of the right posterior limb. The mice were separated into groups and each group received treatment, by intravenous route with either high molecular weight heparin (H), low molecular weight heparin (LMWH), polyvalent antivenom (PAV) or with the combination of PAV plus H or PAV plus LMWH at 15 minutes and 4 hours after the injection of the venom. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours after the injection of the venom. The histological changes in EDL at 1, 3, 7 and 21 days after the injection of the venom were analyzed by light microscopy. In each group the normal and regenerated muscle fibers were quantified using Scion Image computer program. We also evaluated in vitro, the influence of these substances in the proteolytic and phospholipase activities of the venom. Heparins decreased the proteolytic activity of the venom but did not affect its phospholipase activity. However the PAV antagonized both activities. PAV and its combinations showed antimyotoxic activity, according to the magnitude of CK plasma levels. At 21 days the regeneration was observed in all animals, also in those that received only the venom. All treatments, except LMWH, promote a significant increase in the number of muscle fibers.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

Purification of a nontoxic phospholipase A2 from the venom of Indian krait (Bungarus caeruleus).

A nontoxic phospholipase A2 was purified from the venom of Indian krait (Bungarus caeruleus) by a four-step procedure involving electrophoresis, gel filtration and ion-exchange chromatography. The recovery of the enzyme activity was 37% and the purified preparation was 38 times as active as the crude venom. The purified enzyme had a molecular weight of 12,500 and the optimum pH of 7.2. The enzyme showed higher specificity toward phosphatidylethanolamine than phosphatidylcholine. The preparation was not very labile to heat and its activity was dependent on the presence of divalent cations, calcium ions being the most effective activators. The enzyme was completely inhibited by iodoacetic acid but showed high stability against 8 M urea. Purified phospholipase A2 was nontoxic at an iv dose of 5 microgram/g mouse. The high specific activity, the high yield and the nontoxic nature of the enzyme indicate that the major form of phospholipase A2 in Bungarus caeruleus venom is not associated with any toxicity and has properties somewhat similar to that of phospholipase A2 from some other venoms.     

Physicochemical properties of alpha- and beta-fibrinogenases of Trimeresurus mucrosquamatus venom.

alpha- and beta-Fibrinogenases (EC 3.4.21.5) were purified from Trimeresurus mucrosquamatus venom by the technique of recycling chromatography. Both enzymes were single polypeptide chains and homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation constants of alpha- and beta-fibrinogenases were 2.52 and 3.04 respectively. The molecular weight of alpha-fibrinogenase was 21 500--23 400, and that of beta-fibrinogenase was 25 000--26 000. The contents of proline, glycine and tryptophan were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha- and beta-fibrinogenases were pH 8.1 and 5.7 respectively. The optimal pH of alpha-fibrinogenase was about 7.4 and that of beta-fibrinogenase was around 8.5. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.6, 7.4 and 9.0, while that of beta-fibrinogenase was not significantly affected by the same treatment. Both enzymes showed proteolytic activities toward fibrinogen and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities of the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 17 times that of the crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethane sulfonylfluoride and slightly by tosyl-L-lysine chloromethylketone and cysteine.     

A phospholipase A2 with anticoagulant activity. II. Inhibition of the phospholiped activity in coagulation.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Hemorrhagic principles in the venom of Bitis arietans,a viperous snake. I. Purification and characterization

Two hemorrhagic principles (Bitis arietans hemorrhagin a and b: abbreviated as BHRa and BHRb) were purified from the venom of the viperous snake Bitis arietans (puff adder) by gel filtration, ion-exchange and absorption chromatography. A 10-fold purification was achieved for BHRa and 7-fold for BHRb with an overall yield of 6.4% of hemorrhagic activity. The hemorrhagins were homogeneous according to disc- and SDS-polyacrylamide gel electrophoresis and immunodiffusion. BHRa and BHRb consist of 623 and 685 amino-acid residues and their apparent molecular weights were 68 000 and 75 000, respectively. They were also immunologically distinct. The purified hemorrhagins express proteolytic activity with heat-denatured casein and hide powder azure. The proteolytic activity with heat-denatured casein was almost the same as that of the crude venom, but that with hide powder azure was less than one-tenth of that of the crude venom. The purified hemorrhagins were free of arginine esterase and phospholipase A₂ activities and they are acid labile hemorrhagic toxins. Their hemorrhagic activity was inhibited by EDTA, cysteine and by polyvalent anti-snake serum, but not by phenylmethanesulfonyl fluoride or soybean trypsin inhibitor.     

Studies on Habu Snake Venom

It has been found that there are two factors responsible for myolysis in Habu snake venom; heat-labile and heat-stable myolytic factors. As reported previously, the former is a proteinase contained in Habu venom. In this article purification and chemical properties of the heat-stable myolytic factor are presented. Using the method of fractionation with ammonium sulfate and with column chromatography, a heat-stable myolytic factor was obtained ultracentrifugally in a homogeneous state. This factor caused severe myolysis with the aid of both Mg⁺⁺ ion and phospholipase A. It has been found that heat-stable myolytic factor has no activities such as those of proteinase, esterase or ribonuclease. The relationship between the heat-stable myolytic factor and phospholipase A in myolytic activity is discussed.