发形霞水母触手提取物的蛋白稳定性及其溶血活性

目的探讨不同环境因素和处理方法对发形霞水母（Cyanea capillata）触手提取物（tentacle extract,TE）蛋白稳定性及其溶血活性的影响。方法结合蛋白浓度测定、溶血活性检测和SDS-PAGE分析等方法研究不同环境因素和处理方法对TE蛋白稳定性及其溶血活性的影响。结果 TE溶血活性呈明显的剂量依赖关系,其半数溶血分数HU50=226μg/ml;40℃水浴1 h,能去除TE中大量杂蛋白,但仍保持显著溶血活性;TE在4℃放置28 d,其溶血活性保持稳定,25℃下3 d内其溶血活性变化不明显;p H值对TE溶血活性的影响呈钟形曲线,在p H 6.0-11.0之间活性相对稳定,p H 8.0是TE保持溶血活性的最优p H条件;不同缓冲液对TE稳定性及其溶血活性影响显著,浓度大于26%的硫酸铵溶液对TE溶血蛋白具有良好的盐析作用。结论 40℃水浴1 h预处理可显著减少TE中非活性蛋白组分,并降低样品黏性;4℃和p H 8.0是TE保持蛋白稳定性和溶血活性的最适条件;浓度大于26%的硫酸铵盐析有利于溶血蛋白组分富集。     

发形霞水母触手提取物活性的初步研究

目的 初步分离发形霞水母触手提取物并探讨其生物学活性.方法采用自溶、离心的方法去除发形霞水母触手刺丝囊并获取触手提取物.通过阶段梯度阳离子交换色谱,应用0%.20%,40%,100%B液4种比例洗脱液将触手提取物分成4个组分,分别观察分离各组分的溶血、心血管等活性,并与触手提取物平行对比分析.结果成功分离到具有溶血、心血管活性的发形霞水母触手提取物,4个洗脱组分中,0%B液组分无上述3种活性,20%B液组分具有溶血活性、40%B液组分具有心血管活性,而100%B液组分则含有色素.结论通过阳离子交换色谱.初步将发形霞水母触手提取物中具有溶血活性、心血管活性以及色素等组分分离开来.为后续进一步纯化其单一活性组分打下基础.     

蛋白酶抑制剂对霞水母毒素溶血活性的影响

目的研究蛋白酶抑制剂对霞水母毒素溶血活性的影响。方法首先制备霞水母刺丝囊细胞,再利用Mini-Beadbeater组织研磨器破碎细胞提取毒素,然后向获得的霞水母毒素中加入不同种类及剂量的蛋白酶抑制剂,如金属酶抑制剂EDTA、酸性蛋白酶抑制剂Pepstantin A、丝氨酸蛋白酶抑制剂PMSF和Aprotinin,巯基蛋白酶抑制剂Leupeptin,并且测定蛋白酶抑制剂对霞水母毒素溶血活性的影响。结果 EDTA和Pepstantin A能够明显抑制蛋白酶的活性从而增强霞水母毒素的溶血活性,1 mmol.L-1EDTA和4μg.ml-1 Pepstantin A使其溶血率分别从40%上升到93%和5%上升到78%;而PMSF、Aprotinin和Leupeptin却对霞水母毒素溶血活性的影响较小。结论金属蛋白酶抑制剂EDTA和酸性蛋白酶抑制剂Pepstantin A能有效地保护霞水母毒素的溶血活性,为深入研究霞水母毒素提供帮助。     

发形霞水母共附生微生物的分离鉴定及其发酵液的抑菌活性研究

目的对发形霞水母(Cyanea capillata)共附生微生物进行分离鉴定并测定其抑菌活性,以期获得具有较高抑菌活性的菌株。方法利用4种分离培养基从C.capillata各部位分离共附生微生物,首先对挑选的单个菌落进行反复的划线分离,获得各菌株的纯培养;利用形态学观察、生理生化特征检测和16SrDNA序列测定和分析等手段对获得的菌株进行鉴定;以金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌作为指示菌,利用滤纸片扩散法测定菌株发酵液的抑菌活性;通过正交试验方法进一步优化发酵条件,提高菌株发酵产生抑菌活性物质的能力。结果从C.capillata触手、胃囊、伞部、肌肉4个部位共分离出83株菌株,其中放线菌...     

水母毒素心血管活性研究进展

简要介绍水母毒素的心血管毒性及其作用机制,重点讨论了Ca2+通道、儿荼酚胺受体、胆碱能受体等与水母毒素心血管效应的关系.     

水母毒素的研究进展

概述水母毒素的心血管毒性、神经毒性、皮肤与肌肉毒性、细胞毒性、抗氧化活性及多种酶活性等。该毒素成分复杂、不耐热、易氧化,在分离、纯化及生物学活性研究等方面受到限制,因此制备高纯度、高活性水母毒素的新方法有待于探索开发。     

发形霞水母触手提取物对大鼠离体心脏传导系统的影响

目的观察水母触手提取物（TOE）对大鼠离体心脏传导功能的影响,探讨其心脏毒性可能的作用机制。方法72只SD雄性大鼠随机分为6组（n=12）,利用大鼠离体心脏Langendorff模型,分别给予不同剂量的TOE,观察其对大鼠心脏传导系统的影响。结果给予TOE后,与空白组比较,心率减慢和心律失常的几率明显增大,并呈现剂量依赖性。结论TOE对心脏的传导系统具有显著影响,是其心脏毒性的原因之一。     

水母毒素与水母蜇伤

水母蜇伤是最常见的海洋生物伤,其发生率随着人们与海洋环境的密切接触不断上升。水母形态结构虽然简单,但其毒素组成复杂,可造成机体多个系统不同程度的损伤,因此水母蜇伤后临床症状较为复杂,目前尚没有基于毒素作用机制的特效抗毒药物。从水母毒素、水母蜇伤症状以及蜇伤后综合治疗3个方面进行综述,着重阐述近年来水母毒素组分研究的新进展以及针对毒素组分的拮抗药物的研究动态。     

水母毒素及水母来源新功能蛋白研究进展

水母是海洋中一类数量庞大的浮游生物,其体内含有多种高效的生物活性成分,是一个潜在的海洋药物资源宝库。本文通过概述近年来水母毒素及水母来源新功能蛋白的研究进展,对水母生物活性物质的毒性、抗氧化、抗肿瘤和酶活性等作用特点及机理进行了归纳,为开发新型海洋药物提供科学线索。     

水母毒素研究与转化医学

水母蜇伤是最常见的海洋生物伤,其发生率随着近年来环境变化引起的水母爆发性增长在不断上升。水母毒素具有心血管、溶血、神经、肌肉等多种生物毒性,其作用机制不明确,水母蜇伤防治也以对症处理为主。注重水母蜇伤临床症状、救治措施与水母毒素纯化鉴定、作用机制之间的相互联系,将加速水母毒素研究的整体推进。     

Factors influencing hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye.

In this study, hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye and some factors affecting it were assayed. The HU(50) of R. esculentum full venom (RFV) against chicken erythrocytes was 3.40 microg/ml and a Hill coefficient value was 1.73 suggesting at least two molecules participated in hemolytic activity. The hemolytic activity of RFV was affected by some chemical and physical factors such as divalent cations, EDTA, (NH(4))(2)SO(4), pH and temperature. In the presence of Mg(2+), Cu(2+), Zn(2+), Fe(2+), Ca(2+) (>or=2 mM), Mn(2+) ((>or=1 mM), EDTA ((>or=2 mM) and (NH(4))(2)SO(4), the hemolytic activity of RFV was reduced. RFV had strong hemolytic activity at the pH 6-10 and the hemolytic ratios were 0.95-1.19. Hemolytic activity was temperature-sensitive and when RFV was pre-incubated at temperatures over 40 degrees C, it was sharply reduced.     

Partial characterization of the hemolytic activity of the nematocyst venom from the jellyfish Cyanea nozakii Kishinouye

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the hemolytic activity of Cyanea nozakii Kishinouye nematocyst venom on chicken erythrocytes. Venom extract caused a significant concentration-dependent hemolytic effect. The extract could retain its activity at −80 °C but was unstable when kept at 4 °C and −20 °C for 2 days. The hemolytic activity was inhibited by heating within the range of 37–100 °C. The extract was active over a pH range of 5.0–8.63 and the pH optima for the extract was 7.8. Incubation of the venom with sphingomyelin specially inhibited hemolytic activity by up to 70%. Cu²⁺ and Mn²⁺ greatly reduced the hemolytic activity while Mg²⁺, Sr²⁺ and Ba²⁺ produced a relatively low inhibiting effect on the hemolytic activity. Treatment with Ca²⁺ induced a concentration-dependent increase in the hemolytic activity. In the presence of 5 mM EDTA, all the hemolytic activity was lost, however, the venom containing 1.5 mM EDTA was stable in the long-term storage. PLA₂ activity was also found in the nematocyst venom of C. nozakii. These characteristics provide us a fundamental knowledge in the C. nozakii nematocyst venom which would benefit future research.     

The cardiac actions of a toxin-containing material from the jellyfish, Cyanea capillata.

M. J. A. Walker. The cardiac actions of a toxin-containing material from the jellyfish, Cyanea capillata. Toxicon15, 15–27, 1977.—The cardiac actions of a toxin-containing material from the nematocysts of Cyanea capillata have been investigated as previous studies have shown such actions to be responsible for the material's lethality to several species. Electrophysiological, chemical and pharmacological experiments have been performed on various rat cardiac tissues and a probable mechanism of cardiac toxicity elucidated. Cyanea toxin-containing material caused arrhythmias, loss of contractility and contracture in isolated hearts, atria and ventricles. At the concentrations needed to produce the above effects, the material caused a fall in resting membrane potential and action potential (with eventually loss of action potentials) in rat atria. Such profound electrophysiological changes were accompanied in the same tissue by a loss of tissue potassium and a gain in sodium and calcium sufficient to account for the observed changes. Concomitant with the electrophysiological and ionic changes, the oxygen utilization of intact rat atria and isolated pig heart mitochondria were also profoundly affected. It is not known whether effects on metabolism produced the other changes responsible for the cardiac tissue response. C¹⁴-Inulin distribution studies suggested that the tissues are relatively intact. The in vivo cardiotoxicity responsible for lethality (Walker, 1976) can thus be explained in terms of very pronounced changes in ion distribution and subsequent electrophysiological changes.     

In vitro and in vivo haemolytic studies of tentacle-only extract from jellyfish Cyanea capillata

To approach the real haemolytic process of jellyfish toxins, both in vitro and in vivo haemolysis of tentacle-only extract (TOE) from jellyfish Cyanea capillata has been studied. Dose–response curves of the haemolytic activity of TOE in vitro were sigmoid shaped in both erythrocyte suspension and diluted whole blood, with the former more sensitive to TOE. The in vivo haemolysis increased sharply in the first 10 min and was followed by a gradual increase in the following 3 h, with increasing blood potassium and lactic acid accordingly. SC5b-9 complexes were significantly up-regulated in vitro, but not in vivo. These results showed that the haemolysis of TOE in diluted whole blood and in vivo is not totally consistent with that in the erythrocyte suspension, and blood plasma might play a protective role against haemolysis. Thus we suggested that erythrocyte suspension can be used to test the damage of toxin on erythrocyte membrane, while the diluted whole blood may be more suitable to test the haemolysis of toxins.     

Investigations into the cardiotoxicity of a toxin from the nematocysts of the jellyfish, Cyanea capillata.

The action of Cyanea toxin-containing material on active cation transport enzymes from human erythrocyte membranes and on the beating behaviour of cultured neonatal rat heart cells has been examined. The heat labile cardiotoxic principle had no specific effects on the activity of enzymes involved in active transport of Na⁺, K⁺ and Ca²⁺ which would account for the cardiotoxicity previously ascribed to marked disturbances in the distribution of these ions. Exposure to Cyanea toxin produced predictable effects in single cultured heart cells and each treated cell went through the same progression of arrhythmic changes which consisted of an initial increase in rate, followed by variations in rate, rapid fluttering, an intermediate dormant period, fibrillation (asynchronous rapid contractions) and a final cessation of all activity. Arrhythmic changes were compatible with the toxin-induced electrophysiological changes previously described for rat atrial tissue.     

Direct cardiac toxicity of the tentacle-only extract from the jellyfish Cyanea capillata demonstrated in isolated rat heart

Previous studies in our laboratory have shown that the cardiotoxicity is the main reason for rat death caused by tentacle-only extract from jellyfish Cyanea capillata. However, the direct cardiotoxicity in vitro and its mechanisms of toxic action remain unclear. The current studies were performed by using the Langendorff-perfused isolated heart model, which showed a dose-dependent hemodynamic and electrocardiogram changes. Heart injury-related enzymes increased. Histopathological analysis showed early ischemic damage in the myocardium. The Ca channel blockers nifedipine and verapamil led to a marked improvement in recovery of cardiac function, including heart rate, left ventricular developed pressure, positive and negative first derivatives of intraventricular pressure, coronary flow, left ventricular end-diastolic pressure, and electrocardiogram changes. Tentacle-only extract-induced cardiac dysfunction could be partly improved by the pretreatments of both propranolol and phentolamine, but not by either atropine or neostigmine at all. In conclusion, we have verified the direct cardiotoxicity of tentacle-only extract from jellyfish C. capillata by the Langendorff isolated heart model, which consisted of 3 separate parts: sinoatrial node malfunction, cardiomyocyte injury, and coronary spasm. The potential mechanism might be attributed to the overactivation of L-type Ca channel, β- and α-adrenergic receptors, but not cholinergic receptors.     

Factors affecting the hemolytic activity of the Oriental hornet (Vespa orientalis) venom and venom sac extracts

Extracts from the venom sacs of the Oriental hornet (V. orientalis) pupae are devoid of either direct or indirect hemolytic activity. However, in venom sac extract obtained during and following eclosion of the imagines, hemolytic activity of both types is present and gradually rises, reaching maximal levels at days 3 or 4. Complete daily evacuation of the venom sac for 5 consecutive days, results in progressive diminution of its hemolytic activity. Soybean trypsin inhibitor and, to a lesser degree, other protease inhibitors, partially suppress the direct hemolytic activity of V. orientalis venom, thus suggesting the possible role of venom proteases as potentiators of the direct hemolytic activity.     

Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Péron & Léslieur).

Two species of venomous pelagic cnidaria are compared according to their enzymatic, cytotoxic and haemolytic potency. The widely distributed jellyfish Cyanea capillata and Cyanea lamarckii were collected in the North Sea at the coasts of the Orkney Island and the Island of Helgoland. Purified cnidocyst extracts from fishing and mesenteric tentacles were prepared and tested for their bioactivity. The haemolysis induced by toxins of C. capillata was determined with respect to organism size and toxigenic organs. The haemolytic activity of the related species C. lamarckii was documented for the first time. Dose dependent haemolytic activities have been detected by means of protein equivalents at concentrations above 20mug(protein)/mL. Extracts of fishing tentacle cnidocysts showed a less potent haemolytic activity compared to extracts of mesenteric tentacles. In vitro studies with permanent cells of a hepatoma cell line have shown a time and concentration dependent loss of cell vitality up to 90% at 33.3mug(protein)/mL (10mug(protein)/10(5) cells). Supplementing the cell based toxicity tests an enzyme assay was performed to measure a phospholipase A(2) (PLA(2)) activity. A PLA(2)-like activity could be demonstrated in cnidocysts extracts prepared from mesenteric and fishing tentacles of both jellyfish species.