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.     

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。     

Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye

Cyanea nozakii Kishinouye, a jellyfish widely distributed in coastal areas of China, has garnered attention because of its stinging capacity and the resulting public health hazard. We used a recently developed technique to extract jellyfish venom from nematocysts; the present study investigates the lethality of C. nozakii venom. The nematocyst contents were extremely toxic to the grass carp, Ctenopharyngodon idellus, producing typical neurotoxin toxicity. The ID₅₀ was about 0.6 μg protein/g fish. Toxin samples were stable when kept at −80 ^°C, but after 48 h, an 80% decline in lethality occurred at −20 ^°C. Poor stability of the venom was observed within the range of 65-80 ^°C and at pH 3.5. The venom was hydrolyzed by a proteolytic enzyme, trypsin. Fractionation of the venom yielded two protein bands with molecular weights of 60 kDa and 50 kDa. Our results provide the first evidence that C. nozakii produces lethal toxins. These characteristics highlight the need for the isolation and molecular characterization of new active toxins in C. nozakii.     

Isolation and in vitro partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish Stomolophus meleagris

Jellyfish venom contains various toxins and can cause itching, edema, muscle aches, shortness of breath, blood pressure depression, shock or even death after being stung. Hemolytic protein is one of the most hazardous components in the venom. The present study investigated the hemolytic activity of the nematocyst venom from jellyfish Stomolophus meleagris. Anion exchange chromatography, DEAE Sepharose Fast Flow, and gel filtration chromatography, Superdex200 had been employed to isolate hemolytic proteins from the nematocyst venom of jellyfish S. meleagris. Hemolysis of chicken red blood cells was used to quantify hemolytic potency of crude nematocyst venom and chromatography fractions during the purification process. Native-PAGE profile displayed one protein band in the purified hemolytic protein (SmTX); however, two protein bands with apparent molecular weights of ～45 kDa and 52 kDa were observed in the reducing SDS–PAGE analysis. Approximately 70 μg/mL of SmTX caused 50% hemolysis (HU₅₀) of the erythrocyte suspension. The hemolytic activity of SmTX was shown to be temperature and pH dependent, with the optimum temperature and pH being 37 °C and pH 5.0. The present study is the first report of isolation and partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish S. meleagris. The mechanism of the hemolytic activity of SmTX is not clear and deserves further investigation.     

发形霞水母毒素溶血活性研究

目的 探讨发形霞水母毒素(Cyanea capillata full venom,CFV)的体外溶血活性及其影响因素.方法 超声破碎法分离得到CFV,测定其半数溶血分数(HU50),探讨温度、pH、放置时间对溶血活性的影响,并进一步研究不同动物红细胞对溶血活性的敏感性.结果 CFV溶血活性的HU50为5.82 μg·mL-1;溶血活性对温度和pH变化耐受性差,在40℃和pH 8.0时有最大溶血活性;25℃和37℃放置12 h CFV溶血活性迅速下降至18.6%和17.4%;对不同动物红细胞敏感性差异较大,其中以家犬红细胞最为敏感.结论 CFV有显著的溶血活性.     

Partial purification and characterization of a novel neurotoxin and three cytolysins from box jellyfish (Carybdea marsupialis) nematocyst venom

This paper describes one neurotoxin and three cytolysins isolated from the venom of the Caribbean box jellyfish Carybdea marsupialis. To assess the cytolytic and neurotoxic activity of the nematocyst venom, several bioassays were carried out, and to evaluate the effect of the toxin, the dose causing 50% lethality (LD₅₀) was determined in vivo using sea crabs (Ocypode quadrata). The proteins with neurotoxic and cytolytic effects were isolated using low-pressure liquid chromatography. The fraction containing the neurotoxic activity was analyzed by SDS-PAGE and showed a single protein band with an apparent molecular weight of 120 kDa (CmNt). To demonstrate the neurotoxic activity of this protein, a small fraction of the purified protein was injected into a crab, and the typical convulsions, paralysis, and death provoked by neurotoxins were observed. Three fractions containing cytolysins had protein bands in SDS PAGE with apparent molecular weights of 220, 139, and 36 kDa, and their cytolytic activity was confirmed with the haemolysis assay.     

Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana.

In this study, we determined hemolysis activity in human and sheep erythrocytes, and characterized the electrical responses in Xenopus oocyte membrane elicited by the venom of the jellyfish Cassiopea xamachana (Cx). The Cx venom produced hemolysis in both species, being more potent on human red cells. The electrophysiological study showed that the Cx venom elicited three different responses in the oocytes. One current was generated in all the oocytes tested and corresponded with a slow inward current (I(Cx)) associated with an increase in membrane conductance. I(Cx) was concentration-dependent and had a reversal potential of -10.3+/-0.4 mV. Ionic substitution studies indicated that the conductive pathway was mainly permeable to cations and non-selective. The oocyte membrane resistance was completely recovered after washout of the venom, this suggested that the effect was due to generation of a specific membrane conductance as opposed to a possible non-specific membrane breakdown. A comparative study with three distinct native cationic channels present in the oocyte membrane [i.e. (1) hemi-gap-junction channels, (2) mechanosensitive channels, and (3) the ouabain-sensitive channel activated by palytoxin], showed that I(Cx) might correspond to opening of mechanosensitive channels or to activation of an unknown cationic channel located in the oocyte membrane. The bioactive fraction eliciting I(Cx) were peptides and was separated from two other peptidic hemolytic fractions by chromatography.     

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.     

霞水母胶原治疗大鼠佐剂型关节炎的研究

目的 探讨霞水母来源的胶原对大鼠佐剂型关节炎的治疗作用.方法 采用完全弗氏佐剂造成大鼠关节炎模型,致炎后大鼠给予霞水母胶原连续灌胃14 d,观察大鼠双后肢关节肿胀情况,及对血清中一氧化氮(NO),超氧化物歧化酶(SOD)、丙二醛(MDA)的影响.结果 口服霞水母胶原可以降低大鼠的双后肢关节肿胀度,降低大鼠血清中NO和MDA含量,提高大鼠血清中SOD活性.结论 霞水母胶原对佐剂型关节炎有较好的治疗作用,其机理可能与降低体内氧自由基水平及过氧化脂质水平有关.     

Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.     

Beachside preparation of jellyfish nematocyst tentacles.

A comparison of methods for preparing a jellyfish nematocyst suspension from sea nettle (Chrysaora quinquecirrha) fishing tentacles at the beachside was conducted. Autolysis of the tentacle followed by straining and sedimentation on ice was found to be a satisfactory technique. This procedure utilized a tea strainer, plastic cup and conical centrifuge tube, all of which could be made available at a minimally equipped laboratory.     

Preparation and Neutralization Efficacy of Novel Jellyfish Antivenoms against Cyanea nozakii Toxins

Jellyfish stings are a common issue globally, particularly in coastal areas in the summer. Victims can suffer pain, itching, swelling, shock, and even death. Usually, hot water, vinegar, or alumen is used to treat the normal symptoms of a jellyfish sting. However, a specific antivenom may be an effective treatment to deal with severe jellyfish stings. Cyanea nozakii often reach a diameter of 60 cm and are responsible for hundreds of thousands of stings per year in coastal Chinese waters. However, there has been no specific C. nozakii antivenom until now, and so the development of this antivenom is very important. Herein, we collected C. nozakii antisera from tentacle extract venom immunized rabbits and purified the immunoglobulin (IgG) fraction antivenom (AntiCnTXs). Subsequently, two complete procedures to produce a refined F(ab’)₂ type of antivenom (F(ab’)₂-AntiCnTXs) and Fab type of antivenom (Fab-AntiCnTXs) by multiple optimizations and purification were established. The neutralization efficacy of these three types of antivenoms was compared and analyzed in vitro and in vivo, and the results showed that all types of antibodies displayed some neutralization effect on the lethality of C. nozakii venom toxins, with the neutralization efficacy as follows: F(ab’)₂-AntiCnTXs ≥ AntiCnTXs > Fab-AntiCnTXs. This study describes the preparation of novel C. nozakii jellyfish antivenom preparations towards the goal of developing a new, effective treatment for jellyfish stings.     

Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom.

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jellyfish (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and purification procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by D-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20mM EDTA buffer; it was optimal in the presence of 10mM Ca2+ or Mg2+. Two chromatographic purification methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42kDa by SDS-PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG.     

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.     

Stabilization of lethal and hemolytic activities of box jellyfish (Chironex fleckeri) venom

The stability of both the lethal and hemolytic activities of box jellyfish (Chironex fleckeri) tentacle extract was assessed after various extraction procedures. Both activities were higher when no buffers or water were used during the initial extraction. Also, when the extract was first filtered through a Sep-pak C18 cartridge, the residual lethal titre, after incubation for 24 hr at room temperature, was increased 16-fold and hemolysis was increased 2.6-fold. Evidence for proteolytic activity in the extract was also obtained and monitored by size exclusion HPLC.     

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。     

Isolation and partial characterization of rhizolysin, a high molecular weight protein with hemolytic activity, from the jellyfish Rhizostoma pulmo

A new cytolysin has been isolated from the nematocysts of the jellyfish, Rhizostoma pulmo, and named rhizolysin. The hemolysin has a mol. wt of approximately 260,000, a sedimentation coefficient of 10.3 S and is rod-shaped with a calculated axial ratio of about 1:5. It appears to be composed of three subunits with a pI value near 7.8. Rhizolysin shows no phospholipase A activity, nor an induction period for its hemolytic activity and is completely inhibited by sucrose. The optimum pH was 6.75. The mu value calculated from the Arrhenius plot is 5940 cal/mole. Rhizolysin was inhibited by cholesterol and less by sphingomyelin.     

The in vivo cardiovascular effects of the Irukandji jellyfish (Carukia barnesi) nematocyst venom and a tentacle extract in rats

Envenoming by Carukia barnesi may produce life-threatening Irukandji syndrome. There is little published on the activity of C. barnesi venom. This is the first study to investigate the in vivo cardiovascular effects of C. barnesi venom and a tentacle extract (devoid of nematocysts). Venom (50 microg/kg or 100 microg/kg, i.v.) produced a pressor response (42+/-3 and 44+/-6 mmHg, respectively; n=4) and increase in heart rate (31+/-5 and 13+/-2 bpm, respectively; n = 4) in anaesthetised rats. These changes were not dose-dependent and were followed by cardiovascular collapse in one of four rats receiving 50 microg/kg and three of four animals receiving 100 microg/kg. Prazosin (50 microg/kg, i.v.) significantly attenuated the venom (50 microg/kg, i.v.)-induced pressor response (-8+/-3 mmHg; P < 0.05; n = 4) and tachycardia (-9+/-4 bpm; P < 0.05; n = 4). Tentacle extract (100 microg/kg; i.v.) produced a pressor response (51+/-12 mmHg; n = 3) and an increase in heart rate (35+/-1 bpm; n = 3) in anaesthetised rats, with no subsequent cardiovascular collapse. The results of this study are consistent with the effects shown by humans envenomed by C. barnesi which are postulated to be a result of catecholamine release. We show, for the first time, that C. barnesi tentacle extract, free of nematocyst material, produces cardiovascular effects which are distinct from those caused by venom derived from isolated nematocysts.     

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.