Venom from Anemesia species of spider modulates high voltage-activated Ca(2+) currents from rat cultured sensory neurones and excitatory post synaptic currents from rat hippocampal slices

The actions of crude venom from Anemesia species of spider were investigated in cultured dorsal root ganglion neurones from neonatal rats and hippocampal slices. Using mass spectrometry (MALDI-TOF MS), 10-12 distinct peptides with masses between about 3 and 10kDa were identified in the crude spider venom. At a concentration of 5 microg/ml crude Anemesia venom transiently enhanced the mean peak whole cell voltage-activated Ca(2+) current in a voltage-dependent manner and potentiated transient increases in intracellular Ca(2+) triggered by 30mM KCI as measured using Fura-2 fluorescence imaging. Additionally, 5-8 microg/ml Anemesia venom increased the amplitude of glutamatergic excitatory postsynaptic currents evoked in hippocampal slices. Omega-Conotoxin GVIA (1 microM) prevented the increase in voltage-activated Ca(2+) currents produced by Anemesia venom. This attenuation occurred when the cone shell toxin was applied before or after the spider venom. Anemesia venom (5 microg/ml) created no significant change in evoked action potentials but produced modest but significant inhibition of voltage-activated K(+) currents. At a concentration of 50 microg/ml Anemesia venom only produced reversible inhibitory effects, decreasing voltage-activated Ca(2+) currents. However, no significant effects on Ca(2+) currents were observed with a concentration of 0.5 microg/ml. The toxin(s) in the venom that enhanced Ca(2+) influx into sensory neurones was heat-sensitive and was made inactive by boiling or repetitive freeze-thawing. Boiled venom (5 microg/ml) produced significant inhibition of voltage-activated Ca(2+) currents and freeze-thawed venom inhibited Ca(2+) transients measured using Fura-2 fluorescence. Our data suggest that crude Anemesia venom contains components, which increased neuronal excitability and neurotransmission, at least in part this was mediated by enhancing Ca(2+) influx through N-type voltage-activated Ca(2+) channels.     

Bradycardia induced by Mesobuthus tamulus scorpion venom involves muscarinic receptor-G-protein-coupled cell signaling pathways

Indian red scorpion (Mesobuthus tamulus; MBT) envenomation produces various cardio-respiratory abnormalities including cardiac dysrhythmias. The underlying cell signaling pathways for the cardiac dysrhythmias produced by MBT venom are not known. The present study was therefore conducted to delineate the second messenger signaling pathways involved in MBT venom-induced atrial rhythm changes. The effects of venom and various antagonists were examined on spontaneously beating rat right atrial preparations in vitro. The MBT-venom produced an increase (35%), a decrease (45%) and again an increase (50%) in rate at 0.03, 0.3 and 3.0 microg/ml of venom, respectively. On the other hand, force of contraction exhibited a concentration-dependent rise (up to 40%) at all concentrations of venom. Pretreatment with atropine (0.3 microM) blocked the decrease in atrial rate at 0.3 microg/ml concentration of venom while no such blockade was seen in force of contraction. Submaximal concentration of ACh (0.1 nM) decreased the atrial rate by 25%. In the presence of MBT venom (0.3 microg/ml), ACh-induced fall in atrial rate was enhanced. The venom-induced fall in atrial rate and augmentation of ACh response were blocked by pertussis toxin (PTx; a Gi-inhibitor) or methylene blue (a G-cyclase inhibitor). The results indicate that the decrease in atrial rate produced by venom is mediated muscarinic by receptors via Gi-guanylyl cyclase mediated cell signaling pathways.     

Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.     

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。     

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.     

Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea

During oviposition, the endoparasitic wasp Pimpla hypochondriaca injects its pupal hosts with venom. This complex fluid has toxic properties and recently several venom components were characterized. In addition, it was suggested that venom might be involved in host immune suppression. For this to be the case, venom would have to adversely affect hemocytes and this aspect was further addressed in the current study utilizing the larval stage of the tomato moth Lacanobia oleracea as a model system. Using sublethal venom injections we investigated the effects of venom on encapsulation and hemocyte concentration. Additionally, the effects of venom on hemocyte morphology, viability, and phagocytic capability were determined in vitro. Injection of 16 microg of venom protein into sixth instar larvae was sufficient to reduce the ability of hemocytes to encapsulate Sephadex A25 beads by more than 50% in four of five insects examined. Hemocyte concentration in sixth instar larvae 32 h after injection with 16 microg of venom was reduced by 56% compared to that in controls. Damaged hemocytes and cell debris were also observed in hemolymph from venom-treated insects, suggesting that P. hypochondriaca venom has cytotoxic properties. In vitro incubation of washed hemocytes for 20 h with 500 ng/microl venom resulted in disintegration of a high proportion of hemocytes, leaving only parts of the plasma membrane and nucleus intact. Treatment with low concentrations of venom (1.6 ng/microl) resulted in an absence of spread plasmatocytes, which were abundant on control monolayers. High-resolution microscopy of hemocyte cultures exposed to 320 ng/microl venom for 3.5 h on glass slides indicated that venom induced a variety of effects on cellular morphology, including blebbing of the plasma membrane, degranulation, and the formation of cytoplasmic vacuoles. Incubation of hemocytes with 320, 64, or 3.2 ng/microl venom for 3.5 h reduced cell viability to 70, 90, and 92%, respectively, confirming that venom is cytotoxic to hemocytes. Treatment with 320 ng/microl venom reduced the capacity of hemocytes to phagocytose Escherichia coli by 85%. Together, these results demonstrate that at sublethal doses venom has a potent anti-hemocyte action and can impair hemocyte-mediated immune responses.     

川硬皮肿腿蜂毒液对寄主黄粉虫蛹的影响

为揭示川硬皮肿腿蜂寄生对寄主黄粉虫蛹的调控机制,本文通过自然寄生和毒液注射等方法研究了寄生过程中蜂毒液的作用.结果表明: 对人工注射的蛹,寄主被麻痹的程度与毒液注入量呈正相关,恢复活动情况与毒液注入量呈负相关;当人工注射毒液浓度为0.01 VRE时,黄粉虫表现出可逆的轻微麻痹;当浓度提高到0.2 VRE时,黄粉虫蛹表现出不可逆的完全僵化.单独注射土壤菌液,导致蛹大量死亡并出现大范围的黑化现象,而注射毒液和土壤菌液混合物,蛹的黑化作用发生延缓,且死亡率显著下降.对毒液的抑菌测试表明,毒液对金黄葡萄球菌的抑菌作用显著高于大肠杆菌.在一定温度范围内,随着温度升高毒液麻痹活性显著降低,对大肠杆菌抑菌性显著升高,对金黄葡萄球菌抑菌性显著降低.表明川硬皮肿腿蜂毒液对寄主黄粉虫蛹具有麻痹、抑菌、抑制蜕皮和延缓黑化的作用.     

Precipitation of animal plasma proteins by puff-adder venom.

1. Plasma and serum samples obtained from various animals never previously exposed to snakes or snake venom were diffused against different concentrations of puff-adder, Bitis arietans, venom using the double immunodiffusion technique. 2. Depending upon venom concentration, two precipitin arcs could be produced in the case of all plasma samples used. No serum samples showed any arcs except pigeon serum, where one precipitin line was observed. 3. By altering the concentration of snake venom between 1% and 10% when immunodiffusing against plasma a change in position of the precipitin lines was observed and also the disappearance of one or both of the two bands at higher concentrations. This indicates that the arcs observed are in all probability due to precipitation of plasma protein fractions. 4. Previous results indicated that one of the two bands observed when diffusing venom against plasma was due to the precipitation of fibrinogen. By diffusing snake venom against heparin we have now shown that the second band involves this molecule and is not due to another coagulation factor as was suggested previously.     

Neutralization of some hematological and hemostatic alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi pauloensis snake venom, by the aqueous extract from Casearia mariquitensis (Flacourtiaceae)

The aqueous extract from the leaves of Casearia mariquitensis (C. m.), a plant found in Brazilian open pastures, was assayed for its ability to inhibit some hematological and hemostatic effects induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi pauloensis. The aqueous extract from C. m. was able to neutralize the hematological alterations induced by the crude venom (C.V.) upon erythrocytes when the venom was incubated at a ratio of 1:10 (w/w, venom/extract), but it did not neutralize the platelet decreasing ability of C.V. The plasma fibrinogen concentration decreased approximately 36% and 83% when 0.6 LD(50) of the C.V. or neuwiedase, respectively, were injected by i.p. route in mice, and the aqueous extract from C. m. was able to inhibit this effect. The Bbeta fibrinogen chain was protected against degradation caused by crude venom and neuwiedase when the venom or toxin were incubated with C. m. extract. We also observed that this extract exerted a very slight effect on the clotting time, prolonging it only to a little extent. The pulmonary hemorrhage induced by neuwiedase when injected intravenously with 0.6 LD(50) was completely inhibited when this toxin was incubated with the extract at a ratio of 1:10 (w/w, toxin/extract). It is concluded that C. m. displays components able to inhibit some hematological and systemic alterations induced by C.V.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.     

普通离子交换剂用于HPLC柱层析法快速分离眼镜王蛇毒

目的　为了经济快速分离眼镜王蛇(Ophiophagushannah,Oh)蛇毒中的毒素成分。　方法　用普通离子交换剂于高效液相色谱柱 (HPLC) TSKgel SP-Toyopearl 65 0 SF (4× 1 5 0 mm)层析法 ,实验取得最佳分离条件后 ,将蛇毒样品上柱后进行梯度洗脱 ,各洗脱峰收集后在 Cosmosil 5 C4-AR-3 0 0柱 (4 .6× 1 5 0 mm)上进行逆相 HPLC分析。非单峰组分再进行 HPLC凝胶过滤柱TSKgel Toyopearl HW-40 Fine(4× 2 5 0 mm)层析 ,层析峰组分再进行 HPLC逆相分析。　结果　眼镜王蛇毒经HPLC离子交换柱层析获得了 1 6个蛋白组分 ,其中有 5个组分经逆相 HPLC分析单一组分 ;另外的复合性组分再进行 HPLC凝胶过滤柱层析后又得到 5个单峰蛋白组分。　结论　HPLC离子交换柱层析对分离蛇毒蛋白很有实用价值 ,特别是蛇毒样品量少的情况下 (1 0 ug)也能较好分离。还具有分离时间短 (1 h左右 ) ,无须低温条件等优点。HPLC凝胶过滤柱层析可进一步使蛋白组分得到提纯     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom ( Apis mellifera ), three snake venoms ( Naja naja sputatrix, Vipera russellii and Crotalus adamanteus ) and the polypeptide melittin was investigated against Escherichia coli . Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom > melittin > N. naja sputatrix venom ≫ V. russellii venom > C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

江浙蝮蛇(Agkistrodon halys Pallas)蛇毒神经生长因子的分离纯化和特性

神经生长因子（ｎｅｒｖｅ ｇｒｏｗ ｔｈ ｆａｃｔｏｒ, Ｎ Ｇ Ｆ）是第一个被发现,也是迄今为止研究得最为清楚的一种神经营养因子 利用 Ｐ Ｃ１２ 细胞生物活力测定为跟踪检测手段,分别经过 Ｃ Ｍ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ ６ Ｂ、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 及 Ｆ Ｐ Ｌ Ｃ ｍ ｏｎｏ Ｓ层析,从３０ ｇ 江浙蝮蛇粗毒中分离纯化到２００ μｇ Ｎ Ｇ Ｆ,纯化倍数高达１０５经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 测定,该蛋白分子量为 ２６ ｋ Ｄ,由两个亚基通过二硫键交联组成二体形式等电聚焦显示其等电点为６７,与氨基酸组成分析结果相吻合 江浙蝮蛇神经生长因子的生物活力水平与小鼠２５ Ｓ Ｎ Ｇ Ｆ相当,在１～１００ μｇ／ Ｌ 的浓度范围内维持 Ｐ Ｃ１２ 细胞在无血清条件下的存活     

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.     

Isolation and Characterization of Biologically Active Venom Protein from Sea Snake Enhydrina schistosa

The present study is designed to investigate the isolation and characterization of biological and biochemical active venom protein from sea snake, Enhydrina schistosa. The highest purification peaks in ion‐exchange chromatography on DEAE‐cellulose column were obtained for fraction numbers 39–49 when eluted with 0.35–0.45 M NaCl. Eighty per cent purity was obtained in the final stage of purification, and a single protein band of about 44 kDa was visualized in SDS‐polyacrylamide gel under reducing condition. Purified venom protein expressed as haemolytic, cytotoxicity and proteolytic activities with lethal concentration (LC₅₀) at 2.0 μg/mL. Venom protein exhibits enzymatic activity and hydrolyzed casein and gelatin. Gelatinolytic activity was optimal at pH 5–9. In conclusion, the present results suggested that the sea snake venom might be feasible sources for biologically active substances. Thus, this low molecular weight component of the venom protein could be used in potentially serve biological and pharmaceutical aspects.     

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity

BackgroundThe plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study.MethodsE. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC–MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico.ResultsThe bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A₂, hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom.ConclusionFor the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity.General significanceExploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.     

Cost of venom regeneration in Parabuthus transvaalicus (Arachnida: Buthidae)

Scorpion venom has many components, but is mainly made up of water, salts, small molecules, peptides, and proteins. One can reasonably assume that the production and storage of this complex secretion is an expensive metabolic investment. However, to date, no study has addressed the costs associated with the regeneration of venom by scorpions. Using a closed-system respirometer, we examined the difference in oxygen consumption between milked and unmilked scorpions to determine the metabolic costs associated with the first 72 h of subsequent venom synthesis. During this time period, milked scorpions had a significantly higher (39%) metabolic rate than unmilked scorpions. The regenerated venom from a second milking had significantly lower (74%) protein concentration, suggesting that venom regeneration was incomplete after 72 h. The protein content in the regenerated venom was not correlated with oxygen consumption. The significant increase in oxygen consumption after milking supports existing hypotheses about the metabolic cost associated with venom regeneration and provides further insight on why scorpions appear to be judicious in their stinger use.     

In vitro analysis of venom from the wasp Nasonia vitripennis: Susceptibility of different cell lines and venom-induced changes in plasma membrane permeability

Summary The lethal effects of crude venom prepared from the ectoparasitic wasp Nasonia vitripennis were examined with cultured cells from six insect and two vertebrate species. Venom caused cells from Sarcophaga peregrina (NIH SaPe4), Drosophila melanogaster (CRL 1963), Trichoplusia ni (TN-368 and BTI-TN-5B1-4), Spodoptera frugiperda (SF-21AE), and Lymantria dispar (IPL-Ldfbc1) to round up, swell, and eventually die. Despite similar sensitivities and overlapping LC₅₀ values [0.0004–0.0015 venom reservoir equivalents (VRE)/μl], profound differences were noted at the onset of cytotoxicity among the six insect cell lines: over 80% of the NIH SaPe4 and SF21AE cells were nonviable within 1 h after addition of an LC₉₉ dose of venom, whereas the other cells required a 5–10-fold longer incubation period to produce mortality approaching 100%. In contrast, cells from the grass frog, Rana pipiens (ICR-2A), and goldfish, Carassius auratus (CAR), showed little sensitivity to the venom: six venom reservoir equivalents were needed to induce 50% mortality in ICR-2A cells [50% lethal concentration (LC₅₀)=0.067 VRE/μl), and 9 VRE did not yield sufficient mortality in CAR cells for us to calculate an LC₅₀. All susceptible cells showed similar responses when incubated with wasp venom: retraction of cytoplasmic extensions (when present), blebbing of the plasma membrane, swelling of the plasma and nuclear membranes, condensation of nuclear material, and eventual cell death attributed to lysis. The rate of swelling and lysis in NIH SaPe4 and BTI-TN-5B1-4 cells exposed to venom appeared to be dependent on the diffusion potential of extracellular solutes (Na⁺=choline>sucrose≥raffinose>K⁺), which is consistent with a colloid-osmotic lysis mechanism of cell death. When T. ni cells were cotreated with venom and the K⁺ channel blocker 4-aminopyridine, cell swelling and lysis increased with increasing drug concentration. In contrast, cells from S. peregrina were protected from the effects of the venom when treated in a similar manner. Addition of certain divalent cations (Zn⁺² and Ca⁺²) to the extracellular media 1 h postvenom incubation rescued both BTI-TN-5B1-4 and NIH SaPe4 cells, suggesting that protection was gained from closure of open pores rather than prevention of pore formation. Venom from N. vitripennis displayed no hemolytic activity toward sheep erythrocytes, supporting the view that venom intoxication is not by a nondiscriminate mechanism. A possible mode of action of the venom is discussed.     

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.