Venoms of several animals have been used to study various physiopathologic processes, and also to offer opportunity to design and develop new therapeutic drugs. We briefly review certain wasp venom components and their biological effects, which may be potential sources of novel pharmacologically active compounds.

DATA SOURCES We report one case and analyzed 15 Japanese cases concerning multiple organ failure induced by wasp stings. DATA EXTRACTION Thirteen of 15 cases were associated with skin hemorrhage or necrosis after wasp stings. The mean number of stings (+/- standard error) in the patients who died (59 +/- 12) was significantly greater than that in those who survived (28 +/- 4, p=0.01). CONCLUSION The occurrence of skin hemorrhage or necrosis after wasp stings is extremely rare and multiple organ injury after wasp stings is also a rare complication. Thus, cutaneous hemorrhaging or necrosis findings after wasp stings may suggest the development of multiple organ injury. In addition, the number of stings may play an important role in predicting outcome.

Cnidarians such as hydrae and sea anemones are sessile, predatory, soft bodied animals which depend on offensive and defensive allomones for prey capture and survival. These allomones are distributed throughout the entire organism both in specialized stinging cells (nematocytes) and in the body tissues. The cnidarian allomonal system is composed of neurotoxins, cytolysins and toxic phospholipapses. The present bioinformatic survey was motivated by the fact that while hydrae are the most studied model cnidarian, little is known about their allomones. A large-scale EST database from Hydra magnipapillata was searched for orthologs of known cnidarian allomones, as well as for allomones found in other venomous organisms. We show that the hydrae express orthologs of cnidarian phospholipase A2 toxins and cytolysins belonging to the actinoporin family, but could not find orthologs of the 'classic' short chain neurotoxins affecting sodium and potassium conductance. Hydrae also express proteins similar to elapid-like phospholipases, CRISP proteins, Prokineticin-like polypeptides and toxic deoxyribonucleases. Our results illustrate a high level of complexity in the hydra allomonal system, suggest that several toxins represent a basal component of all cnidarian allomones, and raise the intriguing possibility that similar proteins may fulfill both endogenous and allomonal roles in cnidaria.

Bees, fire ants and vespids cause insect sting allergy. These insects have unique as well as common venom allergens. Vespids, including hornets, paper wasps and yellow jackets, have common allergens. Bees and vespids have one common allergen with hyaluronidase activity; they also have unique allergens with different phospholipase activities. Fire ants and vespids have one common allergen, antigen 5 of unknown biologic activity. The common venom allergens with < 70% sequence identity have barely detectable levels of antigenic cross-reactivity. Possible uses of modified allergens for immunotherapy are described.

The developmentally arrested third stage infective larva of hookworms resumes development upon entry into the definitive host. This transition to parasitism can be modeled in vitro by stimulating infective larvae with a low molecular weight ultrafiltrate of host serum together with methylated glutathione analogues. When stimulated to resume development in vitro, activated larvae of the hookworm Ancylostoma caninum released a 42-kDa protein, termed Ancylostoma-secreted protein (ASP). ASP was the major protein released by activated hookworm larvae. Degenerate oligonucleotide primers, based on a partial internal amino acid sequence of the protein, were used together with flanking vector sequence primers to amplify a fragment from a third stage larval cDNA library by polymerase chain reaction. The fragment was used as a probe to isolate a longer clone from the larval cDNA library. The full-length ASP cDNA was found to encode a 424-amino acid protein with homology to the antigen 5/antigen 3 family of proteins from hymenopteran venoms and a family of cysteine-rich secretory proteins. ASP was expressed in bacterial cells, and a polyclonal antiserum against purified recombinant ASP was produced. The antiserum, which was demonstrated to be specific for ASP, was used as a probe to measure the kinetics of ASP release by hookworm larvae. ASP is released within 30 min of stimulation, with the majority released by 4 h. Low levels of ASP were released continuously following activation, but only if the stimuli were present in the incubation medium. The compound 4,7-phenanthroline, previously shown to inhibit larval activation, also inhibited release of ASP. The specific, rapid release of ASP by activated infective larvae suggests that this molecule occupies a critical and central role in the transition from the external environment to parasitism.

Antigen 5, also known as Dol m V, is a major allergen found in the venom of the baldfaced hornet, Dolichovespula maculata. We have inserted the f10 and f17 cDNAs, which encode hornet antigen 5 (HA5) forms 2 (Dol m; VB) and 3 (Dol m VA), respectively, into the genome of the baculovirus, AcMNPV, to produce the recombinant baculovirus gene expression vectors, vEV-HA5f10 and vEV-HA5f17. Insect cells infected with either vEV-HA5f10 or vEV-HA5f17 produce and secrete a novel protein with an electrophoretic mobility which is similar if not identical to authentic mature Dol m V. The gene products also react specifically with a polyclonal antiserum raised to Dol m VB as expected. Dol m V gene products were not acutely toxic when injected into insect larvae. However, infection of fifth instar larvae with vEV-HA5f17 resulted in premature melanization of the larvae and lower weight gain than infection with control virus. Thus, the Dol m V gene product has a subtle, possibly cytotoxic or biochemical effect on insects. The expression systems may prove useful in further structural and functional characterization of these proteins.

The major proteins of yellowjacket venoms have been isolated and characterized immuno-chemically. They consist of hyaluronidase, phospholipase, and antigen 5. Venoms from three species of yellowjacket were studied. Vespula germanica, V. maculifrons, and V. vulgaris. The phospholipases could be isolated in good yield only when affinity chromatography was used to minimize limited proteolysis. A kallikrein-like peptidase was found present in the yellowjacket venom. Phospholipases from these three species were immunochemically indistinguishable from each other, as were their antigen 5s. Sera from individuals sensitive to yellowjacket venom contained IgE and IgG specific for antigen 5 and phospholipase.

Three major venom proteins from different species of wasps have been isolated and characterized. They are hyaluronidase, phospholipase, and antigen 5 of as yet unknown biochemical function. These three proteins are allergens in wasp venom-sensitive persons. The species of wasps studied, of the genus Polistes, were annularis, carolina, exclamans, fuscatus, and instabilis. Antigen 5 and phospholipase from wasp venoms were shown to be antigenically distinct from homologous proteins of yellowjacket venoms. The venom phospholipase from wasp, as well as that from yellowjacket (Vespula germanica), appears to have dual enzymatic specificities of the A1 and B types. That is, hydrolysis takes place at the 1-acyl residue of phosphatidylcholine and at the 1- or 2-acyl residue of lysophosphatidylcholine.

We studied the effect of neurotoxin (JSTX) separated from spider venom on the lobster neuromuscular junction. JSTX selectively suppressed excitatory post-synaptic potentials (e.p.s.p.s) without affecting the inhibitory post-synaptic potentials (i.p.s.p.s). The effect of JSTX was dose-dependent. The threshold dose for suppressing e.p.s.p.s corresponded to a small fraction of the toxin amount in a venom gland. At high concentration, JSTX irreversibly blocked e.p.s.p.s. The reduction in amplitude of extracellularly recorded e.p.s.p.s after JSTX application followed an exponential time course. The rate of suppression increased proportionally with the toxin concentration. JSTX blocked the glutamate potential in the post-synaptic membrane but it failed to affect the aspartate-induced depolarization. Kainic acid potentiated the glutamate-induced depolarization but it was without effect in the presence of JSTX. Depolarization produced by quisqualic acid is suppressed by the toxin. Our results suggest that the spider venom contains specific blockers of glutamate receptors in crustacean neuromuscular junctions.

A venom sac extract selectively and irreversibly suppressed excitatory postsynaptic potentials in the lobster neuromuscular junction without affecting inhibitory postsynaptic potentials. Glutamate-induced depolarization of the postsynaptic membrane was blocked by the venom extract, whereas membrane depolarization by aspartate was unaffected.

We studied the effect of neurotoxin (JSTX) separated from spider venom on the lobster neuromuscular junction. JSTX selectively suppressed excitatory post-synaptic potentials (e.p.s.p.s) without affecting the inhibitory post-synaptic potentials (i.p.s.p.s). The effect of JSTX was dose-dependent. The threshold dose for suppressing e.p.s.p.s corresponded to a small fraction of the toxin amount in a venom gland. At high concentration, JSTX irreversibly blocked e.p.s.p.s. The reduction in amplitude of extracellularly recorded e.p.s.p.s after JSTX application followed an exponential time course. The rate of suppression increased proportionally with the toxin concentration. JSTX blocked the glutamate potential in the post-synaptic membrane but it failed to affect the aspartate-induced depolarization. Kainic acid potentiated the glutamate-induced depolarization but it was without effect in the presence of JSTX. Depolarization produced by quisqualic acid is suppressed by the toxin. Our results suggest that the spider venom contains specific blockers of glutamate receptors in crustacean neuromuscular junctions.

1. We studed Na-independent synaptic transmission in the inhibitory synapse of the walking leg of the spiny lobster (Palinurus japonicus). 2. After loading the preparation with tetrodotoxin (TTX), brief depolarizing current injected in the inhibitory axon produced a small action potential, which propagated to the nerve terminal and gave rise to inhibitory post-synaptic potentials (i.p.s.p.) 3. The presynaptic action potential, in the presence of TTX, failed to propagate after removing Na+ in the solution. The TTX-resistant action potential was decreased, but not blocked by 30 mM-CoCl2. 4. When 4-aminopyridine (4-AP) was added to low Na+ or Na-free solution containing TTX synaptic transmission was restored. When the duration of the current pulse was increased, graded i.p.s.p. were evoked. 5. In high Ca2+ solutions containing K blockers, action potentials with prolonged duration were evoked. 6. The action potential of the presynaptic axon of the lobster neuromuscular junction depends on both Na+ and Ca2+.

In the experiment of electrocardiogram, the crude venom of giant hornet (Vespa mandarinia) showed cardioactive effects on rat heart. The heart rate was accelerated within 5 min after injection of the venom intraperitoneally, then the heart beat was blocked, resulting in conduction delay. The cardioactive constituent was separated into two components by gel filtration. One which was high molecular species such as protein showed complete atrioventricular block. Another component, having a low molecular weight, was fractionated in 5 peaks, which accelerated heart rate.

Ischemia followed by reperfusion of skeletal muscle frequently takes place in trauma surgery. Anoxia followed by reoxygenation leads to reperfusion injury, which damages the involved tissues. However, no information is available about how the neuromuscular junction is affected by ischemia-reperfusion. Tourniquet ischemia of the left hind limb was applied in the anesthetized rat for 2 h. Reperfusion lasted for 2 and 24 h and for 1, 2 and 4 weeks. The extensor digitorum longus and the soleus muscles from both legs were prepared for electron-microscopic analyses. Morphological changes of the neuromuscular junction were investigated. In all cases only the nerve endings (terminals) were affected. The postsynaptic structures were not affected. Changes can be grouped in two categories: degeneration and recovery. Degeneration consisted of the loss of synaptic vesicles, disruption of the presynaptic membrane, degeneration of the mitochondria and the development of vacuoles. It was most severe at 24 h and was still present at 4 weeks of reperfusion. Recovery started at 1 week of reperfusion and lasted at least for 4 weeks. It consisted of the slow reappearance of synaptic vesicles and mitochondria, and restoration of the presynaptic membrane with active zones. Ultrastructure of the skeletal muscle fibers did not show pathological changes. The recovery of the structures may be regulated by the Schwann cells and also by the postsynaptic membrane which is not affected by 2 h of ischemia followed by reperfusion.

In the crayfish opener neuromuscular preparation, regional differences in synaptic transmission are observed among the terminals of a single motoneuron. With a single stimulus, the high-output terminals of the proximal region of the muscle produce a larger excitatory postsynaptic potential than do the low-output terminals of the central region of the muscle. We tested the hypothesis that the low-output terminals exhibit more facilitation than do high-output terminals for twin-pulse, train, and continuous-stimulation paradigms. Previous studies have not employed several stimulation paradigms to induce facilitation among high- and low-output terminals of a single motoneuron. We found that the high-output terminals on the proximal fibers facilitate more than the low-output terminals on the central muscle fibers, in contrast with previous studies on similar muscles. The difference in measured facilitation is dependent on the stimulation paradigm. These results are important because ultrastructural differences between these high- and low-output terminals are known and can be used for correlation with physiological measurements. Short-term facilitation is a form of short-term memory at the synaptic level, and the processes understood at the crayfish neuromuscular junction may well be applicable to all chemical synapses.

A venom sac extract selectively and irreversibly suppressed excitatory postsynaptic potentials in the lobster neuromuscular junction without affecting inhibitory postsynaptic potentials. Glutamate-induced depolarization of the postsynaptic membrane was blocked by the venom extract, whereas membrane depolarization by aspartate was unaffected.

The effects of black widow spider venom (BWSV) on the crayfish stretch receptor and the lobster neuromuscular junction were examined. In crayfish stretch receptor neurons, BWSV caused a slight hyperpolarization followed by a large depolarization. The venom-induced depolarization of the stretch receptor was caused by an increase in membrane conductance to Na+ and Ca2+. Black widow spider venom also caused an increase in the frequency of miniature inhibitory postsynaptic potentials recorded in the stretch receptor. The ability of BWSV to increase the frequency of miniature excitatory postsynaptic potentials (MEPSPs) at the lobster neuromuscular junction was dependent on the divalent cation composition of the bathing medium. Ringer solutions containing Ca2+ supported the greatest venom-induced increase in MEPSP frequency, Mg2+ and Mn2+ supported a moderate increase in MEPSP frequency, while Co2+ and Zn2+ blocked this venom effect entirely. Black widow spider venom did not block axonal conduction in lobster walking leg axons or in the axon of the crayfish stretch receptor. The results suggest that in crustaceans, BWSV interacts specifically with membrane of the soma-dendritic region of the stretch receptor and with nerve terminal membrane, causing an increase in Na+ and Ca2+ conductance.

Diamide[diazine-dicarboxylic acid-bis(dimethylamide)], a thiol-oxidizing agent, has both pre- and postsynaptic actions on the glutaminergic neuromuscular junction of the lobster walking leg. Postsynaptically, diamide produced an increase in the response to exogenously applied glutamate, whereas the effect of diamide on presynaptic transmitter release involved two major changes: 1) a decrease in excitatory junction potential amplitude and 2) an increase in miniature junction potential frequency. Short-term facilitation also decreased. Equilibration with 1,4-dithiothreitol (a sulfhydryl-reducing agent) reversed the decline in excitatory junction potential frequency, and the fall in short-term facilitation. The miniature junction potential frequency increase induced by diamide was independent of external Ca2+, as diamide in a Ca2+-free solution produced a similar response to that in a Ca2+-containing solution. We propose that the action of diamide on transmitter release is similar to the action of polyvalent cations, i.e., diamide has two sites of action, a blockade of inward Ca2+ flux and an increased release of Ca2+ inside the terminal.

Responses to bath-applications of 4-aminopyridine(4-AP) and gamma-aminobutyric acid (GABA) were recorded intracellularly from neurones in the rat isolated superior cervical ganglion. 4-aminopyridine (0.1-1.0 mmol/l) usually induced spontaneous action potentials and excitatory postsynaptic potentials (EPSPs), which were blocked by hexamethonium. Membrane potential was unchanged; spike duration was slightly increased. Vagus nerve B- and C-fibre potentials were prolonged. In 4-AP solution (0.2-0.3 mmol/l), GABA (0.1 mmol/l), 3-aminopropanesulphonic acid or muscimol evoked "bursts" of spikes and EPSPs in addition to a neuronal depolarization. These "bursts", which were not elicited by glycine, glutamate, taurine or (+/-)-baclofen, were completely antagonised by hexamethonium, tetrodotoxin or bicuculline methochloride. It is concluded that:(a) 4-AP has a potent presynaptic action on sympathetic ganglia;(b) presynaptic actions of GABA can be recorded postsynaptically in the presence of 4-AP; and (c) the presynaptic GABA-receptors revealed in this condition are similar to those on the postsynaptic membrane.

1. We studed Na-independent synaptic transmission in the inhibitory synapse of the walking leg of the spiny lobster (Palinurus japonicus). 2. After loading the preparation with tetrodotoxin (TTX), brief depolarizing current injected in the inhibitory axon produced a small action potential, which propagated to the nerve terminal and gave rise to inhibitory post-synaptic potentials (i.p.s.p.) 3. The presynaptic action potential, in the presence of TTX, failed to propagate after removing Na+ in the solution. The TTX-resistant action potential was decreased, but not blocked by 30 mM-CoCl2. 4. When 4-aminopyridine (4-AP) was added to low Na+ or Na-free solution containing TTX synaptic transmission was restored. When the duration of the current pulse was increased, graded i.p.s.p. were evoked. 5. In high Ca2+ solutions containing K blockers, action potentials with prolonged duration were evoked. 6. The action potential of the presynaptic axon of the lobster neuromuscular junction depends on both Na+ and Ca2+.

Uptake of horseradish peroxidase was studied by examining percentages of labelled synaptic vesicles in nerve endings of the excitatory axon innervating the opener muscle of the walking leg in the crayfish (Procambarus clarkii). Terminals on fibres with large excitatory postsynaptic potentials (EPSP) had higher percentage of labelled vesicles than terminals of fibres with small EJPs. The extent of labelling in the synaptic vesicle pool was greater for terminals with higher transmitter output. Evidence for three possible routes of synaptic vesicle formation was found. Movement of vesicles within the terminal as a whole appeared to be constrained, but rapid movement of vesicles within local populations probably occurs.

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.

Intracellular recordings of the membrane potential and evoked excitatory junction potentials were made from the opener muscle in the walking leg of the Hermit crab ( Eupagurus bernhardus ). A variety of amino acid analogues including cis and trans 1-amino-1,3- dicarboxycyclopentane were tested for agonist activity and their potencies were compared with L-glutamate. Quisqualic acid was the most powerful excitant whereas N-methyl-DL-aspartic acid and ibotenic acid were the least active. Threshold concentrations of L-glutamate and quisqualic acid potentiated the ionophoretic L-glutamate potential and the excitatory junction potentials without affecting membrane input resistance. The results are discussed in terms of the structure-activity relationship of the crustacean excitatory receptor compared to other vertebrate and invertebrate nervous systems.