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Hydrolysis of substance P and bradykinin by black widow spider venom gland extract.

Black widow spider venom gland extract was found to contain significant peptidase activity. Aliquots of the venom gland extract incubated at 37 degrees inactivated substance P (SP) and bradykinin but not angiotensin II or the enkephalins. The peptide inactivation was proportional to the duration of the incubation and the amount of extract used. Analysis of the peptides on high pressure liquid chromatography demonstrated that the loss in biological activity of SP and bradykinin in the longitudinal muscle of the guinea pig ileum was correlated with cleavage of the peptides into several fragments. Kinetic studies revealed that SP was initially split into two fragments but that these products underwent further degradation into smaller peptides. The optimal pH for the peptidase activity was 6.5. At 0 degree the enzymatic activity was undetectable, and it was irreversibly destroyed by incubation at 100 degrees for 5 min or by pretreatment of the extract with 100 microM diisopropyl fluorophosphate. In addition, the gland extract preparation hydrolyzed artificial substrates designed to detect trypsin or chymotrypsin-like activity.

Animals↗

Isolation and characterisation of glutamate receptor antagonists from venoms of orb-web spiders.

Two distinct families of low-molecular-weight toxins (argiotoxins) have been isolated from the venom of the orb-web spider. Argiope trifasciata. The toxins have been purified to homogeneity and characterised by spectroscopic, mass spectrometric and microchemical analysis. The major biologically active member of the first family of toxins is 2,4-dihydroxyphenylacetyl-asparagine linked through a C11-tetra-amine to N-terminal arginine; other members of this family are methylene homologues. The second family of toxins possesses an indolic group in place of the 2,4-dihydroxyphenyl chromophore. The toxins act as non-competitive inhibitors at quisqualate-type glutamatergic receptors on a metathoracic retractor unguis nerve-muscle preparation of Schistocerca gregaria. The loss of the N-terminal arginine reduces biological activity of the first family of toxins, but not of the second. The nature of the polyamine appears to be less important, perhaps acting as a spacer between the cationic arginine and the more hydrophobic aromatic tail of the toxins.

Animals↗

Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog motor nerve terminals with FM1-43.

The neurotoxin Black Widow Spider Venom (BWSV) triggers massive release of neurotransmitter at synapses. Here we demonstrate that the action of BWSV on the frog neuromuscular junction can be visualized in vivo by the use of the fluorescent styryl dye FM1-43. This vital dye stains recycled synaptic vesicles upon nerve stimulation. Motor nerve terminals were stained with FM1-43 via electrical stimulation, washed and then exposed to BWSV or alpha-Latrotoxin. All terminals destained completely, independent of external calcium. Exposure of frog nerve terminals to BWSV in the presence of FM1-43 and calcium led to staining of terminals. The staining pattern appeared to be exactly the same as in control preparations, stimulated electrically via the nerve. When the same experiment was performed in the absence of calcium, only a minute quantity of dye was taken up into the nerve terminals, and the synapses looked swollen and puffed. Addition of external calcium to these preparations elicited an immediate shrinking of the nerve terminals, indicating endocytosis. These observations support electron-microscopic data that suggest an important role for extracellular calcium in endocytosis of BWSV poisoned nerve terminals.

Animals↗

Latrodectism: variations in clinical manifestations provoked by Latrodectus species of spiders.

The clinical picture caused by the bite of Latrodectus spp. is described, including the patient's general condition with agonizing pains, cramps, spasms, motor unrest, boardlike abdomen, "facies latrodectismica", sweating, oliguria, hypertension, anxiety, mental excitation and an extended convalescence. The notorious biters are mainly L. mactans, L. variolus, L. hesperus, L. tredecimguttatus, L. hasselti, L. indistinctus and L. menavodi, all of which appear to provoke somewhat similar clinical pictures. For some species, however, there is limited data and for the toxicity of some spiders, such as L. rhodesiensis, no data are available, However, it would appear that the clinical manifestations of the venom are similar in all species.

Animals↗

Chemical characterization of acylpolyamine toxins from venom of a trap-door spider and two tarantulas.

Two acylpolyamines are identified from venom of the trap-door spider, Hebestatis theveniti. These toxins (paralytic to lepidopteran insect larvae) are amides containing 3-(3-indolyl)lactic acid joined to spermine or 1,13-diamino-4,10-diazatridecane (Het389 and Het403, respectively). Het389 is also abundant in venom from a tarantual from Mozambique (Harpactirella sp.). Two additional acylpolyamines (Apc600 and Apc728) are partially characterized from venom of another tarantula, Aphonopelma chalcodes.

Animals↗

Hyperbaric oxygen effects on brown recluse spider (Loxosceles reclusa) envenomation in rabbits.

Human loxoscelism was modeled in albino rabbits by injection of brown recluse spider (Loxosceles reclusa) venom, and the effects of daily or twice-daily hyperbaric oxygen treatment on wound healing were investigated. Lesions similar to those seen in humans were produced in rabbits by intradermal injection of 200 microliters of a venom extract (0.21 microgram protein per microliter), including edema and erythema, ischemia and cyanosis in the first 12 hr, extensive purpura by 24 hr, and crateriform ulcer formation by day four, with induration and eschar formation. Hyperbaric oxygen treatments, consisting of two atmospheres absolute (2 ATA) for 60 min, were applied daily (n = 8) or twice daily (n = 8), while control animals (n = 8) received no treatment. Treatments were initiated 72 hr after venom injection (day 3) to duplicate typical clinical treatment delays, and were administered for seven consecutive days. No significant effects of hyperbaric oxygen treatment on lesion healing were seen as measured by lesion area. However, histologic evaluation of wound tissue collected at euthanasia on day 24 showed clear differences between rabbits receiving twice-daily treatments and those receiving daily or no treatment. The former showed complete re-epithelization or slight ulceration, while the latter usually had necrotic cavities extending into the dermis, with myonecrosis and inflammatory cell accumulation. Thus, no superficial differences were seen between groups, but twice-daily treatments resulted in enhanced recovery at the histologic level.

Animals↗

Isolation of neurotoxic peptides from the venom of the 'armed' spider Phoneutria nigriventer.

Three neurotoxic fractions, lethal to mice, were isolated from the venom of the spider Phoneutria nigriventer, by gel filtration and reverse phase chromatography (Phoneutria toxins 1, 2 and 3). These toxins have mol. wts in the range 6000-9000, and have different amino acid compositions and N-terminal amino acid sequences. The toxins also differ in the lethality and signs they cause in mice after intracerebro-ventricular injection. The median LD50 being respectively for the whole venom, toxins 1, 2 and 3, 47 +/- 5 micrograms, 45 +/- 4 micrograms, 1.7 +/- 0.7 micrograms and 137 +/- 10 micrograms/kg mouse. Toxins 1 and 2 induce excitatory symptoms in mice and toxin 3 a flaccid paralysis with an ED50 of 40 +/- 5 micrograms/kg mouse as measured also by intracerebro-ventricular injection. The presence in the venom of a non-neurotoxic, smooth muscle active peptide is also described.

Amino Acid Sequence↗

Neurotoxic envenoming by an immigrant spider (Steatoda nobilis) in southern England.

A young woman was bitten on the shoulder by a female Steatoda nobilis spider, in Worthing on the south coast of England. She noticed intense radiating pain, local sweating (indicating parasympathetic stimulation) and feverishness. This immigrant species from the Canary Islands has established itself along the south coast of England in recent years. Like the related Mediterranean species S. paykulliana, S. nobilis may be of medical importance and deserves further study.

Adult↗

Effects of black widow spider venom and latrocrustatoxin on crustacean nerve cells: electrophysiological and ultrastructural study.

1. Black widow spider venom (BWSV), or crustacean-specific toxin purified from it (named latrocrustatoxin, LCT) were lethal for the crayfish. 2. Distortions in the EMG and motor activity pattern after LCT injection indicated venom effects on the crayfish nervous system. 3. In the isolated ventral nerve cord, transient activation of the "silent" interneurons and motoneurons occurred after BWSV or LCT application. 4. In the isolated nerve-muscle preparation, LCT application elicited an intense transmitter release resulting in the complete blockade of neuromuscular transmission. 5. Electron microscopic study of the nervous and muscle tissue in venom treated crayfish demonstrated certain destructive changes in the nervous cells especially in the nerve terminals. 6. The results suggest that the LCT acts as a presynaptic neurotoxin in the central and peripheral nervous system of the crayfish.

Action Potentials↗

Selective presynaptic insectotoxin (alpha-latroinsectotoxin) isolated from black widow spider venom.

A homogenous protein of 120,000 mol. wt isolated from black widow spider (Lactrodectus mactans tredecimguttatus) venom and referred to as alpha-latroinsectotoxin was highly potent (4 nM) in the induction of an increase of the frequency of miniature excitatory postsynaptic potentials in blowfly (Calliphora vicina) larvae neuromuscular preparations. In the frog nerve ending, however, even 50 nM alpha-latroinsectotoxin failed to affect transmitter release. Pretreatment of insect preparations with alpha-latrotoxin or frog preparations with alpha-latroinsectotoxin did not prevent the specific effect of consequent applications of alpha-latroinsectotoxin (insect) and alpha-latrotoxin (frog), respectively. The binding of labelled [125I]alpha-latroinsectotoxin to insect and [125I]alpha-latrotoxin to bovine membrane preparations was saturable and highly specific. The presynaptic effect, but not the binding of alpha-latroinsectotoxin, was dependent on the presence of divalent cations in the external medium. Mg2+ could readily substitute for Ca2+ and increase of transmitter release induced by alpha-latroinsectotoxin also occurred in Ca(2+)-free solutions. Pretreatment of preparations with 300 micrograms/ml concanavalin A completely abolished both the presynaptic effect of alpha-latroinsectotoxin and its binding to insect membrane preparations. Thus, the phenomenology of alpha-latroinsectotoxin action on insects resembles in general that described for the action of alpha-latrotoxin on vertebrates. The selectivity of alpha-latrotoxin and alpha-latroinsectotoxin seems to be due to differences in the structure of neurotoxin receptors in nerve endings of vertebrates and insects, although the mode of presynaptic action has a great deal in common.

Animals↗

Isolation and characterization of a protein neurotoxin from the venom glands of the funnel-web spider (Atrax robustus).

1. Ground venom glands from male and female funnel-web spiders (Atrax robustus) were extracted with acetic acid (0.35 M). 2. A protein constituent of these extracts, atraxin, which caused muscle fasciculation when applied to the phrenic nerve-hemidiaphragm of the mouse in vitro, was isolated by the sequential application of ultrafiltration, gel permeation and ion exchange chromatography. 3. When subjected to isoelectricfocusing, amino acid analysis, ultracentrifugation, ultraviolet and 1H nuclear magnetic resonance spectroscopy, it was shown that atraxin had an approximate molecular weight of 9800, an isoelectric point of greater than 9 and that it contained 76 amino acid residues.

Amino Acids↗

A comparative study of properties and toxic constituents of funnel web spider (Atrax) venoms.

The crude venoms of male and female Sydney funnel web spiders, Atrax robustus, were compared by cation-exchange and high-performance liquid chromatography, lethality to new-born mice, polyacrylamide gel isoelectric focusing, immunoelectrophoresis, phospholipase A analysis, effects on the mouse phrenic nerve hemidiaphragm and passive paw oedema in the rat and, except in the case of rat paw oedema, were found to exhibit quite different properties. Polyacrylamide gel isoelectric focusing and high-performance liquid chromatography proved to be suitable for gender and species determination when applied to the venoms of A. formidabilis, A. infensus, A. robustus and A. versutus. These venoms were also compared by lethality, promotion of muscle fasciculation and phospholipase A activity.

Animals↗

The involvement of collagenase in the necrosis induced by the bites of some spiders.

1. The midgut extracts of 13 Australian spider species produced cellular disruption in mouse skin in tissue culture conditions. 2. Microbial collagenase and the venoms of some of these species had similar effects. 3. Five venoms also caused severe dermonecrosis in living mice. 4. Pre-mixing the venoms with L-cysteine caused complete in vivo and partial in vitro inhibition of their effects. 5. It was concluded that collagenase is a major factor in the aetiology of necrotic arachnidism.

Animals↗

Characterization and cloning of insecticidal peptides from the primitive weaving spider Diguetia canities.

Three potent insecticidal peptide toxins were purified from the venom of the primitive weaving spider, Diguetia canities. The toxins share significant homology (> 40%) in their amino acid sequences and are of related size (masses of 6371-7080 Da). In lepidopteran larvae, the toxins cause a progressive spastic paralysis, with 50% paralytic doses (PD50S) ranging from 0.38 to 3.18 nmol/g, suggesting them to be among the most potent insecticidal compounds yet described from arthropod venoms. The most potent of these toxins, DTX9.2, was cloned using a reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encodes a 94 amino acid precursor which is processed to the active 56 amino acid peptide by removal of a signal and propeptide sequence. The gene encoding DTX9.2 was isolated and characterized. The transcriptional unit spans 5.5 kilobases and is segregated into five exons. DNA sequences upstream from the first exon contain a TATA box and two palindromic sequences (one with homology to a CAAT consensus) which together may constitute a functional promoter. The highly segmented gene structure observed for this small peptide suggests that a mechanism such as exon shuffling may have played a role in the evolution of this toxin family.

Amino Acid Sequence↗

Management of spider bites.

A request to transport a patient with an arachnid envenomation may not be one of the most common calls the transport team receives, yet an understanding of how to care for such patients can decrease complications and prevent morbidity and mortality. This case outlines a systemic reaction from a black widow spider bite and discusses its subsequent management from initial assessment in the referring emergency department (ED) to discharge.

Animals↗

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes.

The vitamin B1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(beta-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl2 and KCl solutions via ionic channels produced by latrotoxins (alpha-latrotoxin (alpha-LT) and alpha-latroinsectotoxin (alpha-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl2 inhibited transmembrane current by 31.6+/-3% and by 61.8+/-3% from the trans-side of BLM for alpha-LT channels. Application of DMHT in the solution of 10 mM CaCl2 to the cis-side of BLM decreased the current through the alpha-LIT and RTX channels by 52+/-4% and 50+/-5%, respectively. Addition of Cd2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca2+-current across the alpha-LT channels caused its further decrease by 85+/-5% that coincides favorably with the intensity of Cd2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on alpha-LT channels without considerable influence on the ionogenic groups of Ca2+-selective site inside the channel cavity. The binding kinetics of DMHT with the alpha-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of alpha-LT channel on either side of the BLM. The comparative analysis of effective radii measured for alpha-LT, alpha-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for alpha-LT channel on the trans-side (0.28+/-0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.

Animals↗