Complete amino-acid sequence of the beta-subunit of VTX from venom of the stonefish (Synanceia verrucosa) as identified from cDNA cloning experiments.

A cDNA encoding a subunit of the verrucotoxin (VTX) has been identified from a cDNA library derived from stonefish venom glands. It encodes a polypeptide of 708 amino-acid residues, followed by a 3'-untranslated region of 895 bp long. The ORF contains the complete mature sequence of the beta-subunit of the VTX, as inferred from both the presence of an identical N-terminus sequence and 96% homology among the 506 amino terminus residues found in the partial sequence of the beta-subunit of the stonustoxin from Synanceia horrida (Ghadessy, F.J., Jeyaseelan, K., Chung, M.C.M., Khoo, H.E., and Yuen, R. (1994) Toxicon 32, 1684-1688). Upstream the mature sequence, we noticed the presence of an incomplete peptide of a 13 amino acids, whose unusual primary structure supports the idea of the existence of a propeptide and/or of a new secretion signal.

Origin and function of supporting cells in the venom glands of the lesser weeverfish (Trachinus vipera).

Supporting cells in the venom glands of Trachinus vipera form a network whose meshes are filled with developed glandular cells. These supporting cells are derived from epidermal cells. A connective tissue sheath forms inside the gland, separating it into pockets. A basal lamina surrounding the pockets between the connective tissue and supporting cells can be observed similar to that between connective tissue and epidermis. The pockets are probably formed by epidermal invagination. In view of the ultrastructural observations, the supporting cells not only maintain the cohesiveness of the venom gland, as demonstrated by the presence of numerous interdigitations and desmosomes, but may also play a role in the regeneration of large glandular cells.

Purification of a lethal fraction from the venom of the weever fish, Trachinus vipera C.V.

We report the purification of a lethal fraction of Weever fish venom using preparative electrophoresis. It was found to be composed of four identical subunits with an overall mol. wt of approximately 324,000. A 20 g mouse was killed instantly by an i.v. injection of 2 micrograms.

Cardiotoxicity of verrucotoxin, a protein isolated from the venom of Synanceia verrucosa.

Freshly purified but unstable verrucotoxin (VTX) and a more stable proteic complex of the toxin (p-VTX) were isolated from the venom of the stonefish Synanceia verrucosa and applied to frog atrial fibres. VTX and p-VTX decreased the amplitude and the duration of the stimulated peak tension and accelerated the relaxation phase of the contraction. The negative inotropic effect of p-VTX decreased with increasing the external Ca concentration ([Ca]o) in the Ringer solution. The negative chronotropic effect induced by p-VTX was insensitive to change in [Ca]o. It is reversed by glibenclamide. p-VTX shortened the duration of the plateau and the repolarizing phase of the action potential. Glibenclamide but not tetraethylammonium reversed the p-VTX-induced shortening of the AP repolarizing phase. The data suggest that the toxin isolated from the venom of S. verrucosa inhibits Ca channels and might activate ATP-sensitive potassium channels in frog atrial heart muscle.

Does crude venom of the stonefish (Synanceia verrucosa) activate beta-adrenoceptors in the frog heart muscle?

Venom isolated from the stonefish Synanceia verrucosa was assayed in concentrations of 0.07 and 5.7 micrograms/ml on frog atrial fibres and myocytes. Venom, less than 2.9 micrograms/ml, dose-dependently increased the amplitude and the duration of the stimulated peak tension, lengthened the time constant of the relaxation phase and shortened the duration of the action potential (AP). The concentration of venom 5.7 micrograms/ml decreased the amplitude of the peak tension, induced a contracture, reduced the amplitude of the plateau and shortened its duration as well as the repolarizing phase of the AP. The positive inotropic effect induced by the venom (2.9 micrograms/ml) on the contraction was inhibited dose-dependently by propranolol but was unchanged by the alpha-adrenergic antagonists urapidil and yohimbine, the adenyl cyclase activity remaining sensitive to forskolin. Venom, adrenalin and propranolol competed for a common site. Venom (2.9 micrograms/ml) increased both the Ca and the delayed outward K currents of enzymatically isolated atrial myocytes. The data suggest that the venom activates adrenoceptors, essentially beta-adrenoceptors.

Enzymatic properties of the stonefish (Synanceia verrucosa Bloch and Schneider, 1801) venom and purification of a lethal, hypotensive and cytolytic factor.

The crude venom of the stonefish (Synanceia verrucosa) possesses numerous enzymatic properties (hyaluronidase, eight esterases and ten aminopeptidases). Verrucotoxin was isolated by DEAE and hydroxyapatite chromatography, followed by FPLC gel filtration on Superdex 200 HR 10/30. It was found to be a glycoprotein with a mol. wt of 322,000 +/- 2000, comprising four subunits, 2 alpha (83,000) and 2 beta (78,000). Verrucotoxin, as the crude venom, is lethal for mice, haemolyses washed rabbit erythrocytes and induces a fall in arterial pressure in anaesthetized rats.

Enhancement of oocyte growth in the cockroach Blaberus craniifer by a scorpion toxin, charybdotoxin.

We investigated the ovarian response of Blaberus craniifer to charybdotoxin in both imaginal molt headless females and isolated fed females, at three criteria. 1--Vitellogenesis onset, detected by immunocytochemical localisation of sites binding anti-eggs antibodies in the basal oocyte in headless females: 60% of treated females present a positive response. 2--Oocyte length at D4 during vitellogenesis of isolated fed females which was enhanced by 0.1 - 0.2 microg toxin; doses higher than 0.5 microg/female decrease ovarian growth. 3--Time of oviposition of paired females which was shortened by 24 hrs by the toxin. These results suggest that low doses charybdotoxin enhance vitellogenesis, possibly via the nervous system by means of a substance conditioning both protein synthesis by the fat body and ovarian uptake.

Microtopography of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae): free-living larval stages.

Microtopographic features of the various growth stages of the three free-living larval stages of the rat hookworm Nippostrongylus brasiliensis (Nematoda) were surveyed by scanning electron microscopy. These worms have a rounded anterior end and an elongated tail. Cuticular annulations were observed along the body, which also bore two ribbon-like lateral alae. Two rings of six lip-like lappets were observed around the triradiate oral opening in all larval stages. The cephalic space contained two lateral amphidial pits. The excretory pore in the third anterior part was observed in a ventral view of the larvae. No deirids were observed. The anus with a crescent-shape opening was located posteriorly. Phasmidial apertures, only observed in the third-stage larvae, opened on the lateral alae in the tail region.

Microtopography of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae): parasitic larval stages and adults.

Specimens of the rat hookworm, Nippostrongylus brasiliensis (Nematoda) were recovered from lungs (third- and fourth-stage larvae) and intestine (fourth-stage larvae and adults). The following features were studied in the different stages by scanning electron microscopy: cephalic structures, especially sense organs, synlophe, cervical region, and caudal part. The main differences between the third and fourth stages concerned the lip-like structures around the oral aperture, the appearance of the cephalic space with the presence of a cephalic cap in fourth-stage larvae, the pattern of longitudinal ridges, and sexual differentiation. Pore-like papillae, not seen in third-stage larvae, developed in later stages. Deirids were observed only in adults, and phasmids were poorly discerned. Some of these morphological features, such as the cephalic sense organ apertures and cuticle pores and micropores, can be observed only by scanning electron microscopy. The possible functions of these different structures and their relationship with the behavior of the worms during their life cycle are discussed.

[Pharmacological properties of fish venoms]. / Propriétés pharmacologiques des venins de poissons.

Fish venoms can be lethal for Vertebrates. The effect depends of dose and subject, more than incriminated fish. The most constant symptom is a violent pain; but the serious pharmacological effects are respiratory and heart failure with marked hypotension and cardiac perturbations, neurologic damage, such as seizure and coma. Experimentation is difficult due to venom instability. Activity is lost by distilled water, lyophilisation in buffers, several successive freezing and defreezing. In addition, when venom is broken, other pharmacological effects are evidenced, for instance, with Synanceia verrucosa venom, hypertensive phase takes the place of hypotension. It is difficult to distinguish toxin effect from this of denaturation products of the toxin. Noradrenaline is present in Synanceia venom, and it seems that acetylcholine exists in some venom, at least when diluted in saline solution. Other biological active products are present. Purified toxins allow pharmacological investigations. Stonefish venom is better studied, because venomous glands contain relatively high venom quantity. Stonustoxin from Synanceia horrida exerts its action through NO-synthase liberation, and its primary action can be attributed to its potent vasorelaxant activity, causing a rapid, marked and irreversible hypotension. Trachynilysin, from Synanceia trachynis, causes massive release and depletion of acetylcholine and damage to nerve and muscle fibres, which can account for the inhibition of neuromuscular function, and skeletal paralysis. But the used doses are not compatible with respiratory arrest. Verrucotoxin from Synanceia verrucosa activates potassium channels dependent from ATP; this can explain damage, and probably neurologic and respiratory distress.

Proctolin promotes vitellogenesis onset in the imaginal molt decapitated cockroach Blaberus craniifer.

The ovarian response of the imaginal molt decapitated cockroach, Blaberus craniifer, to nanomolar quantities of synthetic proctolin was evaluated. Sites binding anti-egg proteins were detected in the basal oocyte in 67% (18 out of 27) of the treated females, indicating that vitellogenin uptake is mediated by proctolin. The injected concentration of proctolin was consistent with a physiological role. The results suggest that proctolin, a putative neurotransmitter or neuromodulator, possibly acts on a non-excitable cell, the oocyte. At this level, the precise mechanism is discussed with respect to the calcium-dependent processes involved in both the proctolin myogenic effect and vitellogenin uptake in other species of cockroach.

Indolamines in the cockroach Blaberus craniifer Burm. nervous system--I. Fed and crowded young females.

1. Indolamine levels were determined in the cerebral ganglion, the thoraco-abdominal nerve cord (except the last ganglion), and the 6th abdominal ganglion of females of Blaberus craniifer. 2. Measurements were made at the imaginal molt and on fed and crowded imagos at 10, 20 and 30 post-imaginal days. 3. Indolamines were found in the nervous system of young females, but 5-hydroxytryptophan was only detected in the thoraco-abdominal nerve cord. 4. Amine levels were related to the age of the cockroach, particularly during this period, to post-ecdysis events and ootheca formation.

Biogenic amine levels in the cockroach Blaberus craniifer Burm. nervous system.

1. Apart from octopamine, the same indolamines and catecholamines were detected in the whole nervous system of the cockroach Blaberus craniifer Burm., at the same time. 2. However, levels were found to depend on sex, age, and the anatomic region within the nervous system. 3. Although not established, it is thought that these substances act synchronously from the ganglia. 4. The differences in levels between males and females and between anatomical regions during imaginal life suggest, in this species of cockroach, the physiological importance of the metameric organization in metabolic pathways or functional aspects of biogenic amines.

Indolamines in the cockroach Blaberus craniifer Burm. nervous system--II. Fed and crowded young males in comparison with females.

1. Indolamines were assayed by HPLC-ECD in nervous tissue of fed and crowded young males Blaberus craniifer Burm. 2. In males, as in females housed in the same conditions, levels are depending on both age and region (= ganglia) of the central nervous system. 3. Registered sex differences are discussed in terms of anatomical, physiological and behavioral sexual dimorphism.

Indolamines and onset of vitellogenesis in the imaginal molt-decapitated cockroach Blaberus craniifer Burm.

1. The effects of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and N-acetyl-5-hydroxytryptamine on oocytes of Blaberus craniifer, in which vitellogenesis was prevented by imaginal molt decapitation, were investigated. 2. Sites binding anti-egg-protein antibodies were detected in the periphery of basal oocytes of treated females, with individual variability. 3. In this ovoviviparous cockroach, the onset of vitellogenesis may thus not be triggered solely by juvenile hormone, and indolamines may play a role in the uptake of haemolymphatic proteins by oocytes.

Effects of ecdysteroids on reproductive physiology of Nippostrongylus brasiliensis (Nematoda) in vivo.

1. The influence of ecdysteroids (ecdysone and ecdysterone) was investigated on the control of reproduction in the parasitic nematode Nippostrongylus brasiliensis in vivo. 2. Infestive larvae (L3) were immersed in solutions of ecdysteroids (2.2 microM) at 37 degrees C for 4 hr before injection into the host. The effect on egg-laying was observed two stages later. 3. The treatment increased egg-laying, but had no influence on the timing of the reproductive period. The greatest effect was observed with ecdysone, ecdysterone only inducing a small and non-significant stimulation under our experimental conditions. 4. The physiological role of ecdysteroids in meiosis and gonadal development in nematodes is discussed.

Decapitation or starvation raise haemolymph ecdysteroid titres in the ovoviviparous female cockroach, Blaberus craniifer Burm.

1. Decapitating newly emerged Blaberus craniifer females near the prothorax severs connections between the suboesophageal and prothoracic ganglia, thus depriving them of the neuroendocrine cephalic complex (including brain and suboesophageal ganglion) and the anterior end of prothoracic glands (PGs). 2. As demonstrated by enzyme immunoassay (EIA), headless females have higher levels of ecdysteroids (ECDs) in haemolymph than starved or fed females, indicating that the neuroendocrine cephalic complex influences circulating ECD levels. 3. The time course of hormonal peaks in decapitated females resembles that in starved females during the first post-ecdysial week, suggesting that some as yet unknown regulating mechanism of ECD production lies outside the head. 4. It is suggested that: (a) The PGs are sites for ECDs production in the early post-imaginal period, (b) the prothoracic and suboesophageal ganglia (linked by nerves to PGs) regulate PGs activity, possibly via neural inputs.

Presence of norepinephrine and other biogenic amines in stonefish venom.

Although fish venoms exert a cardiovascular effect, the presence of adrenergic substances was not previously demonstrated. Chromatographic analysis with electrochemical detection showed the presence of substances co-migrating with norepinephrine, dopamine and tryptophan. Serotonin, which was thought to be implicated in the intense pain following fish envenomation, was not detected. Norepinephrine was identified as a component of the stonefish Synanceia verrucosa venom by gas chromatography-mass spectrometry.

Biogenic amines in newly-ecdysed cockroaches.

1. Simultaneous quantification (HPLC and electrochemical detection) of biological extracts have shown dopamine, N-acetyl dopamine, tryptophan, 5-hydroxytryptamine, a 5-hydroxyindolacetic acid-like substance in nervous tissue and hemolymph of Blaberus craniifer and Periplaneta americana. 2. 5-Hydroxytryptophan was only detected in head and thoraco-abdominal nerve cord. 3. Octopamine, but not N-acetyl-5-HT was quantified in the hemolymph.