Scorpion stings represent a medical problem in numerous countries. The scorpion Androctonus australis hector produces three alpha toxins (Aah I to III), which are responsible for most of the lethality in mammals. These toxins act on sodium channel and do not cross-react immunologically. We used RIA and ELISA to measure the concentrations of these three toxins in plasma, urine and different organs after i.v. and s.c. injections of water extracts of venoms in rabbits or mice. In both animals, the toxins rapidly appeared in plasma after s.c. injection as it was previously described for the whole venom. However, the toxins disappeared from the blood more quickly than did other main components of the venom. Thus, serotherapy must be initiated immediately to prevent the toxin from reaching its target. We also detected the toxins in urine, kidneys, heart and lungs, but not in the brain. However, the concentration of Aah II was always lower than that of Aah I. Analysis of five samples of venom collected in different areas of southern Tunisia showed that a large polymorphism exists for the three toxins. This is yet another difficulty for serotherapy as there is no cross-antigenicity between them.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.

OBJECTIIVES To determine the endocrine response to surgical stress in a homogeneous population of 36 women with microprolactinomas, particularly to evaluate the effect of the sharp decrease in plasma prolactin on stress induced hormonal secretion. In addition, the effects of exogenous opiates on prolactin secretion were studied. METHODS The plasma kinetics of cortisol, prolactin, ACTH, GH, and β-endorphin like immunoreactivity (β-ELI) were analysed by including patients operated on with strict anaesthetic and surgical protocols, and by sampling blood every 10 minutes, starting at premedication up to 3 hours after induction. RESULTS AND CONCLUSIONS (a) Surgical stress or opiate administration did not induce prolactin release in patients with microprolactinoma. (b) The dramatic decrease in prolactin concentrations have apparently no effect on the release of other hormones involved in stress. (c) The existence of an early GH peak, independently of any surgical procedure, strongly suggests that GH is released by surgical stress whereas β-endorphin is secreted in response to pain. Thus GH may be a useful marker of surgical stress.

Adenosine is involved in a large number of physiological processes including immune response and vasomotor function. But its precise involvement in renal physiology is poorly understood. We have investigated the putative relationships between cyclosporine A (CsA) and adenosine (ADO) metabolism in kidney transplant recipients (KTR). We first compared ADO plasma levels in three groups of patients and in 10 controls: the first group (N = 14) was composed of CsA-treated KTR; the second group (N = 5) was KTR not treated with CsA, and the third (N = 6) was chronic kidney failure patients. We also measured ADO plasma level in two KTR treated with FK506, a CsA analog. ADO plasma levels in CsA-treated KTR were significantly higher (mean 0.76 microM +/- 0.27) than in the control group (mean 0.31 +/- 0.13; Mean-Whitney test, S = 8.5; P = 2.1 x 10(-4)) and than in the chronic kidney failure group (0.37 +/- 0.16, Mann-Whitney, S = 5.5; P = 1.6 x 10(-3)). In CsA-treated KTR, CsA and ADO plasma levels were significantly correlated (Spearman's, r = 0.8, P = 1.9 x 10(-3)). No significant differences in ADO plasma levels were found between patients with chronic kidney failure and controls (P < 0.05). ADO plasma levels in KTR not treated with CsA were in the same range as those in controls. Finally, the ADO plasma level was increased in the two FK506-treated patients. We also investigated the action of CsA on ADO plasma degradation and uptake by erythrocytes in vitro. No interaction between adenosine deaminase and CsA was found because CsA, in the presence of adenosine deaminase, did not modify the plasma half-life of ADO. Conversely, in the presence of CsA (500 and 1000 ng/ml), the uptake of ADO by erythrocytes was significantly decreased in adenosine deaminase-free samples (analysis of variance, P = 1.8.10(-3) and 1.2 x 10(-4), respectively). We conclude that ADO plasma levels are significantly elevated and correlate with CsA blood level in CsA-treated KTR, and that these high levels are due to CsA inhibition of ADO uptake by red cells. Since ADO and metabolites have well known immunosuppressive and vascular effects, ADO is likely to participate in the immune defect and in the vasoconstriction induced by CsA.

ABSTRACT: Background: Adenosine is a powerful natural vasodilator that participates in the control of cerebral and meningeal blood flow. In this context, it could be involved in the pathophysiology of migraine, since it was previously reported that intravenous adenosine can precipitate crises in migraine patients. Methods: We have investigated circulating adenosine levels in 12 patients suffering from migraine without aura, during crises and in crisis-free periods, and have compared the levels noted to those of a population of 10 controls. To determine if there are interactions between adenosine and serotonin, we examined the effect of adenosine and antagonists on the uptake and the release of (14C) serotonin by platelets. Results and conclusions: We have reached a dual conclusion: 1) during migraine headaches there is an increase (mean 68%) in circulating adenosine levels and this increase may participate in cephalalgia; 2) activation of A2 receptors by adenosine causes a dose-dependent inhibition of calcium-dependent serotonin uptake by platelets. This inhibition of uptake could participate in the rapid elimination of serotonin in migraine sufferers. As a result of this, the use of adenosine antagonists could be an effective complementary treatment for migraine.

Scorpion toxins constitute a family of proteins with a high degree of sequence diversity but a common mode of action. Neutralization of the toxic effects of scorpion stings by serotherapy is limited due to the various serotypes expressed by these proteins. We explored the possibility of raising antibodies to conserved parts of the toxins which could recognize several members of the family. We established the variability profile of a set of 25 scorpion toxin sequences, then evaluated systematically by peptide-scanning methods the antigenicity of one scorpion toxin. The most conserved regions were generally very poorly antigenic. One exception was the N-terminal region, which is both conserved and antigenic. Antibodies were raised in rabbits against an eight-residue synthetic peptide mimicking the N-terminal region. These peptide antibodies were cross-reactive with several scorpion toxins belonging to different serotypes and neutralized both the pharmacological effects (binding to rat brain synaptosomes) and the biological activity (toxicity in mice) of the parent toxin. The molecular model of the toxin indicates that antibody binding to residues 1-8 probably either masks some residue(s) of the N-terminus critical for the biological activity or overlaps with the epitope previously defined by neutralizing monoclonal antibody. These findings could open the way for new therapeutic strategies for the medical care of envenomations.

Hyperthermia and profuse perspiration are rarely absent in severe cases of scorpion envenomation. Based on these observations, the aim of this study was to determine the therapeutic effects of dantrolene, on experimental poisoning by the venom of Androctonus australis hector. Dantrolene is a directly acting muscle relaxant which lowers the body temperature in malignant hyperthermia. The results indicate that the early use of this drug raises the LD(50) in experimentally poisoned mice. If these results are transposable to humans, dantrolene could be a useful therapeutic adjuvant.

Background: In animals and in humans, stress is known to be accompanied by increased beta-endorphin secretion. Methods: Blood samples from 47 patients in a state of stress induced by multiple trauma were assessed for beta-endorphin concentration by radioimmunoassays. Results: We show that there is a clearcut correlation (Spearman's R = 0.72, P = 2.1 x 10(-6)) between the level of consciousness evaluated with the Glasgow score and levels of circulating beta-endorphin. In addition, beta-endorphin levels are higher than normal in patients with Glasgow coma with scores higher than seven, and lower than normal in those with Glasgow coma scores of seven or less. Finally, in the complete absence of stress (shown by the lack of brain activity in six irreversible coma patients), there is a severe drop in the level of circulating beta-endorphin. Conclusion: beta-endorphin serum levels correlate with the state of consciousness of multiple trauma patients.

In addition to pain and stress, endogenous opiates and in particular beta-endorphin could be involved in the modulation of cardiovascular parameters. Several studies have thus shown increases in plasma beta-endorphin levels in the course of septic or hypovolemic shock. Our study involving 44 multiple trauma patients indicates that even in the absence of any hemodynamic disorders, there is a correlation between systolic blood pressure and plasma beta-endorphins. These results argue in favor of the existence of a feedback between systolic blood pressure and plasma beta-endorphins.

A set of 58 overlapping rod-bound peptides was used to map the antigenic reactivity pattern of a 64-residue neurotoxin (AaH II) from the venom of the scorpion Androctonus australis hector. Five anti-toxin rabbit antisera were assayed serially for their capacity to bind to each peptide in the set. Six regions of antigenic reactivity were thus identified (sequences: 1-8, 4-12, 27-35, 39-45, 52-58 and 55-61). When positioned on a 3-D model of the toxin, these regions appeared to correspond to either beta-turn or extended parts of the molecule. The antigenic regions revealed by this technique agree fairly well with those previously mapped on the same toxin by different methods. One discrepancy was, however, that the present study shows the N-terminus to be strongly reactive with anti-toxin antibodies. The antigenicity of this region was confirmed, since rabbit antibodies raised against a synthetic peptide mimicking the sequence 1-8 of the toxin were found to bind the toxin with high efficiency. A fine analysis of the recognition of this region was performed. Alanine-containing analogs of the sequence 1-7 and peptides mimicking the N-terminal of the four main toxins of AaH were probed with anti-toxin and anti-peptide antibodies. Lysine 2, aspartic acid 3 and glycine 4 were shown to be key residues in the recognition of the N-terminal region of the AaH II toxin by anti-toxin antibodies. In contrast, a loose specificity of recognition was shown by one anti-peptide serum which was, in addition, able to recognize the N-termini of all four AaH toxins.

With the aim of developing active protection against the noxious effects provoked in humans by scorpion stings, the possibility of eliciting toxin reactive antibodies by immunization with a short peptide was assessed in mice. The amino acid sequence of residues 50 to 59 of Androctonus australis Hector toxin II was chosen, on the basis of previous results indicating that rabbit anti-(50-59) antibodies neutralize the biological effects of the parent toxin. The peptide was prepared by solid-phase synthesis procedures and used in different forms (free, linearly polymerized, coupled to KLH, coupled to a low-molecular weight B-lymphocyte activator) in order to immunize groups of non-congenic NMRI or congenic C57BL/6 mice. The reactivities of each serum with the peptide and with the toxin were assessed in ELISA. Strong reactivities with both the peptide (mean titer over 1:52,600) and the toxin (mean titer 1:800) were observed in all mice from the group that received the KLH-coupled peptide. However, mouse immune sera failed either to recognize the toxin in a liquid-phase radioimmunoassay or to neutralize the lethal effects of the toxin. The requirements, in terms of affinity and recognition of native conformation, for anti-peptide antibodies to display neutralizing properties are discussed.

Fab fragments of anti‐apamin monoclonal antibodies have been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group P21 with cell dimensions a = 99.0 ± 0.3 Å, b = 137.1 ± 0.4 Å, c = 76.0 ± 0.2 Å and β = 92.9 ± 0.9°. They most likely contain four molecules in the asymmetric unit (V m = 2.39 Å3/Da). The possibility of the existence of non‐crystallographic symmetry is discussed.

Crystals of the fiber protein of adenovirus type 2 have been grown. Analysis of these crystals (type I crystals) showed that they were composed of fiber polypeptide with a lower apparent molecular weight (60 kDa) than that of the soluble or virion-incorporated fiber (62 kDa). N-terminal sequencing revealed that the fiber polypeptide chain of 60 kDa was cleaved at tyrosine17 from the N-end. The C-terminus remained intact. Assays with protease inhibitors suggested that the spontaneous cleavage of the fiber occurring upon its crystallization was due to a cellular, calcium-dependent, chymotrypsin-like protease co-purifying with the fiber and activated during hydroxyapatite chromatography. Crystallization of fiber purified in the presence of chymostatin provided crystals of a different structure under the electron microscope (crystals of type II), composed of 62-kDa fiber polypeptide units. The 62-kDa fiber from the type II crystals, as well as the 62-kDa fiber isolated from infected cell extracts, were able to associate with the penton base in vitro to form a penton capsomer. The 60-kDa fiber has lost this capacity. The accessibility of the N- and C-termini of the fiber inside the penton structure was probed by anti-peptide sera after limited proteolysis. The results are consistent with a polarity of the fiber in which its N-terminus is oriented toward the penton base, the C-terminal domain corresponding to the distal knob.