Lethal Activity In Mice Research Articles

Chemical studies on staphylococcal alpha-toxin and its fragments

THE CRYSTALLIZED staphylococcal alpha-toxin, a single protein with a mol . wt of 36,000, exhibits hemolytic, dermonecrotic and lethal activity (WATANABE and KATO, 1974, 1976) . However, the relationship between its chemical structure and its mechanism of action at the molecular level is not fully understood. Since the purified alpha-toxin is devoid of free SH-groups and disulfide bridges, the toxin was subjected to proteolytic treatment with the aim of obtaining split products which might be more amenable to structural studies . WATANABE and KATO (1978) reported that the purified alpha-toxin treated with trypsin yielded two components by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . A heavy chain fragment (mol . wt 17,000) had lethal activity in mice but lacked hemolytic and dermonecrotic activities, whereas a light fragment (mol . wt 14,000) was nontoxic and unstable . The present effort was undertaken to identify the Nand C-terminal amino acids of alpha-toxin and to correlate the results with the fragment chain structure of the toxin molecule. The crystallized alpha-toxin and heavy and light chain fragments were prepared as described previously (WATANABE and KATO, 1976, 1978). The time course of the digestion of the purified alpha-toxin by trypsin was followed by a slab-polyacrylamide gel electrophoresis (Fig . 1), The position of the heavy and light chain fragments of alpha-toxin was established by staining a few gels . The relevant regions ofthe unstained gels were removed, pooled, homogenized with 0-03% SDS and extracted overnight with the detergent . The extracts were lyophilized and used for the analyses of amino acid compositions and Nterminal amino acid residues . The amino acid compositions of alpha-toxin and its heavy and light chain fragments are summarized in Table 1. Amino acid analyses indicate different contents of lysine, histidine, threonine, methionine and tyrosine in the two fragments. The lethal heavy chain fragment is distinctive in containing only one residue of histidine. In experiments where we wanted to examine the effects of photo-oxidation of the histidine residue (WEIL et al., 1951), exposure of the heavy chain fragment to visible light in the presence of methylene blue at pH 8-0 resulted in a 60% loss of lethal activity in mice . The total of the mol . wts and amino acid residues of the two fragments is 31,000 and 268, respectively (Table 1) . When the alpha-toxin molecule is cleaved simultaneously at more than one position by trypsin, one or several small peptide(s) of no more than ten amino acids each (e .g . 26 amino acid residues in Table 1) are lost during fragment isolation procedures . Amino-terminal analysis, performed by the dansyl chloride method (GRAY, 1967), of alpha-toxin and its heavy chain and light chain fragments indicated alanine, isoleucine and alanine residues, respectively (Table 2). The sequences of the carboxymethylated alpha-

Effects of antiserum on the systemic response in mice caused by a component isolated from an extract of the brown recluse spider ( Loxosceles reclusa) venom apparatus

An extract of brown recluse spider ( Loxosceles reclusa) venom apparatuses causes death in mice when injected i.p., but causes no local response when sublethal doses are injected s.c. An antiserum was produced against the extract by i.v. injection in a rabbit. Preincubation of a component, isolated from the brown recluse venom apparatus extract, with the antiserum resulted in a loss of its lethal activity in mice. Post-treatment of component-challenged mice with the antiserum was successful if accomplished 0·5 hr after challenge, but not at 1·0 and 2·0 hr.

AN ENTEROTOXIN PRODUCED BY CLOSTRIDIUM PERFRINGENS TYPE D. PURIFICATION BY AFFINITY CHROMATOGRAPHY

Clostridium perfringens type D 9867 produced enterotoxin immunologically identical to that produced by types A and C which are responsible for C. perfringens food poisoning. Enterotoxin from type C 5386 and type D 9867 was produced in Duncan og Strong sporulation medium (DS medium). The supernatant fluid from DS-cultures was used for purification of the enterotoxin by affinity chromatography on a monospecific anti-enterotoxin-coupled CNBr activated-Sepharose 4B and activated CH-Sepharose 4B column. The enterotoxin purified by this one-step procedure proved to be of a purity comparable to that obtained by conventional methods, and possessed lethal activity in mice.

Biochemical and Antigenic Properties of a Purified Neurotoxin of Vipera Palestinae Venom

Summary The various activities—neurotoxic, proteolytic and anticoagulant—contained in the DEAE cellulose neurotoxic fraction of Vipera palestinae venom can be adequately separated by means of ammonium sulfate fractionation. The recovery of the neurotoxin, free from protease(s) and anticoagulant, is in the vicinity of 35%. Electrophoretic analysis of the isolated neurotoxin shows three different fractions, of which only one, positively charged, has lethal activity in mice. This component produces a single precipitation line in an agar immune electrophoresis plate. Venom protease(s), free of neurotoxic and anticoagulant activity, enhance the lethal activity of neurotoxin. The nondialyzable neurotoxin is split by trypsin and the dialysate is toxic for mice. Up to 1400 LD50 of the purified neurotoxin may be bound to and detoxified by 1 g of carboxymethyl cellulose (CMC). The CMC-bound isolated neurotoxin is highly immunogenic in rabbits.