Daniel A. Boroff

On the Question of Permeability of the Blood-Brain Barrier to Botulinum Toxin

The clinical symptoms of botulinum intoxication suggest that besides the involvement of the peripheral nervous system, the central nervous system is also affected. Studies were undertaken to determine whether pure toxin of Clostridium botulinum type A could be demonstrated in the brains of poisoned mice. With the aid of autoradiography of the toxin marked with 125-I and indirect fluorescent labeling it was possible to show the presence of the toxin in the parenchyma of the brain.

Purification of Clostridium botulinum type a toxin

Abstract The neurotoxin of Clostridium botulinum Type A has been isolated and purified from a liquid culture. The toxin is homogeneuous by anion and cation exchange chromatography, gel filtration, isoelectric focusing and Ouchterlony gel diffusion technique. The specific toxicity of the purified toxin is 10 · 107 minimum lethal doses/1.0 A 278 nm , the molecular weight is 150 000 by gel filtration method and the iso-electric point is pH 6.1. This preparation could not be distinguished from the α fraction isolated from the crystalline toxin. Results presented here failed to confirm the claim of Gerwing et al.5 that a toxin of 12 200 mol. wt. can be isolated from the liquid culture of this organism.

Method for quantitative determination of free and peptide-linked tryptophan after reaction with 2-hydroxy-5-nitrobenzyl bromide

Abstract A method for quantitative determination of tryptophan in the presence of 20hydroxy-5-nitrobenzyl bromide substituted tryptophan is described. The procedure does not require removal of the excess reagent and can be applied to intact proteins substituted with the reagent. The procedure involves applying mathematically derived correction factors to the analysis data obtained from the Spies and Chambers method for tryptophan determination.

Study of the toxin of Clostridium botulinum. Effects of 2-Hydroxy-5-nitrobenzyl bromide on the biological activity of botulinum toxin

Abstract 2-Hydroxy-5-nitrobenzyl bromide, when reacted with the toxin of Clostridium botulinum Type A, reduced toxicity by 99% and modified 17.8 moles out of 77.2 moles of tryptophan reduced in the toxin. This treatment resulted also in the loss of the ability of the toxin to stimulate protective antibidy formation in rabbits by destroying an antigenic determinant responsible for the formation of neutralizing anti-body in the native toxin. This evidence, in addition to the results obtained by photo-oxidation of the toxin 5 , further supports the thesis previously advanced as to the critical role of tryptophan residues in the toxins reactive sites. 2-Hydroxy-5-nitro-benzyl substitution method yielded data consistent with the estimated number of active tryptophan residues obtained from photooxidation reaction kinetics.

Examination of the possible pathophysiology of the central nervous system during botulinal poisoning

Abstract The effects of α and β components of type A botulinum toxin were tested for their ability to affect the central nervous system of cats, rabbits, and guinea pigs. Components α and β, when administered in lethal doses into the circulatory system of cats and rabbits, do not alter the ongoing electroencephalographic activity. Furthermore, α intoxication of guinea pigs does not result in changes in the brain concentration of acetylcholine or norepinephrine. Ingestion by mice of organs from intoxicated rats causes death only in those instances of organs that are well perfused with blood. The diaphragm and brain, both cholinergic sites, proved not to be toxic.

Cation-exchange chromatography of Clostridium botulinum type A toxin on amberlite IRC-50 resin at pH 5.55.

The hemagglutinin-free toxin (the α fraction) isolated from crystalline toxin of Clostridium botulinum type A, homogeneous by several criteria, was examined on carboxymethyl-Sephadex, sulfoethyl-Sephadex and Amberlite IRC-50 (XE-64) columns to attempt further resolution and to provide additional tests of its homogeneity. Satisfactory conditions for chromatography were obtained only with Amberlite IRC-50 (XE-64) resin. As an analytical tool this resin proved more sensitive than a DEAE-cellulose column since it was able to separate the hemagglutinin when present as trace contaminant in the α fraction, which could not be achieved by anion-exchange chromatography. Although the IRC-50 column separated the 150 000 mol. wt. specie of the α fraction from its aggregated forms, the toxin could not be fractionated into subunits. These observations were consistent with the result of gel filtration of the α fraction and its aggregates on Sephadex columns. The present report also demonstrates for the first time that a protein as large as 150 000 mol. wt. can be successfully chromatographed on IRC-50 (XE-64) resin at pH 5.55.

The Effects of Hydrogen Peroxide and X-Irradiation, Singly and in Combination, on the Growth and Development of Ehrlich Ascites Tumor in Mice

As early as 1934, Crabtree and Cramer reported that x-irradiation inhibition of cell growth is an increasing function of oxygen pressure (2). That the substance active in cell damage is hydrogen peroxide, and that its effect is upon the cell chromosomes, was proposed by Thoday and Read (7). They offered as evidence their observation that the presence of oxygen enhanced the genetic effect produced by roentgen rays. These findings stimulated a wide study of the effectiveness of hydrogen peroxide in cancer therapy. Hollcroft (3) reported that H2O2, administered intravenously to tumor-bearing animals immediately before irradiation, increased the effect of radiotherapy on tumor regression, in comparison with noninjected but irradiated controls. Investigating the action of hydrogen peroxide injected intraperitoneally into rats with Yoshida ascites tumor, Makino (4) found on examination of the ascites fluid that 90 per cent of the tumor cell appeared injured. Stecherl et al. (6) succeeded in prolonging the survi...