Genji Sakaguchi

ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells.

Botulinum ADP-ribosyltransferase C3 (C3 exoenzyme) was purified to homogeneity and added to cultured rat pheochromocytoma PC-12 cells. Incubation with this exoenzyme caused inhibition of cell growth and induced neurites as well as acetylcholine esterase in these cells. These changes were dependent on the amount of the enzyme added to the culture, which correlated with the in situ ADP-ribosylation of the rho/rac proteins in the cells. Preincubation with a specific anti-C3 exoenzyme monoclonal antibody inhibited both the ADP-ribosyltransferase activity and the neurite-inducing activity of the enzyme preparation. These results suggest that C3 exoenzyme affected the cellular function of the rho/rac proteins by ADP-ribosylation to induce these changes in the cells.

Purification, Characterization, and Oral Toxicity of Botulinum Type G Progenitor Toxin

Clostridium botulinum type G (or C. argentinense) strain 2470 was grown overnight at 37°C in chopped meat glucose medium and transferred to a medium consisting of proteose peptone 4%, yeast extract 1%, glucose 1%, and L-cysteine 0.2%, pH 7.3, which was incubated for 6 days at 30°C. The 10-L culture with a potential toxicity of 1.3 x 108 mouse i.p. LD50 was brought to pH 4.0 with sulfuric acid and kept overnight at 4°C. The precipitate was packed by centrifugation and extracted twice with 0.2 M phosphate buffer, pH 6.0. The extract was treated with 0.5% trypsin (Difco 1:250) at 37°C for 60 min. The extract recovered 1.3 × 108 LD50. The residual cells were sonicated and treated with trypsin in a similar way, which recovered 1.1 × 108 LD50. The two extracts were combined, the toxicity of which was set as 100%. Ammonium sulfate was added to the combined extracts to a 0.5 saturation. The precipitate was dissolved in 150 ml of 0.2 M phosphate buffer, pH 6.0, which was clarified by centrifugation. This extract was dialyzed against 0.05 M acetate buffer, pH 4.0, which caused precipitation of the toxin. The precipitate was dissolved in 100 ml of 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. It was clarified by centrifugation, concentrated by salting out, followed by dialysis. The extract of the second salting out recovered 191 mg of protein and 3.3 × 108 LD50 (118%). The extract, divided into 20-ml portions, was subjected to gel filtration on Sephadex G-200 (2.5 × 90 cm). The effluent in the void volume contained 96.3 mg protein and 2.8 × 108 LD50 (100%). The fraction was dialyzed against 0.5 M NaC1-0.05 M acetate buffer, pH 4.0, and added were the same volume of 0.5 M NaCl-0.05 M citrate buffer, pH 4.5, and 0.01% protamine. This was clarified by centrifugation and percolated through SP-Sephadex C-50 (2.5 × 30 cm) equilibrated with 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. The percolate was diluted 2.5-fold with 0.05 M acetate buffer, pH 4.0, applied again to SP-Sephadex C-50 (1.6 × 12 cm) equilibrated with 0.2 M NaC1-0.05 M acetate buffer, pH 4.0, and eluted with NaC1 gradient from 0.2 to 0.7 M. The fractions eluted at 0.34 to 0.48 M NaC1 contained 48.0 mg of protein and 1.9 × 108 LD50 (68%). The toxin fraction was concentrated by salting out and subjected to the second gel filtration on Sephadex G-200 (2.5 × 90 cm) with the same buffer. A single protein peak eluted contained 22.9 mg of protein and 1.1 × 108 LD50 (39%). The specific toxicity was 3.0 × 107 LD50/mg N.

Antigenic Structure of Botulinum Neurotoxins: Similarity and Dissimilarity to the Toxin Associated with Infant Botulism

Clostridium botulinum toxin has been classified into seven immunological types A through G. The toxin consists of a highly potent neurotoxin and a nontoxic component. The neurotoxin exerts its toxic action by inhibition of acetylcholine release, which results in neuromuscular paralysis.1,2 The neurotoxin is produced as a single polypeptide with a molecular weight of about 150 KDa, and is nicked by an endogenous or exogenous protease such as trypsin and other trypsin-like enzymes. The neurotoxin in a nicked form, is made up of two chains, the heavy (ca. 100 KDa) and the light (ca. 50 KDa) chains, which are covalently linked together with at least one disulfide bond.2¨C4 The heavy chain is responsible for binding the neurotoxin to the receptor on neural membrane,5,6 and the light chain is related to blockade of neurotransmitter release in presynaptic nerve endings.7,8 The significance of the nontoxic component in the botulinum toxin molecule as an oral poison has been reviewed by Sakaguchi.9