From lactalbumin hydrolysate (enzymatically) peptide essential to the growth of Pasteurella multocida was isolated by gel filtration using Sephadex G-25 and by column chromatography with the use of Dowex 50W×2. Growth-eliciting effect of effective fraction separated with gel filtration was considerably below that of original material, although that of mixture of the all fractions obtained with gel filtration approximated to that of original material. The addition of various ineffective fractions to the effective one obtained with gel filtration led to different responses; one group of fractions stimulated bacterial growth, the other group of fractions was ineffective or inhibited the growth.
A hypersensitive reaction was observed in three research workers who manage snake venom. Symptoms of those reaction were such as continuous sneezings, serous rhinorrhea, conjunctival itch, dificulty of breething like asthma and general nettle rash. Those hypersensitivity were thought to be induced through inhalation of venom while working in their laboratory. Hypersensitivity appeared within two years in those persons. Steroid, antihistamin and fluid transfusion were effective as treatment.
The cells of Salmonella enteritidis were sonically disintegrated under low temperature, and subjected to a ultracentrifugical procedure. The sediment obtained between the centrifugation at 3, 400g for 30 minutes and at 92, 600g for 60 minutes, was termed particulate fraction, and its supernatant fluid was supernatant fraction. The particulate fraction was treated with sodium deoxycholate as a membrane distrupting agent. By this treatment, it was demonstrated that “unit membrane” structure became to be discernible with difficulty in ultrathin sectioned preparations of the particulte fraction, and, at the same time, its dehydrogenase activities for lactate and succinate were markedly reduced. Moreover, the protective antigenicity for mice of the treated particulate fraction was distinctly inferior to that of the untreated control. However, by the addition of Freund's incomplete adjuvant to the treated particulate fraction, the lowered protective antigenicity was restored in some extent. The protective antigenicity of the supernatant fraction, the principal component of which is thought to be ribosomes, was weak. The addition of the Freund's incomplete adjuvant to the supernatant fraction again could enhance distinctly the antigenicity. From above results a discussion was made on the significance of the unit membrane structure and the ribosomes, which are included simultaneously in the particulate fraction, in its protective antigenicity.
Habu venom treated with the same amount of dihydrothioctic acid (DHTA) at 37°C for 30 minutes lost its hemorrhagic and necrotic action but remained some swelling of foot of a mouse which was inoculated with the toxoided venom subcutaneously into the plantar side of foot. The degree of swelling due to the venom which was harvested from Okinawa was more marked than that of venom from Amami Islands. A precipitation occurred after the mixture of Amami venom with DHTA was more dense than that of venom from Okinawa. The swelling decreased after the freeze-drying of the toxoided venom without loss of its antigenicity.
With a view to exploring the role of alpha toxin in staphylococcal infections, attempts were made to purify the toxin and to clarify pathogenic effects of thus purified preparation. Using supernatant fluid of a brain heart infusion broth culture of Staphylococcvs aureus, strain Wood 46 as starting material, an alpha toxin preparation was obtained by concentration and purification through chromatographic column in various combination including DEAE-Sephadex column; and the preparation thus obtained has proven to be highly homogenous, both physically and immunologically. The hemolytic activity of this toxin was 73, 142.8MHD/Nmg, purification rate was 497.9, and recovery was 1.5 per cent. And S20-w was about 3.2 S. Chemical examination revealed an alpha toxin consisting of a total of 13 amino acid groups, i.e., asparagine, serine, threonine, glutamine, proline, glycine, alanine, valine, isoleucine, leucine, phenylalanine, lysine, and arginine but devoid of saccharide, hexosamine, phosphorus, and lipid. Its hemolytic as well as leucocidin effects were found to be remarkably profound with the blood cells of rabbits, while it affected the cells of mice and man only to a small extent, suggesting its marked species-specificity in these respects. The results of further experiments regarding its lethal effects in mice and dermonecrotic effects in rabbits along with its said behavior as hemolysin all provided evidence in support of the unitarian theory. The alpha toxin preparation produced a characteristic cytopathogenic effect only in rabbit kidney cell cultures in as low a dose as 2 MHD, while it proved to be practically lacking in cytopathogenic effects on cultures of monkey kidney, Hela, L, and FL cell lines. Inoculations with pathogenic strains of the Staphylococci apparently resulted in localised abscesses in rabbits, whereas mice were found undoutedly to be resistant. These experimental findings in all appear to suggest that the biologic activity of the toxin is of cardinal importance in the pathogenesis of staphylococcal infections in rabbits alone, but is extremely minor importance in man.