1. As reported previously, histopathological changes of adrenal glands in Wistar Rats (male, 100-200g) were observed with the injection of purified histamine-sensitizing-factor (HSF): Zona glomerulosa, the outer layer of zona fasciculata, and their lipid content were constant. Marked cytolysis in zona glomerulosa. Extraordinary depletion of lipids in the inner layer of zona fasciculata. Zig-zag formetion from the beneath the capsule reaching the deeper region of the inner layer of zona fasciculata (Fig. 1 and 2).
It is well-known that diphtherial exotoxin induces marked hemorrhages of adrenal gland. In refference to this, histamine-sensitizing-factor (an exotoxin) of Bordetella pertussis might cause any change in the adrenal gland. Under this hypothesis, this experiment was carried out. Consequently, the changes mentioned above (Fig. 1 and 2) were recognized. These changes imply disturbance of secretion of hormones (Cortisol, Aldosteron, Adrenal Androgens). I drew inferences from these facts that these were reported as the results of “stress”. I got a lot of letters from everywhere in the world. Unexpectely, among them, the comment of Prof. M.S. Zakharova at Moscow, USSR, was valuable. She critisized that my findings are not only results of stress, but also they are functional changes of the whole host body. It is of the very importance for establishment of prevention against whooping cough infection. This suggestive comment gave me a hint.
No doubt, the changes (Fig. 1 and 2) express only static stage, followed by rapid repairement, accompanying hyperfunction and production of antitoxin. So that, the forced definition “preventive antigen” is completely useless.Recently, Arai & Munoz could crystallize HA (Fimbrial Hemagglutinin), whick lacks completely preventivity. Also, Arai & Munoz could succeed in crystallization of “pertussigen” (Package of HSF, LPF & thermo-labile lethal antigen). The future of pertussigen deserves the keen interest.
2. Is IC (INTRACEREBRAL CHALLENGE) of mice the one and only as a test?
IC method is spread all over the world like the Bible. Oblivious experiments by Standfast whose work I have learned from Dr. Margeret Pittman of NIH, Md, NSA. Standfast's experiments (1) Usual pertussis vaccine gave good effect on IC mouse protection as well as field trial. (2) Contrary to this, pertussis vaccine heated at 100C for one hour do not any effect on IC mouse protection at all. However, this heated vaccine gave good effect on IN (INTRANASAL CHALLENGE) mouse protection as well as field trial (human being). This undeniable facts are of the very importance, because packkage-pertussigen (a group of exotoxins-thermolabile) was vanished away by heating. Hence, one can obtain the most safe pertussis vaccine without any side effect. It reminded of my heat-stable neurotoxin.
I do not know whether it is an exotoxin or not. In spite of unknown character, it shows
charactristic whooping cough syndromes. My working hypothesis suggests that “heat-stable
antigen” by Standfast might coincident with my “heats-table neurotoxin”.

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Although it is well known that Moraxella bovis produces a hemolytic substance, little work has been done to characterize this substance. This report describes the condition of production of the hemolytic substance and its properties examined with strains of M. bovis isolated from otorrhea of cases of chronic otitis media and ATCC 10900, a type strain of the species.
The growth of ATCC 10900 was enhanced by the addition of yeast extracts (0.5%) to heart infusion medium at pH 7.5, and a sufficient amount of hemolytic substance was produced in this medium. It is not likely that yeast extract directly accelerated the production of the hemolytic substance. Smooth colonies of M. bovis seemed to produce a higher level of the hemolytic substance than rough colonies. Trypsin, when added to the medium at the time of incubation, inhibited the production of the hemolytic substance. None of such other agents as Fe⁺⁺, Mg⁺⁺, oxidizing or reducing agents, surface active agents and lecithin did not affect its production.
The substance of all strains hemolysed horse and sheep red blood cells, however that of 80% of the strains hemolysed human red blood cells and 75% rabbit red blood cells. The production was markedly accelerated by keeping the culture for 24hr at 4C after incubation at 30C.
The hemolytic substance of M. bovis is an extracellular substance, stable to heat, soluble in water, and a ninhydrin-positive, basic substance of low molecular weight.

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Lactobacillus casei ATCC 7469, refractory to egg white lysozyme, was successfully converted to protoplast by treating with endo-N-acetylmuramidase (M-1 enzyme) purified from
the culture supernatant of Streptomyces globisporus. The activity of M-1 enzyme was enhanced by the addition of a urtain disaccharide, e.q. sucrose or lactose as an osmoregulator
for protoplast formation. The protoplast prepared by treating with M-1 enzyme under 0.9M
sucrose for 10min retained the ability of amino acid transport as high as that of the intact
cell. Thus, these protoplasts may be useful for biochemical and genetical studies of Lactobacillus.

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