Pertussigen prepared from Bordetella pertussis strains of various agglutinogen types was found to have similar biological activities in mice, and to give precipitin lines of identity in agar diffusion tests. The doses required to induce histamine hypersensitivity ranged from 0.9 to 9.5ng, and to induce leukocytosis from 124 to 190ng. When pertussigen was detoxified by treatment with glutaraldehyde, it protected mice from intracerebral challenge with B. pertussis at doses of from 12.7 to 24.4μg. The results showed that all smooth strains of B. pertussis produced pertussigen that was biologically and serologically similar, and that it was produced independently of fimbrial hemagglutinin.

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A possible role of intracellular Ca²⁺ and participation of calmodulin in cellular metabolism in endotoxin-poisoned mice were investigated. The levels of calcium in liver cvtosol and liver mitochondria fractions in poisoned mice were markedly higher 18-48hr after endotoxin injection than in the control mice. On the other hand, the levels of serum calcium in the poisoned mice were about 20% lower at 18hr than in the controls. The serum calcium levels in mice injected with 50 and 100μg of endotoxin showed no dose-response effect, but a dose-response effect was observed at a dose of 200-400μg. The serum Ca²⁺ levels in endotoxin-tolerant mice were similar to those in the control mice. The levels in mice injected with glucocorticoid-antagonizing factor mice were about 14% lower at 3hr than in the controls. The mice fed a vitamin D_3- and calcium-free diet showed a higher mortality rate in the early stage (12-18hr) of endotoxication than that of the mice fed a normal diet.
The lipid peroxide levels and Ca²⁺-ATPase activity in the liver mitochondria fraction in endotoxin-poisoned mice showed a higher level than those of the control mice. There was little or no difference in the levels of serum glucose between the mice injected with calmodulin antagonist (trifluoperazine, TFP) plus endotoxin and those given endotoxin alone. However, the liver glycogen levels in TFP plus endotoxin-treated mice were markedly higher than that in mice given endotoxin alone. Furthermore, calcium antagonist (verapamil) plus endotoxin-treated mice had about a 40% higher survival rate after 72hr than those given endotoxin alone.
The findings suggest that there is a possibility of participation of the Ca²⁺-calmodulin system in carbohydrate metabolic disorders during endotoxemia and that the changes in intracellular Ca²⁺ may result in various metabolic disorders.

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The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa.
Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase.
The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0×10^-3M and the activity was inhibited by glycolate, the inhibitor.
The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.

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Cellular polysaccharide fractions of various representative members of genera of the family Spirochaetaceae were obtained by the ammonium hydroxide extraction method. The sugar composition of the polysaccharide preparations was complex and many kinds of sugars such as rhamnose, fucose, ribose, xylose, mannose, galactose, and glucose were detected in all of the spirochetes tested. Of particular interest was the presence of 4-O-methylmannose as a constituent polysaccharide in members of the genus Leptospira. This sugar was not detected in the polysaccharides of Spirochaeta, Borrelia, and Treponema.
The chemical compositions of cell wall fractions were also examined. 4-O-Methylmannose was detected in the cell wall polysaccharides of the genus Leptospira but not in cell walls prepared from the Spirochaeta, Borrelia, and Treponema. The diaminopimelic acid present in cell wall peptidoglycans of the genus Leptospira was meso-diaminopimelic acid (A₂pm). The molar ratios of alanine, glutamic acid, A₂pm, glycine, muramic acid, and glucosamine in leptospiral cell walls were found to be approximately 2:1:1:1:1:1. In contrast to the Leptospira, the peptidoglycans of genera Spirochaeta, Borrelia, and Treponema contained ornithine (Orn) but not A₂pm. Since 4-O-methylmannose and A₂pm were found in the cell wall fractions of genus Leptospira but not in Spirochaeta, Borrelia, or Treponema, it was suggested that the chemical compositions of the cell wall might become an important criterion for the chemotaxonomy of Spirochaetales.

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Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant, which was found to exhibit a very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice, was examined by electron microscopy. When negatively stained with uranyl acetate or ammonium molybdate, the KO3 LPS was found to consist principally of flat ribbon-like structures branching freely (average width 16nm and average thickness 7nm) and to contain a small proportion of spheres (diameter 20-50nm), both structures covered with fine hairy structures (average length approximately 10nm). When the polysaccharide of KO3 LPS was stained by the periodic acid-thiosemicarbazide-silver proteinate procedure, silver granules were deposited on the ribbon-like structures and around the spheres, suggesting that the polysaccharide moiety is located on their surface and that the fine hairy structures consist of the polysaccharide moiety. Comparison by means of preparations stained with uranyl acetate or ammonium molybdate showed that KO3 LPS isolated from the culture supernatant has structural features in common with KO3 LPS isolated from bacterial cells, Escherichia coli O9 LPS isolated from the culture supernatant, and E. coli O127 LPS isolated from bacterial cells. On the basis of the present results, schematic representations of the common physical structure of LPS were drawn; the fine hairy structures attach to the wide surface of the flat ribbon-like structures along their lateral margin.

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Various uniform salt forms of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant were prepared as follows. Basic materials present in KO3 LPS were rigorously removed by electrodialysis and the electrodialyzed KO3 LPS was neutralized with NaOH, KOH, NH₄OH, Ca(OH)₂, tris(hydroxymethyl) aminomethane, or triethylamine. The ultrastructure of the uniform salt forms of KO3 LPS was examined using preparations stained with uranyl acetate. The sodium, potassium, ammonium, and trisaminomethane salt forms were structurally very similar to the natural form of KO3 LPS which consisted of a mixture of flat ribbon-like structures (average width of 16nm and average thickness of 7nm) and spheres with various diameters, both covered with fine hairy structures. When KO3 LPS was converted to the triethylamine salt form, the ribbon-like structures were distrupted into very small granules (7-9nm×9-15nm). The calcium salt form consisted of particles and rods of various sizes and ribbon-like structures which were markedly extended (maximum width of 50nm) and presented irregular shapes. When converted to the calcium salt form, the ribbon-like structures were extended and eventually divided into particles and rods. For reasons still unknown, the sodium salt of KO3 LPS was mostly stained positively with uranyl acetate in contrast to the natural form and the other uniform salt forms which were always negatively stained. In the positively stained preparation of the sodium salt form, it was clearly shown that the ribbon-like structures consisted of a bilayer.

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Guinea pigs were sequentially infected with two closely related flavivituses (Japanese encephalitis and West Nile viruses), and their antibody responses were studied both by enzyme-linked immunosorbent assay (ELISA) and hemagglutination-inhibition (HI). The results by ELISA always showed “original antigenic sin” responses: antibody activity of sera after the second infection was higher to the first infecting virus than to the second infecting virus. However, the results obtained by HI were variable.

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Viral pollution of the river water in Toyama City was surveyed during the two-year period from July 1979 to July 1981, and the ecology of viruses in the river water is discussed. Virus isolation from the river water samples, or from the water squeezed from cotton pads that were immersed in the stream for 3 days, was carried out by the “filter adsorption/elution” method.
River waters were found to be contaminated with various species of enteric viruses, that is, poliovirus, echovirus, coxsackievirus, adenovirus, and reovirus. Poliovirus was isolated during the period immediately after the oral administration of polio vaccine, and coxsackie B virus was frequently isolated all year around. The enterovirus concentration in the river water was significantly high with a maximum of five plaque-forming units of coxsackie B2 virus per 250ml.
The species and type distribution of enteroviruses isolated from the river water coincided well with that of viruses isolated from inhabitants of Toyama Prefecture, with the exception of reovirus which was the largest population of virus species in the river water.

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An attenuated strain L₁₁A of tobacco mosaic virus (TMV) multiplied like wild type strain L at an early stage of infection in tomato leaves. Four days after inoculation, however, multiplication of L₁₁A was drastically reduced (autoregulation) compared with the constant multiplication of L. In mixed infections, L₁₁A strongly inhibited the multiplication of homologous strain L. Experiments with cucumber mosaic virus (CMV) or tobacco plants revealed that the inhibitory mechanism of L₁₁A is not host-specific but virus-specific, and the autoregulatory mechanism is effective only for TMV.
RNA synthesis in L₁₁A infected leaves 4 days after inoculation was studied by polyacrylamide gel electrophoresis. Synthesis of TMV-RNA and its replicative intermediate were strongly inhibited, whereas the replicative form of TMV-RNA and ribosomal RNA were synthesized as in the case of L infection.
Synthesis of non-coat-protein was studied by the incorporation of radioactive histidine into subcellular fractions derived from leaves infected with L or L₁₁A for 4 days. Different patterns of the two strains in protein synthesis were noted. At least three proteins were predominantly synthesized in L₁₁A infection. One of them was observed in the mitochondria fraction. From its position in polyacrylamide gel, it could be viral coded 165K protein which is considered to be involved in viral RNA replication,
These results suggest that the unique nature of attenuated virus L₁₁A, i.e. autoregulation, resulted from the inhibitory mechanism of viral RNA synthesis due to overproduction of 165K protein and is quite distinct from interferon, intrinsic interference or interference by defective virus.

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In mouse spleen cell cultures, TH69, a live Streptococcus faecalis (ATCC 31663) preparation, at a concentration of 20μg/ml induced immune interferon (IFNγ) with molecular weight ranging from 20, 000 to 40, 000 daltons together with a small amount of IFNα/β.
By using nonsensitized mouse spleen cells, the fact that both T-cells and macrophages are required for this IFN production was established. When these spleen cells were obtained from mice sensitized 12 days earlier with 4mg of TH69, twice as much IFN was produced than in cells obtained from nonsensitized mice. This increase was explained by the presence of both sensitized macrophages and T-cells in a reconstitution experiment.

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Ribonucleic acid (immune RNA, iRNA) extracted from the spleens of mice immunized with heterologous red blood cells induced antigen-specific immunologic memory. The type of cells (T cells, B cells) which participate in immunologic memory induced with iRNA was investigated. Immune RNA-primed T cells cooperated with normal, iRNA-primed or antigen-primed B cells and induced a high IgM response. Immune RNA-primed B cells did not cooperate with normal T cells, but did with iRNA- or antigen-primed T cells. The activities of iRNA-primed T and B cells were antigen-specific.

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