Pili were found on the cell surface of non-adhesive Vibrio cholerae O1 Biotype El Tor as well as the adhesive strain. Purified pili of the adhesive and non-adhesive strains were morphologically, electrophoretically, and immunologically, indistinguishable from each other. The molecular weights of both pilin (subunit protein of the pilus) were about 16, 000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These 16kDa pili are different from the pilus colonization factor, which is a 20.5kDa protein, reported by Taylor et al. The 16kDa pili of Vibrio cholerae O1 Biotype El Tor have hemagglutinating activity, but may have no role in colonization, because non-adhesive strains also have such pili.

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Nongrowing Vibrio parahaemolyticus cells rapidly produced putrescine (Put) from added arginine when subjected to a low osmotic stress. This phenomenon was characterized in connection with a regulatory mechanism of the responsible enzymes, arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH). NaCl, KCl, LiCl, sucrose, and glycerol were used as solutes to prepare the resuspending media with various osmolalities. Regardless of whether the solutes were electrolytes or non-electrolytes, exposure of cells to low osmolality brought about instantaneous increases in both intra- and extracellular Put contents without significant changes in the contents of other polyamines. This acceleration in Put production was accompanied by no increases in the specific activities of ADC and AUH. On the other hand, when cells were exposed to the osmolality equivalent to 2 or 5% NaCl, all solutes except for glycerol did not cause a remarkable variation in the intracellular Put content, while the amount of Put in the medium varied depending on the solute used; sucrose and glycerol still greatly prompted Put production, as judged by high Put contents in the media, even at the osmolality equivalent to 5% NaCl. The cation efflux from cells, measured as the K⁺ release, was observed whenever the increase in Put production occurred. Furthermore, in vitro experiments showed that NaCl and KCl inhibited ADC to a similar extent, about 70% inhibition being observed at 200mM. However, AUH was not affected by these compounds. These results suggest that the reduction in the concentrations of Na⁺ and K⁺ predominantly present in cells may cause the increase in activity of the preexisting ADC, which leads to the enhancement of Put production.

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Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320g/cm³ in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95, 000 for L. brevis and L. fermentum and of about 96, 000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.

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To determine the intracellular localization of the Creutzfeldt-Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka-1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.

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A murine macrophage cell line P388D₁ in in vitro culture without any specific stimulation produced both interleukin 1 (IL1) and IL1 inhibitor which inhibits mitogenic response of murine thymocytes to IL1 in the culture fluids. The factor(s) responsible for inhibiting IL1-induced thymocyte proliferation consisted of at least two molecules: factor I (FI) with an isoelectric point of 6.0 and factor II (FII) with an isoelectric point of 5.3, both of which had a similar m.w. of 40-60kDa. FI activity was sensitive to heat (56C) treatment and acid pH (3.0) treatment, while FII was resistant to both treatments. Both FI and FII inhibited mitogenic responses of thymocytes to IL1, but not proliferation of murine lymphoid cells induced by other interleukins, namely, IL2, IL3, or IL4. Neither showed any inhibition of spontaneous proliferation of murine tumor cell lines, suggesting that inhibition was specific for IL1, but not nonspecifically inhibiting for cellular DNA. These IL1 inhibitors were also suggested to be acting in the early phase of interaction between IL1 and lymphoid cells. The possible role of these inhibitors as representatives of regulatory substances, which normally control IL1 activities either in the levels of inflammation or immune responses, was discussed.

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Influence of transglutaminase on the production of interleukin-1 (IL-1) and on the release of active oxygen from mouse peritoneal macrophages was examined using cystamine and methylamine, an enzyme inhibitor and a substrate inhibitor, respectively. Casein-elicited or lipopolysaccharide (LPS)-elicited macrophages have higher levels of transglutaminase activity in comparison with resident macrophages, and there exists a definite correlation between endocytosis of erythrocytes and transglutaminase activity in either group of macrophages. The release of IL-1 by resident macrophages stimulated with LPS in vitro was significantly inhibited by the treatment with both transglutaminase inhibitors. However, these inhibitors were not able to inhibit the release of IL-1 from casein-elicited macrophages stimulated with LPS in vitro. The production of active oxygen from LPS-elicited macrophages was inhibited in a dose-dependent manner by the treatment of macrophages with cystamine, but was not by the treatment with methylamine. However, the treatment of LPS-elicited macrophages with cystamine did not inhibit the uptake of glucose into macrophages. These results suggest that transglutaminase activity in mouse peritoneal macrophages is an important factor for macrophage functions.

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The production of penicillin G and penicillin V amidohydrolases or acylases (E.C.3.5.1.11) was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer. In all the studied fungi, enzymes showing penicillin G and penicillin V amidohydrolase activities were found. Generally, an increase of these activities during fungal autolysis was observed. The presence of phenoxyacetic acid in the medium did not increase these activities. The activities found in the culture fluids were generally higher than that found in the mycelial extracts. Under these conditions, β-lactamases (penicillinases) were not found. The fungi Alternaria alternata, Fusarium culmorum, Penicillium oxalicum, and the species Penicillium 222 were chosen to study penicillin G and penicillin V acylases. The enzymes were precipitated with tannic acid from the culture fluid of their autolyzed cultures. Some kinetic constants of these activities were determined.

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Inhibition of hemagglutinin (HA) activity in a membrane fraction of Bacteroides gingivalis was examined using various compounds. Leupeptin and antipain inhibited the HA activity at nM order. This potency was lost when the aldehyde group of leupeptin was converted to an alcohol moiety. Irreversible protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK), p-chloromercuribenzoate (PCMB), and N-ethylmaleimide (NEM) were also inhibitory. From the inhibition experiments, we speculate that the HA possesses protease activity and that the same site of the molecule participates in the erythrocyte binding and the substrate binding.

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A simple method was developed to visually present the phagocytic activity of leukocytes by using adherent Staphylococcus aureus cells and blood applied on a plastic dish. When heparinized blood was applied on thin-layer of heat-killed S. aureus cells on the plastic dish, plaques due to the phagocytic activity of leukocytes were observed with a microscope under a low magnification. Fewer and smaller plaques were observed when plasma-deprived rather than whole blood was used. Some analyses were made in respect to the fundamental conditions required for optimal results. This method was considered to be useful for conveniently evaluating the serum opsonin activities and phagocytic function of leukocytes in various kinds of diseases.

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