Endotoxic glycolipids (ReGl) extracted from the whole cells (WC) and cell walls of heptose-less Re mutant of Salmonella minnesota with hot phenol-water (PW), phenol-chloroform-petroleum ether (PCP), and chloroform-methanol (CM) were analyzed chemically and examined with an electron microscope. ReGl-PW-(WC) contained mannose and proteins as contaminants, ReGl-PCP(WC) consisted of an excess amount of amino compound (cadaverine), and ReGl-CM(WC) consisted of proteins and cadaverine, in addition to the ReGl constituents.
The ultrastructure of ReGl-PW(WC) resembled onions when stained with uranyl formate, and was spherical when stained with uranyl acetate, sodium phosphotungstate and ammonium molybdate, whereas those of ReGl-PCP(WC) and ReGl-CM(WC) were shaped like ribbons. However, the shadowed ultrastructures of ReGl-PCP(WC) and ReGl-CM(WC) showed small pieces of flat and wide sheets, respectively. ReGl-PCP(WC) re-extracted by the PW method was found to be converted into the onion-like structure which was similar to that of ReGl-PW(WC), while an intermediate form (fingerprint-like) was observed after re-extraction of ReGl-PW(WC) with PCP. It was strongly suggested that the ultrastructural arrangement of ReGl was dependent on the solvent systems used for extraction.

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Endotoxic lipopolysaccharide and glycolipids (RG1) extracted from Salmonella minnesota wild type and R mutant cells (chemotypes Ra, Rb, Re, Rd1, and Rd2), respectively, with hot phenol-water (PW) and phenol-chloroform-petroleum ether (PCP) were analyzed chemically and electron microscopically. All RG1 extracted with PW (RG1-PW) contained excess amounts of phosphate, O-ester linked fatty acids and neutral sugars, while all RG1 extracted with PCP (RG1-PCP) contained excess amounts of free amino groups and fatty acids, in addition to the RG1 constituents. Polyamine (cadaverine), phosphoethanolamine, and an unidentified amino compound were contained in RG1-PCP as free amino groups.
When stained with uranyl formate, the ultrastructure of RG1-PW showed a spherical form (onion-like form), whereas the micrographs of RG1-PCP showed a filamentous structure, regardless of strain differences. On the other hand, the micrographs of RG1-PW represented spherical and doughnut-shaped forms, and the micrographs of RG1-PCP showed filamentous or stick forms, when stained with uranyl acetate. Thus, it is suggested that the ultrastructures of RG1 were dominated by the solvent systems used for extraction, and not by the strains used here.

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Endotoxins extracted from Salmonella minnesotawild strain and R mutants (Ra to Re) were treated with two different alkaline reagents. Treatment with diluted sodium hydroxide, which caused partial removal of O-ester linked fatty acids, changed the ultrastructures of endotoxins from an onion-like structure to monolayer particles (approximately 100Å in diameter) except for endotoxic glycolipids from Rd2 and Re mutants which showed mixed ultrastructures of untreated and treated endotoxins. Treatment with alkaline hydroxylamine, which caused liberation of all O-ester linked fatty acids, changed the ultrastructures of all endotoxins to monolayer particles. The results suggested that the ultrastructures of alkaline-treated endotoxins were dependent on the degree of their hydrophobicity. On the other hand, the micrographs of acid treated endotoxins did not show a constant structure because of the high hydrophobicity.

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The use of continuous free-flow electrophoresis for the purification of extracted lipopolysaccharides (LPSs) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol-water method of Westphal from Salmonella typhimurium and Escherichia coli 0111:B4, were analyzed. Continuous free-flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free-flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free-flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate-polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111:B4 LPS were eliminated by continuous free-flow electrophoresis, but each component of purified E. coli 0111:B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free-flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free-flow electrophoresis is a useful means of purifying extracted crude LPS.

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Galactosamine-6-phosphate was identified as a component of the coat of the Bacillus megaterium QM B1551 spore. It was one of the main constituents of the outermost layer of the spore coat, but it was absent from the other integuments including the cortex. These findings suggest that galactosamine-6-phosphate comprises the phosphorus-containing skeleton structure of the spore coat.

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The presence of glucosephosphate isomerase, one of the key enzymes in carbohydrate metabolism, was confirmed for the first time in the cell-free extract of Leptospira biflexa.
The glucosephosphate isomerase of L. biflexa was heat-labile and its optimum pH was about 8.5. The enzyme showed an optimal temperature of about 45C but was more stable at 30C. K_m value of the enzyme was 5.6×10^-3M. The activity of the enzyme was inhibited by the inhibitor, 6-phosphogluconate.
From this study, the presence of a metabolic pathway, the phosphogluconate pathway, other than non-oxidative pentose phosphate pathway presented by Baseman and Cox was suggested.

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The rate of L-alanine-initiated germination of Bacillus subtilis spore was measured by both loss of heat resistance and loss of turbidity, and the effect of glucose on the germination response to a wide range of concentrations of the germinant was analyzed in the presence and absence of D-alanine, an inhibitor. Glucose stimulated L-alanine germination by means of a cooperative effect: glucose increased the affinity of L-alanine by about 3-fold and the rate of germination by about 1.3-fold. However, glucose had little effect on the binding affinity of D-alanine. The apparent binding constant of L-alanine to the spore, which was determined by the next measurable event in the trigger reaction, was 1.2×10^-5, that of D-alanine was 6×10^-6, and that of glucose was 5×10^-5. The relation between the binding site for glucose and those for L- and D-alanine on the spore is discussed. Effect of glucose analogs was also examined.

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Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice.
In vitro treatment with anti-asialo GM1 antibody plus complement completely abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.

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The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease (VKH) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1⁺ Leu-2a⁺ or Leu-1⁺ Leu-3a⁺ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.

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