Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106. A large amount of cepalycins were produced by growing the cells on 1% glycerin-nutrient agar medium covered with a cellophane membrane. The cell-washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl. Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II. The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption. From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane. Such an interaction may contribute to their hemolytic and antifungal activities.

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Electron microscopy of ruthenium red-stained ultrathin section of strains of Shigella dysenteriae type 1 grown in the Casamino Acids-yeast extract broth medium showed the presence of an extracellular slime layer. The slime appeared as a dense sheath covering bacteria. The presence of slime promoted hemagglutinating activity of the bacteria. The slime polysaccharide (SPS) isolated from the cell-free culture supernatant or the bacterial surface was less than 162, 000 daltons in size and immunochemically similar. The SPS showed cross-reaction with lipopolysaccharide (LPS) antigen in immunological tests; however, it also appeared to be different from LPS since it did not contain 2-keto-3-deoxyoctonate, a core sugar of LPS. A different pattern of separation from LPS was also observed by silver staining of SDS-polyacrylamide gels. From these data it appeared that either LPS and SPS are contaminated with each other or that SPS is the polysaccharide portion of LPS.

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Candida strains were isolated repeatedly from single patients during recurrent episodes of Candida infection in a hospital, and their electrophoretic karyotypes were analyzed by pulsed-field gel electrophoresis using CHEF system. When only C. albicans (in 6 patients) or C. glabrata (in 1 patient) were recurrently isolated, their karyotypes from each patient were almost identical to one another, suggesting that they carried single type of the yeast. When multiple species were recovered from single patients (6 cases), the karyotypes of the most frequently recovered yeast species were almost identical with respect to each patient. The electrophoretic karyotype analysis has been proved to be useful for epidemiological studies because the method can tell not only the species identification but also the differences among the strains of the same species.

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The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80C for 10min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.

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Tetracycline (pp-tet), and kanamycin (pp-kan) resistance genes were cloned from a transferable R plasmid of fish pathogen Pasteurella piscicida, and complete nucleotide sequences were determined. The pp-tet was a class D Tet determinant constructed with the tetA resistance gene of 1, 182bp encoding a protein with a deduced molecular mass of 41kDa and the tetR repressor gene of 654bp encoding a product of 24kDa. The pp-tet was highly homologous to the tet(D) of plasmid RA1 isolated from Aeromonas hydrophila with two nucleotide differences in the tetR, and of plasmid pIP173 from Salmonella ordonez with two nucleotide differences in the tetA. The pp-kan contained 813bp encoding a 31kDa protein of 271 amino acids, and was classified into type aph-Ic. It was identical to the aphA7 in the IAB operon of pBWH77, in which was originally found an isolate of Klebsiella pneumoniae, in its nucleotide sequences and hybrid promoter construction. The genes were connected by an insertion sequence IS26 of 820bp, and were flanked by repeated copies in direct orientation at the 3' flanking region of the pp-tetA and in inverted orientation at the 3' flanking region of the pp-kan. The genetic elements are organized like a complex transposon by close linkage of the IS26 and the pp-tet and -kan.

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Rampant dental caries was induced in hyposalivated rats fed a high sucrose diet without infection of mutans streptococci, in which increased numbers of lactobacilli and S. aureus were demonstrated in the oral flora. Administration of either penicillin or piperacillin, effective against all isolates of lactobacilli, markedly inhibited the caries induction in these rats, while severe dental caries was induced in hyposalivated rats given vancomycin that is inhibitory against S. aureus. These results suggested that certain lactobacilli might induce dental caries in hyposalivated rats fed a sucrose diet. Three strains of Lactobacillus species isolated from the hyposalivated rats were made resistant to erythromycin. The caries-inducing activity of these erythromycin-resistant lactobacilli was studied in hyposalivated rats giving erythromycin in the drinking water at a concentration of 500μg/ml. After a 61-day experimental period, severe dental caries was induced in hyposalivated rats infected with L. fermentum TY1R. On the other hand, low caries incidence was found in hyposalivated rats infected with either L. acidophilus TY7R or L. plantarum TY3R. These results indicate that L. fermentum may be one of causative agents of dental caries in hyposalivated rats fed a sucrose diet.

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Susceptibility to pseudorabies virus (PRV) infection in mice, which were continuously depleted of natural killer (NK) cell activity by injection of anti-asialo GM1, was examined. Effects of serum thymic factor (FTS) on susceptibility of mice to PRV were also investigated. In mice with depleted NK cell activity, the mortality of PRV-infected mice was markedly high, whereas that of FTS-pretreated mice was significantly lower than the controls. Reduced susceptibility to PRV was demonstrated in mice treated with anti-asialo GM1 antisera before the PRV infection. Such a reduced susceptibility was not observed in mice inoculated with the antisera on day 1 post-infection (PI). To analyze the FTS-induced resistance to PRV infection, NK cell activity, macrophage activity, and interferon (IFN) productions were studied. Interferon production and NK cell activity were enhanced in the FTS-pretreated mice, suggesting that interferon may play an important role in this FTS-induced resistance to PRV infection.

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To determine the immunologic characteristics of T-cells in local pulmonary lesions of human T-cell lymphotropic virus type I (HTLV-I) carriers, we investigated lymphocyte surface markers in peripheral blood and bronchoalveolar lavage fluid (BALF) of 38 HTLV-I carriers, 8 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 44 HTLV-I seronegative patients with pulmonary diseases and 7 healthy volunteers using two-color flow cytometric analysis. In peripheral blood, activated T-cells, CD4+HLA-DR+, CD8+HLA-DR+ and CD3+CD25+, and CD4+CD29+ cells increased significantly in carriers and HAM/TSP patients compared with healthy volunteers and seronegative patients. In BALF, T-cells, especially CD25+ cells, increased significantly in carriers and HAM/TSP patients, compared with healthy volunteers and seronegative patients. These findings indicated that T-cells in the lungs, as well as in peripheral blood, are activated in carriers and HAM/TSP patients. Interestingly, there was dissociation between expression of CD3+CD25+ cells in BALF and peripheral blood from these patients. These results suggest that T-cells activated probably by HTLV-I accumulate in the lungs in some carriers and HAM/TSP patients, and HTLV-I may be involved in the immunologic dysfunction in the lungs of these patients. However, we did not find any correlation between the degree of clinical features and the elevation of CD3+CD25+ cells in BALF, or its characteristic features on chest roentgenograms.

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Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzymelinked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the Cε1, Cε2, Cε2/Cε3 junction and Cε3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (FcεRI and FcεRII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-FcεRII binding was clearly inhibited by the mAb recognizing the Cε2/Cε3 junction, suggesting that FcεRII binds to a rather limited area around the Cε2/Cε3 junction. The IgE-FcεRI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in Cε2 was blocked simultaneously with that at the Cε2/Cε3 junction or with that in Cε3, indicating that these three distinct epitopes are related to the FcεRI binding sites. When these three epitopes were shown in the stereograph of human IgE, the FcεRI binding area was spread largely on the groove side between Cε2 and Cε3 domains. These results suggest that FcεRI acquires the high affinity through multiple bindings.

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The effects of high-molecular-weight (HMW) hyaluronic acids (HAs) of 1.9×10⁶ Da, 8× 10⁵ Da and 3×10⁵ Da on the receptor-mediated functions of guinea pig peritoneal phagocytes were studied. HMW-HAs of 1.9×10⁶ Da (HA190) and 8×10⁵ Da (HA80) effectively inhibited the chemotactic activity of polymorphonuclear leukocytes (PMNs) for formyl-Met-Leu-Phe (fMLP). The degree of inhibition was dose-dependent and the concentrations of HA190 and HA80 required for 50% inhibition were 0.5-1.5mg/ml and 1.5-2.5mg/ml, respectively. HMW-HA of 3×10⁵ Da (HA30) hardly affected the chemotaxis within a concentration range of 0.5-5.0mg/ml. The phagocytic activities of PMNs and macrophages (Mφs) for serum-opsonized zymosan (SOZ) and polystyrene latex particles were also inhibited by these HAs in a dose- and molecular-weight-dependent manner and HA190 was again the most inhibitory. By contrast, the release of lysosomal enzyme from Mφs stimulated with SOZ was not significantly affected by HMW-HAs at any concentration used. Furthermore, the binding of [³H]fMLP with PMNs and the rosette formation of Mφs with SOZ were not influenced by the presence of HMW-HAs. These findings suggested that the binding of HMW-HAs to the HA receptors on PMNs and Mφs might produce certain intracellular signals which would be responsible for the suppression of the chemotaxis and the phagocytosis but not for the release of lysosomal enzyme. For the generation of such signals, higher-molecular-weight HMW-HAs would be more effective than lower one.

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The sensitivity and specificity of the polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis, were examined by the use of purified elementary bodies (EBs), cells having inclusions containing reticulate bodies alone and 20 clinical isolates. The numbers of EB and inclusion of C. trachomatis at the detection limit were determined to be approximately 2 to 4 EBs and one inclusion per assay, respectively. No reaction occurred for C. psittaci and C. pneumoniae. All clinical isolates were positively reacted in the PCR assay.

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