Nocardicin A is the antibiotic which was first found to possess a monocyclic β-lactam ring. This antibiotic was inactivated by the cleavage of its β-lactam ring. The direct spectrophotometric assay was applied to measure the rate of enzymatic hydrolysis of Nocardicin A. Nocardicin A was highly stable to both chromosomal and plasmid-mediated β-lactamases. Of the nine β-lactam antibiotics including cefoxitin and cefuroxime, Nocardicin A was the most stable to the β-lactamases tested excluding those from Klebsiella oxytoca and Proteus vulgaris. The latter broad-spectrum β-lactamases hydrolyzed Nocardicin A rather intensively. Extreme stability of Nocardicin A to β-lactamases was suggested to be due to the combination of its low affinity to the enzymes and stabilization of its monocyclic β-lactam ring. Nocardicin A was shown to have inducing ability toward β-lactamases.

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The present study using three isogenic mutants (F⁺P^-, F^-P⁺, F^-P^-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD₅₀) value for burned mice inoculated with non-piliated (P^-) mutant was at least ten times higher than those inoculated with piliated (P⁺) bacteria. Meanwhile the LD₅₀ value for burned mice inoculated with non-flagellated (F^-) mutant was at least 10⁵ times higher than those inoculated with flagellated (F⁺) bacteria. At 24hr after inoculation, the bacterial counts in burned skin of mice inoculated with P⁺ bacteria were ten times higher than those inoculated with P^- bacteria; and at 48hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24hr after inoculation, P⁺ bacteria were isolated from blood, liver (F⁺P⁺), lung (F⁺P⁺), and kidney, while P^- bacteria were not present in these tissues. And at 48hr after inoculation, P⁺ bacteria were isolated from all tissues, while P^- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.

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A simplified and rapid genetic identification method for Campylobacter species without radioisotope was established. Three different amounts of DNA (200, 50, and 12.5ng) extracted from each type strain of Campylobacter species with standard Marmur's procedure were spotted on a nitrocellulose filter. DNA obtained from one ml bacterial suspension at a concentration of McFarland standard turbidity No. 1 of Campylobacter fetus, C. jejuni, C. coli, and C. pylori isolates were sufficiently labeled with photo-biotin within 15min and clearly hybridized with the type strain of the corresponding species within four to six hours. Hybridized spots were visualized with alkaline-phosphatase-conjugated streptavidin color-detection method. The reaction was usually stopped within 30min. Atypical clinical isolates such as a nitrate-negative C. jejuni, two nalidixic acid-resistant C. jejuni, and two strains of C. fetus able to grow at 42C, which were tentatively identified as such, were definitely identified by the simplified DNA hybridization method presented here. This method will be applicable routinely for the definite identification of atypical strains of Campylobacter species and other gram-negative bacteria difficult to identify biochemically.

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The R-form lipopolysaccharide (LPS) from Escherichia coli K-12, from which cationic material had been removed by electrodialysis and the pH of which had fallen to 3.6, formed a rough hexagonal lattice structure with the lattice constant of about 19nm. The rough hexagonal structure was maintained in buffers at pH 5 or lower but disintegrated into the ribbon-like structures in buffers at pH 6 or higher. However, in the presence of 10mM Mg²⁺, the hexagonal lattice structure was not disintegrated even at alkaline pH levels but conversely it became more dense. At pH 8.3 to 8.9, the hexagonal lattice structure with the shortest lattice constant (15nm) was formed. The same optimal pH levels were obtained for formation of the dense hexagonal lattice structure (lattice constant, 14 to 15nm) by the electrodialyzed LPS from Klebsiella pneumoniae strain LEN-111 (O3-:K1-). The ability of Mg²⁺ to induce formation of the dense hexagonal lattice structure of the K-12 LPS depends upon the presence of buffers showing the optimal pH levels, since a very high concentration of Mg²⁺ such as 500mM was required for the lattice formation in distilled water. The amount of the magnesium bound to the K-12 LPS did not significantly differ throughout the pH range of 3 to 9. Therefore, the optimal pH range is another essential factor for formation of the dense hexagonal lattice structure of the LPS in addition to binding of the magnesium to the LPS.

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Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65nm×15-30nm with linkers of 50-60nm long and 6-7nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3. cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.

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We have established two monoclonal antibodies against B-L antigens (chicken Ia-like antigens). The specificity of the antibodies for B-L antigens was determined by two criteria, the cellular expression and the molecular structure of antigens with which they reacted. They reacted with antigens expressed on bursacytes, Con A-blast thymocytes, macrophages, and MDCC MSB1, but not with thymocytes and erythrocytes. In molecular basis, they recognized 64, 000 dalton glycoprotein consisting of two polypeptides, 35, 000 and 32, 000 dalton, which bound non-covalently. To investigate the distribution of B-L antigens on non-lymphoid cells of the bursa of Fabricius, which were thought to play important roles in the differentiation of B cells, anti-B-L antigen and anti-chicken immunoglobulin (Ig) monoclonal antibodies were used. B-L antigen-positive cells were detected in both cortical and medullary areas, whereas Ig-positive lymphoid cells were confined to the medullary areas of normal chicken bursal follicles. In the bursal follicles of cyclophosphamide (CY)-treated chickens, lymphoid cells were depleted but epithelial cells remained intact. And B-L antigen-positive but Ig-negative cells were easily detected in the medullary areas of almost all follicles. These cells were identified to be reticular epithelial cells (REp cells) from the result of their keratin expression.

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A monoclonal antibody, GPT-1, was prepared by fusion of the splenic cells of mice immunized with guinea pig thymocytes with a mouse myeloma cell line. GPT-1 completely inhibited spontaneous rosette formation of T cells with papain-treated rabbit erythrocytes. GPT-1 reacted with 90% of thymocytes, 70% of peripheral blood lymphocytes, and 45% of splenic lymphocytes, but not with B cells. These results indicate that GPT-1 has pan-T reactivity. The antibody specifically bound to a single polypeptide chain with a molecular size of 50-65kD. The surface density of the antigen was higher on thymocytes than on peripheral T cells, suggesting that the antigen is a certain differentiation antigen on T cells. Phytohemagglutinin-activated T cells expressed more antigen molecules than resting T cells. In addition, GPT-1 suppressed the proliferation of T cells induced by the mitogen, indicating that GPT-1 recognizes a T cell-specific surface antigen which is associated with T cell activation. Based on these results, it was concluded that GPT-1 reacts with a guinea pig T cell surface antigen which is similar to the E-receptor protein on human T cells (CD2 molecule).

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In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (FcγRs), one monospecific for IgG2 (Fcγ₂R) and the other bispecific for IgG1 and IgG2 (Fcγ₁/γ₂R), when analyzed by EA-rosette assay. These FcγRs on the cells were further studied by using two monoclonal antibodies toward the FcγRs on guinea pig peritoneal macrophages (anti-Fcγ₁/γ₂R and anti-Fcγ₂R antibody). The anti-Fcγ₁/γ₂R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fcγ₂R but not by anti-Fcγ₁/γ₂R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fcγ₂R and anti-Fcγ₁/γ₂R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-FcγR antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of FcγR, Fcγ₁/γ₂R, and Fcγ₂R, are expressed on the cells. The determination of these FcγRs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fcγ₁/γ₂R alone and 32% of the cells expressed both the FcγRs. On the other hand, about 12% of T lymphocytes was found to express Fcγ₂R alone and the cells expressing Fcγ₁/γ₂R were in the minority (3.8%). T lymphocytes expressing both the FcγRs were not detected. These results show that guinea pig B lymphocytes bear two types of FcγRs and are heterogeneous with regard to their FcγRs and that T lymphocytes express Fcγ₂R mainly.

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Four monoclonal antibodies were prepared against O4 antigen of Vibrio parahaemolyticus. All the antibodies were shown to be specific for O4 antigen by agglutination with heat-killed O-cells of the organism and precipitation with LPS preparations. The inhibition experiments of the precipitations with various sugars and oligosaccharides suggested that the combining sites of these hybridoma antibodies were directed to an antigenic determinant structure containing →3 and →6 linked D-glucose, D-galactose, and N-acetyl-D-galactosamine.

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A large plasmid in a virulent Shigella boydii 5 strain was transferred to plasmid-cured avirulent strains of S. boydii 5, S. boydii 12, S. sonnei form II, and Escherichia coli K12. The transconjugants acquired the ability to invade tissue culture cells, which indicated that the large plasmid in S. boydii is responsible for epithelial cell invasiveness.

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A simplified method for preparation of concentrated exoproteins including protein A and α-toxin produced by Staphylococcus aureus was successfully devised. The concentrated proteins were obtained by cultivating S. aureus organisms on the surface of a liquid medium-containing cellophane bag enclosed in a sterilized glass flask. With the same amount of medium, the total amount of proteins obtained by the method presented here was identical with that obtained by conventional liquid culture. The concentration of proteins obtained by the method, however, was high enough to observe their distinct bands stained on polyacrylamide gel electrophoresis. This method was considered quite useful not only for large-scale cultivation for the purification of staphylococcal proteins but also for small-scale study using the proteins. The precise description of the method was presented and its possible usefulness was discussed.

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Between March and August 1986 in Huangshi City, serum samples were collected from 316 apparently healthy barbers as a study group, as well as from 361 healthy employees of department stores as a control group. They were tested for hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs) and antibody to hepatitis B core antigen (anti-HBc) by enzyme-linked immunoadsorbent assays. Barbers showed a prevalence higher than that in controls for HBsAg (16.8 vs. 9.2%, P<0.01), anti-HBs (67.1 vs. 45.9%, P<0.001), and anti-HBc (39.2 vs. 21.2%, P< 0.001). The prevalence of at least one marker of hepatitis B virus (HBV) infection was significantly higher in barbers than in controls (86.1 vs. 61.7%, P<0.001). Although the socioeconomic status and education level did not correlate with the frequency of HBV markers, the prevalence of HBsAg increased in parallel with the duration of practice. Because of their high risk for HBV infection, barbers need to be screened for markers of HBV infection on a routine basis, and are prime candidates for immunoprophylaxis with hepatitis B vaccine.

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The virulence of five Sendai virus strains (MN, Z, KN, Mol, and Hm) isolated from laboratory rodents was compared, using 3-week-old female Jcl-ICR mice. The virulence of the strains was Mol, MN, KN, Z, and Hm in decreasing order. The 50% lethal dose and 50% lung consolidation inducing dose of the highest virulent strain differed by the order of more than 10³ and 10⁶, respectively, from those of the lowest virulent one. Other properties such as the growth rate in LLC-MK2 cells, neuraminidase activities, and molecular weights of structural proteins also differed among the virus strains. These results indicate that Sendai virus prevailing in laboratory rodents is not homogenous with respect to virulence and some other properties.

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