To observe the possible serological heterogeneity of compact-colony-forming active substance (CCFAS), heat-killed vaccines were prepared by two strains of Staphylococcus aureus, strains SMU 1-46 and SMU 7931, cultured in 0.03M tris-hydrochloride-buffered brain heart infusion, pH 8.4. After immunization with the vaccine in rabbits, antibody responses were observed during a period of six weeks after the immunization either by homologous and heterologous organisms using alkaline serum-soft agar technique. The results showed that remarkable antibody production was shown only against homologous strain, but not against heterologous strain. The antibodies were absorbed out only with highly purified preparation of CCFAS extracted from homologous strain and not with heterologous CCFAS. Differences of the major chemical composition of the substances showed that highly purified CCFAS extracted from strain SMU 7931 contained 2.84 and 2.04 times higher amounts of galactose and 2-amino-2-deoxy-D-galacturonic acid than those of CCFAS obtained from strain SMU 1-46.

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To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.

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Mycoplasma salivarium ATCC 23064 and 24 other oral strains, and Mycoplasma orale ATCC 15539 and 22 other oral strains were examined quantitatively for attachment to glass surfaces by using ELISA method. Although all of the tested strains attached to glass surfaces, M. salivarium attached far more readily than M. orale. The results suggested that electrostatic bonds were involved in the attachment and that bivalent metal ions also played a role.

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We examined the immunochemical structure of the antigenic determinant of S. cerevisiae serotype Ia. The specific factor serum for S. cerevisiae serotype Ia was obtained either from factor 18 serum by adsorption with heat-killed cells of Candida glabrata, or from anti-S. cerevisiae Ia (M 6001) serum by adsorption with heat-killed cells of S. cerevisiae Ib (IFO 0751). We designated this adsorbed serum as factor 18a. Acetolysis of S. cerevisiae cell wall D-mannan gave five oligosaccharides. Signals of ¹H-nuclear magnetic resonance spectra of mannooligosaccharides derived from S. cerevisiae mannan were assigned for their linkages by the aid of those of α-1, 3'-linked mannooligosaccharides derived from glucuronoxylomannan of capsule of Cryptococcus neoformans serotype A-D. Agglutination-inhibition experiments revealed that the mannopentaose from S. cerevisiae mannan was the most effective inhibitor. Moreover, inhibitory activities of α-1, 3'-linked mannotriose, mannotetraose, and mannopentaose which were derived from glucuronoxylomannan of C. neoformans were shown to be higher than those of mannotetraose with one terminal α-(1-3) linkage from homologous S. cerevisiae mannan. These results indicate that mannopentaose with terminal two α-(1-3) linkages is responsible for the specificity of S. cerevisiae Ia.

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Intrauterine infection of murine encephalomyelitis virus GDVII was investigated by the indirect immunofluorescence method. Sixty-day-old pregnant mice were inoculated with 5 gradated doses of the virus ranging from 0.001 to 1.5 MLD₅₀ via i.v. route on the 10th day of gestation. The mice exsanguinated on postinfection day 8 showed high incidences of viral antigen in the uterine walls, placentas, and fetal subcutaneous tissues with doses of 0.1-1.5 MLD₅₀, regardless of the presence of maternal symptoms. Incidence of viral antigen-positive cells in fetal brains was high with a dose of 0.1 MLD₅₀, but not by the other doses. However, the brains of the stillborn and newly-born mice derived from females infected by a dose of 0.5 MLD₅₀ brought about still higher detection rates of viral antigen, as well as in the postpartum uteri. In effect, transplacental transmission of the virus was clearly demonstrated, and appeared to be dose-dependent.

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Synthesis and localization of virus-specific RNA in cells infected with Japanese encephalitis virus (JEV) were examined. To prepare specific RNA probes, we constructed four kinds of plasmids which contained DNA fragments corresponding to JEV genomic RNA. Minus probes, JT18V and JT19III, transcribed by T7 RNA polymerase were able to recognize a negative strand of JEV-specific RNA synthesized in cells as early as 6hr postinfection (p.i.). In the experiments using a plus-strand probe JT19V to hybridize the 3' end of JEV-RNA, not only full-length 42S(+) RNA but also 10S(+) RNA were detected in the infected cells at 24hr p.i. The positive-strand 42S RNA was found in much greater abundance in the membrane fraction than in the supernatant fraction of the infected cells. In contrast, larger amounts of the negative-strand RNAs existed in the supernatant fraction. It is suggested from the data that the JEV-specific negative- and positive-strand RNAs accumulate at different sites in the infected cells.

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In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987)), the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.

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For detection and identification of enteric adenovirus (Ad) types 40 and 41 in stool specimens, enzyme-linked immunosorbent assay (ELISA) was developed with the use of three monoclonal antibodies: Ad group-specific, Ad40 type-specific, and Ad41 type-specific antibodies. Of 860 fecal samples from patients with acute gastroenteritis, 44 strains of Ad were isolated using Graham 293 cell cultures. Of these isolates, 20 were typed as Ad40, 18 were Ad41, and 6 were other Ads by neutralization tests with cell cultures. Results of the ELISA tests on these 860 fecal samples resulted in good agreement to those with the cell culture method. The ELISA tests using Ad type-specific monoclonal antibodies proved to be a specific and rapid technique for laboratory diagnosis of acute gastroenteritis caused by enteric Ads.

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A DNA fragment encoding a hemolytic factor was cloned from the parasitic spirochete Leptospira interrogans serovar autumnalis strain Congo 21-543. Initial clones were isolated by screening a genomic library in pBR322 in Escherichia coli for hemolytic activity. Hemolytic activity was coded by a 4.5 kilobase BamHI-HindIII fragment. Southern hybridization with DNAs from other strains of Leptospira using this gene as a probe showed that DNAs from non-parasitic strains failed to hybridize with the probe, whereas those from all parastic strains tested had the sequence which hybridize to the probe.

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