To identify the causative agent of canine ehrlichiosis that has occurred in the suburbs of Guangzhou, China, since 1998, the 16S rRNA gene was amplified and sequenced. Two sequences of 1, 482 and 1, 483 base pairs were obtained and named as Gzh981 and Gzh982, respectively. The level of similarity of these two was 91.50%, and Gzh981 closely resembled the 16S rRNA gene of Ehrlichia canis, whereas Gzh982 resembled Ehrlichia platys. We therefore conclude that E. canis and E. platys together caused recent outbreaks of canine ehrlichiosis in China.

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The involvement of the virulence plasmid (pSTV) of Salmonella typhimurium in human salmonellosis was examined. Most of the 224 clinical strains isolated from the blood (53) and nonblood samples (171) contained a 90kb or larger plasmid, most of which were pSTV. The rates of pSTV carriage in the isolates showed no statistically significant difference between those derived from the blood and those from other sources (87% vs. 83%; χ²=0.49, 0.1<P<0.9), suggesting that pSTV may not play a critical role in promoting S. typhimurium bacteremia in humans. Nine strains with representative plasmid profiles were tested for the mouse virulence. The result revealed that these clinical isolates contained all three virulent types known: the avirulent, the highly virulent when a pSTV was present, and the moderately virulent regardless of the presence or absence of pSTV. This indicated that mouse virulence of S. typhimurium did not correlate their virulence in humans. Clinical data showed that most patients with primary bacteremia had underlying immunosuppressive diseases, whereas only a few patients with secondary bacteremia had preexisting diseases (87% vs. 13%; χ²=22.73, P<0.005). It is suggested that the contribution of pSTV to S. typhimurium bacteremia in humans is likely to be limited, and both the host factor and the microbial virulence determinants on the chromosome are more important than virulence plasmid in predisposing patients to bacteremia.

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Human beta defensin 2 (HBD-2), the most recently discovered human defensin, has been considered to work as a host defense substance against microbial infection. Using Escherichia coli ATCC 25922, we investigated how some cations and anions influenced the antimicrobial activity of HBD-2. This activity, measured in 10mM phosphate buffer at a concentration of 20μg/ml, reduced significantly in the presence of 100 and 150mM sodium or potassium chloride. The reduction was not significantly different when the total amounts of sodium and potassium ions were equal. The kind and the valence of anions (chlorine and sulfate ions) did not affect the bactericidal activity as long as the concentrations of sodium ions were equal. Divalent ions (calcium and magnesium ions) added to 10mM of Tris buffer significantly inactivated HBD-2 at much lower concentrations (more than or equal to 0.01mM and 0.05mM, respectively) than the monovalent ions did. These findings suggest that HBD-2 kills the bacteria through at least two phases, which are affected independently by either monovalent or divalent ions and unaffected by anions.

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Two tandem genes encoding glucosyltransferase synthesizing water-soluble glucan (GTF-S) were cloned from the lambda gene library of Streptococcus cricetus HS-6 (serotype a) using anti-GTF-S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C-terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF-S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.

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We developed the antigen capture enzyme-linked immunosorbent assay (ELISA) systems for quantification of Borna disease virus (BDV) major antigens, p40 and p24. Using these ELISAs, we quantified the two proteins in various BDV-infected materials, including the cell lysates and culture supernatants as well as the homogenates of experimental animal brains. The ELISAs were also applied to measure the infectious titer of BDV in persistently infected cell lines. Quantitative analysis with these ELISAs allowed us to measure the molecular ratio between the p40 and p24 in infected samples. Interestingly, the ratio of p24 to p40 in persistently infected cells was much higher than that observed in acutely infected cells although the ratios in the supernatants from both cell lines were quite similar. BDV-inoculated gerbil brain cells showed a relatively high ratio of p24 to p40 as compared with acutely infected cells. These observations suggested that the molecular ratio between the proteins strongly depended on the infectious status of BDV in the host cells. The ELISA system developed here could be a convenient method for the quantification of BDV infection and may also be beneficial for understanding viral replication and infectious status in the host cells.

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Colonization by Helicobacter pylori partly depends on acid-dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme-based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.

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Bacteroides forsythus produces species-specific major proteins with high molecular weights of 270 and 230-kDa (270K and 230K). A specific antibody raised against 270K was used for Western blot analysis and immunoelectron microscopy. Western blot analysis showed that the 270K and 230K proteins were immunologically similar. Immunogold labeling of ultrathin-sectioned bacterial cells and biochemical fractionation revealed that these proteins were localized at the outermost cell surface, not in the cytoplasm. These results suggest that major proteins ubiquitous to this species may form the S-layer.

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To evaluate the seropositivity of Chlamydia spp. in human immunodeficiency virus (HIV)-infected subjects in Japan, Chlamydia-specific antibodies in sera collected from 106 HIV-infected subjects were measured by the microimmunofluorescence test. The prevalence of C. pneumoniae-specific IgA, C. trachomatis-specific IgG and IgA and mean titers were significantly higher in the homosexual and heterosexual HIV-infected subjects than in the hemophilic patients and HIV-negative controls. These data indicate that the higher C. pneumoniae and C. trachomatis seroprevalence among HIV-infected subjects is probably due to an HIV risk factor, such as promiscuous sexual behavior, rather than to HIV infection itself.

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We cloned a protease gene of Aeromonas sobria and determined its nucleotide sequence. The protease is composed of 624 amino acid residues and its calculated molecular weight is 66, 737.7. The amino acid sequence showed the characteristic features of a bacterial serine protease. We expressed the protease gene in Vibrio parahaemolyticus from which the synthesized protease is secreted into the culture medium as the mature form, and purified the mature protease by successive column chromatographies. The size of the mature protease is 65, 000 daltons and the amino acid sequence analysis revealed that a 24-amino acid peptide at the amino terminal of the precursor is removed from the mature protease. This peptide might function as a signal peptide in translocation across the inner membrane. Subsequently, we found that the protein, designated ORF2 protein, encoded by the gene lying adjacent to the 3' end of the protease gene plays an important role in production of the protease. Mutation of the ORF2 gene did not affect transcription of the protease gene, but resulted in degradation of the protease in the cell. This shows that ORF2 protein is required for the successful production of the serine protease by cell.

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Glycyrrhizin, an aqueous extract of licorice root, has anti-inflammatory activity and has been used for the treatment of chronic viral hepatitis. In the present study we describe the mechanism by which glycyrrhizin inhibits complement. Glycyrrhizin inhibited the cytolytic activity of complement via the activation of both the classical and alternative pathways, while it had no effect on immune adherence, suggesting that it blocks C5 or a later stage of the complement cascade. Further analysis revealed that glycyrrhizin inhibits the lytic pathway in which the membrane attack complex (MAC) is formed. This mechanism suggests that glycyrrhizin may prevent tissue injury caused by MAC not only in chronic hepatitis but in many autoimmune and inflammatory diseases.

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