In this study, pulsed-field gel electrophoresis (PFGE) was applied to determine if the Vibrio cholerae O1 strains which reappeared after being temporarily displaced in Calcutta by the O139 serogroup were different from those isolated before the advent of the O139 serogroup. NotI digestion generated a total of 11 different patterns among the 24 strains of V. cholerae randomly selected to represent different time frames. Among the V. cholerae O1 strains isolated after July 1993, 4 PFGE banding patterns designated as H through K were observed with pattern H dominating. Pattern H was distinctly different from all other patterns encountered in this study including patterns A, B and C of V. cholerae O1 El Tor, which dominated before November 1992, and pattern F, which was the dominant V. cholerae O139 pattern. Further, pattern H was also different from the NotI banding patterns of the representative strains of the 4 toxigenic clonal groups of V. cholerae O1 El Tor currently prevailing in different parts of the world. NotI fragments of the new clone of V. cholerae O1 did not hybridize with an O139 specific DNA probe, indicating that there was no O139 genetic material in the new clone of V. cholerae O1. Hybridization data with an O1-specific DNA probe again differentiated between the clones of V. cholerae O1 existing before the genesis of the O139 serogroup and the O1 strains currently prevalent.

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Although chemotherapy has been documented to be effective in the treatment of Helicobacter pylori-associated gastritis and gastroduodenal ulcers, some cases are known to have been unsuccessful in the attempt to eradicate this species. In this study, we examined the relation between the susceptibility of H. pylori isolates and the efficacy of chemotherapy. We utilized the modified agar plate dilution method to determine the minimum inhibitory concentrations (MICs) of 63 H. pylori strains isolated before treatment with several drugs routinely used during eradication chemotherapy. Among the drugs tested, amoxicillin (AMPC) and clarithromycin (CAM) demonstrated high degrees of activity with MIC₉₀, 0.39 and 0.2μg/ml, respectively. No highly resistant strain against AMPC was detected among the strains examined, while for CAM and metronidazole (MTZ), 9.5% and 7.9% of the strains, respectively, were resistant before treatment. It should be noted that all of the MICs of the strains from patients with successful therapy were lower than those from patients with unsuccessful therapy. These findings indicate that susceptibility tests should be carried out prior to the commencement of drug administration in order to provide safer and more effective chemotherapy.

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The internal organs of Ixodes ovatus and the ears of wild rodents (Apodemus speciosus, Eothenomys smithii) and an insectivore (Crocidura dsinezumi) were cultured to isolate borreliae; positive samples were examined for the distribution and dissemination of spirochetes in the host tissues using electron microscopy. Seven isolates were derived from the unfed ticks and the three species of mammals. These isolates were identified as Borrelia japonica judging from the outer surface protein profile using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity to a B. japonica-specific monoclonal antibody. Borreliae were found only in the midgut lumen of the tick in close contact with the microvilli on the midgut epithelium; on the other hand, borreliae found in the ears of the mammals existed freely in the collagenous intercellular substances of connective tissues or in close contact with fibrocytes. The ultrastructural disparities between the borreliae in ticks and mammals appeared to correspond to differences in motility. Interestingly, the borrelia which invaded through the perineurium appeared to contact the basement membrane of a Schwann cell that enclosed several nonmyelinated nerve fibers. This may offer important information regarding the involvement of the nervous system in Lyme disease.

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The Porphyromonas gingivalis fimbriae level was examined in the gingival crevicular fluid (GCF) from adult human subjects using an immunoblot assay with a monoclonal antibody. The cytokines, interleukin-1α (IL-1α), IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels in the GCF were quantified by enzyme-linked immunosorbent assay (ELISA). The reactivity of the GCF samples with the monoclonal antibody against P. gingivalis fimbriae was related to the IL-1β, IL-6 and TNF-α levels. Moreover, the fimbriae content was associated with the gingival index (GI). In contrast, no significant correlation was seen between the fimbriae content and IL-1α level. These results suggest that there are possible associations between P. gingivalis fimbriae and IL-1β, IL-6 and TNF-α in the gingival crevicular fluid.

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Through the adoption of a chiral stationary phase in high-performance liquid chromatography and a simple derivatization method for hydroxy fatty acids, it became easy to separate and identify the optical isomers of 2- and 3-hydroxy fatty acids composing several kinds of microbial lipids. The 2- and 3-hydroxy fatty acids were converted with dinitrophenyl isocyanate to their 3, 5-dinitrophenyl urethane derivatives (DU-derivatives), which were analyzable by HPLC using a chiral column. By varying the composition of an eluent, separation of the DU-derivatives of hydroxy fatty acids differing in optical configuration, chain length and position of hydroxyl group was achieved. The general elution orders of these DU-derivatives were determined with authentic 2- and 3-hydroxy fatty acids. Small amounts (-300μg) of ornithine-containing lipids isolated from the Serratia marcescens strains were examined by this method to identify 3-hydroxy fatty acids of the lipids as D isomers.

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We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.

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The genes encoding the glycoproteins H (gH) and L (gL) of human herpesvirus 7 (HHV-7) have been identified. The gH open reading frame (ORF) was 2, 070 base pairs in length and encoded a predicted 690 amino-acid protein. The gH contained characteristics of a transmembrane glycoprotein including 10 consensus N-linked glycosylation sites, 12 cysteine residues, a potential amino-terminal signal sequence and a predicted transmembrane segment located near the carboxyl terminus. The gL ORF was 738 base pairs in length and encoded a predicted 246 amino-acid protein. Four possible N-glycosylation sites and 6 cysteine residues existed within gL. The predicted amino-acid sequences of the HHV-7 gH and human herpesvirus 6 variant A (HHV 6A) gH gene products exhibited 23.6% identity to each other, and those of the gL gene products had 26.0% identity. Upon in vitro translation of the gL gene, the addition of microsomal membranes resulted in two modified products with molecular weights of 32kDa and 35kDa from the unmodified initial translation product of 26kDa. An amino-terminal portion of gH and the full length of gL were expressed as glutathione S-transferase fusion proteins, and these proteins were used to raise immune sera in mice. Lysates of cells infected with HHV-7 were subjected to immunoprecipitation analysis. Approximate molecular weights of 33, 37, 80 and 90kDa polypeptides were immunoprecipitated with antibodies against the gH protein. Antibodies against the gL protein polypeptides with the same molecular weights were also precipitated, and were observed with the antibodies against the gH protein. These results suggest that HHV-7 gH and gL may form a heterodimeric complex with each other in HHV 7 infected cells, as has been reported for other herpesviruses.

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The selective induction of effector functions of a T-cell clone (DB14), specific to pigeon cytochrome c 43-58 (p 43-58) and restricted to I-A^b, was analyzed using a professional antigen-presenting cell, B hybridoma (Th 2.58), and various non-professional antigen-presenting cells (APC), L cells transfected with I-A^b (I-A^b L cells), a medullary thymic epithelial cell line (m-TEC) and a cortical thymic epithelial cell line (c-TEC). The m-TEC and c-TEC expressed I-A^b upon induction with interferon γ (IFN-γ). When stimulated with p 43-58 in the presence of I-A^b L cells as well as Th 2.58 cells, the DB14 cells showed marked proliferation and, after 18hr of culturing, exhibited significant cytotoxicity against the APC. By contrast, in the presence of m, c-TEC, the DB14 cells showed neither proliferation nor cytotoxicity against these TEC but exhibited considerable detachment activity towards them. Furthermore, DB14 cells became expressed activation markers (CD69 or CD44) following stimulation with p 43-58 plus m-TEC or c-TEC. The addition of rIL-2 to the culture of DB14 cells, p 43-58 and m-TEC or c-TEC, restored the proliferative responses. However, it was shown that anergy was not involved in the negligible proliferative responses of DB14 cells after stimulation with p 43-58 plus m, c-TEC. The present findings indicate that differences in APC functions are present among non-professional APC and suggest that the selective induction of T-cell functions can be achieved using the appropriate non-professional APC. The characteristic activation of T cells by TEC may be related to their functional roles in situ.

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Some viruses seem to be capable of suppressing interferon (IFN)-induced 2', 5'-oligoadenylate synthetase (2-5AS) induction. Cells infected with human T-lymphotropic virus type-I (HTLV-I) show different natures for the constitutive production of IFN-γ or sensitivity to IFN. Poor induction of 2-5AS was found in IFN-γ producer cells carrying HTLV-I (MT-1, MT-2 and SMT-1). On the other hand, in non- or low-producing cell lines of IFN-γ such as HUT102 and OKM-2, significant activity of 2-5AS was induced by treatment with IFN-α. A satisfactory level of IFN receptor was detectable in SMT-1 cells in spite of the poor induction of 2-5AS. There were no differences in either the interferon regulatory factor-2 (IRF-2) mRNA transcript or the level of STAT-1α between SMT-1 and HUT102 cells. However, the transcription of IRF-1 mRNA was slightly reduced in SMT-1 cells as compared with that of HUT102 cells.

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A monoclonal antibody (MoAb) to human neutrophils, designated 3H9, was established by screening for the inhibition of neutrophil adherence to plastic plates containing a medium supplemented with fetal calf serum (FCS medium). The antigen recognized by 3H9 was shown to be present on human leukocytes and found at the highest levels on granulocytes. On Western blotting, 3H9 reacted with a molecule having a molecular weight of 80kDa. When this MoAb was added at the same time as a neutrophil stimulant (fMLP), the inhibition of neutrophil adherence to plastic plates in the presence of FCS medium was observed after 60min incubation. Furthermore, this MoAb enhanced not only fMLP-induced chemotaxis but random migration of neutrophils as well. The mechanisms of these phenomena are discussed.

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