Methods for isolation of fecal 7α-dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7-dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C-7α and/or C-7β. All the isolates were strictly anaerobic, Gram-positive rods. Thirteen isolates were non-sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase-positive but indole-, lecithinase-, urease- and oxidase-negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7α-dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C-7α. Nine strains (10, 12, 36S, M-2, M-17, M-18, Y-98, Y-1112, and Y-1113) of the 7α-dehydroxylating bacteria were confirmed to have 7β-dehydroxylation ability, but five strains (O-51, O-52, O-71, O-72, and Y-67) could not transform ursodeoxycholic acid to lithocholic acid.

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Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for β-isopropylmalate dehydrogenase (leuB).

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Carbapenem-hydrolyzing β-lactamase from Serratia marcescens FHSM4055 was purified 926-fold by means of carboxylmethyl Sephadex C-50, Sephacryl S-200, and Mono S column chromatography. The molecular weight was 30, 000 by SDS-PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p-chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo-β-lactamase and that the SH-group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (k_cat/K_m) showed that the enzyme hydrolyzed various β-lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino- and imino-groups, ceftazidime with a carboxypropyloxyimino-group, and cefclidin with a carbamoylquinuclidine-group were poor substrates among the β-lactam antibiotics other than the monobactams tested. The plots of the turnover number (k_cat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK₁, 5.9; pK₂, 9.0; pK₃, 10.8), whereas those of k_cat/K_m gave a bell-shaped curve (pK₁ 5.8; pK₂, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N-terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71-78), the above enzymatic characteristics seem to coincide.

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A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse-transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)-PCR followed by nested PCR. In 5-week-old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76-118 (1.3×10⁵FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post-inoculation (p.i.) by nested PCR and virus-isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods. The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1:203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.

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A murine monoclonal antibody recognizing (1→6)-β-D-glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low-molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.

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The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4⁺ and CD8⁺ T cells and an increase of activated T cells expressing IL-2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-γ plays an important role in the protection of mice from P. carinii infection.

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Production of cholera toxin (CT) in AKI medium and conservation of CT gene (ctx) of 49 strains of Vibrio cholerae O1 were compared by reversed passive latex agglutination (RPLA) and polymerase chain reaction (PCR). The production of CT agreed with conservation of the ctx in 48 out of the 49 strains. Ten strains were positive, and 38 strains were negative by both methods. Only one strain was negative in RPLA and positive in PCR. This suggested that the combination of AKI-SW and RPLA is comparable to PCR to identify CT-producing V. cholerae O1.

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The sera from two patients with murine typhus reacted with whole cells of Rickettsia prowazekii, R. typhi, and Proteus vulgaris OX19, and with lipopolysaccharides (LPS) from the spotted fever group rickettsia strain TT-118 and P. vulgaris OX19 in the enzyme-linked immunosorbent assay. Sera from these patients reacted with ladder-like bands of LPS from R. prowazekii and R. typhi in the immunoblot, whereas the reactivity of these sera with LPS from P. vulgaris OX19 differed from each other. These results indicate that LPS from the typhus group rickettsiae and P. vulgaris OX19 contain similar epitopes.

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Exocellular proteases produced by Vibrio fluvialis, V. furnissii, V. metschnikovii and V. campbellii were characterized and compared to those of V. mimicus protease (VMP) and V. vulnfcus protease (VVP). These proteases possessed both elastolytic and hemagglutinating abilities and were identified, except that of V. metschnikovii, as metalloprotease. Conversely, V. metschnikovii protease failed to exhibit some of the salient features for metalloproteases suggesting the existence of protease(s) other than metalloprotease. However, antibodies against VVP cross-reacted to these proteases and to VMP indicating antigenic relatedness amongst vibrio proteases. This study, thus, demonstrated the prevalent distributions of antigenically related proteases both in pathogenic and non-pathogenic vibrios, bringing their status as a virulence determinant into question.

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The purpose of this study in mono-infected gnotobiotic BALB/cA and C3H/HeN mice was to evaluate the cariogenicity of Enterococcus faecalis. The caries incidence and mean caries score in the BALB/cA mice were significantly higher than those in the C3H/HeN. In both of the mouse strains, the mean number of E. faecalis isolated from the cecum content was almost the same, however, the mean number of E. faecalis from the maxilla of BALB/cA was significantly higher than that of C3H/HeN. These results indicate that C3H/HeN has some factors that prevent E. faecalis from attaching to the tooth surfaces.

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The gene encoding cytochrome c-553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c-553 was synthesized as a precursor protein with an NH₂-terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF-4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.

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To evaluate the relationship between the sugar chain structure and biological activity, fibroblast-derived glycosylated human interferon-β, Chinese hamster ovary cell-derived glycosylated recombinant human interferon-β and Escherichia coli-derived unglycosylated recombinant human interferon-β were evaluated using human hepatoblastoma cells in vitro. Native fibroblast interferon-β expressed more cell-growth inhibitory action, 2'5'-oligoadenylate synthetase induction, and the inhibition of hepatitis B virus DNA replication than its asialoform and two recombinant interferon-βs. These results showed that the sugar chain structure of human interferon-β affects its biological activity on human hepatoblastoma cells.

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