The 16S rRNA primary structure of Eubacterium fossor was determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that E. fossor has a specific phylogenetic association with the Atopobium species and is far from E. limosum, the type species of the genus Eubacterium. Phenotypic characters of E. fossor resemble those of the genus Atopobium. Therefore, we propose that E. fossor should be transferred to the genus Atopobium as Atopobium fossor comb. nov.

View full abstract

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72hr at 35C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration> 8mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.

View full abstract

We examined whether anti-Lewis x (Le^x) and y (Le^y) autoantibodies affect the pathogenesis of Helicobacter pylori-induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti-Le^x and -Le^y immunoglobulin G (IgG) antibodies, and 1 patient had only anti-Le^x antibody. After successful eradication, we measured the serum titer of anti-Le^x and -Le^y antibodies. Six patients had a reduction of the titers of anti-Le^x and/or -Le^y antibodies, whereas no notable changes were detected in 5 patients in the follow-up. This result suggests that anti-Le^x and -Le^y autoantibodies had no critical role in the development of H. pylori-induced peptic ulcer.

View full abstract

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNFα and IL-1β. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNFα or IL-1β production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNFα or IL-1β resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNFα and IL-1β by human adherent AM. Neutralization of TNFα or IL-1β did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5μg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.

View full abstract

A highly conserved 40-nucleotide sequence was identified. Two completely conserved sequences, TAGATT and TAAACT, separated by 17 nucleotides resemble the consensus sequence recognized by the Escherichia coli major sigma factor and sequence found in other chlamydial promoters. In addition, the adenine-rich sequence present in many chlamydial promoters was also conserved upstream of the putative-35 element. These findings suggest that the conserved sequence may play a role in the regulatory function at the transcriptional level. Multiple ATG codons were found at the 5'-terminal region of the chlamydial sigA ORFs except for Chlamydia pneumoniae, although the putative Shine-Dargarno sequence was absent.

View full abstract

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV-6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL-2 and the cells harbor HTLV-I genomes. A severe cytopathic effect (CPE) was observed in many HHV-6B(OK)-infected TaY cells one week after infection. The release of virus from HHV-6B(OK)-infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV-6B(OK) in the supernatant was comparable to the value of 10^3.5 TCID₅₀/ml obtained with PHA-activated cord blood lymphocytes (CBL) that had been infected with HHV-6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus-type capsid- and enveloped-viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV-6A and the Z29 strain of HHV-6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4⁺ T lymphocytes with mitogens such as PHA or IL-2 and the expression of some cellular gene or the HTLV-I gene might be essential for efficient propagation of HHV-6B. TaY cells should play an important role in future investigations of cell-virus interactions and genetic variations or cell tropism of HHV-6 isolates since no cell line that shows propagation of both HHV-6A and HHV-6B has been reported to date.

View full abstract

Vpr, one of the accessory molecules of HIV-1, has been demonstrated to arrest the cell cycle at the G₂ phase. This Vpr-mediated cell cycle arrest is implicated to have an important role in the viral life cycle. In the present study, we quantitate the extent of Vpr-mediated cell cycle arrest with the use of a bicistronic vector consisting of a vpr gene and a green fluorescence protein sequence. Using this system, we examined the effect of several Vprs on cell cycle progression and growth of cells from different species quantitatively. We found that Vpr from the T-cell line-adapted HIV-1_SF2 strain (Vpr2) could not significantly induce G₂ arrest in HeLa cells but was able to induce it in 293T cells. However, strong inhibition of cell proliferation in HeLa cells as well as in 293T cells was observed by Vpr2. This ability of Vpr2 to inhibit cell proliferation without G₂ arrest was also observed when expressed in monkey cell line. Analyses of chimeric Vprs revealed that this species-non-specific growth inhibitory activity of Vpr was not mediated solely by the C-terminal region of Vpr. These results indicated that the growth inhibitory activity of Vpr is independent of its G₂ arresting activity. In addition, the species-non-specific nature of this activity suggests that Vpr has a novel mechanism to retard cell proliferation by influencing basic cellular functions.

View full abstract

We reported previously that the rabies virions contained a 21-kDa cellular transmembrane protein (referred to as VAP21) as a minor component (Sagara, J. et al, Microbiol. Immunol. 41(12): 947-955, 1997). In this study, we further examined the possible interactions of VAP21 with other enveloped viruses, including the vesicular stomatitis virus (VSV; negative-stranded RNA virus), Sindbis virus (positive-stranded RNA virus) and herpes simplex virus type 1 (HSV-1; double-stranded DNA virus). An immunoblot analysis demonstrated that all of these enveloped viruses contained VAP21 in the virion as a minor component. Immunoprecipitation studies suggested that VAP21 was associated with certain viral proteins in the cell, such as the matrix (M) protein of VSV, a capsid protein of, Sindbis virus and at least a capsid protein (VP5) of HSV-1. The association was disrupted by treatment with 0.5% sodium dodecyl sulfate, but resistant to the treatment with 1% NP-40 plus 1% deoxycholate. These results suggest that: 1) VAP21 is not primarily associated with the viral transmembrane glycoprotein but rather with the internal viral protein, and, 2) this association would cause the efficient incorporation of VAP21 into the virion.

View full abstract

Liver injury was induced after infection with Salmonella choleraesuis 31N-1. In T-cell receptor-δ knockout mice, serum alanine transferase level was significantly decreased in comparison with normal control mice after Salmonella infection. On the contrary, in vivo administration of anti-γδ T-cell receptor monoclonal antibody (UC7-13D5) to stimulate γδ-T cells in infected mice significantly increased serum alanine transferase level but decreased bacterial growth compared with infected mice given control antibody (UC8; hamster IgG). These data suggest that γδ-T cells have effector activities not only for protection but also for liver injury during Salmonella infection.

View full abstract

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bag1, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g. HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.

View full abstract

We describe here the identification of sodC gene from enteroaggregative Escherichia coli (EAggEC). A 294bp gene-specific fragment was amplified from the organism by DNA as well as RT-PCR using primers from bacterial sodC sequences. The metal co-factor present in the protein was confirmed by running samples in native gels and inhibiting with 2mM potassium cyanide. However, the nonpathogenic E. coli possesses the gene but does not express it. Thus, the presence of copper-zinc superoxide dismutase encoded by sodC was demonstrated for the first time in EAggEC, which means it could be a novel candidate for a virulence marker.

View full abstract

A PCR technique was used to detect and identify Treponema socranskii associated with periodontitis. A species-specific forward primer was designed for a variable region within its 16S rRNA gene and was used in conjunction with a conserved reverse primer. This primer pair was tested for specificity against 44 oral bacterial strains. Sensitivity was determined using a serial dilution of T. socranskii cells. Amplification products were obtained from all T. socranskii strains tested, but not from other oral bacteria associated with periodontal disease. The detection limit of PCR was 5 T. socranskii cells per PCR. T. socranskii was detected by PCR in subgingival plaque and saliva samples from patients with periodontitis.

View full abstract

Normal mice inoculated intravenously with 50μg trehalose-6, 6'-dimycolate, a glycolipid component of the cell wall of Mycobacterium, in an oil-in-water emulsion (TDM emulsion) acquired a high resistance to intranasal lethal infection of an influenza virus. In contrast, TDM emulsion-treated T-cell receptor δ gene mutant (TCR δ^-/-) mice acquired insufficent resistance against the lethal influenza virus infection. The patterns of insufficient resistance were identical to the results obtained previously with mice which were depleted of T-lymphocytes bearing γδ T -cell receptors (γδ T-cells) by in vivo administration of anti-γδ T-cell receptor monoclonal antibody (Hoq et al, J. Gen. Virol. 78: 1597-1603, 1997). These results strongly suggest that the γδ T-cells play an important non-specific role in resistance against influenza virus infection.

View full abstract