The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum flagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1, 131 nucleotides with a partial calculated flagellin molecular mass of 40, 143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.

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The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen-dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S. typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella. The contribution of pSTV to humam salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.

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The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B. cereus. The addition of high levels (50mM) of leucine, isoleucine and glutamlc acid decreased the toxin production. Other amino acids had no effect at this concentration.

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The heat-shock proteins are coded for the polycistronic operons hsp70 and hsp60 in Staphylococcus aureus. The hsp70 operon is comprised of five genes, hrc37, hsp20, hsp70, hsp40 and orf35, and the hsp60 is comprised of two genes, hsp10 and hsp60. The hsp70 operon transcribed five different sizes of mRNA from three promoters: P1, the most active promoter, transcribed 6.0 and 3.6kb mRNAs; P2 transcribed a single 1.8kb mRNA; and P3 transcribed 4.2 and 2.4kb mRNAs. The hsp60 operon transcribed a single 2.1kb transcript from only one promoter, P1. Both operons had a common structure of inverted repeat element (CIRCE, Controlling Inverted Repeat of Chaperon Expression) at the promoter region. All of the transcripts were heat (46C) inducible. One of the unidentified genes, hrc37, was characterized. The disruptant of the hrc37 in the hsp70 operon enhanced the transcription of both operons at 37C (derepression). Complementation of the disruptant with the cloned hrc37 plasmid recovered the repression of the transcription of both operons at 37C. The product of hrc37, Hrc37, was found to bind to the CIRCE element. These findings indicated that Hrc37 from the hsp70 operon repressed the transcription of both the hsp70 and hsp60 operons by binding to the CIRCE element located at the promoter region.

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The sequence at the amino terminus region of the hemolysin of Aeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser-446 and Ala-447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild-type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.

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The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5μm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain VIO578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V. parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.

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An experimental model for autoimmune enterocolitis was produced in mice by repeated immunization of homologous colon extract together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes in the intestinal lesions were characterized by infiltration with polymorphonuclear leukocytes in the lamina propria, muscularis mucosae and submucosa of repeatedly immunized mice. No such intestinal lesions were produced in mice receiving injections of colon extract alone or KO3 LPS alone. Development of the autoantibody and delayed-type hypersensitivity against colon extract were found in mice immunized with the mixture of colon extract and KO3 LPS. Distinct positive staining was detected specifically on the columnar epithelium of villi. Sera from hyperimmunized mice defined organ-specific antigens present in the intestine. Therefore, it was suggested that the intestinal lesions might be caused by an autoimmune mechanism.

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A human monocytic cell line, THP-1-S, was cultured in a serum-free medium. The effect of the culture supernatant of THP-1-S on the cytotoxicity of rTNF-α to three kinds of cell lines and the binding of rTNF to its receptor were tested. The supernatant inhibited the cytotoxicity of rTNF-α when tested by the neutral red uptake method. In addition, the supernatant blocked the binding of ¹²⁵I-rTNF-α to its receptor. Furthermore, following precipitation with PEG we detected complexes between rTNF-α and the inhibitory factor which formed during incubation with the culture supernatant from THP-1-S cells. However, the supernatant did not bind to or down-regulate the receptor for TNF-α on the cell surface of L-M-2d6 cells. This factor eluted with an apparent molecular mass of 63, 000Da by gel filtration and did not react with antibodies against p55 and p75 TNF receptors. These data suggest that human monocytic cells are capable of releasing an inhibitory factor against rTNF-α in serum-free culture conditions.

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Macrophage migration inhibitory factor (MIF) is involved in the generation of cell-mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B-cell line, WEHI-231, and its stable MIF-antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI-231 cells. Reduced and increased proportions were seen in G1 and S-phase cells, respectively, in WaM2 as compared with WEHI-231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI-231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross-linking sIg. Almost the same amount of p27^kipl expression was detected in WaM2 cells as those in WEHI-231 and vector control cells. Cross-linking of sIg elevated the p27^kipl level equally in these cells irrespective of the MIF-antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross-linking by regulating the cell cycle via a novel intracellular pathway.

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An outbreak of Salmonella enteritidis infections occurred in Otaru, Japan, in September 1997. A total of 143 cases of salmonellosis were reported to the local Public Health Center. In this outbreak, one case had a 214-hr incubation period. We investigated 5 isolates including this case by phage typing and pulsed-field gel electrophoresis (PFGE) to determine the genetic heterogeneity of S. enteritidis. Five isolates were phage typed as reacted, but did not conform (RDNC) with identical reaction patterns and had quite similar PFGE patterns. Thus, the prolonged incubation period may not be attributed to genetic heterogeneity of the organism but rather to other factors.

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To detect neutralization-relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody-binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti-PV1 antibody, 98.3% (118/120) for anti-PV2, and 96.5% (82/85) for anti-PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally-infected populations.

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Vibrio vulnificus protease (VVP), a 45-kDa zinc metalloprotease, consists of two functional domains: an N-terminal 35-kDa polypeptide having endoproteinase activity, and a C-terminal 10-kDa polypeptide that mediates the binding of VVP to the erythrocyte membrane. Therefore, VVP, but not its N-terminal endoproteinase domain alone, has agglutinating activity to rabbit erythrocytes. When a single zinc atom in the catalytic center was substituted by treatment with CuCl₂ or NiCl₂, proteolytic and hemagglutinating activities were reduced by Ni substitution but not by Cu substitution. Cu-treated 35-kDa polypeptide showed sufficient affinity of the catalytic center and weak binding ability to the erythrocyte membrane, but the Ni-treated polypeptide did not. These results suggest that the binding of endoproteinase domain to membrane is also necessary for hemagglutination.

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Theiler's murine encephalomyelitis virus (TMEV) belongs to the Picornaviridae genus. DA subgroup strains of this virus induce early, non-fatal polioencephalomyelitis followed by demyelination in the spinal cord, with virus persistence. We investigated the use of DA strain as a vector for the introduction of a foreign gene into the central nervous system. Human lymphotoxin (LT) gene was inserted in the L region, the most upstream part of the polyprotein coding region of DA genome. Expression of LT was demonstrated by an immunoblot and an enzyme-linked immunosorbent assay on BHK-21 cells that were infected with the recombinant virus. In addition, the expressed LT showed cytotoxicity against L-929 cells.

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To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T. gondii SAG1 gene-transfectants were established by using RMA.S (H-2^b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8⁺ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T. gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.

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To elucidate whether T-cell receptor Jβ gene usage was affected by major histocompatibility complex haplotypes and other genetic backgrounds, we investigated such usage with Jβ-specific probes in four different mouse strains. As a result, (a) frequent usage of Jβ2.1 and Jβ2.6, (b) infrequent usage of Jβ1.3, Jβ1.5 and Jβ1.6, and (c) predominant usage of the Jβ2 cluster compared to the Jβ1 cluster were found. Importantly, these biases were common to almost all the tested Vβ families of the four strains. Thus, TCR Jβ usage would be independent of the major histocompatibility complex haplotypes and other genetic backgrounds.

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