A bacterial strain FP2001 isolated from the exudate of land reclaimed for municipal waste was identified as Pseudomonas paucimobilis. Cells of strain FP2001 were mobile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium. The replacement of rhamnose by arabinose, galactose, glucose or xylose did not lead to the formation of flagella.

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Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial-associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end-point attached heparinized polyethylene (H-PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H-PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non-modified H-PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).

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The clinically isolated heat labile enterotoxin (LT)-producing strains of Escherichia coli can be separated into two groups, namely spontaneous LT-releasing strain and non-spontaneous LT-releasing strain, based on their phenotypes of spontaneous release of LT into the culture medium. The phenotype of spontaneous LT release was observed to correlate closely with the phenotype of the release of numerous small vesicles into the culture medium. Both morphological and biological examinations of the vesicle showed that the vesicle was released from the outer membrane. It can, therefore, be assumed that LT may be released from the cell at the time the vesicles form.

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A 9.8-kbp DNA fragment which contained a neurotoxin gene and its upstream region was cloned from Clostridium botulinum type D strain CB-16. Nucleotide sequencing of the fragment revealed that genes encoding for hemagglutinin (HA) subcomponents and one for a nontoxic-nonhemagglutinin (NTNH) component were located upstream of the neurotoxin gene. This strain produced two toxins of different molecular size (approximately 300kDa and 500kDa) which were designated as progenitor toxins (M and L toxins). The molecular size of the NTNH component of L toxin was approximately 130kDa on SDS-PAGE and its N-terminal amino acid sequence was M-D-I-N-D-D-L-N-I-N-S-P-V-D-N-K-N-V-V-I which agreed with that deduced from the nucleotide sequence. In contrast, the M toxin had a 115-kDa NTNH component whose N-terminal sequence was S-T-I-P-F-P-F-G-G-Y-R-E-T-N-Y-I-E, corresponding to the sequence from Ser141 of the deduced sequence. A 15-kDa fragment, which was found to be associated with an M toxin preparation, possessed the same N-terminal amino acid sequence as that of the 130-kDa NTNH component. Furthermore, five major fragments generated by limited proteolysis with V8 protease were shown to have N-terminal amino acid sequences identical to those deduced from the nucleotide sequence of 130-kDa NTNH. These results indicate that the 130-kDa NTNH of the L toxin is cleaved at a unique site, between Thr and Ser, leading to the 115-kDa NTNH of the M toxin.

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The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O-antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild-type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O-antigen immunity in the host defense against shigellosis.

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HIV-1 Gag protein precursor p55, and its processed products, p17, p24, and p15 were overproduced in Escherichia coli and purified to near homogeneity. To study the antigenic properties and the potentiality as the diagnostic and prognostic reagents, varying amounts of the purified Gag proteins were dotted onto the polyvinylidene difluoride membrane and reacted with 40 sera of HIV-1-infected individuals (35 AC, 1 ARC, and 4 AIDS patients) and 10 sera of normal healthy donors. p55 reacted with 40 (100%) sera of HIV-1 carriers, while p17, p24, and p15 reacted with 37 (92.5%), 35 (87.5%) and 34 (85%) of the 40 sera of HIV-1 carriers, respectively. On the whole, the reaction of p55 was especially strong and that of p15 was the weakest. p55 showed the strongest reaction among the four Gag proteins with all specimens, and it showed a positive reaction with a carrier serum with which none of the processed Gag proteins showed a positive reaction. Therefore, p55 is the most useful antigen among the four Gag proteins for detection of the Gag antibodies and may even be one of the most useful antigens for the diagnosis of HIV-1 infection.

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Peptide VP1 (11-25) of the capsid of hepatitis A virus was synthesized by the Fmoc-polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti-VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti-HAV response was generated with the free peptide, while all the other forms induced both anti-HAV and HAV-neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome-entrapped and KLH peptides.

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In this work, we demonstrate that the simultaneous injection of PC-KLH and Neisseria meningitidis-derived antigens [NMB or PC-(NMB)HI] induced in old NZB/W mice defective responses as does PC-KLH challenge. On the other hand, the simultaneous injection of both immunogenic preparations of N. meningitidis evoked responses similar to those shown by old mice challenged with NMB alone. Alteration in PC-specific PFC responses also affected hapten-free inhibition profiles and their heterogeneities. The increase in PC₅₀S of anti-phosphorylcholine PFC responses and their heterogeneities induced by certain antigens with aging is correlated with a decrease in T15 idiotype expression, suggesting that after the T15 dominant clone disappears no other clone takes control of the anti-PC response. These results suggest that the mechanism(s) involved in the regulation of T15 marker expression play an important role in the inability of old NZB/W mice to mount good anti-PC responses and suggest that regulatory mechanisms induced by PC-KLH dominate those elicited by NMB.

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We investigated the effect of in vivo administration of antibodies against T-cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN-γ) production and granuloma formation in Rhodococcus aurantiacus-infected mice. High titers of endogenous IFN-γ were detected in the extracts of the livers and spleens during 24hr of the infection, reaching the peak at 8hr, and the IFN-γ production was reduced by in vivo administration of anti-NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1⁺ cells. Endogenous IFN-γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN-γ at 1 and 3 weeks was reduced by in vivo administration of anti-CD8 MAb, but not by anti-CD4 MAb or anti-NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti-IFN-γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8⁺ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN-γ is attributable to NK cells in the early phase of the infection and CD8⁺ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8⁺ T cells through the secretion of endogenous IFN-γ.

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We established BALB/c-derived CD8⁺ CTL clones D2-22 (V_β 6⁺), D2-23 (V_β 8⁺) and D2-24 (V_β 8⁺) specific for B10.D2 minor H antigen. D2-22 and D2-23 proliferated without producing IL-2 in response to X-ray-irradiated antigenic cells, Con A, aCD3, PMA and IL-2. Paraformaldehyde-fixed antigenic spleen cells neither induced proliferation in the presence of costimulatory cells nor inhibited responses to irradiated antigenic cells added simultaneously. Unlike the previously reported results with IL-2-producing CTL clones and Th1 clones, the fixed antigenic cells failed to induce antigen-specific unresponsiveness in these IL-2-nonproducing CTL clones. Instead, the responsiveness of these clones to fresh stimulation was found to be reduced severely after 2 days in the culture added with either antigenic or syngeneic fixed cells. Induction of their antigen-nonspecific low responsiveness by the fixed cells was prevented by adding irradiated syngeneic cells into the culture or even by increasing the concentration of responder D2-23 cells. Close contact of D2-23 and irradiated syngeneic cells was required to prevent the reduction of the responsiveness, although this cognate cell-cell interaction could be replaced by exogenously added IL-2 or PMA. Cytolytic and tumor cell growth inhibitory activities of D2-23 were also reduced by incubation with the fixed cells, which was prevented by the addition of irradiated syngeneic cells. These findings showed the unique properties of IL-2-nonproducing CTL clones in signal requirements for maintaining normal responsiveness for proliferation and cytolytic activity.

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The functional contributions of amino acid residue Asp87 of Escherichia coli gyrase A protein (GyrA) was analyzed by site-directed mutagenesis. We generated a series of mutants, in which Asp87 of GyrA was changed to Ala, Val, Phe, Asn, Ser, and Lys. By genetic analysis of gyrA genes in a gyrA temperature-sensitive (Ts) background, it was shown that all these mutations caused the quinolone-resistance. These results indicate that the 87th amino acid of E. coli GyrA must have negative charge in expressing the phenotype of quinolone sensitivity. These findings also suggest that the carboxyl group of Asp87 may interact with quinolone drugs.

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The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.

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The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmalate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1, 061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39, 179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.

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