Tadayori Shimizu

Vibrio trachuri sp. nov., a new species isolated from diseased Japanese horse mackerel.

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4‐diamino‐6,7‐diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.

Anti-Cephalexin Monoclonal Antibodies and Their Cross-Reactivities to Cephems and Penams

Three cell lines producing monoclonal antibodies (MAbs), Cep1-2, 2-2 and 6, against cephalexin were established and the immunoglobulin class of the MAbs was IgM. The cross-reactions of the MAbs with penams and cephems were examined by enzyme-linked immunosorbent assay (ELISA). The cross-reactivities of Cep1-2 and 2-2 were scarcely influenced by the structures of acyl side chains of cephems and penams. The cross-reactivities of Cep1-2 were affected by the presence or absence of dihydrothiazolidine ring of cephem nucleus in hapten-protein conjugates which were prepared by alkaline method, MBS method and activated ester method but the cross-reactivities of Cep2-2 were not. The findings suggest that Cep1-2 recognize the degradate product(s) of cephem nucleus and Cep2-2 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. On the other hand, the cross-reactivities of Cep6 were influenced by the structure of amino acyl side chain. It seems that Cep6 recognize specifically the acyl side chain at the C-7 of cephem. In ELISA inhibition test, three MAbs showed different inhibition pattern. The reaction of Cep1-2 with cephalexin-HSA was inhibited by cephalexin lysate. Cep2-2 and Cep6 were weakly inhibited by the binding to cephalexin-HSA by cephalexin lysate. Furthermore, the reactions of all MAbs were remarkably inhibited by penicillamine. The above results indicate that the MAbs can recognize at least three epitopes of the degradate product(s) of cephem nucleus, NAD and acyl side chain in cephalexin-protein conjugate.

Biological activities and antitumor effects of synthetic lipid a analog linked n-acylated serine

The mitogenicity, lethal toxicity, production of nitric oxide (NO), induction of tumor necrosis factor (TNF) and antitumor activity against Meth A fibrosarcoma by chemically synthesized N-acylated serine-linked non-phosphorylated (A-606 and A607) and phosphorylated (A-608) acylglucosamine-derived lipid A analog were determined. Compounds A-606, A-608 and A-103 [with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3 positions] induced significant incorporation of [3H]thymidine into splenocytes of C3H/He mice at concentrations ranging from 3.13 to 50 microM. However, A-607 [with (R)-3-tetradecanoyloxytetradecanoyl and with tetradecanoyl at the C-2 and C-3 positions] showed most significant incorporation of [3H]thymidine. The compounds A-606, A-608 and A-103 did not exhibit the lethality at doses of 30 and 300 nmol/kg in C57BL/6 mice loaded with D-galactosamine, whereas A-607 caused the death of two out of six mice at a dose of 300 nmol/kg. These compounds, except A-607, exhibited little NO production by macrophages, but did cause NO production in the presence of interferon-gamma (IFN-gamma). Peritoneal macrophages, stimulated with A-606-A-608, caused production of TNF which induce L929 cell lysis in vitro, and A-608 showed high production of TNF. NO-inducible activity and induction of TNF by compound A-103 seemed to be lower than that of serine-linked derivatives. A-607, A-608 and A-103 showed antitumor activity against Meth A fibrosarcoma in BALB/c mice, and furthermore, the enhancement of antitumor activity by a combination of A-608 with muramyl dipeptide (MDP) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined effects of synthetic lipid A analogs and muramyl dipeptide on antitumor activity against meth a fibrosarcoma in mice

Combined effects of chemically synthesized lipid A analogs, the compound A-171 (acylglucosamine-4-phosphate with (R)-3-hydroxytetradecanoyl and (R)-3-hydroxytetradecanoyloxy]tetradecanoyl group at the C-2 and C-3 positions), or the compound A-172 (with (R)-3-hydroxytetradecanoyloxy]tetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl group at the C-2 and C-3 positions), and muramyl dipeptide (MDP) on antitumor activity against Meth A fibrosarcoma, were examined. Meth A fibrosarcoma cells (5 X 10(5)) were inoculated intradermally into BALB/c mice on day 0, compound A-172 and/or MDP were administered intravenously (i.v.) on day 7. Although the antitumor activity by single i.v. injection of A-172 (50 micrograms/mouse) with MDP (10 micrograms) was weaker than that of 50 micrograms of synthetic lipid A analogs (506), or 10 micrograms of bacterial lipopolysaccharide (LPS) with MDP, A-172 alone and with MDP exhibited tumor inhibition rates of 49.0 and 70.6%, respectively. When A-171 (50 micrograms) with MDP (10 micrograms) was administered i.v. twice (days 7 and 10) into mice inoculated Meth A fibrosarcoma, two of five mice caused complete tumor regression. Furthermore, L929 cell lysis by the combination of A-171, A-172 with MDP was higher than that by the analogs or MDP alone, suggesting that the lipid A analogs of monosaccharide type as well as LPS are able to enhance the production of tumor necrosis factor in the presence of MDP.

Biological activities and endotoxic activities of protective antigens (PAgs) of Leptospira interrogans.

The biological and endotoxic activities of protective antigens (PAgs) prepared by the chloroform-methanol-water method from Leptospira interrogans serovars lai, copenhageni and canicola were examined. The PAg preparations did not show a local Shwartzman reaction in the rabbits at doses of 100 micrograms and 50 micrograms/site and lethal toxicity to galactosamine-sensitized mice at the dose of 12.5 micrograms to 50 micrograms/mouse. PAgs exhibited a weak cytotoxic action on peritoneal exudate macrophages of C3H/HeJ and C3H/HeN mice at the dose of 500 micrograms/ml in vitro, but did not show cytotoxicity for BHK-21 cells kidney cells of the Syrian hamster, CHO-K1, ovary cells of the Chinese hamster, and CHL, lung cells of the Chinese hamster, at doses of 5 and 500 micrograms/ml. Gelation activity in the Limulus test was only observed at PAg concentrations over 100 ng/ml, which dose was 10,000 times that of lipopolysaccharide (LPS) of Escherichia coli O55:B5. Furthermore, an adjuvant activity of PAgs was not observed in the production of anti-sheep red blood cell antibody in mice. Mitotic conversion of spleen cells from C3H/HeJ and C3H/HeN mice was observed by the addition of PAgs in vitro. These results indicated that the biological properties of PAgs were different from those of LPS prepared from gram-negative enterobacteria, that PAgs had no endotoxic activity and that the biological safety of PAgs as vaccine was proved.

Rapid and Sensitive PCR Detection of Vibrio trachuri Pathogenic to Japanese Horse Mackerel (Trachurus japonicus)

A polymerase chain reaction (PCR) method was performed for rapid and sensitive detection of pathogenic Vibrio trachuri isolated from cultured Japanese horse mackerel. A set of primers was selected from the base sequence of the Pst I fragment of T9210 chromosomal DNA and used for PCR detection of T9210. This PCR specifically amplified the DNAs from V. trachuri T9210, T9213, and T9216 but not of those other bacterial strains. PCR using a Pst I‐1 primer set made it possible to detect 100 fg of T9210 DNA. The PCR method reported here may be useful for detection and identification of V. trachuri pathogenic to Japanese horse mackerel.

Epitope analysis of Aztreonam by antiaztreonam monoclonal antibodies and possible consequences in beta-lactams hypersensitivity

Three cell lines producing monoclonal antibodies, Az-1 (IgG1), Az-2 (IgG1) and Az-3 (IgM) against aztreonam were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams, which were conjugated with human serum albumin (HSA), were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. In ELISA, Az-1 and Az-2 reacted only with aztreonam and ceftazidime, which have the same acyl side chain. Furthermore, Az-2 showed a strong cross-reaction with carumonam. In the ELISA inhibition test, Az-1 and Az-2 were inhibited from binding to aztreonam-HSA by aztreonam, ceftazidime, aztreonam hydrolysate, aztreonam-epsilon-amino-n-caproic acid (EACA) and ceftazidime-EACA. Az-2 was also inhibited with carumonam. From the above results, it seems that Az-1 can recognize only the degraded structure of monobactam nucleus, and Az-2 can recognize the degraded nucleus moiety and the acyl side chain. On the other hand, Az-3 displayed broad cross-reaction to various beta-lactams in ELISA. Furthermore, the MAb showed no inhibitory reaction with various beta-lactams except aztreonam- and ceftazidime-EACA conjugates in the ELISA inhibition test, suggesting that Az-3 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. The above results indicate that antibodies can recognize at least three epitopes of the degraded product(s) of aztreonam nucleus, acyl side chain and NAD in aztreonam-protein conjugate.

Biological activities of chemically synthesized N-acetylneuraminic acid-(α2→6) -monosaccharide analogs of lipid A

The mitogenicity and lethal toxicity of chemically synthesized lipid A analogs, in which 2,3‐acyloxyacylglucosamine‐4‐phosphate linked to tetraacetyl‐N‐acetylneuraminic acid (compound A‐207) or to N‐acetylneuraminic acid (compound A‐307), were examined. Although the mitogenic activity of the synthetic compounds was weaker than that of bacterial LPS, doses of 10–50 μg/ml of A‐207 and 5–10 μg/ml of A‐307 were capable of increasing incorporation of [3H]thymidine into cultured spleen cells of C57BL/6 mice. Lethal toxicity of A‐207 was observed at 10 μg/mouse in C57BL/6 mice sensitized with D‐galactosamine hydrochloride. However, the attachment of tetraacetyl‐N‐acetylneuraminic acid or N‐acetylneuraminic acid does not appear to enhance the biological activity of acyloxyacylglucosamine‐4‐phosphate.

Biological Activities of Lipopolysaccharide Isolated from Vibrio cholerae O139, a New Epidemic Strain for Recent Cholera in Indian Subcontinent

Biological activities of lipopolysaccharide (LPS) isolated from Vibrio cholerae O139, a new causative agent for recent cholera epidemic in Indian subcontinent, were investigated in comparison with those of LPS from O1 V. cholerae. V. cholerae O139 LPS exerted mitogenic activity, lethal toxicity and Shwartzman reaction to the same extent as those observed for O1 V. cholerae LPS, although these activities except for lethal toxicity were obviously lower than those of Salmonella typhimurium LT‐2 LPS used as a reference. It was, therefore, suggested that O139 LPS does not contribute to the high infective and pathogenic potentials of the V. cholerae O139 strain as in the case of O1 V. cholerae.

Biological activities of chemically synthesized N-acylated serine-linked lipid A analog in mice

The mitogenicity, lethal toxicity and antitumor activity against Meth A fibrosarcoma and the induction of tumor necrosis factor (TNF) of chemically synthesized N-acylated serine-linked nonphosphorylated acylglucosamine-derived lipid A analog (A-601, A-602 and A-603) were determined. Compounds A-603 (with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 position) and A-103 (2,3-acyloxyacylglucosamine-4-phosphate) induced significant incorporations of [3H]thymidine into splenocytes of C3H/He mice at concentrations ranging from 6.25 to 100 microM. The mitogenicity of A-601 and A-602 (with tetradecanoyl at the C-2 position) exhibited a lower activity than of A-603. Compounds A-601 and A-603 showed almost the same lethality at doses from 1 to 50 nmol/mouse in C57BL/6 mice loaded with D-galactosamine, whereas A-103 caused the death of two out of six mice at a dose of 25 nmol/mouse. A-601 and A-603 showed weak antitumor activity against Meth A fibrosarcoma in BALB/c mice, but there was no enhancement of antitumor activity by a combination of A-603 with muramyl dipeptide. Peritoneal macrophages, stimulated with A-601, A-602 or A-603, caused production of TNF which induces L929 cell lysis in vitro. But the activity of A-603 among the compounds on TNF-production was the highest. These findings indicate that the linkage of nonphosphorylated acylglucosamine and N-acylated serine affects the expression of the biological activity.