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A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. 1H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.

In addition to serine (L:D= 68:32), methionine sulfoxide (MSO), L-methionine sulfone (L-MSO2), and disodium γ-cyclic di-L-glutamate were identified in a methanol extract of Bombyx moriL. pupae. MSO was isolated in a diastereomeric mixture of L(+)- and D(+)-MSO in a ratio of 99:1. The presence of these compounds in other developmental stages, including eggs, larvae (1st, 4th, 5th, and mature 5th instar), adults, and excrement (feces and urine) was investigated. The L(+)-isomer of MSO was present in extracts of the 1st and 5th instar larvae, adults, and eggs, but was not detected in feces or urine. The D(+)-isomer was found only in pupal stage extracts, and was excreted into the meconium with L(+)-isomer. L-MSO2and γ-cyclic di-L-glutamate were not detected at other insect life stages or in the insect excrement. γ-Cyclic di-L-glutamate is thought be produced due to blockage of the glutamate synthetic pathway (glutamine synthetase) by L-MSO2and Mg2+. The biochemical role of L-MSO2during the pupal life stage remains unknown, but importantly, the stage-specific expression suggests that it is a candidate molecule for the induction of diapause.

The development of molecular spectroscopy has enabled us to select chlorinated aromatic hydrocarbons very rapidly. In particular, the laser ionization TOFMS (time-of-flight mass spectrometry) method is expected to be useful as an on-line, selective, and sensitive method. In the present work, real-time laser ionization TOFMS measurements were carried out on gaseous chlorinated aromatic hydrocarbons. The laser ionization method used resonance-enhanced two-photon ionization with the direct introduction of gas into the vacuum chamber. This method for analyzing aromatic hydrocarbons was developed using a pulsed supersonic molecular beam method. In the context of developing a highly selective and sensitive method, excitation of monochlorinated benzene at λ= 263.07 nm was found to be effective in the wavelength region from 263 nm to 265 nm. Also the excitation of polychlorinated biphenyls at λ= 266 nm was found to be substantially more effective than at λ= 280, 300 or 320 nm. The achievable sensitivity for real-time (1 min) measurements using the laser ionization TOFMS technique was found to be in the ppbV range.

LI-IT-TOFMS (laser ionization/ion-trap storage/time-of-flight mass spectrometry) is expected to be a powerful tool for environmental monitoring. In the research reported here, real-time LI-IT-TOFMS measurements were carried out on gaseous 2 - 4 chlorinated PCBs in order to evaluate the applicability of an environmental monitoring method. With respect to ion-trap storage for PCBs, we found that the effect was due to the driving RF voltage on the ring electrode in the ion trap. For PCBs ions produced by laser irradiation, we observed that it was more efficient to reach the center of the ion trap by using a gated RF voltage rather than by using a continuous RF voltage. The ion trajectories in the ion trap were simulated by SIMION 7.0. We found that the voltage of the exit end cap electrode affected both the number of ions trapped and the orbit of ions inside the trap cell. Optimization of this parameter was performed using both simulated and experimental results. The achievable PCBs sensitivity for real-time (1 min) measurement using the LI-IT-TOFMS method was found to be in the pptV range (<0.01 mg/m3N) by means of a comparison with the conventional gas sampling/GS-MS method. A satisfactory proportional relationship was confirmed between the laser-based and conventional results.

Background:Alterations of intestinal flora, such as reduction in the concentration of bifidobacteria and increase in that of Bacteroidesspecies, are apparently associated with the severity of ulcerative colitis.Objective:We conducted a randomised clinical trial of the use of a bifidobacteria-fermented milk (BFM) supplement as a dietary adjunct in the treatment of ulcerative colitis.Methods:The subjects were randomly divided into two groups: a group with BFM supplementation (BFM group, 11 subjects) and a control group (control group, 10 subjects). The BFM group was given 100 mL/day of BFM for one year. Colonoscopies, general blood markers and examinations of intestinal flora including the analysis of fecal organic acids were performed at the commencement of the study and after one year.Results:Exacerbation of symptoms was seen in 3 out of 11 subjects in the BFM group and in 9 out of 10 in the control group. Log rank statistic analysis of the cumulative exacerbation rates showed a significant reduction in exacerbations for the BFM group (p= 0.0184). The analysis of microflora and the organic acids in the feces showed a significant reduction in the relative proportion of B. vulgatusin Bacteroidaceae and butyrate concentration, respectively, after supplementation with BFM, in comparison with before.Conclusion:Supplementation with the BFM product was successful in maintaining remission and had possible preventive effects on the relapse of ulcerative colitis.

ABSTRACTSeven Lactobacillusstrains belonging to four species were evaluated for pathogenicity as well as for in vitro sensitivity to the bactericidal mechanisms of macrophages in a rabbit infective endocarditis (IE) model. Two bacteremia-associated strains, L. rhamnosusPHLS A103/70 and L. caseiPHLS A357/84, as well as the L. rhamnosustype strain and the probiotic L. rhamnosusstrain ATCC 53103, showed moderate infectivity, and the virulence of the probiotic L. caseistrain Shirota and type strains such as L. acidophilusATCC 4356Tand L. gasseriDSM 20243Tin the model was negligible. The strains that showed pathogenic potential in the rabbit IE model (PHLS A357/84, PHLS A103/70, and ATCC 53103) were more resistant than strain Shirota to intracellular killing activity by mouse macrophages in vitro and also to bactericidal nitrogen intermediates, such as nitric oxide and NO2-ions. These results suggest that resistance to host innate defense systems, which would function at inflammatory lesions, should be considered in the safety assessment of Lactobacillusstrains.

ABSTRACTWe examined colonization patterns of Shiga toxin-producingEscherichia coli(STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (∼103CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration ofLactobacillus caseifrom the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. caseiadministration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration ofL. caseiincreased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration ofL. caseistrain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.

On the basis of 16S rRNA sequences, species‐ and group‐specific primers for Bifidobacterium adolescentis, B. angulatum, B. bifidum, B. breve, the B. catenulatumgroup (B. catenulatumand B. pseudocatenulatum), and the B. longumgroup (B. longumand B. infantis), which are species commonly found in human intestinal tracts, were developed. The specificity of these primers was confirmed through the use of DNA extracted from 46 strains of 31 Bifidobacteriumspecies, as well as 14 non‐bifidobacterial species that are the predominant bacteria in the human intestinal tract. The present species‐specific primers were applied to the identification of 43 isolated strains, consisting of six strains of B. adolescentis, eight of the B. catenulatumgroup, seven of B. bifidum, nine of B. breve, and 13 of the B. longumgroup.

The receptor structure of Clostridium botulinumneurotoxin type A was analysed by TLC immunostaining. GQ1b was found to be the most potent receptor, and the neurotoxin also bound to GT1b and GD1a, but not to GM3, GM2, GM1, GD3, GD1b and GT1a. Optimum binding of neurotoxin to the ganglioside appeared in 0.01 M phosphate buffer (pH 7.2) containing 0.2% NaCl. Higher and lower NaCl concentrations diminished neurotoxin binding to the ganglioside. In addition, the neurotoxin was able to bind to free fatty acids. Maximum binding was observed on stearic acid and neurotoxin binding to free fatty acids was not affected by NaCl concentration.

TLCGQ1b gangliosideAnti‐neurotoxin antibodySalt effect

A new compound was isolated from the water-soluble fraction of a methyl alcohol extract obtained from the larva of the dobsonfly (Protohermes grandisThunberg). The novel compound had a 12-membered ring and was confirmed to be a cyclic dipeptide of D-ornithine by a chiral analysis, using high-performance liquid chromatography, 2D-nuclear magnetic resonance, and field desorption mass spectrometry.

On the basis of 16S rRNA sequences, species- and group-specific primers for Bifidobacterium adolescentis, B. angulatum, B. bifidum, B. breve, the B. catenulatum group (B. catenulatum and B. pseudocatenulatum), and the B. longum group (B. longum and B. infantis), which are species commonly found in human intestinal tracts, were developed. The specificity of these primers was confirmed through the use of DNA extracted from 46 strains of 31 Bifidobacterium species, as well as 14 non-bifidobacterial species that are the predominant bacteria in the human intestinal tract. The present species-specific primers were applied to the identification of 43 isolated strains, consisting of six strains of B. adolescentis, eight of the B. catenulatum group, seven of B. bifidum, nine of B. breve, and 13 of the B. longum group.