An outbreak of food-borne listeriosis due to cheese in Japan, during 2001

Food-borne outbreaks caused by Listeria monocytogenes have been recognized in US and European countries. Only sporadic cases, of neonatal listeriosis, have been reported in Japan. Since L. monocytogenes has been often isolated from foods in Japan, food-borne outbreaks potentially could have occurred. In February 2001, L. monocytogenes serotype 1/2b was isolated from a washed-type cheese during routine Listeria monitoring of 123 domestic cheeses. Further samples from products and the environments at the plant that produced the contaminated cheese were examined for L. monocytogenes. L. monocytogenes serotype 1/2b was detected in 15 cheese samples, at most probable number that ranged from <30 to 4.6 x 10(9)/100 g, and in environmental samples. Studies with people who had consumed cheese from the plant revealed 86 persons who had been infected with L. monocytogenes. Thirty-eight of those people had developed clinical symptoms of gastroenteritis or the common cold type after the consumption of cheese. Isolates from those patients exhibited the same serotype, pathogenicity for mice and HeLa cells, DNA fingerprinting patterns and PCR amplification patterns. From the epidemiological and genetic evidence, it appeared that the outbreak was caused by cheese. This is the first documented incidence of food-borne listeriosis in Japan.     

Antimicrobial activity of nutmeg against Escherichia coli O157

We examined the difference between Escherichia coli O157 and non-pathogenic E. coli in their tolerance to spices. Various spices (5 g each) were homogenized at 25 degrees C for 10 min with 5 ml of 70% ethyl alcohol, and the supernatant solutions obtained by centrifugation were used as spice extracts. When the E. coli strains were incubated with each spice extract at concentrations of 0.01% and 0.1%, a noteworthy difference was observed between the O157 and non-pathogenic strains in their tolerance to nutmeg. The populations of the non-pathogenic strains could not be reduced, but those of the O157 strains were remarkably reduced. Antibacterial activity by the nutmeg extract was also found against the enteropathogenic E. coli O111, but not against enterotoxigenic (O6 and O148) and enteroinvasive (O29 and O124) E. coli. When we examined the antibacterial effect of volatile oils in nutmeg on the O157 and non-pathogenic E. coli strains, all O157 strains tested were found to be more sensitive to beta-pinene than non-pathogenic E. coli strains.     

Novel sugar phosphotransferase system applicable to the efficient labeling of the compounds synthesized via the non-mevalonate pathway in Escherichia coli

It is known that 1-deoxy-D-xylulose taken up by Escherichia coli is used as the precursor of the compounds synthesized via the non-mevalonate pathway, such as isoprenoids, probably after conversion into 1-deoxy-D-xylulose 5-phosphate. In this report, we show that a novel phospho(enol) pyruvate-dependent phosphotransferase system catalyzes the uptake and phosphorylation of 1-deoxy-D-xylulose.     

Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.     

Steroid hormone profiles of urban and tidal rivers using LC/MS/MS equipped with electrospray ionization and atmospheric pressure photoionization sources

A highly sensitive and uncomplicated method of analyzing steroidal hormones in river and estuarine water samples was developed using a liquid chromatography tandem mass spectrometer equipped with an electrospray ionization (ESI) source and atmospheric pressure photoionization (APPI) source. Steroidal hormones included not only estrogen but also androgen and conjugates of these two. APPI displayed greater sensitivity than ESI for most of the unconjugated steroids examined, with very high sensitivity for testosterone and 4-androstene-3,17-dione in particular. For conjugated hormones, in contrast, ESI was more effective. The method developed was applied to the determination of hormones in the rivers of Osaka City and their estuaries, where the hormones detected were affected by the effluent from municipal wastewater treatment plants (WWTPs), and hormone concentration values were comparable to those reported in previous studies of such effluent. Because of the two-way flow and stagnancy of streams and watercourses, continuous input of steroidal hormones from WWTPs seems to bring about local accumulation. Levels of androgen were 1 order of magnitude lower than those of estrogen. Estrone, estrone 3-sulfate, and 4-androstene-3,17-dione were detected in almost all water samples, with maxima of 51, 5.1, and 6.4 ng L(-1), respectively.     

Novel poly-gamma-glutamate-processing enzyme catalyzing gamma-glutamyl DD-amidohydrolysis

The pgdS gene product of Bacillus subtilis, PgdS, cleaves poly-gamma-glutamate (PGA) in an endo-peptidase-like fashion. However, its catalytic property remains obscure. In this study, a simple assay for the PgdS enzyme using 1-fluoro-2,4-dinitrobenzene was developed, and some characteristics of PgdS, such as optimal pH, were examined. The enzyme was strongly inhibited by a thiol-modifying reagent, suggesting that it possesses essential cysteine residue(s) in catalysis. PgdS exhibited a high affinity to PGA that consisted mainly of D-glutamate residues, but no affinity to PGA composed only of L-glutamate residues (L-PGA). The enzyme processed DL-copolymer-type PGA (DL-PGA) with an average molecular mass of 1,000 kDa to a high-molecular-mass L-glutamate-rich fragment (average 200 kDa), the L-rich PGA fragment, and low-molecular-mass fragment composed mostly of D-glutamate residues (average 5 kDa), D-fragment. To deepen our understanding of the catalytic property of the PgdS enzyme, we analyzed the structures of the N- and C-terminal regions and found that D-glutamyl residues successively lie even at both ends of the L-rich PGA fragment. Our observations indicate that PgdS is a novel endo-peptidase that specifically cleaves the gamma-amide linkage between two D-glutamate residues in PGA, i.e., gamma-glutamyl DD-amidohydrolase. The enzyme is possibly useful in the biochemical processing of B. subtilis DL-PGA.     

Analysis of essential amino acid residues for catalytic activity of glutaminase from Micrococcus luteus K-3

Structural-based mutational analysis of salt-tolerant glutaminase from Micrococcus luteus K-3 (Micrococcus glutaminase) revealed that three amino acid residues, S64, K67, and E160, were essential to a catalytic reaction. The result suggested that Micrococcus glutaminase had a possible catalytic mechanism similar to class A beta-lactamase rather than glutaminase-asparaginase from Pseudomonas 7A.     

Anthocyanins,but not Anthocyanidins,from Bilberry (Vaccinium myrtillus L.) Alleviate Pruritus via Inhibition of Mast Cell Degranulation

Abstract: We have previously reported that bilberry anthocyanins exhibit an anti‐pruritic effect in a mouse model of allergic contact dermatitis. It has been reported that anthocyanins are particularly sensitive to thermal treatment and are easily hydrolyzed to anthocyanidins when exposed to high temperatures. The objective of this study was to compare the anti‐pruritic effect of anthocyanin‐rich quality‐controlled bilberry extract and anthocyanidin‐rich degraded extract using a mouse model of allergic contact dermatitis. BALB/c mice with allergic contact dermatitis induced by 4 weeks of repeated application of 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) were administered Bilberon‐25 orally for 4 weeks after sensitization with TNCB. The effect of Bilberon‐25 on pruritus was evaluated by measurement of scratching behavior. RBL‐2H3 mast cells were used to investigate the effect of Bilberon‐25 on degranulation in 48/80‐stimulated mast cells. Compared with nonheated Bilberon‐25, the proportion of anthocyanins in heated Bilberon‐25 decreased, and the proportion of anthocyanidins was increased in heated‐time dependent manner. Treatment with non‐heated Bilberon‐25 significantly attenuated the TNCB‐induced increase in scratching behavior, whereas treatment with 2 h‐heated Bilberon‐25 did not. Moreover, 300 μg/mL nonheated Bilberon‐25 showed significant inhibition of degranulation in RBL‐2H3 mast cells, whereas 2 h‐heated Bilberon‐25 had no effect at any concentration studied. It is assumed that the inhibitory effect of bilberry anthocyanins on pruritus might be mediated, at least in part, by its inhibitory effect on mast cell degranulation. In conclusion, the anthocyanin‐rich but not anthocyanidin‐rich bilberry extract may be a useful dietary supplement for skin diseases involving pruritic symptoms, such as chronic allergic contact dermatitis, atopic dermatitis, and rhinitis.     

New clean-up method with dispersive solid-phase extraction for simultaneous determination of pesticide residues in livestock products by GC-MS/MS

A simple clean-up method was developed for the simultaneous determination of pesticide residues in livestock products by GC-MS/MS. The pesticide residues were extracted with acetonitrile-ethanol (1 : 1), and matrix components such as adipose were effectively eliminated by a combination of refrigerated centrifugation, dispersive solid-phase extraction, and multifunctional column chromatography. In this method, samples are treated quickly and easily without the need for gel-permeation chromatography. Among 131 pesticides tested, 115 showed recovery within the range from 70 to 120%, with relative standard deviations of less than 15%. The quantification limits for the 115 pesticides in livestock products were 0.001 to 0.01 μg/g.