Theasaponin E1 destroys the salt tolerance of yeasts

Cells of Zygosaccharomyces rouxii in a medium containing a high concentration of NaCl were killed during incubation for 2-4 h with a low concentration of a mixture of saponins from tea seeds (TSS). The higher the concentration of NaCl in the medium, the higher the inhibitory effect of TSS on the growth of the yeast. The above inhibitory effect of TSS on the growth of the yeast was not observed when cells were incubated in hypertonic media composed of nonionic substances such as sugars. The ATPase activity of plasma membrane preparations from the yeast cells was slightly affected by the addition of TSS. It is shown that TSS facilitates leakage of glycerol from the yeast cells under NaCl-hypertonic conditions. The major inhibitor in the mixture of saponins was isolated and identified as theasaponin E1. Its isomer, theasaponin E2, did not have any effect on the salt tolerance of Z. rouxii or Saccharomyces cerevisiae.     

Molecular biology of oxygen tolerance in lactic acid bacteria: Functions of NADH oxidases and Dpr in oxidative stress

Lactic acid bacteria including Streptococcus mutans lack cytochromes and heme-containing proteins. Most lactic acid bacteria also lack catalase. However, they can grow in the presence of air. In view of the defense against oxygen toxicity, the lack of catalase in lactic acid bacteria is not always consistent with its aerotolerance. Mechanisms, by which lactic acid bacteria establish their growth in air, are therefore an active area of investigation. We identified two kinds of NADH oxidase genes, nox-1 and nox-2 for H2O2-forming NADH oxidase (Nox-1) and H2O-forming NADH oxidase (Nox-2), respectively, in S. mutans and found that Nox-1 is homologous with flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase (AhpR), consisting of AhpF and AhpC. We also identified ahpC which is homologous with ahpC of S. typhimurium, upstream of nox-1 in S. mutans. In the first and second parts of this article, we will refer to the role of Nox-1 which acts together with AhpC as bi-component peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohol and/or H2O. We will also refer to the role of Nox-2 in carbohydrate metabolism of S. mutans in its aerobic life. Nox-2 was found to be involved in regenerating NAD+, which is required for glycolysis in S. mutans. While studying nox-1 and ahpC double deletion mutant of S. mutans, we found that the mutant still showed the same level peroxide tolerance as did the wild-type strain. The finding suggested the existence of another antioxidant system in addition of Nox-1 and AhpC in S. mutans. We identified a new gene, dpr (for Dps-like Peroxide Resistance gene) and its product, Dpr, as an iron-binding protein which is responsible for oxygen tolerance in S. mutans. In the third part of this article, we will refer to the current status of knowledge of molecular cloning of dpr, the characteristics of dpr-disruption mutants, and a mechanism by which Dpr confers aerotolerance to S. mutans.     

Aggregate formation of rCHO cells and its maintenance in repeated batch culture in the absence of cell adhesion materials

Aggregate formation of recombinant Chinese hamster ovary (rCHO) cells capable of producing granulocyte colony-stimulating factor (G-CSF), using medium lacking cell adhesion materials in a repeated batch culture, was examined together with cell growth, cell viability and G-CSF production. The rCHO culture was conducted in a rotary shaker and the medium was changed every five days. The formation of stable cell aggregates with high reproducibility was observed after the first medium change. The size of the cell aggregates (consisting of several 10s to 40,000 cells) formed during the repeated batch culture ranged from 30 to 600 microm. The cell density of the aggregates reached as high as 2 x 10(6) cells/ml and the viability was maintained at more than 80% for 19 d. Changing the medium to avoid glucose exhaustion effectively maintained the cell density, cell viability and G-CSF productivity at high levels.     

In vivo visual reporter system for detection of estrogen-like substances by transgenic medaka

Detection of endocrine disrupting chemicals, in particular, environmental estrogens with living organisms, has many advantages if compared to chemical analysis. The screening of novel pollutants with meaningful endpoints, the integration of uptake, bioconcentration, and excretion as well as the evaluation of endocrine disrupting effects with respect to toxicity require in vivo biotests for estrogen-like substances (ELSs). Critical disadvantages of whole organism biotests are their low sensitivity and the need for laborious and time-consuming work. To overcome these problems, we have developed a transgenic medaka strain harboring the green fluorescence protein (GFP) gene driven by choriogenin H gene regulatory elements. Choriogenin H is an egg envelope protein induced by estrogens in the liver. With yolk sac larvae of this strain, GFP induction in liver was observed 24 h after onset of aqueous exposure to 0.63 nM 17beta-estradiol (E2), 0.34 nM ethynylestradiol, or 14.8 nM estrone. Furthermore, concentrated sewage treatment effluent induced GFP expression. Comparison of E2 equivalents estimated by GFP-induction in transgenic medaka, a YES assay, and GC/MS showed detection limits in the same order of magnitude. These results indicated that the sensitivity of the transgenic medaka strain was sufficient for application as an alternative model in monitoring environmental water samples for ELSs.     

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.     

Efficient photochemical decomposition of long-chain perfluorocarboxylic acids by means of an aqueous/liquid CO2 biphasic system

Photochemical decomposition of persistent and bioaccumulative long-chain (C9-C11) perfluorocarboxylic acids (PFCAs) with persulfate ion (S2O8(2-)) in an aqueous/liquid CO2 biphasic system was examined to develop a technique to neutralize stationary sources of the long-chain PFCAs. The long-chain PFCAs, namely, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUA), which are used as emulsifying agents and as surface treatment agents in industry, are relatively insoluble in water but are soluble in liquid CO2; therefore, introduction of liquid CO2 to the aqueous photoreaction system reduces the interference of colloidal PFCA particles. When the biphasic system was used to decompose these PFCAs, the extent of reaction was 6.4-51 times as high as that achieved in the absence of CO2. In the biphasic system, PFNA, PFDA, and PFUA (33.5-33.6 micromol) in 25.0 mL of water were 100%, 100%, and 77.1% decomposed, respectively, after 12 h of irradiation with a 200-W xenon-mercury lamp; F- ions were produced as a major product, and short-chain PFCAs, which are less bioaccumulative than the original PFCAs, were minor products. All of the initial S2O8(2-) was transformed to SO42-. The system also efficiently decomposed PFCAs at lower concentrations (e.g., 4.28-16.7 micromol of PFDA in 25.0 mL) and was successfully applied to decompose PFNA in floor wax.     

Severe ethanol stress induces assembly of stress granules in Saccharomyces cerevisiae

Stress granules (SGs) and processing bodies (P bodies) are cytoplasmic domains and play a role in the control of translation and mRNA turnover in mammalian cells subjected to environmental stress. Recent studies have revealed that SGs also form in the budding yeast Saccharomyces cerevisiae in response to glucose depletion and robust heat shock. However, information about the types of stress that cause budding yeast SGs is quite limited. Here we demonstrate that severe ethanol stress generates budding yeast SGs in a manner independent of the phosphorylation of eIF2α. The concentration that generated budding yeast SGs (>10%) was higher than that causing P bodies (>6%), and P bodies were assembled prior to SGs. As well as mammalian SGs, the assembly of budding yeast SGs under ethanol stress was blocked by cycloheximide. On the other hand, the budding yeast SGs caused by ethanol stress contained eIF3c but not eIF3a and eIF3b, although the eIF3 complex is a core constituent of mammalian SGs. Moreover, null mutants (pbp1Δ, pub1Δ and tif4632Δ) with a strong reduction in SG formation did not resume proliferation after the elimination of ethanol stress, indicating that the formation of budding yeast SGs might play a role in sufficient recovery from ethanol stress.     

Acceptor recognition of kojibiose phosphorylase from Thermoanaerobacter brockii: syntheses of glycosyl glycerol and myo-inositol

The glucosyl transfer reaction of kojibiose phosphorylase (KP; EC 2.4.1.230) was examined using glycerol or myo-inositol as an acceptor. In the case of glycerol, KP produced two main transfer products: saccharides A and B. The structure of saccharide A was O-alpha-D-glucopyranosyl-(1-->1)-glycerol and that of saccharide B was O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)-glycerol. These results show that KP transferred a glucose residue to the hydroxyl group at position 1 of glycerol. On the other hand, when myo-inositol was used as an acceptor, KP produced four transfer products: saccharides 1-4. The structures of saccharides 1 and 2 were O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively; those of saccharides 3 and 4 were O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively. KP transferred a glucose residue to the hydroxyl group at position 1 or 5 of myo-inositol. On the basis of the structures of their glucosyl transfer products, glycerol and myo-inositol were found to have a common structure with three hydroxyl groups corresponding to the hydroxyl group of the glucose molecule at positions 2, 3 and 4. The conformation of these three hydroxyl groups in the structure is equatorial. This structure is the substrate recognition site of KP. It has been suggested that KP strictly recognizes the structures of glycerol and myo-inositol, and catalyzes the transfer reaction of a glucose residue to the hydroxyl group at position 1 in glycerol, and at position 1 or 5 in myo-inositol, corresponding to position 2 in glucose.     

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.