To prevent environmental mercury poisoning incidents, an effective technology for treating mercury-polluted environments is urgent. Recently, with advances in biotechnology, bioremediation utilizing microorganisms to remove mercurials from contaminated sites has become one of the most rapidly developing fields of environmental restoration. A number of bioremediation strategies, including biotransformation, biosorption, and bioprecipitation of mercurials, have been developed to treat mercurial-polluted environments and mercury-containing waste. To construct bacteria that are capable of specifically accumulating mercury, we have genetically engineered Escherichia coli to express a mercury transport system and organomercurial lyase enzyme simultaneously, and overexpress polyphosphate, a strong chelator of essential divalent metals. The mercury accumulation system was designed so that overexpressed polyphosphate would serve as a mercury accumulator; the mercury transport system would make the bacterial cell specifically accumulate mercury; and the intracellular accumulation process would allow the bioaccumulation system to be less sensitive to ambient conditions. The applicability of the new engineered bacteria in the environmental remediation of mercurials is evaluated and discussed in this review.

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Atherosclerosis is a vascular lesion that is a common health problem in advanced countries. Functional damage of the vascular endothelial cells, vascular smooth muscle cell hyperplasia, and procoagulant/antifibrinolytic state of blood are generally observed in the lesion. Since polysaccharides such as heparin modulate vascular cell behavior through interaction with cytokines/growth factors, we hypothesized that polysaccharides from natural sources may possess beneficial biological activities that prevent atherosclerosis. Changes in cultured aortic endothelial and smooth muscle cells were investigated after treatment with the polysaccharides sodium spirulan (Na-SP) — a sulfated polysaccharide obtained from a hot water extract of the blue-green alga Spirulina platensis — and colominic acid (CA)-prepared as a homopolymer of N-acetylneuraminic acid produced by Escherichia coli K1. The experiments suggest that depolymerized Na-SP and sulfated CA can function as precursors of the agents that prevent atherosclerosis. In particular, both the chemically modified polysaccharides significantly inhibit the proliferation of the arterial smooth muscle cells without exhibiting any toxic effects on the integrity of the vascular endothelial cell layers. The results also indicate that chemical modifications, for example, depolymerization of Na-SP and sulfation of CA, can control the biological effects of these polysaccharides on vascular cells.

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The effects of repeatedly brewed green tea infusion on the formation of nitrosamine in vitro and in vivo, and on cancer mortality were examined. The first and second brews of green tea infusion inhibited the formation of nitrosomorpholine in the presence of morpholine and nitrite (nitrosation of morpholine), but the third to eighth brews accelerated it. The green tea infusion brewed from 5 g of leaves in 200 ml hot water (strong tea infusion) inhibited the nitrosation of morpholine, but that brewed from 2.5 g or less (weak tea infusion) promoted the nitrosation. The brewed green tea infusion that inhibited nitrosation of morpholine contained catechins at a high concentration, and that that promoted nitrosation contained catechins at a low concentration. The effects of green tea administered to Wistar male rats and that consumed by humans on the formation of nitrosamines were also examined. In both rats and humans, nitrosamine formation was inhibited by strong green tea extract but was increased by weak green tea extract. The concentration of catechins in the green tea infusion brewed by the general households in tea-producing areas was significantly higher than that brewed in non-producing areas. We examined the relationship between the concentration of catechins in green tea infusion brewed in different areas and the standardized mortality ratio (SMR) of cancer in respective areas, and found that the catechin concentration in green tea infusion correlated inversely and significantly with the SMR of cancer in that area. We concluded that strong green tea might inhibit the formation of nitrosamines and decrease the risk of carcinogenesis.

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We earlier reported that malathion residue in wheat kernels is enzymatically eliminated by adding water to swell the sample as pretreatment in the official method set forth in the Food Sanitation Law of Japan. We examined the activities of monooxygenase, organophosphorus hydrolase, methyltransferase, and carboxylesterase as possible malathion-degradable enzymes in wheat kernels. GC/MS analysis resulted in no activity of monooxygenase in wheat kernels, because malaoxon was not produced in the reaction mixture of the homogenate incubated with malathion for 4 hr. When five organophosphorus pesticides that have a thioether group (P-S-C) were reacted with the wheat kernel homogenate, no formation of thiol groups was detected with the 5,5′-Dithiobis(2-nitro-benzoic acid) (DTNB) reagent, indicating that there was no activity of organophosphorus hydrolase in the kernels. Pesticides that do not have a carboxylester group but do have a dimethyl thio- or dimethyl dithio-phosphate group were not decomposed by the homogenate, suggesting that there is no contribution of methyltransferase to malathion degradation. Among the organophosphorus pesticides with a carboxylester group, only the thion compounds malathion, phenthoate, and methacrifos were enzymatically decomposed by the homogenate. Malathion was also demonstrated to be the substrate of carboxylesterase, as the pesticide competitively inhibited the activity with p-nitrophenyl acetate as the typical substrate. Conversely, oxon organophosphorus pesticides were not degraded in the homogenate, the carboxylesterase of which was noncompetitively inhibited. These results suggest that malathion degradation in wheat kernels is caused by the thion organophosphorus pesticide-specific carboxylesterase.

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A simple and rapid method for the determination of linear alkylbenzene sulfonates (LAS) in aqueous samples was developed by using in-tube solid-phase microextraction (SPME) coupled with liquid chromatography (LC). Introducing a short section of open-tubular gas chromatography column as the extraction device, in-tube SPME method has been developed by Pawliszyn et al. for an effective on-line coupling of sample preparation and LC separation. As an open tubular capillary column for the in-tube SPME, we used a porous-layer open-tabular (PLOT) column with a porous layer on the inner wall and a larger surface area. The separation of LAS was carried out by using a mobile phase containing 65% acetonitrile and 35% sodium perchlorate aqueous solution (287 mM) with a Wakopak^® WS AS-Aqua column. LAS in the sample solution was enriched through the capillary sequentially by using a microsyringe pump equipped with a gastight syringe and analyzed by using high performance liquid chromatography with fluorescence detection. The fluorescence excitation and emission were fixed at λ_ex = 221 nm and λ_em =284 nm. With the optimum conditions, linear calibration curves were obtained from 0.2 to 25 μg/l. The detection limits based on 3σ were between 0.02 and 0.10 μg/l and the relative standard deviations of the method were between 1.6 and 12% (n = 5). The method was successfully applied to the determination of LAS in natural water samples. This method was applied to the determination of LAS in some urban river waters where domestic wastewater was discharged.

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A method was developed for determining illudin S, a naturally occurring toxin in the mushroom Lampteromyces japonicus in food samples with the use of gas chromatography-mass spectrometry (GC-MS). Samples of a model food, 'Ton-jiru' (miso soup), were spiked with authentic illudin S, which was then extracted from the samples by a two-step solvent extraction with ethyl acetate and acetonitrile-n-hexane, followed by solid phase extraction using a normal phase Bond Elut SI cartridge. The recovery of illudin S from the samples was in the range of 61-74%. Illudin S was identified by its GC retention time and both electron and chemical ionization mass spectra. In the splitless injection mode, the calibration curves for peak area ratio of illudin S to anthracene (internal standard) were linear, in the range from 150 pg to 150 ng with correlation coefficients exceeding 0.997, and the detection limit was 36 pg per injection volume (1 μl).

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A bioluminescent assay using Vibrio fischeri Deutsche Sammlung von Mikroorganismen (DSM) 7151 was applied to evaluate the toxicities of some new antifouling chemicals. First, the efficient concentration, led to 50% inhibition of bioluminescence (EC₅₀) values of the single antifouling chemicals zinc 2-pyridinethiol-1-oxide (Zn-pt), copper 2-pyridinethiol-1-oxide (Cu-pt), CuSO₄, zinc bis(N,N′-dimethyl)-dithiocarbamate (Ziram), 4,5-dichloro-2-(n-octyl)-3(2H)-isothiazolone (SeaNine 211), pyridine triphenylboron (PTPB), 3-iodo-2-propynyl butylcarbamate (IPBC), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (Diuron), dichlofluanid (N-dichlorofluoromthylthio-N′,N′-dimethyl-N-phynylsulfamide) (DCF), and 2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine (Irgarol 1051) were determined to be 0.08, 0.12, 0.22, 0.31, 0.35, 0.75, 8.49, 12.74, 39 and > 40 mg/l at 30 min of incubation, respectively. Then, 45 different combinations composed of two antifoulants each were evaluated. Based on the EC₅₀ values at 30 min of incubation, typical patterns of interaction for the combinations were classified into three groups based on the comparison of inhibition difference between single chemicals and their mixtures. Mixture toxicity indices were also introduced to examine the interaction effect of each combination. The results showed that most combinations were partially additive, and there was no antagonistic effect among the present combinations of chemicals. Additive effects were observed in the case of Diuron, PTPB, or SeaNine 211 when mixed with IPBC. Marked synergistic effects were observed for Irgarol 1051, Ziram, Zn-pt, and Cu-pt when mixed with Cu²⁺, which will make these chemicals more toxic against organisms in marine environments.

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We investigated the effect of BRAND'S Essence of Chicken (BEC) on the basic metabolism of blood glucose in mice loaded with restraint stress. In the glucose intravenously treated mice, the stress prolonged the half-life period (T_1/2) of elimination for blood glucose from 51.7 to 62.2 min. The elimination rate decreased to 82.5% per min in stressed mice compared with a starved control. The T_1/2 of blood glucose was 55.2 min in the BEC treated group. BEC alleviated a stress-induced decrease in blood glucose. The improved glucose metabolism was well explained by the findings that insulin levels were elevated and glycogen synthesis in liver was remarkably activated by BEC. Our study demonstrates that BEC improves metabolic dysfunction of blood glucose by elevating insulin levels reduced by restraint stress.

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Elastic fibers play an important role in characteristic elastic properties of tissues such as skin, lungs, ligaments, and blood vessels. Many of the disorders related to elastic fibers are due to errors in assembly, yet the basic mechanisms of this process remain unclear. Cutis laxa, which is caused by an elastin gene mutation, includes a rare and heterogeneous group of the disorders characterized by lax, inelastic skin. To investigate whether elastic fiber assembly insufficiency is a possible factor in cutis laxa, we compared normal tropoelastin (nTE) and frameshift-mutated tropoelastin (fmTE) from a patient with autosomal dominant cutis laxa using an in vitro elastic fiber assembly model, in which purified recombinant tropoelastin was added to the culture medium. Assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Immunofluorescence microscopy indicated an approximate 50% decrease in the deposition of fmTE in the extracelluar matrix, in contrast to that of nTE. The amount of cross-linked amino acids unique to mature insoluble elastin in the fmTE assembly was also decreased by approximately 20%. In addition, we clarified that the molecular interaction between fmTE and the amino-terminal domain of fibrillin-1 or full-length fibulin-5 was significantly decreased. Our results suggest that the defect of fmTE fiber assembly is due, at least in part, to decreased molecular interactions between tropoelastin and the microfibrillar components fibulin-5 and fibrillin.

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The effects of bee pollen extract on osteoclastic bone resorption in vitro were investigated. The water-solubilized extracts were obtained from the bee pollen of Cistus ladaniferus. Femoral-diaphyseal or -metaphyseal tissues of rats were cultured for 48 hr in medium containing either vehicle, bone-resorbing factor, or bone-resorbing factor plus bee pollen extracts (10, 100, or 1000 μg/ml of medium). Culture with parathyroid hormone [human (1-34) PTH; 10^-7 M], prostaglandin E₂ (PGE₂; 10^-5 M), or 1,25-dihydroxyvitamin D₃ (10^-6 M) caused a significant decrease in calcium content in the diaphyseal or metaphyseal tissues. These decreases were completely prevented in the presence of bee pollen extracts (10, 100, or 1000 μg/ml). The presence of PTH (10^-7 M) caused a significant increase in medium glucose consumption and lactic acid production in the diaphyseal or metaphyseal tissues. These increases were significantly inhibited by culture with bee pollen extracts (10, 100, or 1000 μg/ml). Mouse bone marrow cells were cultured for 7 days in the presence of bone-resorbing factor in vitro. Culture with PTH (10^-7 M), PGE₂ (10^-5 M), tumor necrosis factor-α(10 ng/ml of medium), or lypopolysaccharide (10 μg/ml of medium) caused a significant increase in osteoclast-like cell formation. These increases were significantly inhibited in the presence of bee pollen extracts (10, 50, or 100 μg/ml of medium). This study demonstrates that bee pollen extract has inhibitory effects on osteoclastic bone resorption in vitro.

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The polycyclic musks (PCMs), 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphtha-lene (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-γ-2-benzopyran (HHCB), are widely used as fragrance compounds in laundry detergents, soaps and cosmetics. To assess the potential toxicological effects associated with AHTN and HHCB, Caenorhabditis elegans (C. elegans) was used as a model organism for eco-toxicity testing. We examined acute toxicity using 50% lethal concentrations (LC₅₀) after 24 hr PCM exposure and also examined changes in the test endpoints of growth and maturation such as body length, percentage of gravid worms and fecundity. The LC₅₀ for C. elegans was found to be more than 255.2 mg/l for AHTN and 194.6 mg/l for HHCB. In growth tests, the lowest observed effect concentrations (LOEC) in C. elegans for AHTN and HHCB were 12.8 mg/l and 9.8 mg/l, respectively. In maturation tests, LOECs were estimated at 6.4 mg/l for AHTN and 9.8 mg/l for HHCB. In reproduction tests, while maximum LOECs of 19.5 mg/l were observed for HHCB, concentrations of more than 25.5 mg/l were obtained for AHTN.

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The present study was aimed to evaluate the anti-diabetic potential of Terminalia chebula (T. chebula) fruits on streptozotocin (STZ)-induced experimental diabetes in rats. Oral administration of ethanolic extract of the fruits (200 mg/kg body weight/rat/day) for 30 days significantly reduced the levels of blood glucose and glycosylated hemoglobin in diabetic rats. Determination of plasma insulin levels revealed the insulin stimulating action of the fruit extract. Also, the alterations observed in the activities of carbohydrate and glycogen metabolising enzymes were reverted back to near normal after 30 days of treatment with the extract. Electron microscopic studies showed significant morphological changes in the mitochondria and endoplasmic reticulum of pancreatic β cells of STZ-induced diabetic rats. Also, a decrease in the number of secretory granules of β-cells was observed in the STZ-induced diabetic rats and a these pathological abnormalities were normalized after treatment with T. chebula extract. The efficacy of the fruit extract was comparable with glibenclamide, a well known hypoglycemic drug.

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To understand metallothionein (MT)-independent mechanisms for cadmium (Cd) resistance in mammalian cells, we previously established MT-null Cd-resistant cells from embryonic fibroblast cells of MT-I and -II knockout mice. The Cd resistance of these cells was conferred primarily by a marked reduction of Cd accumulation. To identify genes responsible for Cd resistance as well as Cd transport, we carried out several DNA microarray analyses using cDNAs obtained from two clones of Cd-resistant cells and parental cells. A competitive hybridization of Cy3- and Cy5-probed cDNAs on a DNA chip was carried out with dye-swapping. After a careful examination of the reproducibility and reliability of the data obtained using five different chips, it was found that the expression of 78 genes was enhanced and that of 48 genes was reduced in Cd-resistant cells compared with those in parental cells. These genes include those involved in signal transduction, ubiquitin pathway, and cell-to-cell interactions. Several genes for transporters including solute carrier family transporters and ATP-binding cassette transporters were up- or down-regulated. The examination of mRNA levels using quantitative real-time PCR revealed that the expression of Slc39a14 encoding ZIP14, a member of the zinc transporter ZIP (ZRT-, IRT-like protein) family, was markedly down-regulated in both clones of Cd-resistant cells. Although it is not yet clear whether ZIP14 has the ability to transport Cd, these results suggest that the lowered expression of ZIP14 may be involved in Cd resistance in MT-null cells.

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The deposition processes of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in Kanazawa, Japan were studied by examination of the concentrations of PCDDs and PCDFs (PCDD/DFs) and their tetra- to octa-chlorinated homologues in the rain. The weighted mean deposition flux was 360 pg/m²/day [7.7 pg-toxicity equivalency quantity (TEQ)/m²/day]. Deposition fluxes obtained in this study were lower than the average value of Japan, suggesting that Kanazawa is a less polluted area. The seasons with the highest and next highest deposition fluxes were winter and spring, respectively, possibly due to the presence of an inversion layer in winter and spring that reduced the atmospheric dilution of pollutants. In addition, the ratio of PCDFs to total deposition flux in winter was larger than the ratios in the other seasons, possibly due to the burning of fossil fuels for residential heating. Deposition flux of each tetra- to octa-chlorinated homologue of PCDD/DFs was negatively correlated with surface temperature. Other meteorological parameters were positively correlated with almost all tetra- to octa-chlorinated homologues, except for heptachloro dibenzo-p-dioxins (HpCDDs) and octachloro dibenzo-p-dioxin (OCDD), possibly as a result of the photochemical reaction of pentachlorophenol (PCP), which produced mainly OCDD and traces of HpCDDs. In ambient air, the two most dominant homologues were tetrachloro dibenzo-p-dioxins (TeCDDs) and OCDD, while, in soil, the two most dominant homologues were OCDD and TeCDDs. The washout ratios increased with the increase in chlorine substitution. Thus, the difference in washout ratios between homologues might be one of the reasons for the difference of the homologue profile between the air and soil.

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The effects of phytocomponent p-hydroxycinnamic acid (HCA) on biochemical components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecullar bone) tissues of rats in vivo were investigated. Rats were orally administered HCA 1, 2, or 5 mg/100 g body weight once daily for 7 days. The administration of HCA did not cause a significant change in body weight or serum calcium and inorganic phosphorus levels. Calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal and metaphyseal tissues were significantly increased with the administration of HCA 2 or 5 mg/100 g. Diaphyseal calcium and metaphyseal DNA contents were significantly increased with the dose of HCA 1 mg/100 g. The activity of tartrate-resistant acid phosphatase, which is a marker enzyme in osteoclastic bone resorption, was significantly decreased with the administration of HCA 1, 2, or 5 mg/100 g. This study demonstrates that the administration of HCA has anabolic effects on bone calcification in the femoral tissues of rats in vivo.

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We established a method for the simultaneous quantitative analysis of nine organophosphorus pesticides (OPs) and their active oxon forms in water samples using gas chromatography with mass spectrometric detection with solid-phase extraction (SPE). In this method, the lower limit of detection for the nine oxons ranged from 0.5 to 20 ng/ml. Each calibration curve had good linearity, with correlation coefficients (R²) greater than 0.991. In comparing three SPE cartridges, the recovery rate of these compounds extracted from water was highly reproducible using a cartridge of packed silica bonded with C₁₈. The limit of quantification ranged from 2.5 to 200 ng/ml at 500-fold concentrations. When the OPs were examined after chlorination treatment to simulate the water treatment process, they decomposed rapidly and were converted to their oxon forms as primary reaction products of chlorination. Under these established analytical conditions, the behavior of oxons formed in the environment and after water treatment can be determined accurately.

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The efficiencies of rice-bran for removing pesticides from vegetables was studied. Chlorothalonil was successfully removed from commercial eggplant and cucumber with an average removal efficiency of 95% after 5 min when vegetables were pickled in rice-bran paste. Moreover, tetradifon was removed from commercial eggplant, commercial cucumber, and cultivated cucumber with an average removal efficiency of 80% after 10 min. The mechanism of pesticides removal by rice bran was attributed to the uptake of pesticides into intracellular particles called spherosomes.

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Povidone-iodine (PVP-I) gargling solution is used to prevent bacterial infection, but because of its unpleasant smell and metallic taste, people often drink a flavoring agent, such as green tea, immediately after gargling. Green tea beverages, however, have antioxidant activity and may decrease the antibacterial activity of PVP-I. In this study, we investigated the interaction between green tea beverages and PVP-I. Addition of O-i ocha^® or O-i ocha koiaji^® caused color changes of PVP-I from amber to yellow-brown or to orange-yellow, respectively. Ascorbic acid (0.03 mg/ml) caused no color change in PVP-I. The antioxidant activity was highest in O-i ocha koiaji^®, followed by Hajime^®, Nama cha^®, O-i ocha^®, and Flavancha^® in that order. Sou ken bi cha^® and ascorbic acid (0.03 mg/ml) showed poor antioxidant activity. All six green tea beverages that we examined inhibited the antibacterial activity of PVP-I towards Streptococcus salivarius, Streptococcus mutans (S. mutans) and Streptococcus sanguis (S. sanguis). O-i ocha koiaji^® showed the most potent inhibitory activity. The antioxidant activity was significantly correlated with the minimum inhibitory dilution ratio of green tea beverages for S. sanguis (r = 0.763, p < 0.05) and S. mutans (r = 0.854, p < 0.01). These findings indicate that green tea beverages reduce the antibacterial activity of PVP-I in proportion to their antioxidant activity. In conclusion, beverages with antioxidant activity, such as green tea beverages, should not be consumed immediately after PVP-I gargling.

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Liquid coffee and milk based liquid coffee, both containing 1.0 g of mannooligosaccharides (MOS) from coffee mannan, were administered to two groups of six subjects each. The subjects consumed one or the other of the beverage everyday for two weeks. The level of fat in their excrement was subsequently analyzed. In both liquid coffee (p < 0.05, respectively) and milk based liquid coffee (p < 0.05, respectively), the concentration of the beverage containing MOS intake showed a significant decrease in comparison with the placebo and interval or those who did not drink any coffee. The result suggests that the intake of MOS can increase the level of fat excreted from the body irrespective.

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Mice were fed either a high fat diet or a high fat diet containing 1% mannooligosaccharides (MOS) for twelve weeks. The effects of MOS on fat accumulation and excretion were examined. After twelve weeks, the percentage weight of the fat and hepatic triglyceride level were significantly lower in the MOS group than that of the control group (p < 0.05 and p < 0.01 respectively). Furthermore, the serum triglyceride level had a decreasing tendency in the MOS group (p = 0.058). On the other hand, the fecal triglyceride level as well as the amount of fat excreted significantly increased in the MOS group (p < 0.05). This study showed that the administration of MOS lessened the fat accumulation in the parametrial adipose tissue and the liver while at the same time increased the amount of fat being excreted. These results indicate that MOS may prevent the fat storage through inhibiting the intestinal absorption of dietary fat in a high fat diet.

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