Recent intensive research has demonstrated that vanadate (VO₃^-, oxidation state +5) and vanadyl (VO²⁺. oxidation state +4) both mimic almost various actions of insulin in cellular systems. An interest in vanadium emerged in 1985 that vanadate administrated orally as a drinking water to streptozotocin-induced heperglycemic diabetic rats (STZ-rats) reduced the high levels of blood glucose down to normal levels and ameliorated many of the aberrations induced by hyperglycemia. In 1990, we proposed first orally active vanadyl complexes such as vanadyl-cysteinemethylester and vanadyl-oxalate complexes in STZ-rats. Since then, we have developed orally active vanadyl complexes with different coordination modes such as VO(O₄), VO(O₂N₂). VO(N₄), VO(S₄) and VO(S₂N₂). Among them, vanadyl-picolinatecomplexes with VO (N₂N₂) coordination mode have been found to be potent insulin-mimetic agents, on the basis of the results on in vitro test using isolated rat adipocytes with respect to the inhibition of the release of free fatty acids (FFA) from the cells and in vivo evaluation (intraperitoneal injection and oral administration) using STZ-rats. Based on the results, the usefulness of vanadyl in treating and preventing diabetes mellitus has been proposed.

References

1) H. Sakurai and A. Tsuji, in "Vanadium in the Environment" ed by J. 0. Nriagu, John Wiley & Sons, New York, 1998, 297

2) H. Sakurai, K. Fujii, S. Fujimoto, Y. Fujisawa, K. Takechi, and H. Yasui, in “Vanadium Compounds : Chemistry, Biochemistry, and Therapeutic Applications, ACS Sym. Ser. 711”ed by A. S. Tracey and D. C. Crans, ACS. Washington, 1998, 344

We have purified and characterized three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, which appear to be involved in iron-sulfur cluster formation and/or biosynthesis of selenophosphate. All of them catalyze eliminations of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. We substituted Ala for each of Cys358 of CSD, Cys364 of CsdB, and Cys328 of IscS, which correspond to catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of the decomposition of L-selenocysteine and that the conserved cysteine residues play a critical role only for L-cysteine desulfurization.

Uptake and distribution of various trace elements in the Se-deficient (I) , (II) , and control rats were examined by the multitracer technique, which can be used to evaluate the behavior of many elements under the same experimental condition. Wistar male rats born to Se-deficient darm were fed with Se-deficient diet for 12 weeks after birth to make them Se-deficient rats (I) . Wistar male rats (4 weeks old) were fed with Se-deficient diet for 8 weeks to make them Se-deficient rats (II) . And 4-week-old rats were fed with Se-adequate diet (0.2ppm of Se) for 8 weeks to use them control ones. The multitracer solution was injected intravenously into each rat. The Se-deficient (I) , (II) , and control rats were sacrificed at 3, 12, 24, and 72h after injection, and the radioactivity in their organs was measured using high-purity Ge detectors. The uptake of Se was higher in the brain of the Se-deficient rats (I) and (II) than in that of the control ones. The uptake of Se was higher in the testicles of the Se-deficient rats (II) than in that of the control ones. The uptake of As and Fe was larger in the liver of the Se-deficient rats (I) than in that of the Se-deficient (II) and control ones. Selenium enhances As excretion to bile in the rats. The observed accumulation of As in Se-deficient rats (I) suggests that bile excretion of As was decreased by the severe Se-deficiency. The increase of Fe uptake in the liver of Se-deficient rats (I) suggests an increase of Fe-binding proteins, such as ferritin in the liver.

Cadmium (Cd) is a toxic heavy metal which causes cell impairment at higher concentration and acts as endocrine disruptor at extremely low concentration which may cause adverse effect against ecological systems involving humans. Studying toxic effect of heavy metals from the aspect of the cellular function by using a single cell eukaryotic protist Euglena gracilis, we revealed that an inorganic form Cd, CdCl₂ caused abnormal cell division at the ppb level that used to be regarded as safe level. In the present study, Euglena gracilis Z grown in the Koren-Butner(KH) medium for 6 days at 28°C under 2,800 lx light/dark cycle with every 12 hours interval was used as a biomarker organism. To the Euglena cells of 10⁴ cell/ml in the Zn-deficient KH medium CdCl₂ as 200ppb to 20ppm was added, and cultured for 2 to 7 days under the same condition as above. The occurrence of teratogenic cell division was checked under Allen’s video-enhanced contrast microscopy ARGUS-100. The effect of Zn addition on the prevention of CdCl₂-induced teratogenicity was examined with addition of ZnSO₄ from 1 to 200ppm by keeping CdCl₂ at 10ppm. Exposure of CdCl₂ on the Euglena gracilis Z cells caused abnormal cell proliferation under the Zn-deficient Koren-Butner medium. More abnormal cell proliferation was observed at ppb level rather than at ppm level. As far as incubating the cells in the presence of CdCl₂ suppression of the outbreak of abnormal cell proliferation could not be accomplished even by the addition of Zn as 10 times as much molar as Cd.

In our previous paper the authors reported the restoration promoting effect of physiologically functional materials processed water ( functional water) on tributyltin chloride (TBTCl) -intoxicated Euglena gracilis. Among the ‘functional waters’ examined, water processed with some mineral-encaging zeolites gave remarkable restoration effect on the TBTCl-intoxicated Euglenαcells. Our present study was to investigate the behavior of TBTCl in Euglena cells incubated with/without Fe-encaging zeolite processed water (Fe-zeolite-water). By subcellular fractionation of TBTCl-intoxicated Euglena gracilis Z cells, atomic absorption spectrophotometry, GLC and GC-MS,it was revealed that TBTCl was readily incorporated into the cell and localized in cytosol, nuclei and cell membrane fractions. After incubation with Fe-zeolite”water for 3 hrs, tin was found in the extracellular fraction, and intracellular distribution analysis revealed that tin was detected mainly in the microsomal fraction, which suggest participation of phase I biotransformation enzyme system. Whereas in the cells incubated without Fe-zeolite water, tin still remained in the cytosol fraction. GC-MS study revealed that dibutyl tin dichloride was the major metabolite of TBTCl. Those results indicate that the Fe-zeolite-water promoted biotransformation of TBTCl directly or indirectly.

Organotin exposure induced severe olfactory lesions. The mechanism was kinetically analyzed from the viewpoints of the movement of trace elements, an inhibition of signal transduction and toxic cell death (necrosis or apoptosis) in the olfactory system. Organotin exposure induced excessive accumulation of calcium in the olfactory system such as olfactory epithelium and olfactory bulb. This calcium localization depended upon the number and species of ligand of organotin compounds and also was found to be due to the processes such as the activation of adenylcyclase by an excessive increase of PTH in the special regions of the brain, excessive increase in the formation of cAMP by the activation of adenylcyclase, disorder of cAMP-activated channels by the excessive increase of cAMP, and an excessive influx of extracellular Ca²⁺ by the disorder of the channels. Moreover, this rapid and excessive increase in the concentration of intracellular Ca²⁺ seems to inhibit CaM kinase II functions followed by leading to necrosis or apoptosis in vivo or toxic cell death in vitro. On the other hand, this excessive accumulation of calcium was suppressed by feeding oyster (Crassostrea gigas) extract and vitamin E (α-Tocopherol). This mechanism was suggested to include the association of beyond antioxidant function from the studies on trace element-mediated oxidative stress or peroxide scavenger systems.

It is well-known that the oyster extract contains a large amount of zinc. Zinc is an essential nutrient required for normal reproductive development and function in mammals. Zinc deficiency in males has profound effects on spermatogenesis. Therefore, we studied the effects of oyster extract on sperm maturation and function in zinc-deficient mice. Zinc deficiency for 12 weeks induced the decrease in body weight, testis weight and sperm counts in epididymis. After zinc deficiency for 6 weeks, an increase in sperm counts was induced in the zinc carbonate-supplemented group. On the other hand, the sperm motility significantly increased in the oyster extract-supplemented group. Some nutrients containing oyster extract, such as taurin and glycogen, may be directly or indirectly related with the sperm function. From these findings, it is suggested that the oyster extract may be a useful diet which can prevent the defects of spermatogenesis in the zinc-deficient status.

Zinc is a very important nutrient in normal reproductive and embryonic developments of mammalians. It has been reported that the oyster extract contains a high level of zinc. The trace elements in the oyster extract as well as shell were determined by neutron activation analysis (NAA). The oyster extract and shell were prepared from Oyster (Crassostrea gigas). All samples were lyophilized and portions of the samples were pulverized for NAA at Research Reactor Institute of Kyoto University. Each element in the samples was determined with the corresponding peak area of γ energy after (n, γ) reaction. On the other hand, the oyster extract, that contains the antioxidants such as natural radical scavengers and metalloproteins, is strongly suggested to scavenge reactive oxygen species (ROS) including superoxide anion radical (・O₂^-) and hydroxyl radical (・OH) and thus inhibit the lipid”peroxidation in the biological systems such as microsomes. The ROS scavenging and anti-lipid-peroxidative activities of oyster extract (high molecular weight fraction : HOE) were studied with cytochrome c reduction and ESR-spin trapping methods, and TBA assay using rat liver microsomes, respectively. The amounts of the element in oyster extract were observed to be in the following order: Cl > Na > Zn > Br > Fe > Cr > Mn > Se, while those in oyster shell were found to be in the order of Ca> Sr> Na > Fe > Cl > Al > Cr > Zn > Br > Mn. Zn was determined in both oyster extract and shell, in which HOE contains the highest level of Zn ( 0.38mg Zn/1g of dry weight sample) . HOE scavenged dose-dependently both superoxide anion and hydroxyl radicals, and inhibited the Fe³⁺-ADP-induced NADPH-dependent lipid peroxidation in rat liver microsomes, although they were very week. From these results, it is suggested that the oyster extract is a useful diet which not only involves many important nutrients but prevents oxidative stresses in mammals.

The effects of Crassostrea gigas extract (JCOE) on endoethial injury were investigated using a cell line HAEC, human aortic human aortic endothelial cell. Cells were subjected to hypoxia in a chamber with 95% nitrogen and 5% carbon dioxide for 4 hours. Reoxygenation was initiated by replacing the media and putting the cells in an environment of room air plus 5% carbon dioxide. Anoxia-reoxygenation induced cellular oxidative stress, which was determined by the levels of reduced glutathione as well as the levels of intracellular reactive oxygen species. Cell viability of HAEC cells subjected to anoxia-reoxygenation significantly decreased. Preteatment of JCOE for 24 hours at a dose of 1000 μg/ml siginificantly reversed the decrease of cell viability induced by anoxia-reoxygenation,and also reversed the decrease in cellular glutathione levels. These results show that JCOE contains one of effecting functional food factors for reducing the oxidative stress in endothelial cells.

Vitamin A and D are essential nutrients for normal skeletogenesis and bone growth in mammals. In growing calves, however, excess amounts of these vitamins synergistically cause growth inhibition of the hind limb. In this study, we investigated the adverse effect of these vitamins on a chondrocyte-like cell line, ATDC5.

All-trans retinoic acid (RA ; a vitamin A metabolite) or 1,25-dihydroxyvitaminD3 (1,25-D; a bioactive form of vitamin D) inhibited cell proliferation and these vitamins synergistically inhibited cell proliferation. RA or 1,25-D also reduced proteoglycan accumulation to the cell layer in a dose dependent manner and these vitamins synergistically reduced proteoglycan accumulation. Although either 100nM of RA or 10nM of 1,25-D increased cellular alkaline phosphatase activity (an of cellular hypertrophy), co-treatment with these vitamins did not affect the activity.

These results suggest that alterations of cell proliferation and differentiation by the synergism of VA and VD are associated with growth inhibition of the limbs in growing calves.

Recently, we have found that administration of the nitric oxide (NO) synthase inhibitor to rats caused hyperlipidemia and higher body fat, implying the fat accumulation by inhibiting NO synthesis in vivo. In this study, we investigated the alterations in lipid metabolism of rats received the NO donor, sodium nitroprusside (SNP). Rats were received SNP in drinking water at 0.03% for 5 weeks. The rats were fed the same amount of the high fat diet between the two groups. Growth was unaffected by SNP treatment. The weight of abdominal adipose tissue was reduced by SNP. Serum total cholesterol and free fatty acid were also reduced by SNP, while serum triglyceride was unaffected by SNP. The results suggest that higher NO is associated with lower body fat and serum cholesterol.

Cysteine dioxygenase (CDO) catalyzes the first step reaction of L-cysteine metabolic pathway. This enzyme regulates taurine synthesis in mammals. A dose-dependent decrease in CDO mRNA level was observed by treatment of HepG2 cells with IL-1β. The CDO mRNA level decreased after 2h and reached a minimum at 6h -16h after the IL-1β treatment. H7 completely blocked the IL-1β-induced down-regulation of CDO gene. HA-1004 also blocked the down“regulation only minimally. These results suggest that the protein kinase C (PKC) mediated signaling pathway may act mainly in the IL-1β-induced down-regulation of CDO gene. In contrast. treatment of HepG2 cells with each of cycloheximide or anisomycin increased the CDO mRNA level. The IL-1β-induced down-regulation of CDO gene was also observed in the cells treated with each of cycloheximide or anisomycin. These results suggest that the two independent regulatory mechanisms may exist in the regulation of CDO gene.

A novel tumoricidal protein from Tricholoma matsutake, induced the morphological changes typical to apoptosis such as nuclear condensation, aneuploidity and DNA fragmentation at concentrations as low as 5-20ng/ml to cancer cells. Molecular alterations related to cell cycle, especially G1/S transition were investigated with a human keratinocyte transformed with oncoproteins, E6 and E7 of human pappiloma virus (HPV) -16. TTM didn’t alter significantly an oncosuppressor p53 level, but induced hyperphosphorylation of pRB. Time-dependent change of G1 cyclins, cdk2 and cdk4 after addition of TTM showed that expression level of cdk inhibitors, INK4 family and p27^Kipl did not altered, while that of p21^Waf1 was downregulated.

To study bone growth and its effective factors, we measured broadband ultrasound attenuation (BUA) and speed of sound (SOS) on 81 healthy girls in the elementary school and 425 women’s college students, who were 18 years old, by means of ultrasound bone densitometry. The elementary school girls were measured twice ; first at grade 4 when their mean age was 9.5, and second at grade 5. General data on all the girls and female students were also obtained : 1) physical characteristics-height, weight, body mass index (BMI), lean body mass (LBM), body fat percentage (%Fat) ; 2) physical fitness-grip strength, back strength, vertical jump ; 3)other details-frequency of sports activity, menstruation cycle, milk and milk product intake. Finally, we statistically analyzed all data by using paired and Student' t-test, and simple regression analysis.

Results were as follows : 1) almost all factors except vertical jump showed a significantly increase for the year from grade 4 to 5; 2) significantly positive correlations existed between BUA and SOS in both groups of girls and students, between BUA and all factors in the female students, but BUA and all physical characteristics and fitness except vertical jump in the girls ; 3)there was no significant difference among correlation coefficients between BUA and SOS of either group ; 4) there were no significant correlations between SOS and all factors except BUA in the girls of irade 4, but significantly positive correlations appeared between SOS and all physical characteristics, except %fat. and left hand and mean grip strength in those of grade 5 ; 5) in the college students, there were significantly positive correlations between SOS and all physical characteristics except height. and vertical jump ; 6) the frequency of weekly sports club activity of the high SOS group was higher than that of the low ; 7) the frequency of milk and milk product intake of the elementary school girls was significantly higher than that of the college students.

In conclusion, significant correlations appeared between SOS, which shows the quantity of bone minerals, and physical characteristics and sports activity on beginning of the acute development. These are one of the characteristics of the growth period.

Contents of arsenic (As) and selenium (Se) in 129 black tea infusion samples were measured by inductively coupled plasma mass spectrometry. The 129 samples consisted of Ceylon tea (n=53) , Assam tea (n=38) and Darjeeling tea (n=38). Significant (p<0.001) differences in contents of the both two elements among Assam tea (As, 1.15 ± 0.61ppb ; Se , 2.20 ± 0.80ppb),Ceylon tea (As, 0.37 ± 0.15ppb ; Se, 1.03 ± 0.32ppb) and Darjeeling tea (As, 0.76 ± 0.24ppb; Se, 1.69 ± 0.51ppb) were observed. Effect of piece size of tea leaf on the contents of As and Se in the infusion samples was insignificant. These results indicate that the original place of black tea is probably identified by As and/or Se contents in the infusion.

Antioxidant action of pyridine compounds was analyzed in relation to the metal coordination. Iron-mediated lipid peroxidation, determined as the formation of thiobarbituric acid-reactive substances, was inhibited by dipicolinic acid (pyridine 2,6-dicarboxylic acid), but not by other pyridine dicarboxylates including quinolinic acid, lutidinic acid, cinchomeronic acid, isocinchomeronic acid. Dipicolinic acid further protected some enzymes against copper-mediated oxidative inactivation. Copper-catalyzed formation of hydroxyl radical causing oxidative inactivation of AMP deaminase was inhibited by dipicolinic acid under the in situ conditions of yeast cells. Dipicolinic acid further attenuated the inhibition by copper ion of glutathione reductase. However, other pyridine dicarboxylates did not show any protective effect. Dipicolinic acid enhanced the autooxidation of Fe²⁺ ion, whereas other pyridine carboxylates rather inhibited the autooxidation of ferrous ion. Ascorbate-catalyzed production of Cu⁺ ion, a potent prooxidant, from Cu²⁺ ion was completely inhibited by dipicolinic acid.

Antioxidant effect of dipicolinic acid can be explained by enhancement of the oxidation of ferrous ion, and by the inhibition of the formation of cuprous ion as a prooxidant: this may be due to the electron-deficient nature of pyridine ring with dicarboxylic acid at ortho position followed by binding of iron and copper ions.

Seasonal variation in the composition of Oyster (Crassostrea gigas) was studied. Oyster cultured in Hiroshima bay was monthly collected on April to December in 1998. Moisture was significantly increased on August to October. Glycogen content was remarkably decreased on July to October and then moderately restored. Oyster samples collected on June showed significantly higher zinc content (443 ± 63ppm) than those collected on other months (227 -329ppm). Seasonal variation was not observed in cadmium content. Difference was not observed in a ratio of soluble zinc/insoluble zinc between oyster samples collected on April and those on June. In a Sephadex G-75gel chromatography, soluble zinc of oyster collected on April was separated into two fractions while that on June was separated into three fractions. These results indicate that zinc in oyster seasonally varied in quality and quantity.

Beta D-galactose binding lectin was purified from the phosphate-saline (PBS) extract of fruiting body of Agaricus blazei by precipitation with 0.6 sat. ammonium sulfate follwed by affinity chromatography on either asiaofetuin-. N-acetyllactosamine or β-aminoethyl D-galactosidesepharose column, and gel filtration. The purified lectin consists of tetrametric glycopeptides with subunit of 16kDa.

N-terminal amino acid sequences up to 30 amino acid residues suggested that there was essentially no homology with hitherto known lectins. The Agaricus blazei lectin (AGbA) interacted with glycans and glycoproteins containing terminal β-galactose residues, such as asialofetuin, human salivary mucin, lactose-BSA. plant xyloglucan. Lincorice AGP etc . These precipitation reactions and the inhibition studies indicated that AGbA must recognize the β-galactose at the terminal end. AGbA was shown to be a glycoprotein (neutral carbohydrate, 5.2% ; Man : Fuc=4.8 : 1.0), and the specific interaction with α-mannose-binding Crocuss lectin suggested a possible glycopeptide chain with (1,3) α-mannosyl mannose terminals.

We have reported that zinc promotes differentiation of 3T3-L1 preadipocytes. The objective of the present experiment was to investigate the effect of zinc on NO production and the effect of NO on the differentiation of 3T3-L1 preadipocytes. NO metabolites concentration was decreased by the addition of 1μM zinc into the medium. NO donors such as SNP and NOC18 suppressed glycerophosphate dehydrogenase (GPDH) activity, a marker of adipocyte differentiation,in dose-dependent manner. A NOS inhibitor (L-NAME) significantly increased GPDH activity. These results suggested that preadipocytes produced NO during differentiation,NO autocrinically inhibited adipocyte differentiation, and zinc promoted adipocyte differentiation through inhibiting NO production of preadipocytes.

Oyster extracts contain important trace nutrients such as taurine and zinc abundantly, and that it has various physiological function such as platelet aggregation inhibitory function or blood glucose improvement effect is reported . Further, antioxidative activity of oyster extracts has been confirmed using model system.

In this study, we examined the effect of oyster extracts supplementation on oxidizability of rats plasma and red blood cells (RBC) membrane. Rats were fed AIN93G diet containing oyster extracts (0.5, w/w) for 5 weeks. The oxidizability of plasma and RBC membrane, as evaluated in vitro by the duration of the lag-phase in hemin-hydrogen peroxide induced oxidation, were significantly decreased. In addition, the total antioxidative activity were significantly increased. On the other hand, a major antioxidative substances and peroxidizability index in plasma and RBC membrane were not changed by oyster extracts supplementation. These results suggest that a certain factors, such as polyphenol, flavonoid, play an important role in antioxidative effects of oyster extracts supplementation.