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BACKGROUND Skin sensitizers induce allergic reactions through the induction of reactive oxygen species. Allyl nitrile from cruciferous vegetables has been reported to induce antioxidants and phase II detoxification enzymes in various tissues. We assessed the effects of repeated exposure to allyl nitrile on sensitizer-induced allergic reactions. MATERIAL AND METHODS Mice were dosed with allyl nitrile (0-200 µmol/kg), and then received a dermal application of 1 of 3 sensitizers on the left ear or 1 of 2 vehicles on the right ear. Quantitative assessment of edema was carried out by measuring the difference in weight between the portions taken from the right and left ears. We tested enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and thiobarbituric acid reactive substances (TBARS) in ears. RESULTS Repeated exposure to allyl nitrile reduced edemas induced by glutaraldehyde and by 2, 4-dinitrochlorobenzene (DNCB), but not by formaldehyde. The repeated exposure decreased levels of TBARS, a marker of oxidative stress, induced by glutaraldehyde and by DNCB, but not by formaldehyde. Allyl nitrile elevated SOD levels for the 3 sensitizers, and CAT levels for formaldehyde and DNCB. Allyl nitrile also increased GPx levels for formaldehyde and DNCB, but not for glutaraldehyde. The reduced edemas were associated with changes in oxidative stress levels and antioxidant enzymes. CONCLUSIONS Repeated exposure to allyl nitrile reduced allergic reactions induced by glutaraldehyde and by DNCB, but not by formaldehyde. This reduction was associated with changes in ROS levels and antioxidant enzyme activities.

Vitamin K2 (VK2) has been used as a therapeutic agent for osteoporosis, since it has been suggested to be able to reduce the frequency of fractures by improving bone quality; however, bone turnover is strictly regulated by various cytokines and hormones. In the present study, the effect of menaquinone-4 (MK-4) on bone turnover was investigated using the senescence-accelerated mouse prone 6 (SAMP6) strain. Since water-immersion restraint stress (WRS) causes a significant decrease in bone mineral density (BMD), WRS was used as the bone resorption model in the SAMP6 strain. Six-week-old SAMP6 male mice were divided into the following three groups: Control, WRS and WRS + MK-4. WRS was performed for 6 h per day, 5 times a week, for 4 weeks. Following WRS, MK-4 (30 mg/kg) was injected subcutaneously 3 times a week for 4 weeks. No growth retardation was observed in the WRS groups as compared with the control group. In the WRS groups, the BMD was significantly lower than that in the control group. The levels of bone formation and resorption markers were increased in the WRS groups, indicating that WRS reduced the BMD by promoting high bone turnover. A bone histomorphometrical examination showed that the trabecular (Tb) bone mass in the secondary spongiosa at the distal femur was significantly reduced in the WRS mice, and this reduction was abrogated by MK-4 treatment. Specifically, the Tb bone reduction was caused by the activation of osteoclasts (Ocs), and Oc activity was suppressed by MK-4. The number of osteoblasts and the mineral apposition rate were significantly increased in the WRS and WRS + MK-4 mice, suggesting that WRS triggered a significantly higher mineral apposition rate. These results indicate that MK-4 can induce recovery from the bone mineral loss caused by WRS treatment. Further studies are required to clarify the association between bone quality and MK-4.

金沢大学医薬保健研究域医学系, Asian dust, known as kosa in Japanese, is a major public health concern. In this panel study, we evaluated the effects of exposure to kosa on daily cough occurrence. The study subjects were 86 patients being treated for asthma, cough variant asthma, or atopic cough in Kanazawa University Hospital from January 2011 to June 2011. Daily mean concentrations of kosa and spherical particles were obtained from light detection and ranging (LIDAR) measurements, and were categorized from Grade 1 (0 μg/m3) to 5 (over 100 μg/m3). The association between kosa and cough was analyzed by logistic regression with a generalized estimating equation. Kosa effects on cough were seen for all Grades with potential time lag effect. Particularly at Lag 0 (the day of exposure), a dose-response relationship was observed: the odds ratios for Grades 2, 3, 4, and 5 above the referent (Grade 1) were 1.111 (95% confidence interval (CI): 0.995-1.239), 1.171 (95% CI: 1.006-1.363), 1.357 (95% CI: 1.029-1.788), and 1.414 (95% CI: 0.983-2.036), respectively. Among the patients without asthma, the association was higher: the odds ratios for Grades 2, 3, 4 and 5 were 1.223 (95% CI: 0.999-1.497), 1.309 (95% CI: 0.987-1.737), 1.738 (95% CI: 1.029-2.935) and 2.403 (95% CI: 1.158-4.985), respectively. These associations remained after adjusting for the concentration of spherical particles or particulate matter with an aerodynamic diameter of less than 2.5 μm (PM2.5). Our findings demonstrate that kosa is an environmental factor which induces cough in a dose-response relationship. © 2014 Elsevier B.V., Embargo Period 12 months

The health effects associated with Asian dust have attracted attention due to the rapid increase in the number of Asian dust events in East Asia in recent years. The aim of this study was to investigate the associations between Asian dust and daily cough, as well as allergic symptoms, in adult patients who suffer from chronic cough. We enrolled 86 adult patients from Kanazawa University Hospital, Japan, who were diagnosed with asthma, cough variant asthma, atopic cough or a combination of these conditions. From January to June 2011, subjects recorded their symptoms in a diary every day. Asian dust and non-Asian dust periods were defined according to the dust extinction coefficient, measured using the light detection and ranging (LIDAR). The daily levels of total suspended particulates, polycyclic aromatic hydrocarbons (PAHs) and coexisting factors related to allergies, such as the Japanese cedar pollen count, were measured. McNemar's test showed that there were significantly more cough-positive patients during Asian dust periods than during the non-Asian dust period (p=0.022). In addition, during Asian dust periods when the daily levels of Japanese cedar pollen, Japanese cypress pollen and PAHs were elevated, there were significantly more patients who experienced itchy eyes than during the non-Asian dust period (p

Asian dust, known as kosa in Japanese, is a major public health concern. In this panel study, we evaluated the effects of exposure to kosa on daily cough occurrence. The study subjects were 86 patients being treated for asthma, cough variant asthma, or atopic cough in Kanazawa University Hospital from January 2011 to June 2011. Daily mean concentrations of kosa and spherical particles were obtained from light detection and ranging (LIDAR) measurements, and were categorized from Grade 1 (0μg/m3) to 5 (over 100μg/m3). The association between kosa and cough was analyzed by logistic regression with a generalized estimating equation. Kosa effects on cough were seen for all Grades with potential time lag effect. Particularly at Lag 0 (the day of exposure), a dose-response relationship was observed: the odds ratios for Grades 2, 3, 4, and 5 above the referent (Grade 1) were 1.111 (95% confidence interval (CI): 0.995-1.239), 1.171 (95% CI: 1.006-1.363), 1.357 (95% CI: 1.029-1.788), and 1.414 (95% CI: 0.983-2.036), respectively. Among the patients without asthma, the association was higher: the odds ratios for Grades 2, 3, 4 and 5 were 1.223 (95% CI: 0.999-1.497), 1.309 (95% CI: 0.987-1.737), 1.738 (95% CI: 1.029-2.935) and 2.403 (95% CI: 1.158-4.985), respectively. These associations remained after adjusting for the concentration of spherical particles or particulate matter with an aerodynamic diameter of less than 2.5μm (PM2.5). Our findings demonstrate that kosa is an environmental factor which induces cough in a dose-response relationship. © 2014 Elsevier Ltd.

Objectives Serine protease inhibitor Kazal type-5 (SPINK5) plays a crucial role in deciding the timing of desquamation of the skin. Its gene expression is limited at the very surface of the stratum granulosum (SG), whereas expression of kallikreins (KLKs) encoding proteases is usually found throughout the stratum spinosum and SG. Methods To explore the difference in expression regulation of these proteases/inhibitors, the function of SPINK5 promoter was examined using luciferase assay. Results Luciferase assay targeting the SPINK5 promoters (nucleotide −676/−532 and −318/−146 from the major transcription start site) showed high intensity in NHEK human keratinocyte. These two sites had neither common cis-elements nor GATA3 element but electrophoretic mobility shift assay showed similar retardation bands. Moreover, DNA footprinting did not display specific protected bands. Thus, we could not identify cis-element(s) that controlled these elements. Differentiation induced by high Ca2+ medium failed to alter their luciferase activities. Transfection of GATA3 expressing vector significantly but slightly increased them and that of vector expressing its dominant negative form decreased. Conclusions Although GATA3 is reportedly important for inhibition of proliferation and induction of differentiation of keratinocytes, its effect on SPINK5 expression was indirect and GATA3 alone was insufficient for final differentiation of keratinocytes where full SPINK5 expression was observed.

DNA methylation is the covalent modification of DNA that affects its function, without altering DNA sequences. Three important roles of DNA methylation include intrauterine programming, acquired predisposition, and transgenerational inheritance. A wide variety of factors can affect DNA methylation. Intrauterine programming involves drastic changes in DNA methylation patterns during cellular development and differentiation, which have a long-lasting effect on the predisposition of offspring. Influences from the mother, including maternal nutritional status, modify intrauterine epigenetic programming. In contrast to the rapid and drastic changes in utero, postnatal factors in daily life can also continue to slowly and dynamically change DNA methylation patterns in both somatic and germ cells. Epigenetic changes occurring in germ cell DNA exert a transgenerational impact on the phenotype of future generations, thus providing a means for ancestral transmission of environmental experiences. Despite adaptive ability, mismatch effect of transgenerational inheritance could be potentially harmful to health if environment has changed, and the acquired acclimatization is no longer beneficial. Increasing evidence from both human and animal studies indicates that DNA methylation exerts a causal impact on the development of hypertension. Therefore, an adverse outcome of maternal malnutrition could be the development of hypertension in offspring, whereby nutritional factors or disease conditions could induce phenotypes susceptible to hypertension through alteration of DNA methylation patterns. These factors are likely to alter DNA methylation patterns in all tissues including germ cells, and despite no direct evidence of an association between transgenerational epigenetic inheritance and hypertension, it is likely to play a role.

The aim of the present study was to determine the molecular basis of the beneficial effects of genistein and/or menaquinone‑4 (MK‑4) on bone quality. Initially, 1 µM genistein was applied to MC3T3‑E1 cells for 24 h and the upregulated mRNAs that were detected by microarray were selected for further examination by reverse transcription‑quantitative‑polymerase chain reaction. Among them, alterations were observed in the level of GATA‑binding protein 6 (GATA6), Notch gene homolog 2 (NOTCH2), Wnt family member 5A (WNT5A), bone γ‑carboxyglutamate protein (BGLAP), chondroadherin (CHAD), dipeptidyl peptidase 4 (DPP4), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), alkaline phosphatase (ALP) 3 and ATPase phospholipid‑transporting 11A (ATP11A) in response to treatment with 0.1 µM 17‑β‑estradiol, 1 µM genistein, and/or 1 µM MK‑4. GATA6, NOTCH2 and WNT5A are considered to be associated with osteoclast, but not osteoblast, function; however, increases in osteoblastic mRNAs, including BGLAP and CHAD, were observed in each of the treatment groups at 48 h. Immunocytochemical analysis confirmed an increase in CHAD and DPP4 proteins following the administration of genistein + MK‑4. Furthermore, genistein + MK‑4 led to alterations in cell morphology to spindle or oval shapes, and increased the intensity of ALP staining. Although the level of ALP mRNA was not consistently altered in response to the treatments, a marked increase in ALP activity was observed following 96 h treatment with genistein + MK‑4. Therefore, the simultaneous intake of genistein and MK‑4 appears to be beneficial for the maintenance of bone quality.

Repeated exposure to cruciferous allyl nitrile can induce antioxidant and phase 2 detoxification enzymes in various tissues. In the present study, we examined the effect of five days repeated exposure to allyl nitrile at subtoxic levels (0 - 400 μmol/kg/day) on the mouse ear. There was an increase in catalase activity in the ear at 100 - 400 μmol/kg/day, while elevated quinone reductase activity was observed at 400 μmol/kg/day only. Next, after repeated allyl nitrile exposure (0 - 400 μmol/kg/day), the skin irritant croton oil was applied to the ear to induce skin acute inflammation (oedema). Compared with the 0 μmol/kg/day group, animals in the 100 and 400 μmol/kg/day pre-treatment groups showed reduced oedematous response to croton oil. The reduced oedematous response was inversely associated with enhanced myeloperoxidase activity used as index of the presence of neutrophils. These data suggest that repeated exposure to allyl nitrile at subtoxic levels contributes to protection against croton oil-induced ear dermatitis, potentially through decreasing reactive oxygen species and through infiltration of neutrophils.

金沢大学環日本海域環境研究センター, The effect of fugu parathyroid hormone 1 (fugu PTH1) on osteoblasts and osteoclasts in teleosts was examined with an assay system using teleost scale and the following markers: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Synthetic fugu PTH1 (1-34) (100 pg/ml-10 ng/ml) significantly increased ALP activity at 6 h of incubation. High-dose (10 ng/ml) fugu PTH1 significantly increased ALP activity even after 18 h of incubation. In the case of TRAP activity, fugu PTH1 did not change at 6 h of incubation, but fugu PTH1 (100 pg/ml-10 ng/ml) significantly increased TRAP activity at 18 h. Similar results were obtained for human PTH (1-34), but there was an even greater response with fugu PTH1 than with human PTH. In vitro, we demonstrated that both the receptor activator of the NF-κB ligand in osteoblasts and the receptor activator NF-κB mRNA expression in osteoclasts increased significantly by fugu PTH1 treatment. In an in vivo experiment, fugu PTH1 induced hypercalcemia resulted from the increase of both osteoblastic and osteoclastic activities in the scale as well as the decrease of scale calcium contents after fugu PTH1 injection. In addition, an in vitro experiment with intramuscular autotransplanted scale indicated that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher than that in the control scales. Thus, we concluded that PTH acts on osteoblasts and osteoclasts in the scales and regulates calcium metabolism in goldfish. © 2011 Elsevier Inc. All rights reserved.

Inpatient satisfaction, job satisfaction/stress of medical workers, and hospital profitability under the 7:1 nursing care system (in which 1 nurse cares for 7 patients at a time) were compared with those under the 10:1 system at a hospital with the diagnosis procedure combination (DPC) payment system.A total of 202 inpatients discharged from the Departments of Cardiology and Metabolism completed an inpatient satisfaction questionnaire. A total of 108 medical workers were recruited to survey their job satisfaction/stress and to estimate the effects of the DPC. The profits for 10 cardiac and metabolic diseases in 2008 were compared with those in 2007.Mean inpatient satisfaction scores were around 4 ("somewhat satisfied") under both the 10:1 and 7:1 systems, and increased significantly to 4.14-4.38 under the 7:1 system. Excluding workload of physicians, the other stresses of physicians/nurses remained unaltered, as did their low job satisfaction. They estimated their understanding of the DPC as insufficient but felt that introducing the DPC neither shortened length of stay nor improved "the quality of medical/nursing care," regardless of the system. Total percentage profit in 2008 was almost the same as that in 2007, whereas diseases with deficits increased from 3 to 4. [corrected]The 7:1 system was somewhat beneficial for inpatients but not always for medical worker quality of life (QOL) or for hospital income, which are important to maintain high quality of medical/nursing care. It is important to further explore factors increasing QOL of medical workers and hospital income.

金沢大学医薬保健研究域医学系, High-dose cruciferous allyl nitrile can induce behavioral abnormalities in rodents, while repeated exposure to allyl nitrile at subneurotoxic levels can increase phase 2 detoxification enzymes in many tissues, although the brain has not been investigated yet. In the present study, we examined the effect of 5 days repeated exposure to subneurotoxic allyl nitrile (0-400 μmol/kg/day) on the brain. Elevated glutathione S-transferase activity was recorded in the striatum, hippocampus, medulla oblongata plus pons, and cortex. Enhancement of quinone reductase activity was observed in the medulla oblongata plus pons, hippocampus, and cortex. In the medulla oblongata plus pons, elevated glutathione levels were recorded. Following repeated subneurotoxic allyl nitrile exposure (0-400 μmol/kg/day), mice were administered a high-dose allyl nitrile (1.2 mmol/kg) which alone led to appearance of behavioral abnormalities. Compared with the 0 μmol/kg/day group, animals in the 200 and 400 μmol/kg/day pre-treatment groups exhibited decreased behavioral abnormalities and elevated GABA-positive cell counts in the substantia nigra pars reticulata and the interpeduncular nucleus. These data suggest that repeated exposure to subneurotoxic levels of allyl nitrile can induce phase 2 enzymes in the brain, which together with induction in other tissues, may contribute to protection against allyl nitrile neurotoxicity. © 2009 Elsevier Ltd. All rights reserved.

Among Tissue kallikrein genes (KLKs), KLK1 is abundantly expressed in human skin. Although its putative promoter is known to have various cis-elements, they have not been functionally tested. In the present study, the regulation mechanism of KLK1 promoter supporting such abundant expression was examined. Luciferase assay targeting the KLK1 promoter (nucleotide − 1153/+ 40 from the major transcriptional start site) was performed on NHEK human keratinocyte. − 954/− 855, − 428/− 236, and − 100/+ 40 had the induction activity. The motif search program failed to find unique binding motifs in − 428/− 236, whereas both − 954/− 855 and − 100/+ 40 had a unique GATAs binding motif. Electrophoretic mobility shift assay (EMSA) and DNA footprinting confirmed the binding of NHEK nuclear protein to these motifs that were supershifted by anti-GATA3 antibody. Among GATA isoforms, GATA3 alone could be amplified in RNA obtained from NHEK. Moreover, introduction of GATA3 into fibroblastic NIH3T3 cells enhanced the activity of KLK1 promoter containing − 954/+ 40, while that of GATA3 dominant negative mutant to NHEK cells impaired the same promoter's activity. Thus, GATA3 was found to bind the site located at − 954/− 855 and to be a key regulator of abundant KLK1 expression in human keratinocyte.

Class II alcohol dehydrogenase (pi-ADH), encoded by alcohol dehydrogenase (ADH4), is considered to contribute to ethanol (EtOH) oxidation in the liver at high concentration. Four single nucleotide polymorphisms (SNPs) were found in the promoter region of this gene. Analysis of genotype distribution in 102 unrelated Japanese subjects revealed that four loci were in strong linkage disequilibrium and could be classified into three haplotypes. The effects of these polymorphisms on transcriptional activity were investigated in HepG2 cells. Transcriptional activity was significantly higher in cells with the -136A allele than in those with the -136C allele. To investigate whether this difference in transcriptional activity caused a difference in EtOH elimination, previous data on blood EtOH changes after 0.4 g/kg body weight alcohol ingestion were analyzed. When analyzed based on aldehyde dehydrogenase-2 gene (ALDH2) (487)Glu/Lys genotype, the significantly lower level of EtOH at peak in subjects with -136C/A and -136A/A genotype compared with subjects with -136C/C genotype indicated that -136 bp was a suggestive locus for differences in EtOH oxidation. This effect was observed only in subjects with ALDH2 (487)Glu/Glu. These results suggested that the SNP at -136bp in the ADH4 promoter had an effect on transcriptional regulation, and that the higher activity of the -136A allele compared with the -136C allele caused a lower level of blood EtOH after alcohol ingestion; that is, individuals with the -136A allele may consume more EtOH and might have a higher risk for development of alcohol dependence than those without the -136A allele.

DNA methylation is the covalent modification of DNA that affects its function, without altering DNA sequences. Three important roles of DNA methylation include intrauterine programming, acquired predisposition, and transgenerational inheritance. A wide variety of factors can affect DNA methylation. Intrauterine programming involves drastic changes in DNA methylation patterns during cellular development and differentiation, which have a long-lasting effect on the predisposition of offspring. Influences from the mother, including maternal nutritional status, modify intrauterine epigenetic programming. In contrast to the rapid and drastic changes in utero, postnatal factors in daily life can also continue to slowly and dynamically change DNA methylation patterns in both somatic and germ cells. Epigenetic changes occurring in germ cell DNA exert a transgenerational impact on the phenotype of future generations, thus providing a means for ancestral transmission of environmental experiences. Despite adaptive ability, mismatch effect of transgenerational inheritance could be potentially harmful to health if environment has changed, and the acquired acclimatization is no longer beneficial. Increasing evidence from both human and animal studies indicates that DNA methylation exerts a causal impact on the development of hypertension. Therefore, an adverse outcome of maternal malnutrition could be the development of hypertension in offspring, whereby nutritional factors or disease conditions could induce phenotypes susceptible to hypertension through alteration of DNA methylation patterns. These factors are likely to alter DNA methylation patterns in all tissues including germ cells, and despite no direct evidence of an association between transgenerational epigenetic inheritance and hypertension, it is likely to play a role.

The balance between serine proteases, kallikreins (KLKs), and their inhibitor, Kazal type-5 (SPINK5), is important to maintain steady desquamation. To explore regulation of SPINK5 expression in skin, its promoters (nucleotide -676/-532 and -318/-146 from the major transcription start site) were subjected to luciferase assay because these two sides displayed high luciferase intensity in NHEK human keratinocyte. Electrophoretic mobility shift assay (EMSA) appeared specific nuclear protein binding to these two sides with almost the same size shift. However, in DNA foot printing, -676/-532 and -318/-146 had no specific sequence protection. Thus, at this moment, we could not confirm the major regulator of SPNK5 gene expression in skin. We, at least, acknowledge that the exploration of the relationship between KLKs and SPINK5 will give crucial information related to skin differentiation/proliferation. (partially supported by Grant-in-Aid C-264607944).

Latent transforming growth factor (TGF)-β binding proteins (LTBPs) play important roles in the secretion and activation of TGF-β. We previously reported that LTBP-1L is overexpressed in some patients with ovarian cancer. To clarify the molecular mechanism of LTBP-1L regulation, we analyzed DNA sequences in the promoter region of LTBP-1L and identified two novel single nucleotide polymorphisms, −202G/C and +20A/C. While the alleles with −202C and +20C were initially reported, our data demonstrated that −202G and +20A are common in both ovarian cancer patients and healthy patients in the Japanese population. Luciferase reporter assays revealed that the G-A haplotype induced transcriptional activation in a Sp1-dependent manner. Electrophoretic mobility shift assays showed that increased binding affinity of Sp1 to the promoter with −202G and +20A. Interestingly, ovarian cancer patients (n = 42) with G-A/G-A homozygous genotype had increased expression of LTBP-1 and apparently poorer survival than those with other genotypes (P = 0.02). These findings suggest that the single nucleotide polymorphisms −202G/C and +20A/C on the LTBP-1L promoter may affect the clinical outcome of ovarian cancer patients, probably via up-regulating protein expression. Further studies using a larger number of samples will definitively determine the correlation between LTBP-1 haplotype and clinical behavior of ovarian cancer.

In contrast to selenoprotein Ps (SeIPs) from other animal species, bovine selenoprotein P-like-protein (SeIPLP) was found to contain a tandem repeat of (CAYYCC)(11). During an investigation into whether SeIPLP was a bovine substitute for SeIP or uniquely bovine, its mRNA was found to consist of multiple variants with different length tandem repeat, namely p(0) with (CAYYCC)(11), p(-4) lacking (CAYYCC)(4), p(-8) lacking (CAYYCC)(8), and p(-9) lacking (CAYYCC)(9). Although they were encoded on a single gene locus, neither classicalGT-AG: nor minor classAT-AC: donator-acceptor sequences for alternative splicing were identified. A subsequent S1 protection assay using oligonucleotides, whose sequence may occur as variants, performed against bovine poly(A)(+)RNA identified a total of nine variants. Judging from the sequence of these variants and the branch point mapping, the consensus sequence for recognition of the donator was CACCCCCAC: and of the acceptor and the branch point A nucleotide,ACCCC: CAT orACCCC: CATCCCCAT. Furthermore, when the p(0) insert mRNA was expressed in COS-7 cells derived from an African green monkey kidney, cDNAs corresponding to p(-8) and p(-9) could be isolated. Therefore, the bovine SeIPLP mRNAs consisted of multiple variants probably due to a novel splicing mechanism which was not bovine-specific but common to other mammals.

The objective of the present study was to examine the possible generation of allylnitrile from commonly consumed cruciferous vegetables, and to determine the long-term behavioral effects of its oral administration at levels comparable to or greater than human dietary exposures. On the basis of gaschromatographic and mass spectrometric analyses, allylnitrile generation was observed in eight out of twelve vegetables, broccoli, broccoli (young stems and leaves), brussels sprouts, cabbage, cauliflower, chinese cabbage, komatsuna and kaiware-daikon (young stems and leaves). The daily dietary intake of allylnitrile was estimated to be at least 0.12 micromol/kg body weight for Japanese, based on its generation from the vegetables, broccoli, cabbage, cauliflower and Chinese cabbage and their daily dietary consumption. Mice received oral doses of 2, 20, 200, 500 and 1,100 micromol/kg allylnitrile once a day, 5 days per week for 13 weeks. Mice in the lower dosage groups of 2, 20 and 200 micromol/kg exhibited no behavioral changes. Mice dosed at the level of 500 micromol/kg showed restlessness, and one of them displayed alteration in tail hanging. These abnormalities were seen around seven days following the beginning of the treatment period. Animals in the highest dosage group elicited behavioral abnormalities, and their degree increased with increasing dosage. These results suggest that allylnitrile intake levels through daily vegetable consumption is below the level producing behavioral abnormalities.

A therapeutic agent of vitamin K2 is approved for the treatment of osteoporosis in Japan. However, little is known about the efficacy of dietary intake of vitamin K2 for bone health. We compared the effects of various levels of fermented soybeans (Natto) intake, which contains plenty of vitamin K2, on bone stiffness and bone turnover markers in healthy premenopausal women. Seventy-three healthy premenopausal women were randomly divided into four groups matched for age and parity categories. Natto was supplied as follows: Group 1 (no intake), Group 2 (once per month), Group 3 (once per week) and Group 4 (three times per week). Subjects took Natto at a lunch for 1 y, and the stiffness index by quantitative ultrasound and bone turnover markers were assessed at baseline, 6 mo and 1 y. There was no statistical difference in the stiffness index during the 1 y observation. However, bone specific alkaline phosphatase (BAP) in Group 4 was higher than that in Group 3 at 1 y and undercarboxylated osteocalcin (Glu) in Group 4 was significantly lower than those in Groups 1, 2 and 3 at 6 mo. Logistic regression analysis showed that the risk of reduction of bone formation markers declined to 0.07 in Group 4 based on that in Group 1. In premenopausal women who had to keep the stiffness index as high as possible before menopause, Natto intake may have contributed to the promotion of bone formation.

Background: Increasing lifespan has forced elderly persons and researchers alike to look at life and aging in a new way: at adding life to years rather than years to life. With increasing lifestyle choices for the elderly, the present study was undertaken to determine and compare the factors associated with life satisfaction in elderly living in a residential care home and in the community in Japan. Methods: This was a cross-sectional, self-reported, questionnaire-based study. Elderly persons from the residential care home in Kyoto City (n = 261, mean age ± SD = 79.6 ± 6.6 years, male : female = 1 : 3.1) and from the town of Urausu, Hokkaido (n = 733, mean age ± SD = 74.8 ± 6.8 years, male : female = 1 : 1.3), participated in the study. This represented 95.3% and 89.5% of all eligible elderly ≥ 65 years in the home and town, respectively. Activities of daily living, higher functions, medical and social history, geriatric depression scale and quality of life were studied. Using multiple logistic regression models, factors associated with high satisfaction of life were identified. Results: In community-dwelling elderly, relationship with friends, as opposed to relationship with family for elderly in residential care home, were factors related to life satisfaction. Conclusions: This study demonstrates the relative importance of social factors versus medical and functional factors as determinants of life of elderly. We have also shown how these factors differ for elderly living in different environments. Understanding these different as well as common determinants of life satisfaction from a whole array of diverse ones allows us to make effective strategy to improve the quality of life of elderly living in different conditions.

Background: Since the start of Long Term Nursing Insurance in Japan in April 2000, there has been much discussion about and emphasis on leading healthy and active twilight years, without ever being bedridden if possible. In an attempt to address some of these concerns, this comparative study examines the factors associated with dependence in the activities of daily living (ADL) in two samples of elderly people living in two different environments. Methods: In this cross-sectional, self-reported study, 262 and 968 elderly people from a residential care home and Yogo town completed a 65-item questionnaire. Questions pertaining to ADL, medical and social history, quality of life (QOL) and the 15-item Geriatric Depression Scale were included in the questionnaire. Using logistic regression model, factors associated with ADL dependence were determined. Results: In the residential care home group, age, female gender, instrumental ADL, communication-related ability, history of falls, stroke, and osteoarthropathy, depressive tendency and low satisfaction with life emerged as the factors associated with ADL dependence, after multivariate logistic regression. The corresponding factors in the community-dwelling elderly group were age, depressive tendency, history of osteoarthropathy, and low sense of health. Conclusion: Age and female gender were the two non-modifiable risk factors associated with ADL dependence. Stroke, followed by osteoarthropathy and falls were the main medical conditions predisposing to functional dependence. Dependence in instrumental ADL, independence in communication-related ADL, depressive tendency and components of QOL were factors associated with ADL dependence. Awareness of these factors not only helps to identify at-risk patients, to initiate preventive measures and to promote disability-delaying activities, but also helps in the holistic management of geriatric patients.

Fermented soybeans (Natto), a traditional Japanese food, contain more than 100 times as much vitamin K2 as various cheeses and are considered to promote gamma-carboxylation. Thus it is conceivable that Natto may play a preventive role in the development of osteoporosis. In this study, the relationships between the bone stiffness index measured by ultrasound, bone turnover markers, and lifestyle factors, including Natto intake, were examined in relation to vitamin D receptor (VDR) polymorphism. Among 117 premenopausal volunteers, approximately 75% were bb homozygotes, 20% were Bb heterozygotes, and only 5% were BB homozygotes. The B allele group and the bb group were subdivided according to Natto intake. In a monovariate analysis, no significant differences in indices for dietary intake, including Ca and vitamin D intake, were observed. The stiffness index in the B allele group, however, was slightly lower than in the bb groups when there was no Natto intake. There were no significant differences in serum ALP and Gla-osteocalcin, bone formation markers, or NTx and Ca in urine, bone resorption markers. A logistic regression test, including the interactional effect of Natto intake and VDR RFLP, indicated that the B allele group was a risk factor of bone mineral loss and that Natto was effective in maintaining bone stiffness in this group. Although the present study was cross sectional and requires longitudinal investigation, Natto may improve the bone health of people who have a low affinity receptor for vitamin D.

Angiotensinogen (AGT) has a central role in maintaining blood pressure and fluid balance. DNA methylation is an epigenomic modification maintaining a steady pattern in somatic cells. Herein we summarize the link between AGT regulation and DNA methylation. DNA methylation negatively regulates AGT expression and dynamically changes in response to continuous AGT promoter stimulation. High-salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT enhancer-binding protein-binding sites, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue. Salt-dependent hypertension may be partially affected by increased adipose AGT expression. Because angiotensin II is a well-established aldosterone-releasing hormone, stimulation of adipose AGT by aldosterone creates a positive feedback loop. This effect is pathologically associated with obesity-related hypertension, although it would be physiologically favorable for humans to efficiently retain their body fluid. The clear difference in DNA demethylation patterns between aldosterone and cortisol indicates a difference in the respective target DNA-binding sites between mineralocorticoid and glucocorticoid receptors in the AGT promoter. Stimulation-induced interactions between transcription factors and target DNA-binding sites trigger DNA demethylation. Dynamic changes in DNA methylation occur in relaxed chromatin regions both where transcription factors actively interact and where transcription is initiated. In contrast to rapid histone modifications, DNA demethylation and remethylation will progress relatively slowly over days or years. A wide variety of stimuli in daily life will continue to slowly and dynamically change DNA methylation patterns throughout life. Wise choices of beneficial stimuli will improve health.

Indonesia is ranked as the 4th highest contributor to tuberculosis (TB) in the world. Semarang District in Central Java displays extremely low case detection rate (CDR), possibly contributing to the local prevalence of TB.A case-control study was performed to explore the factors that cause such low CDR. We recruited 129 TB cases and 83 controls that visited the same centers and were not diagnosed with TB.The cases had 7.5 ± 2.3 symptoms/person on average, indicating the delay in diagnosis because the controls only displayed 1.0 ± 1.7. The multiple logistic regression analysis comparing the cases/controls extracted following factors as a risk to have TB: farmer, close contact with TB patients, ignorance of whether Bacillus Calmette-Guérin (BCG) was accepted or no, smoking, low income, a lot of people living in the same room, irregular hand wash before meals, not wash hands after blow, soil floor, and no sunlight and no ventilation in the house.Neither the cases nor the controls knew the symptoms and how to avoid TB infection, which probably caused the delay in diagnosis. It is difficult to change the current living conditions. Thus, the amendment of the community-based education program of TB seems to be required.

Vitamin K2 (VK2) has been used as a therapeutic agent for osteoporosis, and might reduce the frequency of fractures by improving bone quality. In the present study, effect of VK2 on bone turnover w...

Using the differential display method, latent transforming growth factor-beta 1 (TGF-beta 1) binding protein 1 (LTBP-1) mRNA was identified as one of the enriched mRNAs in ovarian carcinoma tissues after isolation of genes responsible for the development of ovarian cancer. Semi-quantitative reverse transcription (RT)-PCR analysis showed that expression of LTBP-1 and TGF-beta 1 mRNAs was much higher in both serous and mucinous adenocarcinomas than in their benign counterparts, including serous and mucinous cystadenomas and cystadenomas of low malignant potential (LMPs). Immunohistochemical analysis demonstrated that only proliferating benign adenoma cells were immunoreactive for both LTBP-1 and TGF-beta 1 proteins. In contrast, most serous and mucinous adenocarcinoma cells and their surrounding stroma were intensely immunoreactive for LTBP-1 and TGF-beta 1. LTBP-1 and TGF-beta 1 proteins, and their complex forms were identified in ovarian carcinoma cell lines and in their culture media by western blot analysis, suggesting these products were produced in ovarian carcinoma cells. RT-PCR analysis demonstrated that LTBP-1L, one of the LTBP-1 transcripts that has a strong activity in targeting the latent form of TGF-beta 1 to extracellular matrix (ECM), was predominantly expressed in ovarian carcinomas. Taken together, the results suggest that upregulation of LTBP-1 in ovarian carcinoma cells may have an important role in distributing TGF-beta1 in the stromal tissues surrounding carcinoma cells.

Differentially expressed genes in human esophageal carcinoma/normal tissue pairs were identified by means of a modified differential display technique to overcome the limitations of the conventional technique. Out of the eight cDNAs isolated, three were novel. The other five clones consisted of cDNA encoding cytokeratin 13, complement 7, KIAA1160, expression sequence tag (EST)-Hs110855 and EST-Hs13662. Quantitative reverse transcription-PCR (RT-PCR) for 10 carcinoma/normal tissue pairs confirmed down-regulation of cytokeratin 13, complement 7 and KIAA1160 mRNAs in carcinoma tissue and up-regulation of EST-Hs110855 mRNA. EST-Hs13662 mRNA also seemed to increase in carcinoma tissues but not to a statistically significant extent. In situ hybridization confirmed that cytokeratin 13 mRNAs localized in differentiating keratinocytes of normal epithelia and had disappeared in carcinomas, suggesting that this down-regulation reflects de-differentiation during carcinogenesis. The functions of complement 7 and KIAA1160 mRNAs are unclear in normal tissue but their down-regulation in carcinoma tissue may help the development of esophageal carcinoma. The expression of EST-Hs110855 mRNA reportedly observed in carcinomas of different origins suggests that this EST is a carcinogenesis-related gene. Our modified technique, which eliminates the source of false-positives, reduces the screening time, and dispense with the radioisotope, was found to be useful for isolating differentially expressed genes from clinical specimens with apparently genetically distant cellular populations and a very limited mass.

The isolation of differentially expressed genes in the nucleus accumbens (NA) from chronically ethanol-administered rats may help in understanding the underlying molecular mechanisms for the development and reinforcement of ethanol addiction. The differential display indicated that around 0.1-0.2% of mRNA could be considered to be affected by chronic ethanol-administration in the NA, regardless of whether ethanol directly affected gene expression in the NA or the gene alteration was secondary to changes in neuronal activity caused by ethanol. Forty-six clones were successfully reamplified, and screening by reverse Northern blot analysis resulted in the isolation of five up-regulated and three down-regulated genes. One of the up-regulated cDNAs was homologous to human TGFβ1 and its preferential expression was also observed in the cerebellum and locus coeruleus (LC). Since clone c10 displayed an extremely strong preferential expression in the ethanol-administered NA, its upstream sequence was analyzed by 5′ rapid amplification of DNA ends (5′RACE) but the coding sequence has not yet been isolated. c118 showed enrichment in the ethanol-administered NA and displayed strong homology to the mouse KH domain RNA binding protein QKI-5A. The 5′RACE analysis confirmed that this clone encoded rat QKI-5A. Since QKI proteins are considered to be regulators of myelination and their absence causes dysmyelination, its up-regulation may offer protection against ethanol-induced dysmyelination. Another 12 cDNAs were registered as expression sequence tags (ESTs) or novel with their functions unknown. It is considered important, however, that to their upstream sequences including coding regions and promoter sequences are identified not only to estimate the roles of these differentially expressed genes in ethanol addiction but also to clarify whether ethanol-dependent gene-regulation can occur or not.

Allylnitrile and crotononitrile induce behavioral abnormalities in mice. To explore the possible involvement of the vestibular system in these behavioral abnormalities, the expression of Fos protein, used as an indicator of neuronal activity, was examined within various brain structures in allylnitrile-, crotononitrile- and vehicle-treated mice. In each nitrile-treated mouse, Fos expression was observed in brain structures, which were divided into two groups. The structures in group 1 showed Fos expression between 1.5 h and 2 days postdosings, and in those in group 2 expression remained for up to 30 days postdosing. As most of these structures, especially in group 2, were identical to some Fos-positive structures observed after unilabyrinthectomy, the present results indicate that each nitrile induces Fos expression by causing a change in the peripheral vestibular system, resulting in behavioral abnormalities.

A single dose of allylnitrile in mice might induce persistent behavioral abnormalities, of which the mechanism is not yet known. The present study was undertaken to explore the relationship between behavioral abnormalities and pathological changes in the brain of mice following exposure to allylnitrile. Exposure to allylnitrile (63, 84, and 112 mg/kg, p.o.) resulted in dose-dependent changes in behavioral abnormalities, including increased locomotor activity, circling, retropulsion, head twitching, and alteration in reflexive behavior, which appeared at day 2 postdosing and were persistent throughout the experimental period (60 days) at the higher dose levels. Allylnitrile produced neuronal retraction including hyperchromasia of the nuclei in the raphe nuclei, cerebral cortex, hypothalamus, hippocampal CA1 and dentate gyrus later than 30 days. No gliosis was observed in these regions. Not all but a significant number of neurons in the hippocampal CA1, medial habenula and raphe nuclei were immuno-reactive to CPP32 (Caspase-3) even at day 2. These neurons were also positive to Hoechst 33258 staining, indicating allylnitrile caused apoptotic changes in specific neurons when neuronal behaviors became apparent. These apoptotic changes were persistent even in the area without neuronal contraction such as medial habenula. However, almost all neurons in these areas were also positive to terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). It is conceivable that allylnitrile caused apoptotic changes in neurons but did not always lead them to cell death immediately. Moreover, even when neuronal contraction resulted in retention of behavioral abnormalities, onset of these abnormalities seems to be associated with the impairment in the habenulo-raphe relay due to activation of apoptotic cascade in neurons.

Occupational Exposure to Low Concentrations of Organic Solvents in Shipyards: Hironobu KATSUYAMA, et al. Department of Public Health, Kawasaki Medical School—Because shipbuilders have to use organic solvents in very confined spaces, they have a higher risk of inhaling the solvents than workers working in an open space. The working environment at 14 workplaces with very limited space was examined and biological monitoring was performed on 18 workers who worked in such places. Out of 14 workplaces, only 4 did not exceed the assessment criterion. Although exhaust ventilation fans and chemical cartridge respirators were used to avoid exposure to solvent vapor irrespective of the concentrations of solvents, biological samples revealed inhalation of solvent vapor. Urinary excretion of hippuric acid and methylhippuric acid varied from 0.07 to 0.92 g/ g Cr and 0.02 to 0.42 g/g Cr at the end of the shift, respectively. Concentration of toluene and xylene in the end-exhaled air varied from

To clarify whether nitric oxide synthase (NOS) is involved in development of myopia, we examined the influence of form deprivation on the expressions of NOS isoform mRNA.NOS isoform cDNAs were amplified from total RNA extracted from control and 7-day-form-deprived chick retina-RPE (retinal pigment epithelium)-choroid, using competitive RT-PCR (reverse-transcription polymerase chain reaction). Each NOS isoform protein was also analyzed by Western blotting and immunohistochemistry.Expression of inducible NOS (iNOS) mRNA was highest in the control chick retina-RPE-choroid, followed by the expression of brain NOS (bNOS) mRNA. Expression of endothelial NOS (eNOS) mRNA was faint. The iNOS protein level, however, was only slightly higher than the levels of the bNOS and eNOS proteins and was found mainly in the outer part of the photoreceptor layer and inner and outer parts of RPE and choroid. bNOS alone was found in the outer nuclear layer. Although form deprivation reduced the iNOS and bNOS mRNA expressions, only the iNOS protein showed significant reduction.All three NOS isoforms were expressed in chick retina-RPE-choroid. Predominant expression of iNOS, instead of bNOS and eNOS, suggested the existence of ocular tissue-specific regulation of the iNOS gene. In addition to differences in expression level, bNOS displayed regional differential expression. Moreover, only iNOS was reduced in response to form deprivation. It is suggested that NOS isoforms may be differentially involved in the mechanisms regulating the posterior eye tissues, including myopic eye growth.

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2–3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120–160% of their initial amplitudes. A selective A1 adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A1 adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A1 adenosine receptors.

Prevention of a Malaria Outbreak among Non-Immune Japanese Workers Engaged in the Construction of a Thermal Power Plant in Sonebhadra, India: Hironobu KATSUYAMA, et al. Department of Health, Mitsubishi Heavy Industries—Continuous consumption of anti-malarial drugs is not always recognized as the first choice for prevention of malaria among workers residing in malarious areas for long periods. In Japan, personal protective measures have been primarily recommended. However, a high incidence of malaria was observed among workers engaged in construction of a thermal power plant in the Sonebhadra district, Uttar Pradesh, India, during the period from February to August, 1991. On-site inspection revealed the difficulties of preventing mosquito bites among personnel who had lived in industrialized countries and were not immune to malaria. Repeated education regarding the danger of malaria and rigorous precautions from mosquito bites dramatically reduced the incidence of malaria among such personnel. Not only proper usage of personal protection measures such as long sleeved shirts, mosquito repellents, mosquito nets etc., but also an effort to reduce the number of mosquitoes in living and working areas by such means as insecticides, spraying operations, etc. should be emphasized. On the other hand, chemoprophylaxis still seemed to be the most effective protective measure for workers compelled to remain outdoors until late at night. The utility and availability of protective divices may depend on the working status and circumstances, so that on-site inspection helps to ensure occupational health, and also affords an opportunity to instruct the workers on malaria prevention. (J Occup Health 1997; 39; 105-112)

A case control design was used to investigate human papillomavirus (HPV) prevalence and risk factors associated with development of cervical squamous intraepithelial lesion (SIL) and cervical cancer (CC) in Japan. One hundred and twenty-three women with histologically confirmed SIL or CC were compared to a control group of 778 cytologically normal women. With the use of a polymerase chain reaction (PCR)-based method for detection of low-risk (types 6 and 11) and high-risk (types 16, 18, 31, 33, 35, 52 and 58) HPVs, a high prevalence of HPV infection was observed in smokers among the controls. Logistic regression analysis demonstrated that high-risk HPV infection was the most significant risk determinant for LSIL (OR =9.4, 95% CI =4.5-19), HSIL (OR =77,95% CI=28-217) and CC (OR = 97,95% Cl =35-269). It also showed that unmarried women, women married for 5 to 19 years and smokers represented high risk groups for SIL, while smokers and women with a history of many pregnancies/parities had increased risk for CC. Smoking was the only HPV infection-independent factor for CC, suggesting that smoking may have a carcinogenic effect on the cervix. Since neither history of other cancer nor family cancer history was associated with SIL or CC, genetic factors appear to play little role in cervical carcinogenesis. The risk for cervical neoplasia due to HPV infection increased after marriage in Japan, suggesting a role for husbands as carriers of HPV transmission. Protection from high-risk HPV infection may be of greatest importance for prevention of cervical cancer.

DNA methylation patterns are maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not preserved. We demonstrate that stimulatory signals can change the DNA methylation status at a CCAAT/enhancer binding protein (CEBP) binding site and a transcription start site and activate expression of the angiotensinogen gene ( AGT ). A CEBP binding site in the human AGT promoter was hypomethylated in tissues with high expression of AGT , but not in those with low expression. The transcriptional activity of AGT promoter sequences cloned into a reporter plasmid depended on DNA methylation. In cultured human cells, interleukin 6 stimulation caused DNA demethylation around a CEBP binding site and a transcription start site; demethylation was accompanied by increased CEBP-β recruitment and chromatin accessibility of the AGT promoter. DNA methylation activity decreased in the nucleus. Excess circulating aldosterone upregulated AGT expression and was accompanied by DNA hypomethylation around a CEBP binding site and a transcription start site in human visceral adipose tissue. High salt intake led to upregulation of Agt expression, DNA hypomethylation around 2 CEBP binding sites and a transcription start site, and decreased DNA methylation activity in rat visceral adipose tissue. Taken together, CEBP binding initiates chromatin relaxation and transcription, which are followed by DNA demethylation around a CEBP binding site and a transcription start site in the AGT promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of AGT expression from an inactive to an active state.

Chemical pollution of the Amur River has seriously damaged traditions and caused posttraumatic stress disorder (PTSD) among the Nanai, the indigenous people living along this river. This study was performed to clarify the ethnographic characteristics of PTSD in this unique population. The study group consisted of 75 male and 112 female randomly selected volunteers. PTSD severity measured using scores of the Impact of Event Scale – Revised (Total-I) and Clinical-Administered PTSD Scale (Total-C) was estimated according to demographic and ethnocultural backgrounds, clinical status, and ethnopsychological attitudes toward the Amur River. The differences in averages of Total-I and Total-C were not always the same in the groups divided by ethnographic information. Logistic regression analysis with a dependent variable, possibly without PTSD (Total-I

When cDNA containing proteins enriched in the bovine cerebellar cortex were cloned, a clone which seemed to encode a selenoprotein P-like protein was isolated. The coding nucleotide sequence of its cDNA insert displayed high homology to rat and human selenoprotein P cDNA but contained 12 rather than 10 TGAs (12 rather than 10 selenocysteines in deduced amino acids), a tandem repeat of one CACTCC (His-Ser) and seven CATCCCs (His-Pro), and a 3′ untranslated region approximately 890 bases shorter than that of rat liver selenoprotein P. RT-PCR using a set of primers flanking to the repeat displayed the existense of mRNA without the repeat. The tandem repeat and its adjacent region consisted of a similar motif of CAC/TCC/AC/T. Thus, these proteins included a (His-Pro) rich domain with a slightly negative free energy change irrespective of having the tandem repeat or not. Such His-Pro repeats reportedly exist in the segmentation gene paired or homeobox protein Om ( 1D ) of Drosophila . Moreover, both this selenoprotein P-like protein mRNA and selenoprotein P mRNA were expressed in all the areas of the brain but most prominently in the cerebellar cortex, hippocampus, and olfactory bulb. These findings suggest the possibility that these selenoproteins are major selenium carriers in the brain and play a role in the morphological response of nerve or glial cells.

In Japan, the medical examiner system was enforced only in three large cities, Tokyo metropolitan, Osaka and Kobe Cities. In other areas without this system, autopsy rates are much lower than in the areas with the system. Since the population of the aged (>/=65 years old) has been increasing recently, the subjects for medicolegal investigations seem to be also increasing. In the present study, between Mie Prefecture and those three Medical Examiner's Offices in Japan, the inquest results during the 5-year-period from 1996 to 2000, especially on the aged, were compared. The aged accounted for approximately 50% of all inquest cases in those areas. Autopsy rates for the aged were 16, 24 and 75% in Tokyo, Osaka and Kobe, respectively. Seventy-five to eighty percent was classified in deaths due to disease. Seventy to seventy-five percent of death due to disease was subclassified in circulatory diseases. The highest incidence of vascular diseases was observed in Kobe whose autopsy ratio was the highest. On the contrary, ambiguous causes of deaths, e.g. heart failure or unknown, were still frequent in Mie Prefecture.

Metallothionein (MT) gene expression in the brain has been most thoroughly studied using rodents. Although MT is considered to be a ''housekeeping'' protein even in the brain, the basal MT mRNA expression level is not always high. Differences in the responses of rats and mice have made it difficult to interpret the data. Moreover, the response to inducers is not always apparent, probably because the brain is protected by the blood-brain barrier and initial responses to inducers in peripheral tissues modulate their accumulation in the brain. A relatively high content of MT protein in the brain might be sufficient to elicit minute alterations in the level of inducers. Nonetheless, regulation of MT gene expression in the brain seems to be important in e.g. maintaining the levels of trace elements and controlling redox potentials. The localization and utilization of trans elements such as MTF-I and MEP-I in the brain will provide new aspects for study. The high homology among MT isoforms with respect to nucleotide as well as amino acid sequences has made it difficult to obtain cDNA probes or antibodies capable of distinguishing MT isoforms. Thus, their cross-reactivity might make changes in MT mRNAs appear minimal when MT isoforms are differently regulated. The rapid developments in methodology permitting sensitive, rapid, high-resolution analysis could clarify the background of tissue- and cell-specific gene regulation as well as differential induction.