Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis.

Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 °C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 °C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with >10-times higher proteolytic activity digested <20% of collagen. Following thermolysin treatments, almost all GAGs were extracted from the cartilage, whereas only 56% of GAGs were extracted after collagenase digestion. Disaccharide analyses of extracted GAG chains revealed >98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with <60% extraction efficiencies using collagenase, depending on GAG classes. These results indicate that thermolysin allows complete GAG extraction from solid articular cartilage and that complete solubilization is required for accurate and reproducible analyses of cartilage GAGs. Hence, thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive GAGomic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels.

Inverse association between soya food consumption and insulin resistance in Japanese adults.

OBJECTIVE: The purpose of the present study was to examine the association between soya food consumption and insulin resistance using baseline data of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study in Tokushima, Japan. DESIGN: This cross-sectional study included 1274 subjects, aged 34-70 years at baseline, living in Tokushima Prefecture between 2008 and 2013. Fasting blood samples were collected and information on lifestyle characteristics including soya food intake and medical history were obtained using a structured self-administered questionnaire. The homeostasis model assessment of insulin resistance (HOMA-IR) was measured and those with HOMA-IR ≥ 2.5 were defined as having insulin resistance. Multiple logistic regression models were used to analyse the association between soya product intake and the prevalence of insulin resistance. SETTING: Rural communities located in Tokushima Prefecture, Japan, between 2008 and 2013. SUBJECTS: A total of 1148 adults (565 men and 583 women), aged 34-70 years. RESULTS: The frequency of intake of miso soup, total non-fried soya products and total soya products showed significant inverse dose-response relationships with insulin resistance, after adjustments for potential confounders. When soya product intake was calculated as soya protein and isoflavone, the odds ratios of insulin resistance decreased significantly as the estimated intake of soya protein increased. Furthermore, significant inverse dose-response relationships were observed for total non-fried soya products and total soya products, after adjustment for total vegetable or total fibre consumption. CONCLUSIONS: The present results indicate that the intake of soya products and non-fried soya products is associated with reduced insulin resistance in the Japanese population.

A brain-specific Grb2-associated regulator of extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) (GAREM) subtype, GAREM2, contributes to neurite outgrowth of neuroblastoma cells by regulating Erk signaling.

Grb2-associated regulator of Erk/MAPK1 (GAREM) is an adaptor molecule in the EGF-mediated signaling pathway. GAREM is expressed ubiquitously in human organs and cultured cells. Two GAREM homologues are encoded by the human genome. Therefore, previously identified GAREM is named GAREM1. Here we characterized a new subtype of GAREM, GAREM2, that is specifically expressed in the mouse, rat, and human brain. Three GAREM2 tyrosines (Tyr-102, Tyr-429, and Tyr-551) are phosphorylated upon EGF stimulation and are necessary for binding to Grb2. Furthermore, GAREM2 and Shp2 regulate Erk activity in EGF-stimulated cells. These characteristics are similar to those of GAREM1. GAREM2 is expressed in some neuroblastoma cell lines and is also tyrosine-phosphorylated and bound to Grb2 after treatment with EGF. Eventually, GAREM2 regulates Erk activation in the presence of EGF or insulin like growth factor 1. GAREM2 also regulates insulin-like growth factor 1-induced neuronal differentiation of the SH-SY5Y neuroblastoma cell line. Although the structure and function of both GAREM subtypes are similar, GAREM1 is recruited into the nucleus and GAREM2 is not. Nuclear localization of GAREM1 might be controlled by a GAREM1-specific nuclear localization sequence and 14-3-3ε binding. The N-terminal 20 amino acids of GAREM1 make up its nuclear localization sequence that is also a 14-3-3ε binding site. The GAREM family is a new class of adaptor molecules with subtype-specific biological functions.

Association between dietary calcium intake and arterial stiffness according to dietary vitamin D intake in men.

Studies on the associations of dietary Ca and vitamin D intakes with arterial stiffness are scarce. In the present study, these associations were evaluated in Japanese men. Data from a total of 535 eligible men, aged 35-69 years, who participated in the baseline survey of a cohort study in Tokushima Prefecture, Japan, and underwent brachial-ankle pulse wave velocity (ba-PWV) measurements were analysed. ba-PWV is a measure of arterial stiffness and is recognised as a marker of atherosclerotic vascular damage. Information regarding the cohort's lifestyle characteristics including dietary behaviour over the past year was obtained from a structured self-administered questionnaire. Dietary Ca and vitamin D intakes were adjusted for total energy intake using the residual method and divided into quartiles; the highest quartile was used as the reference. General linear models were used to evaluate the associations between dietary Ca and vitamin D intakes and ba-PWV values adjusted for probable covariates. The association between dietary Ca intake and ba-PWV was further evaluated using similar general linear models stratified by dietary vitamin D intake (median or below/above median). Dietary Ca intake was found to be significantly inversely associated with ba-PWV after adjusting for probable covariates (P for trend = 0·020). However, no such association was observed between dietary vitamin D intake and ba-PWV. The inverse association between dietary Ca intake and ba-PWV was striking in subjects with higher dietary vitamin D intake. However, no association was found in subjects with lower dietary vitamin D intake. These results indicate that adequate dietary Ca and vitamin D intakes may be protective against the development of arterial stiffness in Japanese men.

Inverse correlation between coffee consumption and prevalence of metabolic syndrome: baseline survey of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study in Tokushima, Japan.

BACKGROUND: It is unclear whether consumption of coffee and green tea is associated with metabolic syndrome. METHODS: This cross-sectional study enrolled 554 adults who had participated in the baseline survey of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study in Tokushima Prefecture, Japan. Consumption of coffee and green tea was assessed using a questionnaire. Metabolic syndrome was diagnosed using the criteria of the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) and the Japan Society for the Study of Obesity (JASSO). Logistic regression analysis was used to examine the association between consumption of coffee and green tea and prevalence of metabolic syndrome and its components. RESULTS: After adjustment for sex, age, and other potential confounders, greater coffee consumption was associated with a significantly lower prevalence of metabolic syndrome, as defined by NCEP ATP III criteria (P for trend = 0.03). Participants who drank more coffee had a lower odds ratio (OR) for high serum triglycerides (P for trend = 0.02), but not for increased waist circumference or high blood pressure. Using JASSO criteria, moderate coffee consumption (1.5 to <3 cups/day) was associated with a significantly lower OR for high plasma glucose (OR = 0.51, 95% CI 0.28-0.93). Green tea consumption was not associated with the prevalence of metabolic syndrome or any of its components. CONCLUSIONS: Coffee consumption was inversely correlated with metabolic syndrome diagnosed using NCEP ATP III criteria, mainly because it was associated with lower serum triglyceride levels. This association highlights the need for further prospective studies of the causality of these relationships.

Intrauterine sensitization of allergen-specific IgE analyzed by a highly sensitive new allergen microarray.

BACKGROUND: To design a rational allergy prevention program, it is important to determine whether allergic sensitization starts in utero under the maternal immune system. OBJECTIVE: To investigate the origin of allergen-specific IgE antibodies in cord blood (CB) and maternofetal transfer of immunoglobulins. METHODS: The levels of food and inhalant allergen-specific IgE, IgA, IgG, and IgG(4) antibodies in CB and maternal blood (MB) from 92 paired neonates and mothers were measured by using a novel allergen microarray of diamond-like-carbon-coated chip, with high-sensitivity detection of allergen-specific antibodies and allergen profiles. RESULTS: The levels of allergen-specific IgE antibodies against food and inhalant allergens and allergen profiles were identical in CB and newborn blood, but the levels and profiles, specifically against inhalant allergens, were different from those in MB. The level of allergen-specific IgA antibodies was below the detection levels in CB despite clear detection in MB. Therefore, contamination with MB in CB was excluded on the basis of extremely low levels of IgA antibodies in CB and the obvious mismatch of the allergen-specific IgE and IgA profiles between CB and MB. However, the levels of allergen-specific IgG and IgG(4) antibodies and their allergen profiles were almost identical in both MB and CB. CONCLUSION: Allergen-specific levels of IgE and IgA antibodies and their allergen profiles analyzed by the diamond-like-carbon allergen chip indicate that IgE antibodies in CB are of fetal origin. Food-allergen specific IgE antibodies were detected more often than inhalant-allergen specific IgE antibodies in CB, the reason of which remains unclarified.

Detection of a glutathionyl-carbonylated group (GS-CO-) on D-dopachrome tautomerase with preferential binding of GS-CO- to MIF proteins in rat livers damaged by carbon tetrachloride.

Liver damage has been induced in animal experiments using carbon tetrachloride (CCl4), a potent hepatotoxin. CCl4 is activated by cytochrome P450 2E1, which results in the formation of various metabolites including phosgene. Although D-dopachrome tautomerase (DDT) is abundant in the liver, its role currently remains unclear. The biological activity of DDT, for which the N-terminal proline is a key site, has been detected in various tissues. We herein incidentally detected a 333 Da modification to the N-terminal proline of DDT in rat livers damaged by CCl4. We identified that this modification as glutathionyl carbonylated group, which was formed by condensation of phosgene and reduced glutathione (GSH). We examined other glutathionyl-carbonylated proteins using two dimensional-polyacrylamide gel electrophoresis, mass spectrometry, and Western blotting for GSH, and detected only one glutathionyl-carbonylated protein, macrophage migration inhibitory factor (MIF). DDT belongs to the MIF family of proteins, and amino acid sequence identity between DDT and MIF is 33%. We concluded that MIF family proteins are major targets for glutathionyl carbonylation.

Decrease in Glycosaminoglycan with Aging in Normal Rat Articular Cartilage Is Greater in Females than in Males.

OBJECTIVE: Osteoarthritis (OA) is more prevalent in females. We hypothesized that changes in articular cartilage (AC) constituents with aging may cause differences. Herein, we aimed to compare the changes in AC constituents with aging in male and female normal rats. DESIGN: The glycosaminoglycan (GAG) and collagen (COL) contents of the AC in knee, hip, and shoulder joints of male and female rats were quantified and compared between age groups and sexes. RESULTS: The amount of GAG was decreased in multiple joints in both males and females with aging. In females, it had a significant decrease in all joints measured. The decrease in GAG with aging was more severe in females than in males. Even in young rats, the amount of knee joint GAG was significantly less in females than in males. The amount of COL in the AC was unchanged with aging in both sexes. CONCLUSIONS: The drastic GAG decrease with aging in female normal rats may explain the higher prevalence and more severe OA in females.

Role of a tyrosine phosphorylation of SMG-9 in binding of SMG-9 to IQGAP and the NMD complex.

SMG-9 is a component of the NMD complex, a heterotetramer that also includes SMG-1 and SMG-8 in the complex. SMG-9 was also originally identified as a tyrosine-phosphorylated protein but the role of the phosphorylation is not yet known. In this study, we determined that IQGAP protein, an actin cytoskeleton modifier acts as a binding partner with SMG-9 and this binding is regulated by phosphorylation of SMG-9 at Tyr-41. SMG-9 is co-localized with IQGAP1 as a part of the process of actin enrichment in non-stimulated cells, but not in the EGF-stimulated cells. Furthermore, an increase in the ability of SMG-9 to bind to SMG-8 occurs in response to EGF stimulation. These results suggest that tyrosine phosphorylation of SMG-9 may play a role in the formation of the NMD complex in the cells stimulated by the growth factor.

Correlations of fish intake and plasma docosahexaenoic acid levels with each congener of PCDDs/PCDFs/dioxin-like PCBs in blood from the Japanese population.

PURPOSE: The purpose of the present study was to investigate the factors associated with blood levels of each congener of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) in the Japanese population. METHODS: A cross-sectional study was performed on 1,656 subjects (755 men and 901 women) aged 15-73 years, who were living in 90 different areas of 30 prefectures in Japan. Blood levels of 29 PCDD, PCDF, and DL-PCB congeners were determined by high-resolution gas chromatography/mass spectrometry. In addition, a questionnaire survey on life style, including dietary habit, was carried out. RESULTS: The median total toxicity equivalent (TEQ) was 17 pgTEQ/g lipid. After adjustment for age, sex, body mass index, smoking habit, and consumption of other food groups, six PCDDs/PCDFs with 4-6 substituted chlorine atoms and 10 DL-PCBs, but not HeptaCDD/F or OctaCDD, showed significant positive correlations with the frequency of intake of fish and shellfish. Furthermore, significant positive relationships were also found between plasma levels of docosahexaenoic acid (DHA), a biomarker of fish intake, and 10 PCDDs/PCDFs with 4-6 chlorine atoms and 10 DL-PCBs. The partial correlation coefficients with plasma DHA were significantly higher for DL-PCBs than for PCDDs/PCDFs, and partial correlation coefficients for PCDDs/PCDFs significantly decreased with increasing number of chlorine atoms (Spearman r = -0.80, P = 0.001). CONCLUSIONS: Blood levels of PCDDs/PCDFs with 4-6 chlorine atoms and DL-PCBs were positively associated with fish intake in the Japanese population. These results may be explained by the higher degree of bioaccumulation of these congeners in fish and shellfish in the ecosystem, and the high consumption of fish among the Japanese population.

Gene expression signatures in peripheral blood cells from Japanese women exposed to environmental cadmium.

The objective of this study was to examine the effects of environmental cadmium (Cd) exposure on the gene expression profile of peripheral blood cells, using an original oligoDNA microarray. The study population consisted of 20 female residents in a Cd-polluted area (Cd-exposed group) and 20 female residents in a non-Cd-polluted area individually matched for age (control group). The mRNA levels in Cd-exposed subjects were compared with those in respective controls, using a microarray containing oligoDNA probes for 1867 genes. Median Cd concentrations in blood (3.55 microg/l) and urine (8.25 microg/g creatinine) from the Cd-exposed group were 2.4- and 1.9-times higher than those of the control group, respectively. Microarray analysis revealed that the Cd-exposed group significantly up-regulated 137 genes and down-regulated 80 genes, compared with the control group. The Ingenuity Pathway Analysis Application (IPA) revealed that differentially expressed genes were likely to modify oxidative stress and mitochondria-dependent apoptosis pathways. Among differentially expressed genes, the expression of five genes was positively correlated with Cd concentrations in blood or urine. Quantitative real-time PCR (RT-PCR) analysis validated the significant up-regulation of CASP9, TNFRSF1B, GPX3, HYOU1, SLC3A2, SLC19A1, SLC35A4 and ITGAL, and down-regulation of BCL2A1 and COX7B. After adjustment for differences in the background characteristics of the two groups, we finally identified seven Cd-responsive genes (CASP9, TNFRSF1B, GPX3, SLC3A2, ITGAL, BCL2A1, and COX7B), all of which constituted a network that controls oxidative stress response by IPA. These seven genes may be marker genes useful for the health risk assessment of chronic low level exposure to Cd.

D-Dopachrome tautomerase is a candidate for key proteins to protect the rat liver damaged by carbon tetrachloride.

Carbon tetrachloride (CCl4) is known to induce liver damage. Animal experiments with CCl4 injections have revealed many findings, especially mechanisms of liver damage and liver regeneration. Recently, proteomic approaches have been introduced in various studies to evaluate the quantitative and qualitative changes in the comprehensive proteome level. The aim of this research is to elucidate the key protein for liver damage, liver protection and liver regeneration by using proteomic techniques. 50 % (v/v) CCl4 in corn oil was administered intraperitoneally to adult male rats at a dose of 4ml/kg body weight. Approximately 24h after the injection, the liver was removed and extracted proteins were analyzed with cleavable isotope coded affinity tag (cICAT) reagents, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). A twelvefold increase in D-dopachrome tautomerase (DDT) was indicated. This enzyme has been reported to be involved in the biosynthesis of melanin, an antioxidant. According to the histological analysis, melanin levels were increased in un-damaged hepatocytes of CCl4-treated rats. These results suggest that the increase in DDT is a response to liver damage, accelerates melanin biosynthesis and protects the liver from oxidative stress induced by CCl4.

Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.

NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (µM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400-700 µM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown.

Circulating osteoprotegerin is associated with age and systolic blood pressure, but not with lipid profile or fasting glucose, in postmenopausal women.

OBJECTIVE: Osteoprotegerin (OPG), an inhibitor of osteoclastogenesis and osteoclast activation, has been reported to be linked to vascular biology. The aim of this study was to clarify the relationships between circulating OPG and the risk factors for vascular disorders in postmenopausal women. DESIGN: Eighty Japanese postmenopausal women were enrolled in this cross-sectional study. Clinical parameters (age, number of years since menopause, body mass index, systolic and diastolic blood pressure); serum concentrations of OPG, creatinine, calcium, and phosphorus; serum lipid profile; plasma glucose; and bone mineral density of the L2-4 vertebral bodies were determined for each woman. RESULTS: In rank-order correlation analysis, serum OPG concentrations had significant positive correlations with age (r = 0.29, P = 0.03), systolic blood pressure (r = 0.45, P < 0.01), diastolic blood pressure (r = 0.34, P < 0.01), and serum creatinine (r = 0.29, P = 0.04). Serum OPG concentration also had a marginally significant negative correlation with bone mineral density of the L2-4 vertebral bodies (r = -0.25, P = 0.06). However, serum OPG did not correlate with body mass index, serum lipid profile, or plasma glucose. The correlation of serum OPG with systolic blood pressure persisted after adjustment for both age and serum creatinine. CONCLUSIONS: These results suggest that increased circulating OPG in postmenopausal women is closely related to higher systolic blood pressure, which could cause atherosclerosis.

Associations of environmental exposure to dioxins with prevalent diabetes among general inhabitants in Japan.

The aim of this study was to evaluate the associations of environmental exposure to dioxins with diabetes among general inhabitants in Japan. A cross-sectional study was performed on 1374 participants, who were not occupationally exposed to dioxins, aged 15-73 years, living widely in 75 different residential areas of 25 prefectures in Japan through 2002-2006. Seven polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs), 12 dioxin-like polychlorinated biphenyls (PCBs), which are assigned a toxic equivalency factor, and biochemical factors were determined in fasting blood. A questionnaire survey on life-style including past history of diseases and treatments was also performed. We examined the associations of the accumulated toxic equivalents (TEQs) of PCDDs+PCDFs, dioxin-like PCBs and total dioxins with prevalent diabetes. Simple and partial correlation analyses revealed that HbA1c correlated with the accumulated TEQs of PCDDs+PCDFs, dioxin-like PCBs and total dioxins. In logistic regression analyses, the third and the highest quartiles of dioxin-like PCBs had adjusted odds ratios (ORs) of 3.07 (95% CI 1.16-8.81) and 6.82 (95% CI 2.59-20.1) compared to the reference (first plus second quartiles). On the other hand, the highest but not the third quartiles of PCDDs+PCDFs and total dioxins had significantly higher adjusted ORs compared to the respective references. These associations persisted when the subjects with poor liver or poor renal function were removed from the analysis. This recent representative data from general inhabitants in Japan showed associations of environmental exposure to dioxins, especially dioxin-like PCBs, with diabetes.

Dietary intake of PCDDs/PCDFs and coplanar PCBs among the Japanese population estimated by duplicate portion analysis: a low proportion of adults exceed the tolerable daily intake.

Dietary intake of dioxins was estimated by duplicate portion analysis of consecutive 3-day food samples among 86 men and 288 women (aged 17-72 years), who were living in 75 different areas of 25 prefectures in Japan. The mean (median) intake of PCDDs+PCDFs, coplanar PCBs (co-PCBs), and total dioxins, expressed on the basis of toxicity equivalents (TEQ), was 0.46 (0.34), 0.59 (0.39), and 1.06 (0.79)pg/kg body weight/day, respectively. Dietary intake was highest in fishing areas, followed by farming and urban areas. In multiple regression analysis, TEQs of PCDDs+PCDFs, co-PCBs, and total dioxins were positively associated with age and intake amount of fish and shellfish, and milk and dairy products, and negatively associated with survey year. There were significant positive correlations between dietary intake and blood levels for TEQs of PCDDs+PCDFs, co-PCBs, and total dioxins (Pearson r=0.35-0.36). The proportion of those whose dietary intake exceeded the tolerable daily intake (TDI), set by the World Health Organization (4pg TEQ/kg/day) and European Union (2pg TEQ/kg/day), was estimated at 2.1% and 10.4%, respectively. However, these proportions were considered to be overestimated because of the effect of day-to-day within-person variation. Therefore, the ratio of within- and between-person variance was estimated by applying random effects one-way analysis of variance to repeated measurements for another group of 35 persons. When the effect of within-person variation of dietary intake was accounted for, the proportion of subjects whose long-term intake exceeded the TDI of WHO and EU decreased to 0.06% and 2.9%, respectively.

Cadmium-induced renal dysfunction and mortality in two cohorts: disappearance of the association in a generation born later.

The association between exposure to environmental cadmium and mortality was investigated in two cohorts. The study population consisted of 275 (cohort I) and 329 (cohort II) residents (aged >or=40 years) in a cadmium-polluted area, Nagasaki Prefecture, Japan, who had participated in health surveys conducted in 1982 and 1992, respectively. The follow-up period extended from 1982 or 1992 to 2005. In the study area, the dietary cadmium intake had decreased after 1980-1983 because of the restoration of cadmium-polluted paddy fields. In cohort I, the mortality rate among those with urinary beta2-microglobulin (beta2-MG) concentration >or=1000 microg/g creatinine (cr.) was 1.41 times higher than the regional reference rate (95% confidence interval [CI] 1.07-1.83). After adjusting for age and other variables, in men, urinary N-acetyl-beta-D-glucosaminidase, and in women, serum creatinine, beta2-MG clearance, and urinary beta2-MG were significantly associated with increased mortality. However, in cohort II, urinary beta2-MG or total protein was not significantly associated with survival. These findings indicate that cadmium-induced renal dysfunction was a significant predictor of mortality, but that such an association is disappearing, probably because of the selective loss of advanced cases and reduced exposure and body burden.

Cause-specific mortality and cancer incidence rates in relation to urinary beta2-microglobulin: 23-year follow-up study in a cadmium-polluted area.

A longitudinal study was performed to investigate the associations of exposure to environmental cadmium (Cd) with cause-specific mortality and cancer incidence rates. The study population comprised 275 adults living in a Cd-polluted area, Nagasaki Prefecture, Japan. The follow-up period extended from 1982 to 2005 for the analysis of cancer mortality, and from 1985 to 2002 for the analysis of cancer incidence. In the study area, the daily Cd intake from foods had decreased after 1980-1983 because of the restoration of Cd-polluted rice fields. The mortality rate among those with urinary beta2-microglobulin (U-beta2M)>/=1000 microg/g creatinine was significantly higher than that of the Japanese population for death from causes other than cancer, but not for cancers (177 at the 95% confidence interval [CI] 94-303). From analysis within the Cd-polluted area, the age-adjusted rate ratio of cancer deaths associated with increased U-beta2M was 2.58 (95% CI 1.25-5.36). The incidence rate of cancer among those with U-beta2M>/=1000 microg/g creatinine was 1.38 (95% CI 0.69-2.47) times that of the regional reference rate. Within the Cd-polluted area, the age-adjusted rate ratio of developing cancer associated with high U-beta2M was 1.79 (95% CI 0.84-3.82). In summary, there was a significant association between U-beta2M excretion and cancer mortality. However, there was neither a significantly increased standardized incidence ratio of cancer, nor significant relationship between U-beta2M and cancer incidence rate, though the point estimates were higher than unity. Continued follow-up and investigation of a larger cohort may be required before drawing a conclusion for the association between exposure to environmental Cd and cancer risk.

A liver-derived immunosuppressive factor is an arginase: identification and mechanism of immunosuppression.

We found a substance in culture medium of neonatal pig liver fragments, which suppresses an immune response monitored by (3)H-thymidine incorporation using phytohemagglutinin (PHA)-stimulated lymphocytes. We named it as an immunosuppressive factor (ISF). To purify ISF, ammonium sulfate fractionation, DE52, SP-Sephadex, hydroxyapatite, blue Sepharose, heparin Sepharose and Superdex gel filtration columns were used. Using these purification procedures, ISF was purified 1,254-fold, with 9.2% recovery, from the culture medium of neonatal pig liver fragments, and was identified as arginase by its biochemical characteristics including molecular size, amino acid sequences of digested peptides and expression of arginase activity. The addition of ISF caused to decrease in arginine concentration in culture medium and at the same time DNA synthesis was suppressed dose-dependently, both of which were recovered by the addition of NOHA (N(G)-hydroxy-L-arginine), an arginase inhibitor. In addition, the depletion of arginine in culture medium also led to the inhibition of DNA synthesis. These results led us to the conclusion that immunosuppressive effect of ISF was due to arginase activity that decreased arginine concentration in culture medium, not to another function of ISF.

[Introduction of proteomic approach to environmental medicine].

Recent progress in life science technology and the availability of much information on genes obtained by genome analysis has enabled us to analyze the changes of proteins on a large scale. Sets of proteins are called proteomes, and proteomics is the scientific field of proteome analysis including differential, post translational modification and interaction analyses. Various proteomic techniques, particularly two-dimensional gel electrophoresis (2-DE), mass spectrometry, protein chip methods, and surface plasmon resonance (SPR), are very useful for acquiring proteomes in cells, tissues and body fluid, and for analyzing interactions between a protein and other biofactors including proteins. A proteomic approach is also useful for determining biomarkers of diseases and key proteins involved in various stages of metabolism such as differentiation, cell cycle and apoptosis. Environmental pollutants including endocrine disruptors inhibit activities of various organs in wild animals and humans. Proteomic approaches could be very useful tools for elucidating the mechanisms of damage caused by environmental pollutants. In this review, we describe the application of a proteomic approach to the field of environmental medicine.