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Abstract Background Osteoporosis and osteoporotic fracture in men are significant public health problems in an aging society. However, information on male osteoporosis remains impressively lacking, especially for Asians. We designed the Fujiwara-kyo Osteoporosis Risk in Men (FORMEN) study as an ancillary study of a cohort study, the Fujiwara-kyo study, to determine risk factors for osteoporotic fractures in Japanese men. Methods/Design Design: A community-based single-centre prospective cohort study with at least a 5-year follow-up Subjects: All the male participants of the Fujiwara-kyo study who were living in the four cities studied, aged 65 years and older, and able to walk without aid from others. Primary outcome: Incidence of osteoporotic fractures including vertebral and clinical non-vertebral fractures. Additional outcomes: Change in bone mineral density (BMD), change in hip geometry, onset of receiving benefits from Long-term Care Insurance (LCI), health-related quality of life, and mortality. Baseline measurements: BMD at the lumbar spine (LS) and hip (TH), hip geometry, vertebral deformity assessment, bone turnover markers, physical and cognitive performance, various medical and lifestyle factors, and geriatric psychosocial measures confirmed by interviews based on self-administrated questionnaires. Outcome surveillance: Annual mail surveys and a follow-up survey at the fifth year comprising similar items to the baseline study will be used to determine the outcomes. Receipt of benefits from LCI and mortality will be obtained from the city governments. Current status: The baseline study was conducted for 2174 eligible men, and 2012 completed the study and were eligible for follow-up. Prevalence rates of osteoporosis (BMD 2.5 SD or more below the young adult mean (YAM)) and low BMD (BMD 1 SD or more below YAM) in at least one of LS and TH were calculated to be 4.4% and 41.8%, respectively. The proportion of men with low BMD only at TH showed a significant increasing trend with aging (p < 0.0001) while that only at LS showed a decreasing trend (p = 0.0386). The prevalence rate of osteoporosis was underestimated when diagnosed using only BMD at LS. Other baseline measurements were successfully obtained. Discussion FORMEN baseline study was performed as designed and the FORMEN cohort study was successfully launched.

Abstract Background Vertebral fractures are the most common type of osteoporotic fracture. Although often asymptomatic, each vertebral fracture increases the risk of additional fractures. Development of a safe and simple screening method is necessary to identify individuals with asymptomatic vertebral fractures. Methods Lateral imaging of the spine by single energy X-ray absorptiometry and vertebral morphometry were conducted in 116 Japanese women (mean age: 69.9 ± 9.3 yr). Vertebral deformities were diagnosed by the McCloskey-Kanis criteria and were used as a proxy for vertebral fractures. We evaluated whether anthropometric parameters including arm span-height difference (AHD), wall-occiput distance (WOD), and rib-pelvis distance (RPD) were related to vertebral deformities. Positive findings were defined for AHD as ≥ 4.0 cm, for WOD as ≥ 5 mm, and for RPD as ≤ two fingerbreadths. Receiver operating characteristics curves analysis was performed, and cut-off values were determined to give maximum difference between sensitivity and false-positive rate. Expected probabilities for vertebral deformities were calculated using logistic regression analysis. Results The mean AHD for those participants with and without vertebral deformities were 7.0 ± 4.1 cm and 4.2 ± 4.2 cm (p < 0.01), respectively. Sensitivity and specificity for use of AHD-positive, WOD-positive and RPD-positive values in predicting vertebral deformities were 0.85 (95% CI: 0.69, 1.01) and 0.52 (95% CI: 0.42, 0.62); 0.70 (95% CI: 0.50, 0.90) and 0.67 (95% CI: 0.57, 0.76); and 0.67 (95% CI: 0.47, 0.87) and 0.59 (95% CI: 0.50, 0.69), respectively. The sensitivity, specificity, and likelihood ratio for a positive result (LR) for use of combined AHD-positive and WOD-positive values were 0.65 (95% CI: 0.44, 0.86), 0.81 (95% CI: 0.73, 0.89), and 3.47 (95% CI: 3.01, 3.99), respectively. The expected probability of vertebral deformities (P) was obtained by the equation; P = 1-(exp [-1.327-0.040 × body weight +1.332 × WOD-positive + 1.623 × AHD-positive])-1. The sensitivity, specificity and LR for use of a 0.306 cut-off value for probability of vertebral fractures were 0.65 (95% CI: 0.44, 0.86), 0.87 (95% CI: 0.80, 0.93), and 4.82 (95% CI: 4.00, 5.77), respectively. Conclusion Both WOD and AHD effectively predicted vertebral deformities. This screening method could be used in a strategy to prevent additional vertebral fractures, even when X-ray technology is not available.

Astrocytes, the most common glial cells in the central nervous system, maintain neuronal functions and have roles in neurological diseases. Acetylcholine (ACh) is one of the most essential neurotransmitters, and ACh receptor (AChR) ligands were recently reported to influence astrocyte functions. However, the functions of ACh, the only endogenous agonist of AChR, in astrocytogenesis and in the expression of astrocytic marker genes have not been known. We previously demonstrated that the inhibition of acetylcholine esterase (AChE) suppressed the differentiation of rat glioma C6 cells, an astrocyte differentiation model, and we observed a suppressive effect of ACh agonists on astrocyte differentiation. Our present study revealed that in the cAMP-induced differentiation of C6 cells, an AChR antagonist alleviated the expression of glia fibrillary acidic protein (GFAP) that had been suppressed by dichlorvos (DDVP), an organophosphate and an AChE inhibitor. Our findings also demonstrated a direct effect of ACh on the GFAP expression, and that muscarinic AChR is involved in the suppressive effect of ACh on the GFAP expression in differentiation-induced C6 cells. This is the first report indicating that ACh the only endogenous agonist for AChRs functions as a mediator of astrocyte differentiation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) is a principal factor for neurogenesis, neurodevelopment and neural survival through a BDNF receptor, tropomyosin-related kinase (Trk) B, while BDNF can also cause a decrease in the intracellular glutathione (GSH) level. We investigated the exacerbation of methylmercury-induced death of rat cerebellar granular neurons (CGNs) by BDNF in vitro. Since methylmercury can decrease intracellular GSH levels, we hypothesized that a further decrease of the intracellular GSH level is involved in the process of the exacerbation of neuronal cell death. In the present study, we established that in CGN culture, a decrease of the intracellular GSH level was further potentiated with BDNF in the process of the methylmercury-induced neuronal death and also in GSH reducer-induced neuronal death. BDNF treatment promoted the decrease in GSH levels induced by methylmercury and also by L-buthionine sulfoximine (BSO) and diethyl maleate (DEM). The promoting effect of BDNF was observed in a TrkB-vector transformant of the rat neuroblastoma B35 cell line but not in the mock-vector transformant. These results indicate that the exacerbating effect of BDNF on methylmercury-induced neuronal death in cultures of CGNs includes a further decrease of intracellular GSH levels, for which TrkB is essential.

Bone strength is predominantly determined by bone density, but bone microarchitecture also plays an important role. We examined whether trabecular bone score (TBS) predicts the risk of vertebral fractures in a Japanese female cohort. Of 1950 randomly selected women aged 15 to 79 years, we analyzed data from 665 women aged 50 years and older, who completed the baseline study and at least one follow-up survey over 10 years, and who had no conditions affecting bone metabolism. Each survey included spinal imaging by dual-energy X-ray absorptiometry (DXA) for vertebral fracture assessment and spine areal bone mineral density (aBMD) measurement. TBS was obtained from spine DXA scans archived in the baseline study. Incident vertebral fracture was determined when vertebral height was reduced by 20% or more and satisfied McCloskey-Kanis criteria or Genant's grade 2 fracture at follow-up. Among eligible women (mean age 64.1 ± 8.1 years), 92 suffered incident vertebral fractures (16.7/10(3) person-years). These women were older with lower aBMD and TBS values relative to those without fractures. The unadjusted odds ratio of vertebral fractures for one standard deviation decrease in TBS was 1.98 (95% confidence interval [CI] 1.56, 2.51) and remained significant (1.64, 95% CI 1.25, 2.15) after adjusting for aBMD. The area under the receiver operating characteristic curve of TBS and aBMD combined was 0.700 for vertebral fracture prediction and was not significantly greater than that of aBMD alone (0.673). However, reclassification improvement measures indicated that TBS and aBMD combined significantly improved risk prediction accuracy compared with aBMD alone. Further inclusion of age and prevalent vertebral deformity in the model improved vertebral fracture prediction, and TBS remained significant in the model. Thus, lower TBS was associated with higher risk of vertebral fracture over 10 years independently of aBMD and clinical risk factors including prevalent vertebral deformity. TBS could effectively improve fracture risk assessment in clinical settings., (© 2014 American Society for Bone and Mineral Research.)

The main target of neurotoxins is neurons because they comprise the main part of neural function, but glial cells may be indirect targets because they support the function of neurons. Among the glial cells, astrocytes in particular act as "nurse cells", regulating neuronal survival and functions. In the present study, to reveal whether a known neurotoxic substance, organophosphate dichlorvos (DDVP), affects the differentiation of astrocytes, we used an astrocyte differentiation model in rat glioma C6 cells. Morphological change and induction of GFAP expression in the differentiating C6 cells were suppressed by DDVP treatment. The known potential targets of DDVP are acetylcholine esterase (AChE), fatty acid amide hydrolase and methyl guanine methyl transferase. Among the specific inhibitors against these enzymes, the AChE inhibitor paraoxon successfully suppressed the cellular morphological changes and the induction of GFAP expression in differentiating C6 cells. These results indicate that DDVP inhibits differentiation in the C6 astrocyte-differentiation model, in which at least AChE inhibition is involved and that AChE is a potent regulator of the differentiation. Furthermore, considering that the main substrate of AChE is ACh, thus, ACh may act as regulators of astrocyte differentiation., (© 2013 Elsevier B.V. All rights reserved.)

End-stage renal failure deteriorates bone mass and increases fracture risk. However, there are conflicting reports in the literature regarding the effects of mild to moderate renal dysfunction on bone mineral density (BMD). We investigated the association between renal function and BMD at the spine and hip and bone metabolism markers in community-dwelling elderly Japanese men. From 2174 male volunteers aged ≥65 years, we examined 1477 men after excluding those with diseases or medications known to affect bone metabolism. Renal function was assessed by serum cystatin C and estimated glomerular filtration rate (eGFR) calculated using the Modification of Diet in Renal Disease Study equation. Bone metabolism was evaluated using levels of serum amino-terminal propeptide of type I procollagen (PINP) and tartrate-resistant acid phosphatase isoenzyme 5b (TRACP-5b), which represent bone metabolic status independent of renal function. eGFR was inversely associated with BMD after adjusting for potential confounders (P < 0.01). Cystatin C showed a weaker but significant association with BMD. eGFR was modestly positively associated with PINP levels (P = 0.04), although cystatin C concentrations were neither associated with PINP nor TRACP-5b levels. Since BMD integrates bone metabolism from the past to present, inverse associations between renal function and BMD may be attributed to past factors, such as obesity. Our findings suggest that low renal function does not affect bone metabolism in a population of community-dwelling elderly Japanese men. Longitudinal studies will be necessary to clarify whether low renal function affects bone loss., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hepatic stellate cells (HSCs) intracellularly preserve vitamin A in the normal liver. When the liver is damaged, HSCs transform into myofibroblast-like cells, and then proliferate and increase their expression of collagen. Cultured on a plastic plate, HSCs spontaneously activate. To maintain HSCs in a quiescent state with low expression of collagen, coating methods with extracellular matrixes (ECMs) such as Matrigel-coating or laminin-rich coating are commonly used for HSC cultivation. Kishimoto et al. [14] reported that Fragmin®/protamine microparticles (F/P-MPs) have the ability to absorb heparin-binding cytokines like ECMs. Therefore, we examined whether the cultivation on an F/P-MPs-coated plate maintains the quiescent state of RI-T cells (derived from rat HSCs) including the suppression of collagen expression. We found that the mRNA levels of collagen type IαI and TGF-β1 in RI-T cells were significantly suppressed in the cultivation on F/P-MPs-coated plates compared to cultures on noncoated and Matrigel-coated plates. We conclude that the F/P-MPs coating method is useful for maintaining with low expressions of collagen IαI and TGF-β 1 mRNA levels in HSCs.

There are no data concerning a relationship between alcohol and bone status from a large-scale community-based study of elderly Japanese men. The baseline survey for the Fujiwara-kyo Osteoporosis Risk in Men Study was performed in 2174 male participants during the period from 2007 to 2008 in Nara Prefecture, Japan. Among them 1665 fitted the following inclusion criteria: (a) age ≥65years, (b) no diseases or drug therapy that could affect bone mineral density (BMD). We analyzed 1421 men with complete information about alcohol intake. We found that alcohol intake and BMD were positively correlated after adjustment for age, body mass index, natto intake, milk intake, smoking, physical activity, education, marital status, and hypertension. Adjusted total hip BMD of men with alcohol intake >39g/day was 0.90g/cm(2) and that of abstainers was 0.85g/cm(2). With regard to bone turnover markers, alcohol intake was inversely associated with serum levels of osteocalcin and tartrate-resistant acid phosphatase isoenzyme 5b. A two-piece linear regression model revealed a positive relationship between alcohol intake and crude mean BMD for the total hip in those with alcohol intake of less than 55g/day. In contrast, alcohol intake and BMD in those with an alcohol intake of 55g/day or more was inversely correlated. The present large-scale study of elderly Japanese men revealed that although an alcohol intake of <55g/day was positively correlated to BMD, alcohol intake of ≥55g/day was inversely correlated to BMD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Vitamin K (VK) has a protective effect on neural cells. Methylmercury is a neurotoxicant that directly induces neuronal death in vivo and in vitro. Therefore, in the present study, we hypothesized that VK inhibits the neurotoxicity of methylmercury. To prove our hypothesis in vitro, we investigated the protective effects of VKs (phylloquinone, vitamin K(1); menaquinone-4, vitamin K(2) ) on methylmercury-induced death in primary cultured neurons from the cerebella of rat pups. As expected, VKs inhibited the death of the primary cultured neurons. It has been reported that the mechanisms underlying methylmercury toxicity involve a decrement of intracellular glutathione (GSH). Actually, treatment with GSH and a GSH inducer, N-acetyl cysteine, inhibited methylmercury-induced neuronal death in the present study. Thus, we investigated whether VKs also have protective effects against GSH-depletion-induced cell death by employing two GSH reducers, L-buthionine sulfoximine (BSO) and diethyl maleate (DEM), in primary cultured neurons and human neuroblastoma IMR-32 cells. Treatment with VKs affected BSO- and DEM-induced cell death in both cultures. On the other hand, the intracellular GSH assay showed that VK(2), menaquinone-4, did not restore the reduced GSH amount induced by methylmercury or BSO treatments. These results indicate that VKs have the potential to protect neurons against the cytotoxicity of methylmercury and agents that deplete GSH, without increasing intracellular GSH levels. The protective effect of VKs may lead to the development of treatments for neural diseases involving GSH depletion., (Copyright © 2011 Wiley-Liss, Inc.)

Objective: We examined anthropometric indicators to improve predictive ability of asymptomatic vertebral fracture screening models., Study Design and Setting: Data were obtained from the 1996 Japanese Population-based Osteoporosis (JPOS) Study. McCloskey-Kanis criteria diagnosed vertebral deformities on X-ray absorptiometric images in 693 women aged > or =50.The multiple logistic regression model included age, height, weight, postmenopausal status, total hip BMD, and arm span (AS) or sitting height as explanatory variables. Akaike's information criterion (AIC) evaluated model goodness-of-fit., Results: Age-adjusted AS and sitting height in subjects with and without vertebral deformities were 147.2+/-0.6 cm and 148.5+/-0.2 cm (P=0.055), 78.5+/-0.5 cm and 79.9+/-0.2 cm (P=0.007), respectively. Every 5-cm increase in AS indicated 1.5-fold increased risk of prevalent vertebral deformity in the model including age, height, weight, postmenopausal status, and BMD. Including the explanatory variable AS in models yielded better predictive accuracy than excluding AS (AIC, 441.7 vs 446.6, respectively). Sitting height did not significantly influence model predictive ability., Conclusion: Predictive accuracy of model for vertebral fracture including age, height, weight, postmenopausal status, and BMD improved when AS was added as an explanatory variable. Models to screen for asymptomatic vertebral fractures should include AS.

Beta-sitosteryl-D-glucoside and oleuropein isolated from the olive tree (Olea europaea) and their hydrolysed derivatives were tested by a feeding stimulative activity bioassay using the olive weevil (Dyscerus perforatus). Although the steroidal glucoside showed potent feeding stimulative activity, the activity of the aglycone (beta-sitosterol) was significantly lower than that of the glucoside. On the other hand, the difference in the activity between oleuropein, a secoiridoid glucoside, and the hydrolysed derivatives was not significant.

Guided by a feeding stimulant activity test on the olive weevil (Dyscerus perforatus), two compounds that showed potent feeding stimulant activity were isolated from the olive tree (Olea europaea). Based on their spectral data and a literature survey, they were identified as (-)-olivil (1) and (+ )-1-acetoxypinoresinol (2). The activities of these minor lignans were significantly higher for the female than for the male weevil.

A compound having structures of both vitamins E and C, L-ascorbic acid dl-alpha-tocopherol phosphoric acid diester potassium salt (EPC-K), which was proven to have both anti-oxidative3%!d moisturizing effects, has been formulated in the quasi drug hair growing products to mainly prevent dandruff and itching which may be one of the causes for the hair loss. Stabilities of EPC-K in 75% ethanolic solutions with various pHs (2-10) were examined extensively by storing them at 50 degrees C for 30 d and by sunlight exposure for 30 d. Decomposition of EPC-K was only observed under pH 2 at a level of 50% and under sunlight exposure at 25% level. A main decomposition product was identified as tocopheryl phosphate (EP), suggesting that a decomposition route was through hydrolysis. EPC-K was found to decompose by as much as 20% under 30 d storage at 50 degrees C when the concentration of the aqueous ethanolic solution was 0-30%. 1H, 13C and 31P-NMR studies in addition to a micelle formation test using pyrene fluorescent probe revealed that EPC-K formed a micelle at such low concentration of ethanol, which was assumed to be a cause for unstableness of EPC-K in that range. Hydrolytic decomposition of EPC-K was found by the reaction rate study to be a pseudo first order reaction with activation energy of 16.98 kcal/mol.

Glycyrrhizinates such as monoammonium and dipotassium glycyrrhizinates which are extracted from licorice, converted to easily water-soluble salts and refined have been formulated in many quasi-drug products as an anti-inflammatory agent. A quantitative analysis of glycyrrhizinates in quasi-drug products has usually and successfully been carried out by high performance liquid chromatography (HPLC). However, a slightly higher value than the actual content has sometimes been observed in daily analysis. As the result of our various studies to investigate these phenomena, we finally found that a minor constituent in commercial glycyrrhizinates, which was identified as monomethyl glycyrrhizinate, was hydrolyzed after they were formulated in quasi-drug products and converted to glycyrrhizin which in turn was determined by HPLC as a higher value.