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Accumulating evidence suggests that vitamin D has anticarcinogenic effects. However, it is unclear whether the nutrient is involved in the early stage of colorectal carcinogenesis. We examined the association between circulating vitamin D concentrations and colorectal adenomas in Japanese men. The study subjects comprised 656 cases of colorectal adenomas and 648 controls with normal colonoscopy among male self defense officials receiving a pre-retirement health examination between 1997 and 2004. Plasma or serum levels of 25-hydroxyvitamin D [25(OH)D] were measured using a radioimmunoassay method. Logistic regression analysis was used to obtain odds ratios (OR) and 95% confidence intervals (CI) with adjustment for potential confounding variables. Overall, there was no measurable association between circulating 25(OH)D concentrations and colorectal adenomas. When the analysis was restricted to subjects whose blood was taken during the winter season (November-April), the prevalence odds of colorectal adenomas for the highest versus lowest quartile of 25(OH)D was statistically significantly decreased (OR = 0.58; 95% CI = 0.34-0.99). The reduction was more pronounced for the rectum (OR = 0.22) and distal colon (OR = 0.47) than for proximal colon (OR = 0.70). During the summer season (May-October), higher levels of 25(OH)D were associated with an increased odds of small, but not large, adenomas. The present study adds to evidence that high levels of circulating vitamin D measured during darker season is associated with decreased prevalence of adenomas in the distal sites of the colorectum.

Background— Two consortium-based genome-wide association studies have recently identified robust and significant associations of common variants with systolic and diastolic blood pressures in populations of European descent, warranting further investigation in populations of non-European descent. Methods and Results— We examined the associations at 27 loci reported by the genome-wide association studies on Europeans in a screening panel of Japanese subjects (n=1526) and chose 11 loci showing association signals (1-tailed test in the screening, P CASZ1 , MTHFR, ITGA9 , FGF5 , CYP17A1-CNNM2 , ATP2B1 , and CSK-ULK3 —with any or all of these 3 traits: systolic blood pressure ( P =1.4×10 −14 to 0.05), diastolic blood pressure ( P =1.9×10 −12 to 0.05), and hypertension ( P =2.0×10 −14 to 0.006; odds ratio, 1.10 to 1.29). The strongest association was observed for FGF5 . In the whole study panel, the variance ( R 2 ) for blood pressure explained by the 7 single-nucleotide polymorphism loci was calculated to be R 2 =0.003 for male and 0.006 for female participants. Stratified analysis implied the potential presence of a gene-age-sex interaction, although it did not reach a conclusive level of statistical significance after adjustment for multiple testing. Conclusions— We have confirmed 7 loci associated with blood pressure and/or hypertension in the Japanese. These loci can guide fine-mapping efforts to pinpoint causal variants and causal genes with the integration of multiethnic results.

Background: Circulating high-sensitivity C-reactive protein (CRP) is a good marker of chronic low-grade inflammation. The few studies that have addressed the relationship between coffee consumption and CRP concentrations report inconsistent findings. The authors of this study examined the relationship between coffee and green tea consumption and serum concentrations of CRP, and the interaction with alcohol consumption, smoking, and obesity in a large population of free-living Japanese men and women. Methods: Study subjects were 10,325 men and women, 49–76 years of age, living in Fukuoka City who participated in a baseline survey of a cohort study on lifestyle-related diseases. Coffee and green tea consumption and other lifestyle characteristics were assessed using a structured questionnaire. Anthropometric measurements and venous blood samples were also included. Results: CRP concentrations were progressively lower with increasing levels of coffee consumption, after adjustment for smoking and other covariates (p for trend=0.03) in men, but not in women. Stratified analysis indicated that this inverse association was primarily limited to men with a high consumption of alcohol (≥50 g/day). Green tea consumption showed no measurable relationship with CRP concentrations in either men or women. Conclusions: Coffee may be protective specifically against alcohol-induced hepatic inflammation. Further studies are warranted in different populations. Clin Chem Lab Med 2010;48:849–54.

Objective. Few studies have suggested an inverse relation between coffee intake and serum concentrations of uric acid (UA), but none has addressed the relation in men and women separately. We examined the relation between coffee intake and serum UA levels in free-living middle-aged and elderly men and women in Fukuoka, Japan.Methods. Study subjects were derived from the baseline survey of a cohort study on lifestyle-related diseases, and included 11.662 men and women aged 49–76 years; excluded were those with medication for gout and hyperuricemia, use of diuretic drugs, and medical care for cancer or chronic kidney disease. Statistical adjustment was made for body mass index, alcohol use, hypertension, diabetes mellitus, and other factors.Results. There were inverse associations of coffee consumption with serum UA concentrations and hyperuricemia in men regardless of adjustment for covariates. Women showed a statistically significant, but weaker, inverse association between coffee and serum UA levels after allowance for the confounding factors.Conclusion. The findings add to evidence for a protective association between coffee intake and hyperuricemia.

Using two oligonucleotide probes which were synthesized based on the amino acid sequences of tryptic fragments obtained from purified bovine lung endothelin(ET) receptor, we isolated a cDNA encoding non-isopeptide-selective type of ET receptor (ETB) from a human liver cDNA library. The cDNA encoded a protein of 426 amino acid residues with putative seven transmembrane segments, exhibiting a significant similarity with the G-protein-coupled receptor superfamily. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and human ET-1-specific(ETA) receptor, respectivaly. COS cells transfected with the cloned cDNA expressed the binding sites with high affinity for every ET or sarafotoxin(SRT) isopeptide. Modification of disulfide bridges, a N-terminal amino acid or C-terminal hydrophobic residues of the isopeptides markedly reduced their binding affinity for the expressed ETB receptor (Fig.), suggesting that ETB receptor recognizes the common structure...

Aim The aim of this study was to examine the association of serum bilirubin, an endogenous antioxidant, with serum high sensitivity C-reactive protein (hs-CRP) level, HbA 1c , and the prevalence of type 2 diabetes in middle-aged and elderly Japanese men and women ( n = 12,400). Methods Analysis of covariance and logistic regression analysis were used to estimate geometric means of hs-CRP and HbA 1c and odds ratios of prevalent diabetes according to bilirubin concentrations, respectively, with statistical adjustment for behavioral factors and liver enzymes. Results Geometric means of hs-CRP and HbA 1c were progressively lower with increasing concentrations of serum bilirubin in men and women each. An inverse association between serum bilirubin and HbA 1c was slightly attenuated after adjustment for hs-CRP, but still remained highly significant (trend P = 0.0004 in men and trend P = 10 −5 in women). Multivariate-adjusted odds ratios of prevalent diabetes for the lowest to highest quintiles of serum total bilirubin were 1.00, 1.00, 0.73, 0.80, and 0.73 (trend P = 0.002), without adjustment for hs-CRP, and 1.00, 1.04, 0.76, 0.86, and 0.79 (trend P = 0.01), with adjustment for hs-CRP. Conclusions Higher concentrations of serum bilirubin probably confer protection against the development of type 2 diabetes.

Aim: Recent studies have demonstrated that the Wnt signaling pathway plays an important role in bone metabolism. The purpose of this study was to examine whether the gene of secreted frizzled-related protein 1 (SFRP1), a Wnt antagonist, is involved in the etiology of osteoporosis using association study. Methods: Seven single nucleotide polymorphisms (SNP) in the SFRP1 gene were genotyped and analyzed for association with bone mineral density (BMD) in 931 Japanese women (63.5 ± 6.7 years old, mean ± standard deviation). Results: One SNP (rs16890444) located in intron and another (rs3242) located in the 3′-untranslated region of the sFRP1 gene were significantly associated with the lumbar spine BMD value, and BMD values for both the femoral neck and the total hip, respectively. Women with the T/T genotype of the former SNP had a lower BMD value of the lumbar spine (L2–L4) compared with those with C/C or C/T (BMD value adjusted for age, duration after menopause, and body mass index: 0.781 vs 0.830, P = 0.037), while women with the T/T genotype of the latter SNP had higher BMD values of femoral neck and total hip compared with those with C/C or C/T (adjusted BMD value: femoral neck, 0.721 vs 0.633, P = 0.025; total hip, 0.834 vs 0.737, P = 0.027). Conclusion: These results suggest that the SFRP1 may be a candidate gene for a BMD determinant, but further studies need to consolidate the present findings.

Both androgen and estrogen play important roles in bone metabolism. As estrogen has a potent inhibitory effect on bone resorption, bone turnover increases in postmenopausal osteoprosis caused by estrogen deficiency. Treatment with selective estrogen receptor modulator (SERM) as well as hormone replacement therapy (HRT) improve bone turnover increased in postmenopausal women to normal range before menopause. Although the decrease of androgen level with aging is involved in pathogenesis of male osteoporosis, the detailed mechanism remains to be clarified.

Raloxifene (RLX), a selective estrogen receptor modulator (SERM), binds to the estrogen receptor alpha (ERalpha) and acts as an agonist in some tissues, and as an antagonist in others. To clarify the molecular mechanism underlying the tissue specificity of SERMs, we examined the intracellular localization of ERalpha using a green fluorescent protein (GFP)-tagged protein in culture cells from various tissues. Although ERalpha formed intranuclear foci in the presence of estradiol (E(2)), RLX translocated ERalpha into the nucleoli in breast cancer cell lines. This phenomenon was not observed in cells from other tissues. Immunofluorescence staining revealed that endogenous ERalpha was also translocated into the nucleoli in the presence of RLX. Mutation analyses demonstrate that helix 12 of ERalpha is essential to the nucleolar translocation of ERalpha. These results suggest that translocation of ERalpha into the nucleoli is RLX-specific and is a key event for RLX-induced growth repression of mammary gland cells.

OBJECTIVE To identify novel type 2 diabetes gene variants and confirm previously identified ones, a three-staged genome-wide association study was performed in the Japanese population. RESEARCH DESIGN AND METHODS In the stage 1 scan, we genotyped 519 case and 503 control subjects with 482,625 single nucleotide polymorphism (SNP) markers; in the stage 2 panel comprising 1,110 case subjects and 1,014 control subjects, we assessed 1,456 SNPs (P < 0.0025, stage 1); additionally to direct genotyping, 964 healthy control subjects formed the in silico control panel. Along with genome-wide exploration, we aimed to replicate the disease association of 17 SNPs from 16 candidate loci previously identified in Europeans. The associated and/or replicated loci (23 SNPs; P < 7 × 10–5 for genome-wide exploration and P < 0.05 for replication) were examined in the stage 3 panel comprising 4,000 case subjects and 12,569 population-based samples, from which 4,889 nondiabetic control subjects were preselected. The 12,569 subjects were used for overall risk assessment in the general population. RESULTS Four loci—1 novel with suggestive evidence (PEPD on 19q13, P = 1.4 × 10–5) and three previously reported—were identified; the association of CDKAL1, CDKN2A/CDKN2B, and KCNQ1 were confirmed (P < 10–19). Moreover, significant associations were replicated in five other candidate loci: TCF7L2, IGF2BP2, SLC30A8, HHEX, and KCNJ11. There was substantial overlap of type 2 diabetes susceptibility genes between the two populations, whereas effect size and explained variance tended to be higher in the Japanese population. CONCLUSIONS The strength of association was more prominent in the Japanese population than in Europeans for more than half of the confirmed type 2 diabetes loci.

The three chimera peptides of sarafotoxins S6b (SRTb) and S6c (SRTc), [Thr 2 SRTb, [Asn 4 ]SRTb and [Glu 9 ]SRTb, were synthesized chemically. From the comparisons of lethality, vasoconstrictor activity and receptor binding activity of SRTb, SRTa ([Asn 13 ]SRTb), SRTc ([Thr 2 Asn 4 Glu 9 Asn 13 ]SRTb), [Thr 2 SRTb, [Asn 4 ]SRTb and [Glu 9 SRTb, it appears that the Lys 9 to Glu 9 substitution greatly diminishes these activities while the Lys 4 to Asn 4 substitution does not affect them, and the Ser 2 to Thr 2 substitution or the Tyr 13 to Asn 13 substitution slightly diminishes these activities. There results suggest that the very low activities of SRTc are caused mainly by the Lys 9 to Glu 9 substitution, but not by the Ser 2 to Thr 2 substitution, which was suggested to be responsible for the weak bioactivities of SRTd ([Thr 2 ,Ile 19 ]SRTb).

Adiponectin is an adipocyte-derived protein with an insulin-sensitizing action. Circulating levels of adiponectin are inversely correlated with obesity, especially abdominal obesity. Some studies have suggested that low levels of circulating adiponectin might be related to increased risk of colorectal cancer and adenomas. The present study examined the relationship between total and high-molecular-weight (HMW) adiponectin to colorectal adenomas in the Self Defense Forces (SDF) Health Study. The study subjects comprised 656 cases of colorectal adenomas and 648 controls with normal colonoscopy among men receiving a preretirement health examination at two Self Defense Forces hospitals. Total and HMW adiponectin were slightly lower in adenoma cases than in controls; geometric means of total adiponectin were 5.42 µg/mL in cases and 5.63 µg/mL in controls (P = 0.13), and the corresponding values of HMW adiponectin were 2.47 µg/mL and 2.57 µg/mL, respectively (P = 0.29). Regardless of adjustment for body mass index and other lifestyle factors, total adiponectin was unrelated to the risk of colorectal adenomas. Total adiponectin levels were inversely related to the risk of large adenomas (≥ 5 mm), but not of small adenomas, with a nearly statistically significant decreasing trend (P = 0.06). However, the inverse association was largely ascribed to body mass index and other lifestyle factors. HMW adiponectin showed no clear association with either overall or size-specific risk of colorectal adenomas. The study provided suggestive evidence for a protective association between adiponectin and large adenomas, but did not indicate a protective association independent of adiposity. (Cancer Sci 2008; 99: 781–786)

Using a specific and sensitive radioimmunoassay (RIA) for the carboxyl terminal tail of endothelin (ET) (His16-Trp21), we have confirmed the presence of the converting activity from synthetic human big ET-1 to ET-1 in the homogenate of cultured bovine aortic endothelial cells. The optimal pHs for the converting activities were found at pH 3.0 and pH 7.0. The activity at pH 3.0 was completely inhibited by pepstatin A, whereas the activity at pH 7.0 was not affected by known various protease inhibitors except EDTA and EGTA. When the products from big ET-1 were analyzed on an ODS and a CN columns, only ET-1 was detected at pH 7.0, but various ET-like immunoreactivities other than ET-1 were detected at pH 3.0. These findings strongly suggest that mature ET-1 is formed from big ET-1 in the endothelial cells by a metal-dependent neutral protease.

Wnt signaling plays an important role not only in development and morphogenesis of embryos but in pathogenesis of various disorders including cancers. Analyses of the mutations in LDL receptor-related protein 5 (LRP5), a Wnt co-receptor, revealed that the Wnt signaling pathway is important for bone formation. Glucocorticoids suppress the canonical Wnt signaling pathway by such mechanisms as the enhancement the expression of dickkopf-1 (Dkk-1) and secreted frizzled-related protein 1 (sFRP1), and activation of the glycogen synthase kinase-3beta (GSK-3beta) activity. The inhibition of the canonical Wnt signal by glucocorticoid may be involved in the pathogenesis of glucocorticoid-induced osteoporosis.

Steroid hormones play important roles not only in the reproductive system but also in bone metabolism. We examined the functional relationship between steroid hormone receptors and the Runx2 transcription factor that is essential for osteoblast differentiation and proliferation. A functional reporter assay using promoters carrying steroid hormone-responsive elements revealed that Runx2 suppressed ligand-dependent transcriptional activation mediated by receptors. To examine intracellular localization of these proteins, a three-dimensional imaging study was performed by laser scanning confocal microscopy of green fluorescent protein (GFP)-fused proteins. As previously reported, ligand-bound human androgen receptor (AR) was translocated from the cytoplasm to the nucleus and formed subnuclear fine foci. Coexpression of human Runx2 disrupted the AR subnuclear fine foci formation, and the intranuclear fluorescent pattern of AR became similar to that of Runx2. On the other hand, ligand-bound ARs repressed the Runx2-mediated transactivation function. Runx2 was also extracted from its original compartment by ligand-bound ARs. These results suggest that both Runx2 and ARs repress the transactivation function of the other protein by extracting it from its original compartment. The AR and Runx2 may play a mutual role in transcriptional activation in osteoblasts.

We previously demonstrated that testicular zinc-finger protein (TZF) was a corepressor of the androgen receptor (AR). In the present study, we further showed that TZF-L, an alternative spliced variant of TZF, enhanced transactivation function of AR. Deletion analysis of TZF-L revealed that its N-terminus, which almost corresponded to that of TZF, but not its C-terminus was able to interact with AR. Additional analysis suggested that TZF and TZF-L were able to form both homodimers and heterodimers. TZF-L inhibited the homodimer formation of TZF and the intranuclear dot formation of TZF. We propose that in the unique regulation system of AR-mediated transactivation, two spliced isoforms of TZF act as coactivator and corepressor, respectively.

Vascular endothelium plays important roles in vascular relaxing, anti-atherosclerotic, or anti-thrombotic effect through production of vasoactive substances such as nitric oxide. Accumulated evidences suggest that endothelial dysfunction is the initial step in the development of atherosclerosis. Recently, less invasive or non-invasive method such as venous occlusion plethysmography and measurement of flow-mediated dilatation has been developed to evaluate endothelial function. Several investigations using these methods showed that endothelial function is impaired with aging. The mechanism of endothelial dysfunction with aging remains to be fully elucidated, although oxidative stress may be involved in impairment of endothelial function.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by chronic unovulation, hyperandrogenism and polycystic change in ovary. Hyperinsulinemia is so often accompanied with PCOS that insulin resistance may play important roles in pathogenesis of PCOS. Recent studies reported the effectiveness of insulin-sensitizing drugs on treatment of patients with PCOS. Thiazolidinedione, an agonist of PPARgamma receptor, improves not only insulin sensitivity but also hyperandrogenism and ovulatory dysfunction in patients with PCOS. Insulin-sensitizing drugs such as thiazolidinediones are expected to be a novel therapy for PCOS, although further studies on the effectiveness and safety should be required.

Glucocorticoid-induced osteoporosis(GIO)is the major cause of secondary osteoporosis, and prevention of GIO is thought to be essential to avoid bone fracture. Direct inhibitory effect of glucocorticoid on bone formation is considered as the main mechanism of GIO. Clinical guidelines have recommended that bisphosphonate is the first choice for the treatment of GIO. Bone mineral density in patients with GIO has recently been reported to be increased by the treatment with parathyroid hormone(PTH)at a low dose and by intermittent administration.

To explore the mechanism of glucocorticoid-induced osteoporosis, we investigated the effect of glucocorticoid on canonical Wnt signaling that emerged as a novel key pathway for promoting bone formation. Wnt3a increased the T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-dependent transcriptional activity in primary cultured human osteoblasts. Dexamethasone suppressed this transcriptional activity in a dose-dependent manner, while 1,25-dihydroxyvitamin D3 increased this transcriptional activity. LiCl, an inhibitor of glycogen synthase kinase-3beta, also enhanced the Tcf/Lef-dependent transcriptional activity, which was, however, not inhibited by dexamethasone. The addition of anti-dickkopf-1 antibody partially restored the transcriptional activity suppressed by dexamethasone. Dexamethasone decreased the cytosolic amount of beta-catenin accumulated by Wnt3a and also inhibited the nuclear translocation of beta-catenin induced by Wnt3a. These data suggest that glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts, partially through the enhancement of the dickkopf-1 production.

Bone morphogenetic proteins (BMP) are cytokines that promote differentiation of mesenchymal stem cells into differentiated osteoblasts and bone formation. Recently, HMG-CoA reductase inhibitors (statins) emerged as a candidate for the treatment of osteoporosis, because they stimulate BMP-2 expression and bone formation. Inhibition of Rho/Rho-kinase pathway may be involved in statin effect on bone formation.

The pyrrolizidine alkaloid plant toxin monocrotaline pyrrole (MCTP) causes pulmonary hypertension in experimental animals. The present study aimed to examine the effects of MCTP on the endothelium-dependent relaxation. We constructed an in vitro disease model of pulmonary hypertension by overlaying MCTP-treated bovine pulmonary artery endothelial cells (CPAEs) onto pulmonary artery smooth muscle cell-embedded collagen gel lattice. Acetylcholine (Ach) induced a relaxation of the control CPAEs-overlaid gels that were pre-contracted with noradrenaline, and the relaxation was inhibited by L-NAME, an inhibitor of NO synthase (NOS). In contrast, when MCTP-treated CPAEs were overlaid, the pre-contracted gels did not show a relaxation in response to Ach in the presence of 0.5 mM l-arginine. Expression of endothelial NOS protein, Ach-induced Ca2+ transients and cellular uptake of l-[3H]arginine were significantly smaller in MCTP-treated CPAEs than in control cells, indicating that these changes were responsible for the impaired NO production in MCTP-treated CPAEs. Since cellular uptake of l-[3H]arginine linearly increased according to its extracellular concentration, we hypothesized that the excess concentration of extracellular l-arginine might restore NO production in MCTP-treated CPAEs. As expected, in the presence of 10 mM l-arginine, Ach showed a relaxation of the MCTP-treated CPAEs-overlaid gels. These results indicate that the impaired NO production in damaged endothelial cells can be reversed by supplying excess l-arginine.

Although sex steroid hormones have significant effects on bone metabolism, the molecular mechanisms of these actions have not been fully elucidated yet. We examined the functional relationship between steroid hormone receptors and Tob, a member of an anti-proliferative protein family and a negative regulator of osteoblast proliferation and differentiation. Luciferase assay using promoters carrying hormone-responsive elements revealed that both Tob1 and Tob2 proteins but not PC3 suppressed steroid hormone receptor-dependent transcriptional activation in MC3T3-E1 osteoblastic cells. Mutated Tob proteins carrying amino acid substitutions at an LXXLL motif also showed the same degree of inhibition of the transcriptional activation as the wild type. By observation of androgen receptor (AR)-tagged with green fluorescent protein under a confocal laser scanning microscope, we found that Tob1 inhibits the nuclear foci formation of dihydrotestosterone-bound AR. These results indicate that Tob family proteins may negatively regulate sex steroid hormone action in bone formation.

To clarify the underlying mechanism of glucocorticoid-induced osteoporosis, we investigated the effect of glucocorticoid on the expression of dickkopf-1 (Dkk-1), an antagonist of Wnt signaling, in primary cultured human osteoblasts. Dexamethasone markedly induced the expression of mRNA for Dkk-1 in a dose- and time-dependent manner. The expression of Kremen1, a receptor for Dkk, did not change by the treatment with dexamethasone, while that of low-density lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor, slightly decreased by the treatment with dexamethasone. Dexamethasone increased the transcriptional activity of the Dkk-1 gene promoter in human osteoblasts. Serial deletion and mutation analyses of the Dkk-1 promoter showed that one putative glucocorticoid responsive element-like sequence located from -788 to -774bp is essential for the enhancement of the Dkk-1 promoter activity by dexamethasone in human osteoblasts. Since the Wnt signal is now recognized as a crucial regulator for bone formation, the Dkk-1 enhanced by glucocorticoid may inhibit the Wnt signal in osteoblasts, which may be involved in the pathogenesis of glucocorticoid-induced osteoporosis.

Vascular endothelium constitutively generates nitric oxide (NO) in large vessels and induces a relaxation of smooth muscle cells. However, little is known about the production of NO in microvessels, where smooth muscle layers are thin or absent. In this study, we have compared the constitutive production of NO in bovine brain microvascular endothelial cells (BBECs) with that in bovine aortic endothelial cells (BAECs). ATP, acetylcholine (ACh) and A23187 induced Ca(2+) transients both in BBECs and BAECs. In contrast, although ATP and A23187 evoked a similar degree of [Ca(2+)](i) increase in both types of cell, they failed to induce NO production in BBECs, as measured with an NO-sensitive fluorescent dye DAF-2, whereas in BAECs there was an increase in DAF-2 fluorescence. Hypotonic stress induced ATP release and subsequent NO production in BAECs, but not in BBECs. We have developed an in vitro model vessel system that consists of aortic smooth muscle cells embedded in a collagen gel lattice and overlaid with endothelial cells. Precontracted gels showed relaxation in response to ACh, when BAECs were overlaid. However, ACh-induced relaxation was not observed in BBEC-overlaid gels. Expression of eNOS protein as well as cellular uptake of l-[(3)H]arginine were significantly lower in BBECs than in BAECs. These results indicate that Ca(2+)-dependent NO production is at an undetectable level in BBEC, for which at least two factors, i.e. low levels of eNOS expression and l-arginine uptake, are responsible.

Both the soluble and membrane fractions prepared from cultured bovine endothelial cells (ECs) possessed the converting activities to metabolize big endothelin-1 (big ET-1) to endothelin-1 (ET-1) at neutral pH. Metal chelators inhibited the activities of both fractions, whereas phosphoramidon, a metalloprotease inhibitor, strongly inhibited only the activity of the membrane fraction. Phosphoramidon reduced the secretion of ET-1 and concomitantly enhanced the release of big ET-1 from cultured ECs. The incubations of big ET-1, big ET-2, and big ET-3 with cultured ECs resulted in their conversions to mature ETs. Phosphoramidon also abolished these conversions. These results indicate that vascular endothelium can convert not only endogenous big ET-1 but also exogenous big ET isopeptides to their mature ETs through a phosphoramidon-sensitive neutral metalloprotease.

Big endothelin-1 (big ET-1) is converted into an active form, ET-1, by endothelin-converting enzyme-1 (ECE-1). To assess the mechanism of substrate recognition by ECE-1, we examined the effects of variously substituted analogues of big ET-1 on ECE-1 activity, using solubilized membranes prepared from human ECE-1-expressed CHO-KI cells. Among the big ET-I analogues tested, [ 21 Phe]big ET-1[18-34] and [ 31 Ala]big ET-1[18-34] exhibited significant inhibition of ECE-I. A kinetic analysis revealed [ 21 Phe]big ET-1[18-34] to be a competitive inhibitor (K i = 21 μM) and [ 31 Ala]big ET-I [18-34] to be a noncompetitive inhibitor (K i = 36 μM). These results suggested that ECE-1 recognizes big ET- I at the P1 position and the C-terminal region in a different manner and that modification of these regions can produce ECE-I inhibitors.

Endothelin (ET) was originally isolated from culture media of aortic endothelial cells as a potent vasoconstrictive peptide (1). An active form of ET, consisting of 21 amino-acid residues, is generated from an inactive form of big ET-1 by a specific enzyme, called endothelin-converting enzyme (ECE). Accelerated production of ET-1 in damaged vascular endothelial cells was strongly suggested to be involved in the development of various fatal cardiovascular disorders, such as acute myocardial infarction, acute renal failure, and posthemorrhagic cerebral vasospasm. The design of specific inhibitors of ECE, along with those of ET receptors, may lead to the development of new treatments for these diseases. Accordingly, many investigators have focused on the identification and characterization of ECE. This chapter describes a history from the discovery of ECE to molecular characterization of this enzyme.

To investigate the role of functional genetic polymorphisms of metabolic enzymes of tobacco carcinogens in the development of colorectal adenomas.The study subjects were 455 patients with colorectal adenomas and 1052 controls with no polyps who underwent total colonoscopy in a preretirement health examination at two Self Defense Forces hospitals. The genetic polymorphisms studied were CYP1A1 2A (rs 4646903), CYP1A1 2C (rs 1048943), GSTM1 (null or non-null genotype), GSTT1 (null or non-null genotype) and NQO1 C609T (rs 1800566). Genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR method using genomic DNA extracted from the buffy coat. Cigarette smoking and other lifestyle factors were ascertained by a self-administered questionnaire. The associations of the polymorphisms with colorectal adenomas were examined by means of OR and 95%CI, which were derived from logistic regression analysis. Statistical adjustment was made for smoking, alcohol use, body mass index and other factors. The gene-gene interaction and effect modification of smoking were evaluated by the likelihood ratio test.None of the five polymorphisms showed a significant association with colorectal adenomas, nor was the combination of GSTM1 and GSTT1. A borderline significant interaction was observed for the combination of CYP1A1 2C and NQO1 (P = 0.051). The OR associated with CYP1A1 2C was significantly lower than unity among individuals with the NQO1 609CC genotype. The adjusted OR for the combination of the CYP1A1 2C allele and NQO1 609CC genotype was 0.61 (95%CI: 0.42-0.91). Although the interaction was not statistically significant (P = 0.24), the OR for individuals carrying the CYP1A1 2C allele and GSTT1 null genotype decreased significantly compared with those who had neither CYP1A1 2C allele nor GSTT1 null genotype (adjusted OR: 0.69, 95%CI: 0.49-0.97). Smoking did not modify the associations of the individual polymorphisms with colorectal adenomas. There was no measurable effect modification of smoking even regarding the combination of the genetic polymorphisms of the phase I and phase II enzymes.Combination of the CYP1A1 2C and NQO1 609CC genotypes was associated with a decreased risk of colorectal adenomas regardless of smoking status.

AIM: To investigate the associations between dietary intake of polyphenols and colorectal cancer. METHODS: The study subjects were derived from the Fukuoka colorectal cancer study, a community-based case-control study. The study subjects were 816 cases of colorectal cancer and 815 community-based controls. The consumption of 148 food items was assessed by a computer-assisted interview. We used the consumption of 97 food items to estimate dietary intakes of total, tea and coffee polyphenols. The Phenol-Explorer database was used for 92 food items. Of the 5 foods which were not listed in the Phenol-Explorer Database, polyphenol contents of 3 foods (sweet potatoes, satoimo and daikon) were based on a Japanese study and 2 foods (soybeans and fried potatoes) were estimated by ORAC-based polyphenol contents in the United States Department of Agriculture Database. Odds ratios (OR) and 95%CI of colorectal cancer risk according to quintile categories of intake were obtained by using logistic regression models with adjustment for age, sex, residential area, parental history of colorectal cancer, smoking, alcohol consumption, body mass index 10 years before, type of job, leisure-time physical activity and dietary intakes of calcium and n-3 polyunsaturated fatty acids. RESULTS: There was no measurable difference in total or tea polyphenol intake between cases and controls, but intake of coffee polyphenols was lower in cases than in controls. The multivariate-adjusted OR of colorectal cancer according to quintile categories of coffee polyphenols (from the first to top quintile) were 1.00 (referent), 0.81 (95%CI: 0.60-1.10), 0.65 (95%CI: 0.47-0.89), 0.65 (95%CI: 0.46-0.89) and 0.82 (95%CI: 0.60-1.10), respectively (Ptrend = 0.07). Similar, but less pronounced, decreases in the OR were also noted for the third and fourth quintiles of total polyphenol intake. Tea polyphenols and non-coffee polyphenols showed no association with colorectal cancer risk. The site-specific analysis, based on 463 colon cancer cases and 340 rectal cancer cases, showed an inverse association between coffee polyphenols and colon cancer. The multivariate-adjusted OR of colon cancer for the first to top quintiles of coffee polyphenols were 1.00 (referent), 0.92 (95%CI: 0.64-1.31), 0.75 (95%CI: 0.52-1.08), 0.69 (95%CI: 0.47-1.01), and 0.68 (95%CI: 0.46-1.00), respectively (Ptrend = 0.02). Distal colon cancer showed a more evident inverse association with coffee polyphenols than proximal colon cancer. The association between coffee polyphenols and rectal cancer risk was U-shaped, with significant decreases in the OR at the second to fourth quintile categories. There was also a tendency that the OR of colon and rectal cancer decreased in the intermediate categories of total polyphenols. The decrease in the OR in the intermediate categories of total polyphenols was most pronounced for distal colon cancer. Intake of tea polyphenols was not associated with either colon or rectal cancer. The associations of coffee consumption with colorectal, colon and rectal cancers were almost the same as observed for coffee polyphenols. The trend of the association between coffee consumption and colorectal cancer was statistically significant. CONCLUSION: The present findings suggest a decreased risk of colorectal cancer associated with coffee consumption.

A membrane-bound endothelin converting enzyme (ECE) of porcine aortic endothelial cells (ECs) was solubilized with Lubrol PX with high efficiency and stability. The solubilized ECE was bound to Ricinus communis agglutinin (RCA) but not to peanut agglutinin (PNA) or wheat germ agglutinin (WGA), suggesting that the ECE has a galactosylated structure possessing a high affinity for RCA. The sequential chromatography on RCA-agarose, PNA-agarose and a TSKgel DEAE-5PW column attained 2,100-fold purification for the ECE over the membrane fractions. The purified ECE was sensitively inhibited by phosphoramidon but not by thiorphan. The present RCA and PNA affinity column procedures may be a powerful approach to isolation of ECE of EC origin.

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.

Background Visceral obesity is positively related to insulin resistance. The nature of the relationship between waist circumference and insulin resistance has not been known in Japanese populations. This study examined the relationship between waist circumference and insulin resistance and evaluated the optimal cutoff point for waist circumference in relation to insulin resistance in middle-aged Japanese men. Methods Study subjects included 4800 Japanese men aged 39 to 60 years. Insulin resistance was evaluated by the homeostasis model assessment of insulin resistance (HOMA-IR). The relationship of waist circumference with HOMA-IR was assessed by use of adjusted means of HOMA-IR and odds ratios of elevated HOMA-IR defined as the highest quintile (≥2.00). Receiver operating characteristics (ROC) curve analysis using Youden index and the area under curve (AUC) was employed to determine optimal cutoffs of waist circumference in relation to HOMA-IR. Results Adjusted geometric means of HOMA-IR and prevalence odds of elevated HOMA-IR were progressively higher with increasing levels of waist circumference. In the ROC curve analysis, the highest value of Youden index was obtained for a cutoff point of 85 cm in waist circumference across different values of HOMA-IR. Multiple logistic regression analysis also indicated that the AUC was consistently the largest for a waist circumference of 85 cm. Conclusion Waist circumference is linearly related to insulin resistance, and 85 cm in waist circumference is an optimal cutoff in predicting insulin resistance in middle-aged Japanese men.

To clarify the existence and the distribution of endothelin (ET) receptor subtypes, we have examined the pharmacological properties and the molecular weight ( M r ) of 125 I-ET-1 and 125 I-ET-3 binding sites in various tissues of pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound 125 I-ET-1 in all the tissues examined. ET-3, sarafotoxin S6b (SRT-b) and sarafotoxin S6c (SRT-c) displaced the 125 I-ET-1 with the same sensitivity as ET-1 (IC 50 = 0.1–1.4 nM) in brain, kidney, liver and adrenal, whereas the three peptides showed very weak competition (IC 50 = 40–500 nM) against 125 I-ET-1 binding in cardiac atria, aorta, lung, stomach and uterus. The computer analyses of the binding data suggested the presence of high ( K d 1 = 0.04–0.29 nM) and low ( K d 2 = 60–190 nM) affinity binding sites for ET-3 and SRT-b in lung and stomach. 125 I-ET-3 bound to the high affinity sites in lung and stomach was displaced by ET/SRT isopeptides almost equipotently. Two proteins with M r of 47,000 and 35,000 were affinity-labeled with 125 I-ET-1 in cerebellum, while a protein with M r of 123,000, in addition to the two proteins, was predominantly labeled in lung. The above findings indicated that two distinct subclasses of ET receptors, namely, ET-1-specific and ET/SRT family-common receptors were distributed in various proportions in mammalian tissues, and suggested that their molecular forms are also different.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.

To characterize the properties and the distribution of endothelin (ET) receptor subtypes, we have examined the ligand selectivity and the molecular weight (Mr) of [125I]ET-1 and [125I]ET-3 binding sites in various tissues of human and pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound [125I]ET-1 in all of the tissues examined. ET-3, sarafotoxin S6b (SRTX-b), and sarafotoxin S6C (SRTX-c) displaced the [125I]ET-1 with nearly the same sensitivity as ET-1 (IC50 = 0.07-3.0 nM) in brain, kidney, liver, and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against [125I]ET-1 binding in atria, aorta, lung, stomach, and uterus. The Bmax value for [125I]ET-3 was 83% of that for [125I]ET-1 in human liver membranes, whereas the Bmax for [125I]ET-3 was only 12% of that for [125I]ET-1 in human atrial membranes. [125I]ET-3 bound to liver and atrial membranes was displaced by ET/SRTX isopeptides almost equipotently. Two proteins with Mr of 110 and 50 kDa were specifically affinity-labeled with [125I]ET-1 in porcine lung membranes. The above findings indicated that two distinct subclasses of ET receptors, namely ET-1/ET-2-specific and ET/SRT family common receptors, were distributed in various proportions in mammalian tissues.