Association between serum uric acid and lumbar spine bone mineral density in peri- and postmenopausal Japanese women.

SUMMARY: Previous studies on the association between uric acid and bone mineral density yielded conflicting results. In this study, we demonstrated positive association between uric acid and lumbar spine bone mineral density in peri- and postmenopausal Japanese women. Further research is needed to elucidate the underlying mechanism. INTRODUCTION: Oxidative stress has been implicated in the pathogenesis of osteoporosis. Uric acid, a potent antioxidant substance, has been associated with bone mineral density but previous studies have yielded conflicting results. The objective of the study was to examine the association between serum uric acid and lumbar spine bone mineral density (BMD). METHODS: This was a retrospective analysis of medical records of 615 women, aged 45-75 years, who had lumbar spine BMD measurement by dual-energy X-ray absorptiometry as a part of health checkup from August 2011 to July 2012. RESULTS: Mean serum uric acid level was 4.7 mg/dL. Serum uric acid level was positively and significantly associated with lumbar spine BMD independent of age, body mass index, smoking, drinking, physical activity, years after menopause, diabetes mellitus, hypertension, serum calcium, estimated glomerular filtration rate, plasma C-reactive protein, and serum alkaline phosphatase (standardized beta = 0.078, p = 0.049). Uric acid rapidly increased until the age of 60 years, and then decelerated but continued to increase thereafter. The association between lumbar spine BMD and uric acid remained significantly positive after excluding women older than 60 years. CONCLUSION: The present study showed that higher uric acid levels were linearly associated with higher lumbar spine BMD in peri- and postmenopausal Japanese women. Further research is needed to elucidate the underlying mechanism of the association between uric acid and BMD.

Inter- and intra-rater reliability in fibroptic endoscopic evaluation of swallowing.

Fibroptic endoscopic evaluation of swallowing (FEES) is a useful way for dentists to evaluate oropharyngeal dysfunction. However, no study has paid attention to inter- and intra-rater reliability of FEES evaluation about oropharyngeal dysfunction. The purpose of this study is to verify whether dentist who trained and experienced for evaluation of dysphagia could diagnose oropharyngeal function with FEES. Nine dentists independently evaluated FEES images of 10 cases four times each. At first, evaluators performed the first evaluation without consulting the evaluative criteria. Subsequently, evaluators independently re-evaluated at 1-week intervals for three consecutive weeks, consulting the evaluative criteria. And then, inter- and intra-rater reliability was calculated. Cohen's Kappa was used to assess reliability. The results found that overall inter-rater reliability was 0·35±0·04 (first evaluation), 0·45±0·05 (s), 0·44±0·05 (third) and 0·46±0·04 (fourth). Most of inter-rater reliability related to aspiration was moderate to high, but lower for categories that evaluated timing of swallowing and mastication. In contrast, intra-rater reliability was moderate to high for overall categories, at 0·53±0·04 (first vs. second evaluation), 0·55±0·04 (first vs. third), 0·53±0·04 (first vs. fourth), 0·55±0·03 (second vs. third), 0·60±0·03 (second vs. fourth) and 0·78±0·03 (third vs. fourth). FEES is reliable for experienced dentists to diagnose oropharyngeal function. Moreover, repeated evaluation with the aids of evaluative criteria is useful to improve the reliability of FEES.

Identification of novel androgen response genes in prostate cancer cells by coupling chromatin immunoprecipitation and genomic microarray analysis.

The androgen receptor (AR) plays a key role as a transcriptional factor in prostate development and carcinogenesis. Identification of androgen-regulated genes is essential to elucidate the AR pathophysiology in prostate cancer. Here, we identified androgen target genes that are directly regulated by AR in LNCaP cells, by combining chromatin immunoprecipitation (ChIP) with tiling microarrays (ChIP-chip). ChIP-enriched or control DNAs from the cells treated with R1881 were hybridized with the ENCODE array, in which a set of regions representing approximately 1% of the whole genome. We chose 10 bona fide AR-binding sites (ARBSs) (P<1e-5) and validated their significant AR recruitment ligand dependently. Eight upregulated genes by R1881 were identified in the vicinity of the ARBSs. Among the upregulated genes, we focused on UGT1A and CDH2 as AR target genes, because the ARBSs close to these genes (in UGT1A distal promoter and CDH2 intron 1) were most significantly associated with acetylated histone H3/H4, RNA polymerase II and p160 family co-activators. Luciferase reporter constructs including those two ARBSs exhibited ligand-dependent transcriptional regulator/enhancer activities. The present study would be powerful to extend our knowledge of the diversity of androgen genetic network and steroid action in prostate cancer cells.

Anomalous electronic structure of ionic liquids determined by soft x-ray emission spectroscopy: contributions from the cations and anions to the occupied electronic structure.

Soft x-ray emission spectroscopy was used for elucidating the electronic structure of ionic liquids [C(4)mim](+)PF(6)(-) and [C(4)mim](+)OTf(-), where [C(4)mim](+) stands for methylbutylimidazolium cation and OTf(-) for the trifluoromethanesulfonate anion. Nonresonant spectra measured above N, O, and F 1s edges selectively probed the molecular orbitals (MOs) of the cation and anions. They give a clear evidence that the highest occupied molecular orbital of the [C(4)mim](+) cation contributes to the topmost occupied states of the ionic liquids [C(4)mim](+)PF(6)(-), while both cationic and anionic MOs contribute for the case of [C(4)mim](+)OTf(-). Resonant soft x-ray emission spectra at the N 1s edge of these ionic liquids revealed that the energy gap of [C(4)mim](+)PF(6)(-) is solely determined by the [C(4)mim](+) cation, in contrast to usual ionic crystals. The ionic liquids form a new class of the ionic materials from the viewpoint of the electronic structure.

Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells.

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice.

Adult respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and the molecular mechanisms underlying it are poorly understood. Acid aspiration-induced lung injury is one of the most common causes of ARDS, characterized by an increase in lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration, and respiratory failure. Here, we investigated the role of platelet-activating factor (PAF) and the PAF receptor (PAFR) gene in a murine model of acid aspiration-induced lung injury. Overexpression of the PAFR gene in transgenic mice enhanced lung injury, pulmonary edema, and deterioration of gas exchange caused by HCl aspiration. Conversely, mice carrying a targeted disruption of the PAFR gene experienced significantly less acid-induced injury, edema, and respiratory failure. Nevertheless, the efficiency of PMN sequestration in response to acid aspiration was unaffected by differences in PAFR expression level. The current observations suggest that PAF is involved in the pathogenesis of acute lung injury caused by acid aspiration. Thus, inhibition of this pathway might provide a novel therapeutic approach to acute lung injury, for which no specific pharmaceutical agents are currently available.

Characteristics of leachate from pyrolysis residue of sewage sludge.

The pyrolysis residue (SP) of sewage sludge (SS) produced at 500 degrees C was subjected to batch and column leaching tests to investigate the release of its organic and inorganic constituents and metals. For comparison, incineration ash (SI) obtained from a SS incinerator was also tested. Pyrolysis and incineration reduced organic matter of SS from 0.78 kg kg(-1)-dry SS to 0.16 and 0.01 kg kg(-1)-dry SS, respectively. Heavy metals remained in SP without being volatilized, although Cd and Pb were transferred into the off-gas during incineration. In the batch leaching test with the leaching liquid-to-solid mass ratio (L/S)=10, the pH of the SS, SP, and SI filtrates was 6.3, 7.9, and 11.0, respectively. The total organic carbon concentrations were in the order SS (877 l mg l(-1))>>SP (99 mg l(-1))>SI (26 mg l(-1)). The SP and SI filtrates met the landfill standard for the Cd and Pb concentrations (<0.3 mg l(-1)). In the column tests, although the SP contained more organic matter than that of SI, its carbon discharge into the leachate under aerobic conditions was similar to that of SI under anaerobic conditions. The leaching of heavy metals, such as Cd, Cr, Pb, and Zn, was also suppressed in SP during the active decomposition of organic matter. We demonstrated that pyrolysis reduces the potential release of pollutants from sewage sludge in landfill, making it a promising method of treating sewage sludge before landfilling.

Restricted nutrition-induced low birth weight, low number of nephrons and glomerular mesangium injury in Japanese quail.

Insufficient nutrition during the perinatal period causes structural alterations in humans and experimental animals, leading to increased vulnerability to diseases in later life. Japanese quail, Coturnix japonica, in which partial (8-10%) egg white was withdrawn (EwW) from eggs before incubation had lower birth weights than controls (CTs). EwW birds also had reduced hatching rates, smaller glomeruli and lower embryo weight. In EwW embryos, the surface condensate area containing mesenchymal cells was larger, suggesting that delayed but active nephrogenesis takes place. In mature EwW quail, the number of glomeruli in the cortical region (mm2) was significantly lower (CT 34.7±1.4, EwW 21.0±1.2); capillary loops showed focal ballooning, and mesangial areas were distinctly expanded. Immunoreactive cell junction proteins, N-cadherin and podocin, and slit diaphragms were clearly seen. With aging, the mesangial area and glomerular size continued to increase and were significantly larger in EwW quail, suggesting compensatory hypertrophy. Furthermore, apoptosis measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis was higher in EwWs than in CTs on embryonic day 15 and postnatal day 4 (D4). Similarly, plasma glucocorticoid (corticosterone) was higher (P<0.01) on D4 in EwW quail. These results suggest that although nephrogenic activity is high in low-nutrition quail during the perinatal period, delayed development and increased apoptosis may result in a lower number of mature nephrons. Damaged or incompletely mature mesangium may trigger glomerular injury, leading in later life to nephrosclerosis. The present study shows that birds serve as a model for 'fetal programming,' which appears to have evolved phylogenetically early.

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7.

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.

Inhibitory effect of angiotensin II type 2 receptor on coronary arterial remodeling after aortic banding in mice.

BACKGROUND: The renin-angiotensin system is thought to be critical for the development of cardiac hypertrophy, whereas the role of the angiotensin II type 2 (AT(2)) receptor in the process is not defined. Using the AT(2) receptor-null (Agtr2-) mouse, we tested the hypothesis that the AT(2) receptor could exert an antigrowth effect in cardiac hypertrophy. METHODS AND RESULTS: Cardiac hypertrophy was induced by suprarenal abdominal aortic banding in 10- to 12-week-old Agtr2- and wild-type (Agtr2+) mice for 6 or 12 weeks. Carotid arterial pressure was not different between the strains, although aortic banding increased arterial pressure by approximately 40 mm Hg. Aortic banding increased the heart-weight/body-weight ratio and the cross-sectional area of cardiomyocytes by 15%, resulting in comparable cardiomyocyte hypertrophy in the 2 strains. In contrast, coronary arterial thickening and perivascular fibrosis, determined by the media/lumen-area ratio and the collagen/vessel-area ratio, respectively, were 50% greater in Agtr2- than in Agtr2+ mice after banding, whereas these parameters were similar in sham-operated mice. Radioligand binding studies using the whole heart and immunohistochemistry showed that AT(2) receptor expression was limited and localized in the coronary artery and perivascular region. CONCLUSIONS: These results suggest that the AT(2) receptor mediates an inhibitory effect on coronary arterial remodeling, such as medial hypertrophy and perivascular fibrosis in response to pressure overload, and an activation of the renin-angiotensin system.

Red wine polyphenols inhibit proliferation of vascular smooth muscle cells and downregulate expression of cyclin A gene.

BACKGROUND: Red wine polyphenols have been shown to contribute to the "French paradox" phenomenon, which consists of lower morbidity and mortality from coronary heart disease in the French population. Although vascular smooth muscle cell (VSMC) proliferation plays an important role in the progression of atherosclerotic lesions, the effects of red wine polyphenols on VSMC proliferation have not been elucidated. METHODS AND RESULTS: We extracted the total polyphenolic fraction from red wine (RW-PF) by column chromatography. Treatment with RW-PF showed a potent inhibitory effect on the proliferation and DNA synthesis of cultured rat aortic smooth muscle cells (RASMCs). In contrast, the inhibitory effect of RW-PF on the proliferation of bovine carotid endothelial cells was observed only at much higher concentrations. To elucidate the molecular mechanisms of this antiproliferative effect of RW-PF on RASMCs, we investigated the effects of RW-PF on cell cycle regulation. RW-PF downregulated the expression of cyclin A mRNA and cyclin A promoter activity. In addition, RW-PF decreased the binding of nuclear proteins to the activating transcription factor (ATF) site in the cyclin A promoter and downregulated the mRNA levels of transcription factors, cAMP-responsive element-binding protein (CREB), and ATF-1. CONCLUSIONS: These results suggest that the downregulation of cyclin A gene expression may contribute to the antiproliferative effect of red wine polyphenols on RASMCs through the inhibition of transcription factor expression.

Estrogen prevents oxidative stress-induced endothelial cell apoptosis in rats.

BACKGROUND: Estrogen replacement attenuates the increased risk of cardiovascular disease in postmenopausal women. Recent studies using an in vitro culture system have shown that estrogen inhibits endothelial cell (EC) apoptosis. The in vivo relevance of this finding, however, is not defined. To do so, we have developed a rat vascular injury model in which EC apoptosis induced by hydrogen peroxide plays a role. METHODS AND RESULTS: Intracarotid arterial administration of 0.01 mmol/L hydrogen peroxide for 5 minutes evoked EC apoptosis after 6 to 24 hours, determined by nuclear staining with Hoechst 33342, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and electron microscopy. Apoptosis was associated with EC loss and was followed by EC regeneration at 72 hours and neointima formation at 1 to 2 weeks. Estradiol replacement in ovariectomized female Wistar rats decreased the rate of apoptotic ECs by approximately 50%, assayed by nuclear morphology of en face specimens, resulting in increased remaining ECs and decreased neointima formation. Progesterone did not influence the effects of estradiol on EC apoptosis. CONCLUSIONS: These results provide new insight into the cardioprotective action of estrogen as well as a paradigm of the response-to-injury hypothesis.

Augmented contractile function and abnormal Ca2+ handling in the aorta of Zucker obese rats with insulin resistance.

To investigate the role of hyperinsulinemia/insulin resistance in vasomotor tone regulation, we studied the effects of vasoactive substances on tension and intracellular free calcium concentration ([Ca2+]i) of aortic smooth muscle derived from rats that were made hyperinsulinemic by insulin infusion and from Zucker obese rats with insulin resistance. The tension and [Ca2+]i of fura 2-loaded aortic strip preparations without endothelium were simultaneously measured by using a fluorimeter. Ten male Wistar rats received a continuous subcutaneous infusion of insulin (18 nmol x kg(-1) x day(-1)) for 2 weeks with osmotic minipumps (INS group). A control group of 10 rats received vehicle. The plasma immunoreactive insulin concentration in the INS group increased to 930 +/- 54 pmol/l. The increase in [Ca2+]i and tension by KCl and phenylephrine (PE) were lower in the INS group without alteration of the [Ca2+]i-tension relationship. The responses to serotonin (5-HT) in the INS group were similar to those in the control group. In contrast, responses to KCl, PE and 5-HT were markedly enhanced in Zucker obese rats compared with those in Zucker lean rats. The pretreatment of aortic preparations from lean rats with Bay K8644 significantly enhanced the responses to KCl to the level observed in the preparations from obese rats; however, Bay K 8644 failed to affect the responses to KCl in obese rats. These results suggest that enhanced vascular contractile responses to vasoactive substances, possibly due to altered function of the voltage-dependent Ca2+ channel in vascular smooth muscle, may play an important role in the pathogenesis of hypertension in the insulin resistance syndrome.

Risk factor analyses for macrovascular complication in nonobese NIDDM patients. Multiclinical Study for Diabetic Macroangiopathy (MSDM).

To examine the characteristic features of risk factors for macroangiopathy (MA) in nonobese Japanese NIDDM patients, 899 NIDDM patients with and without MA were registered from 40 facilities. Of these, 386 subjects were identified as having any form of MA (total MA); these included 211 with ischemic heart disease (IHD), 163 with cerebrovascular disease (CVD), and 77 with peripheral vascular disease (PVD). Univariate analyses revealed the following common risk factors for total MA, IHD, CVD, and PVD: age, hypertension, systolic blood pressure (sBP) or diastolic blood pressure (dBP), duration of diabetes, diabetic microangiopathy (retinopathy, nephropathy, and neuropathy), low HDL cholesterol level, and higher LDL cholesterol/HDL cholesterol ratio. Additional significant risk factors for specific conditions were also identified, respectively, as male sex for total MA, IHD, and PVD, smoking for IHD and PVD, and high fasting plasma glucose level for total MA and CVD. With stepwise multivariate logistic regression analysis, older age, duration of diabetes, smoking, and low LDL cholesterol/HDL cholesterol ratio were identified as significant and independent risk factors for total MA, IHD, CVD, and PVD. Other risk factors identified were high dBP for IHD, CVD, and PVD, high sBP for total MA, and low BMI for PVD. These results clearly demonstrated that duration of diabetes, smoking, hypertension, and dyslipidemia are major risk factors for MA in NIDDM patients. Since the mean BMI was similar for both groups (approximately 23 kg/m2) and there were no significant differences in immunoreactive insulin levels before and after 75-g oral glucose challenge testing, obesity and hyperinsulinism at the time of the analyses were not considered to play an important role for the pathogenesis of MA in Japanese NIDDM patients. By using the chi 2 test, cutoff points were determined for six of the most commonly measured risk factors. The cutoff point was the level beyond which a significantly higher prevalence of MA occurred. The cutoff points (rounded slightly upward in some cases) for fasting plasma glucose, sBP, dBP, serum total cholesterol level, serum triglyceride level, and BMI were 140 mg/dl, 140 mmHg, 80 mmHg, 180 mg/dl, 120 mg/dl, and 23 kg/m2, respectively. When these cutoff points were used as control criteria, the prevalence of MA was significantly lower in subjects whose risk factor measurements remained under the proposed control criteria for four or more of the six variables. In conclusion, in nonobese NIDDM patients, age, hypertension, and dyslipidemia were found to be risk factors for MA. Duration of diabetes was also demonstrated as an independent risk factor, indicating the close association of deranged glucose metabolism with the pathogenesis of MA in NIDDM patients. It seems to be crucial to control these risk factors for the prevention of MA in NIDDM patients.

Endothelial nitric oxide synthase is essential for the HMG-CoA reductase inhibitor cerivastatin to promote collateral growth in response to ischemia.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, or statins, are prescribed widely to lower cholesterol. Accumulating evidence indicates that statins have various effects on vascular cells, which are independent of their lipid-lowering effect. Here, we tested the hypothesis that statins may augment collateral flow to ischemic tissues. We induced hind-limb ischemia in wild-type mice and treated them with either saline or cerivastatin. Cerivastatin enhanced the blood flow recovery dramatically as determined by Laser Doppler imaging. The mice treated with saline displayed frequent autoamputation of the ischemic toe, which was prevented completely by cerivastatin. Anti-CD31 immunostaining revealed that cerivastatin significantly increased the capillary density. Endothelial nitric oxide synthase (eNOS) activity was enhanced markedly in the mice treated with cerivastatin. The angiogenic effect of cerivastatin was abrogated in eNOS deficient (eNOS-/-) mice. These results indicate that eNOS is essential for cerivastatin to promote collateral growth in response to ischemia.

Acute and chronic smooth muscle cell apoptosis after mechanical vascular injury can occur independently of the Fas-death pathway.

Vascular smooth muscle cell (VSMC) apoptosis has been demonstrated in vascular lesions, such as atherosclerotic and postangioplasty restenotic lesions. Balloon injury also induces VSMC apoptosis. Fas is a death factor that mediates apoptosis when it is activated by its ligand, FasL. Fas-mediated apoptosis was found to be implicated in the pathogenesis of vascular diseases in which Fas/FasL expression was detected. We investigated whether the Fas/FasL interaction mediated acute and chronic VSMC apoptosis and lesion formation in a vascular injury model that may resemble balloon angioplasty. A large spring wire was inserted into the femoral artery of C3H/HeJ (wild-type), C3H-gld (Fas ligand-/-), and C3H-lpr (Fas-/-) mice. The wire was left in place for 1 minute to denude and expand the artery. Massive apoptosis was observed in medial VSMCs from 1 to 7 hours later. There was no difference in the number of apoptotic cells among the 3 groups of mice 4 hours after injury. At 4 weeks, the injured arteries presented signs of concentric neointimal hyperplasia composed exclusively of VSMCs. There was no difference in the degree of neointima hyperplasia (intima/media ratios were as follows: wild type 1.4+/-0.3, gld 1.0+/-0.2, and lpr 1.3+/-0.2) or in the number of apoptotic nuclei among the 3 groups. These findings suggest the existence of other signaling pathways for acute and chronic VSMC apoptosis, at least that induced by mechanical vascular injury.

Induction of nuclear orphan receptor NGFI-B gene and apoptosis in rat vascular smooth muscle cells treated with pyrrolidinedithiocarbamate.

NGFI-B is one of the orphan nuclear receptors, and its gene is implicated in the apoptosis of T cells. The aim of this study was to investigate the expression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Pyrrolidinedithiocarbamate (PDTC) is a modulator of an oxidative state and is reported to induce apoptosis only when the density of VSMCs is low. Under low VSMC density (10 000 cells/cm(2)), addition of PDTC (0.1 to 10 micromol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining under fluorescence microscopy. At low VSMC density, expression of NGFI-B mRNA was induced 1 hour after the addition of PDTC, peaking at 6 hours, and persisted for up to 12 hours. The protein level of NGFI-B was increased 4 hours after PDTC addition and persisted for up to 12 hours. Under low VSMC density, PDTC-induced expression of NGFI-B mRNA was correlated with the magnitude of apoptosis, which was quantified by enzyme immunoassay for histone-associated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm(2)), PDTC did not induce apoptosis, and the expression of NGFI-B was only transient. This transient expression pattern was also seen when VSMCs were treated with phorbol ester, calcium ionophore, hydrogen peroxide, or angiotensin II, even at low cell density. We next investigated whether the NGFI-B gene may act as a transcription factor under treatment with PDTC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B-responsive elements. The PDTC-induced transcriptional activity of NGFI-B was 2-fold higher at low cell density than at high cell density. These data demonstrate that NGFI-B can be induced in VSMCs and suggest that NGFI-B may play a role in PDTC-induced VSMC apoptosis.

Immunohistochemical detection and northern blot analysis of estrogen receptor in osteoblastic cells.

Expression of estrogen receptor (ER) was studied in MC3T3-E1 cells (mouse osteoblastic cell line), HOS TE85 cells (human osteosarcoma cell line), and primary osteoblastic cells derived from mouse calvaria with immunohistochemical techniques. The staining of ER was readily detectable in MC3T3-E1 cells, HOS TE85 cells, and primary osteoblastic cells by using a monoclonal anti-ER antibody that recognizes the DNA binding domain of ER. The immunoreactivity was distributed in the cytoplasm as well as in the nuclei. 17 beta-Estradiol (10(-8) M) did not alter this staining pattern. The expression of ER was confirmed by Northern blot analysis using rat ER cDNA probe, which revealed a 6.5 kb band in MC3T3-E1 cells and a 6.2 kb band in HOS TE85 cells. The mRNA level of ER was not altered by 17 beta-estradiol (10(-8) M). The immunohistochemical studies showed that ER was not detectable in all cells but in a small population of each cell type. This study is the first report to demonstrate the presence of ER immunohistochemically, and our results suggest the heterogeneity of ER expression among osteoblastic cells.

Association of bone mineral density with apolipoprotein E phenotype.

The phenotypes of apolipoprotein E (Apo E) and their relationship with the bone mineral density (BMD) were examined in 284 unrelated postmenopausal Japanese women aged 47-82 years (64.0 +/- 1.0 years, mean +/- SE). The Apo E phenotype was analyzed by the isoelectric focusing method, followed by immunoblotting. The relationship between the Apo E phenotype and the vitamin D receptor (VDR) gene or estrogen receptor (ER) gene genotypes was also studied in the same population. The Apo E phenotypic frequencies in our population were 9.9% for E3/2, 66.5% for E3/3, 1.8% for E4/2, 19.7% for E4/3, and 2.1% for E4/4. We classified these phenotypes into three categories: Apo E4-/- (E3/2 and E3/3, n = 217, Apo E4 +/- (E4/3 and E4/2, n = 61), and Apo E4+/+ (E4/4, n = 6). The age, body weight, body height, and years since menopause were not significantly different among these three categories. The lumbar BMD values in these three groups were significantly different in the order of E4-/- (0.91 +/- 0.01 g/cm2), E4 +/- (0.85 +/- 0.02 g/cm2), and E4+/+ (0.83 +/- 0.06 g/cm2) (p = 0.031). The same trend was also observed for the Z score of the total BMD (p = 0.022). The serum level of intact osteocalcin in E4+/+ (15.2 +/- 5.7 ng/ml) was higher than in E4-/- (7.7 +/- 0.3 ng/ml) or E4 +/- (7.7 +/- 0.7 ng/ml) (p = 0.004 by analysis of variance). However, there were no other significant differences in the serum or urinary levels of bone turnover markers. Serum cholesterol in the E4+/+ group tended to be higher than in the other two groups (p = 0.05). There were no significant associations of the VDR and ER genotypes with the Apo E4 phenotype. A multivariate linear regression analysis revealed Apo E4 to be a significant, independent predictor of the Z score of the lumbar BMD. The effect of the Apo E4 allele on the Z score of the lumbar BMD (-0.493 +/- 0.152) was not significantly different from that in the AAB of VDR (-0.616 +/- 0.225) or PPxx of ER (-0.785 +/- 0.314). In conclusion, the Apo E4 allele is associated with a low bone mass in postmenopausal Japanese.