bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of androgens on parathyroid hormone and interleukin-1-stimulated prostaglandin production in cultured neonatal mouse calvariae
Authors: Dr. Pilbeam Carol C. , Raisz Lawrence G.
Published: February 2010

In this study we show direct inhibitory effects on prostaglandin E2 (PGE2) production by the androgens, testosterone (T) and dihydrotestosterone (DHT), in cultured neonatal mouse calvariae. After 24 h of preculture with or without androgens, bones were treated with bovine (1–34)-parathyroid hormone (PTH) or recombinant human interleukin-1α (IL-1). During preculture androgens decreased PGE2 release only in those experiments in which control PGE2 was high. PTH increased medium PGE2 9-fold at 24 h, and 10−11 M T inhibited this increase by 50%. Treatment with IL-1 for 24 h increased medium PGE2 19- to 22-fold, and 10−10 M T and DHT inhibited this increase by 60 and 70%, respectively. T did not significantly affect the PTH-stimulated release of previously incorporated 45Ca or alter the PTH inhibition of incorporation of [3H]proline into collagenase-digestible protein. IL-1 stimulated 45Ca release by 60–80%, and small but significant reductions of 20–30% were seen with T and DHT. This study shows that T and DHT have direct effects on bone at physiologic concentrations, similar to our previous study in which PTH-stimulated PGE2 production in the same culture system was inhibited by physiologic concentrations of 17β-estradiol, and suggests that prostaglandins may mediate some of the effects of androgens in vivo.
Lysozyme synthesis in osteoclasts
Authors: Hilliard Thomas J. , Meadows Gary , Dr. Kahn Arnold J.
Published: February 2010

Osteoclasts may or may not be directly related to monocytes and macrophages, but it is well established that these cell types share a number of features in common.(1) In the present study we sought to extend this comparison by assessing lysozyme synthesis in osteoclasts, an enzyme known to be produced and secreted in large amounts by monocytes and macrophages.(2,3) Our data show that freshly isolated chicken osteoclasts and osteoclasts in situ contain an abundant amount of lysozyme and correspondingly high steady-state levels of the enzyme's messenger RNA. Marrow macrophages, at various stages of in vitro maturation, also possess lysozyme mRNA but in amounts approximately two to four times lower than osteoclasts. These observations reaffirm the monocyte-macrophage nature of the osteoclast but raise questions about the function of the lysozyme in this cell. At present, the role of the lysozyme in osteoclast activity remains unexplained.
Improved vertebral fracture threshold in postmenopausal osteoporosis by radiogrametric measurements: Its usefulness in selection for preventive therapy
Authors: Dr. Meema H. Erik
Published: February 2010

Measurements of the combined cortical thickness at the midshafts of the second metacarpal and the proximal radius, as well as their sums, were made from fine-grained radiographs in 107 normal premenopausal and 227 postmenopausal women and in 57 women with at least two spontaneous vertebral compressions. The −2 SD level of the premenopausal women was found to be an excellent vertebral fracture threshold for the radius measuring site and for the sums of metacarpal and radial measurements. Only 2 of 57 (4%) and 3 of 57 (5%), respectively, of values in the vertebral compression group fell at or above this level. At −3 SD a “secondary fracture threshold” was found for the sums in that only 7 of 57 (12%) of values in women with vertebral compressions were above this limit, but −3 SD was less satisfactory for the radius.

We conclude that this method discriminates as well between nonfracture and fracture cases as other radiologic methods for measurement of bone mass or density. Because it is less costly it seems better suited for mass screening in selecting postmenopausal women for preventive therapy than the less widely available high-technology methods.
Calcitonin secretion in idiopathic renal stone formers
Authors: Dr. Fuss Michel , Pepersack Thierry , Corvilain Jacques , Bergmann Pierre , Simon Jacques , Body Jean-Jacques
Published: February 2010

Several studies demonstrated a reduction in bone mineral content (BMC) in idiopathic renal stone formers (RSF). We found this reduction in association with a chronic low-calcium diet. Low calcium intake could theoretically result in calcitonin deficiency, responsible for increased bone resorption. This hypothesis was tested in 22 male RSF eating a low-calcium diet (350 ± 72 SD mg/day) for 2 years or more, who showed a significant reduction in their BMC. When compared to 15 normal male subjects eating a free diet, RSF showed increases in serum alkaline phosphatase activity and fasting urinary excretion of hydroxyproline and calcium, suggesting increased bone turnover. Plasma calcitonin levels were measured by radioimmunoassay following an extraction-concentration technique (exCT). Basal plasma exCT levels were higher (P < 0.005) in RSF (4.1 * 0.8 SEM pg/ml) than in normal subjects (2.8 ± 0.4). Following a 5 minute infusion of 2 mg elemental calcium per kg, levels of plasma exCT tended to increase more, although not significantly, in RSF (51.3 ± 9.4 pg/ml) than in normal subjects (36.6 ± 9.7). The CT secretory response, taking into account changes in serum calcium concentration (AexCT/ACa), was higher (P < 0.05) in RSF (50.0 ± 10.0) than in normal subjects (25.6 ± 6.6). Our study thus demonstrates that RSF chronically fed a low-calcium diet have increased basal plasma CT levels and increased CT cells responsiveness. CT deficiency cannot therefore be considered a cause for the low BMC associated with a chronic low-calcium diet in RSF.
In vivo osteogenic activity of isolated human bone cells
Authors: Yamamoto Takehisa , Ecarot Brigitte , Glorieux Francis H.
Published: February 2010

Human bone cells were obtained as the outgrowth from cancellous bone fragments pretreated with collage-nase and DNase. The osteogenic potential of cells in primary culture was assessed upon intramuscular transplantation into young mice pretreated with cortisone. Transplants were recovered after 2 weeks and examined by light microscopy. Of 34 transplants, 6 showed evidence of osteogenesis and 12 the production of unmineralized matrix. Only cells were observed in the other transplants. In an attempt to find a biochemical marker for osteogenic cells we have assayed medium osteocalcin and alkaline phosphatase activity levels in cultures before transplantation. No correlation was found between the level of expression of the two osteoblast markers and the osteogenic potential of the cells.
Effects of interleukin-6 on cellular function in UMR-106–01 osteoblastlike cells
Authors: Fang Meika A. , Hahn Theodore J.
Published: February 2010

High levels of interleukin-6 (IL-6) have been detected in synovial fluid from patients with inflammatory arthropathies associated with local bone resorption, suggesting a role for IL-6 as a local regulator of bone resorption and remodeling. In the present study we examined the effects of IL-6 on [3H]thymidine ([3H]TdR) incorporation, collagen synthesis, and alkaline phosphatase activity in UMR-106–01 rat osteoblastic osteosarcoma cells. IL-6 stimulated a dose-dependent increase in [3H]TdR incorporation that was maximal at 1000 U/ml (+147% of basal, p < 0.005) in osteoblastlike cells that were in a logarithmic phase of growth. The increase in [3H]TdR incorporation was maximal between 12 and 24 h and was neutralized by pretreatment with the polyclonal rabbit antibody to IL-6. IL-6 also increased cell number and the secretion of prostaglandin E2 in UMR-106–01 cells in logarithmic growth phase. The stimulation of [3H]TdR incorporation and release of PGE2 into the culture medium by IL-6 was inhibited by indomethacin. A 24 h exposure of the osteoblastlike cells to 1000 U/ml of IL-6 reduced [3H]proline incorporation into collagenase-digestible (CDP) protein to 73% of control values (p < 0.01). Noncollagen protein (NCP) synthesis was inhibited to 80% of control values (p < 0.01) by 1000 U/ml of IL-6. The inhibitory effect was relatively greater on CDP than on NCP and consequently resulted in a decrease in the percentage of collagen synthesis. Alkaline phosphatase activity was not altered in these cells after a 24 h exposure to 1–1000 U/ml of IL-6. These results indicate that IL-6 has a direct effect on osteoblastlike cells, stimulating DNA synthesis via a prostaglandin-dependent mechanism and suppressing collagen and noncollagen protein synthesis.
Effects of transforming growth factor β1 on the regulation of osteoclastic development and function
Authors: Hattersley Gary , Chambers Timothy J.
Published: February 2010

Transforming growth factor (TGF) β1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-β1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-β1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-β1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-β1 in osteoclast generation.
Features of the renal parathyroid hormone-parathyroid hormone-related protein receptor derived from structural studies of receptor fragments
Authors: Karpf David B. , Bambino Thomas , Alford Gay , Nissenson Robert A.
Published: February 2010

Our earlier results indicated that the binding moiety of the renal PTH receptor is an 85 kD protein that is susceptible to proteolytic cleavage to a 70 kD form that supports high-affinity binding and Gs coupling, and to a 50–55 kD form that contains the ligand binding domain but does not couple to Gs. In the present study we used [125I]hPTHrP-(1–34)amide and a chemical cross-linking technique to discern the structural features of the intact 85 kD PTH/PTHrP receptor that are retained in the proteolyzed forms to “structurally map” the receptor. The results of lectin chromatography and endoglycosidase treatment show that the paritally proteolyzed receptor forms retain the complex, N-linked glycans present on the intact receptor. This conclusion is further supported by the finding that wheat germ agglutinin was equally effective at competitively inhibiting specific [125I]hPTHrP-(1–34)A binding to the 70 kD form and the intact 85 kD receptor. Specific binding of [125I]hPTHrP-(1–34)A to the intact 85 kD receptor or to the 70 kD form was completely abolished by treatment with disulfide reducing agents, and both partially proteolyzed receptor forms (70- and 50 kD) were shown to retain the small (≤ 14 kD) labeled fragment that is released from the intact receptor by disulfide reduction. Lectin chromatography and endoglycosidase treatment revealed that the ≤ 14 kD receptor component is not glycoslyated. The ≤ 14 kD fragment does not contain a transmembrane spanning region, as its release from the membrane can be affected without detergent solubilization. Identical partial proteolytic maps of the receptor were obtained whether the receptor was covalently labeled with [125I]hPTHrP-(1–34)amide or [125I]bPTH-(1–34). These results suggest a model of the renal PTH/PTHrP receptor binding moiety as a single-chain protein in which the sites of glycosylation, ligand binding, and the functionally critical disulfide bonds are in extracellular domains near one end of the protein and the sites of proteolysis reside near the other end of the protein. These studies also provide further confirmation that PTH and PTHrP bind to a structurally indistinguishable renal receptor and validate the use of PTHrP as a ligand for studies designed to characterize and purify the PTH receptor.
Role of prostaglandins in interleukin-1-induced bone resorption in mice in vitro
Authors: Akatsu Takuhiko , Takahashi Naoyuki , Udagawa Nobuyuki , Imamura Kazunobu , Yamaguchi Akira , Sato Kanji , Nagata Naokazu , Dr. Suda Tatsuo
Published: February 2010

The mechanism of bone resorption induced by interleukin 1 (IL-1) was examined in mice using three different in vitro assay systems: a fetal long bone organ culture system, a bone marrow culture system, and a co-culture system of primary osteoblastic cell populations and spleen cells. In the organ culture system, recombinant human IL-1α (rhIL-1α) increased both bone resorption and osteoclast number. Both were partially suppressed in the presence of indomethacin. In the marrow culture, both rhIL-1α and rhIL-1β stimulated osteoclastlike cell formation, which was completely inhibited by adding indomethacin concurrently. Furthermore, there was a good correlation between the number of osteoclastlike cells formed and the amount of prostaglandin E2 (PGE2) released into the culture media. This indicates that PGE2 is involved in the mechanism of IL-1-mediated osteoclastlike cell formation. In the coculture of primary osteoblastic cell populations and spleen cells, rhIL-1 again stimulated osteoclastlike cell formation, which was inhibited by adding indomethacin. In the cocultures in which direct interaction between osteoblastic cells and spleen cells was inhibited, PGE2 synthesis was similarly increased but no osteoclastlike cells were formed. These results indicate that IL-1 induces osteoclast formation by a mechanism involving PG (most likely PGE2). Furthermore, direct interaction between osteoclast progenitors and osteoblastic cells is required in the osteoclast recruitment induced by IL-1.
Longitudinal precision of dual-energy X-ray absorptiometry in a multicenter study
Authors: Orwoll Eric S. , Oviatt Shelia K. ,   The Nafarelin/Bone Study
Published: February 2010

Reproducibility is a key issue in both clinical and research applications of bone mineral density (BMD) measurements. To examine the longitudinal precision of dual-energy x-ray absorptiometry (DEXA) for the measurement of mineral density in vivo and in vitro, the performance of a group of instruments in the course of a multicenter longitudinal clinical trial was monitored. Measures were performed on eight identical machines (Hologic QDR1000) and analyzed using the same automated software program. Short-term precision was good in vitro, [anthropomorphic spine phantoms; mean intrasite coefficient of variation (CV) 0.42 ± 0.1% (SD)] and in vivo (lumbar spine; CV 1.1 ± 0.5%). Intersite measures of a single spine phantom (specified mineral content −57.8 g) revealed a range of 57.3–58.4 g (CV 0.7%). In two subjects intersite CV in vivo were 3.7 and 2.1% (spine) and 1.8 and 3.2% (femoral neck). At five sites frequent phantom measures were performed over a 1 year period (mean number of measures 196) and revealed a mean all-point CV of 0.43% (range 0.35–0.53%). Longitudinal precision in vivo was somewhat less (mean CV of spinal measures 1.1%, femoral neck 1.2%, trochanter 1.3%, and Ward's area 2.4%). At one additional site large variations in phantom measures heralded repeated mechanical failures that eventually required machine replacement. In summary, DEXA demonstrates good in vitro and in vivo longitudinal precision, providing the basis for expanded clinical and research usefulness. Nevertheless, stringent quality assurance measures are required to detect and respond to system malfunctions.
Normocalcemia without hyperparathyroidism in vitamin D-deficient rats
Authors: Kollenkirchen Uwe , Fox John , Walters Marian R.
Published: February 2010

Despite numerous attempts, no reliable dietary regimen exists to achieve vitamin D deficiency (-D) in rats without attendant changes in plasma parathyroid hormone (PTH), Ca, or phosphate. This represents an important obstacle to proper investigations of the physiologic role(s) of vitamin D metabolites in the function of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] target tissues. This paper describes the successful development of such a diet, which uses a combination of high Ca content, properly controlled Ca/P ratio, and lactose. Normal weanling rats were fed diets containing A, 0.8% Ca, 0.5% P, +D3, or -D diets containing B, 0.8% Ca and 0.5% P; C, 2.0% Ca and 1.25% P; or D, 2.0% Ca, 1.25% P, and 20% lactose. After 6 diet weeks group D rats remained normocalcemic and normophosphatemic, but diet groups B and C became hypocalcemic (6.9 ± 0.8 and 7.2 ± 0.4 mg/dl, respectively). Thus high dietary Ca and P was incapable of maintaining normal plasma Ca levels in the absence of dietary lactose. The normocalcemia in group D was not maintained by elevated PTH secretion because N-terminal PTH levels were also normal (14 ± 3 versus 20 ± 5 pg/ml). In contrast, PTH levels were markedly elevated in hypocalcemic groups B and C (47 ± 7 and 48 ± 10 pg/ml, respectively). Plasma 25-OHD3 and 1,25-(OH)2D3 levels were reduced to < 120 and < 12 pg/ml, respectively, in all -D groups. Thus the high-Ca diet and the use of normal weanlings did not impede the development of vitamin D deficiency. This rat model of normocalcemic vitamin D deficiency will greatly facilitate elucidation of the physiologic role(s) of vitamin D metabolites in the normal functioning of 1,25-(OH)2D3 targets.
Androgen treatment prevents loss of cancellous bone in the orchidectomized rat
Authors: Wakley Glenn K. , Schutte Harold D. , Hannon Kathleen S. , Turner Russell T.
Published: February 2010

This report describes histomorphometric evidence for an important role of androgens in maintaining cancellous bone balance at a remodeling site in vivo. Rats were orchiectomized (ORX) at 7 weeks of age and received either androgens or vehicle 1 week later (testosterone, 1-dehydrotestosterone, or 5-dihydrotestosterone) by subcutaneous pellet, producing controlled release of the drug for 3 weeks. Intact male rats and untreated ORX animals served as controls. After 4 weeks untreated ORX resulted in undetectable serum testosterone levels and marked atrophy of seminal vesicles compared with intact controls. Histomorphometry revealed severe cancellous osteopenia in the secondary spongiosa of the proximal tibial metaphysis. The length of bone surface lined by apparently “active” osteoblasts and number of osteoclasts per length of cancellous bone surface were increased following ORX. Testosterone treatment at 5 mg (per 21 days) produced subphysiologic serum testosterone levels. In contrast, 10 and 25 mg pellets resulted in serum testosterone levels comparable to those in intact rats. Testosterone treatment of ORX rats prevented the bone effects of ORX, and the degree of protection was dose dependent. 1-Dehydro- and 5-dihydrotestosterones displayed a bone-protective effect similar to that of testosterone. The results demonstrate that gonadal insufficiency results in a cancellous osteopenia that is preventable by testosterone treatment. Further, because a similar protective action was achieved using the nonaromatizable androgen 5-dihydrotestosterone, the results suggest that this bone-sparing effect is mediated by androgen rather than by metabolism of the androgen to an estrogen.
Smoking and bone loss among postmenopausal women
Authors: Krall Elizabeth A. , Dawson-Hughes Bess
Published: February 2010

We examined the effect of smoking on bone mineral density (BMD), rates of bone loss, and fractional whole-body retention of 47Ca in healthy postmenopausal women enrolled in a 2-year calcium supplementation trial. Bone density was measured by single- and dual-photon absorptiometry.

BMD of the radius at the study baseline was inversely related to pack-years of exposure when controlled for body mass index and years since menopause (partial r = -0.18, p = 0.05, n = 125). The adjusted mean (±SD) annualized rate of bone change from the radius was greater among smokers than nonsmokers (-0.914 ± 2.624%/year, n = 34, versus 0.004 ± 2.568%/year, n = 278, respectively; p = 0.05). Similar trends were observed at the femoral neck, os calcis, and spine. Rates were adjusted for caffeine intake, alcohol use, supplement type, and, at the spine only, menopausal status. At entry into the trial higher serum levels of alkaline phosphatase and lower levels of total and ionized calcium were found in smokers compared to nonsmokers. These differences did not persist with supplementation. In 44 women studied fractional 47Ca retention was lower in the 8 smokers than the 36 nonsmokers (16.6 versus 19.1%, respectively; p = 0.03).

These results demonstrate an increased rate of bone loss at the radius after menopause and suggest that smoking is associated with decreased calcium absorption.
The giant cells recruited by subcutaneous implants of mineralized bone particles and slices in rabbits are not osteoclasts
Authors: Dr. Marks Sandy C. , Chambers Timothy J.
Published: February 2010

We have compared structural and functional characteristics of native osteoclasts and the multinucleated giant cells (MNGC) recruited by subcutaneous implants of mineralized bone particles and slices in normal rabbits. Weekly evaluation of the implants for 5 weeks showed distinct differences between MNGC and osteoclasts in the host with respect to morphology and the ability to stain for tartrate-resistant acid phosphatase and acid ATPase. An osteoclast-specific monoclonal antibody bound strongly to osteoclasts but not MNGC. Ground bone slices similarly implanted were surrounded by MNGC but did not show resorption pits by scanning electron microscopy. These data show that the MNGC recruited to subcutaneous implants of mineralized bone particles and slices lack the enzymatic, cell surface, and functional features of osteoclasts.
Familial aggregation of paget's disease of bone
Authors: Siris Ethel S. , Ottman Ruth , Flaster Edith , Kelsey Jennifer L.
Published: February 2010

This epidemiologic study of Paget's disease of bone used data from 788 cases and 387 spouse controls to investigate the following: (1) the extent to which this disorder aggregates in families; (2) the cumulative incidence of the disease in first-degree relatives of patients throughout life; and (3) the influence of age at diagnosis (<55 versus 55+ years) and presence of bone deformity in the case on risk of Paget's disease in relatives.

A positive family history in parents or siblings was reported by 12.3% of cases and 2.1% of controls. The rate of Paget's disease was approximately seven times as high in relatives of cases as in relatives of controls, and this increased rate did not differ according to gender of case or control or gender of relatives. Cumulative incidence of Paget's disease to age 90 was much higher in relatives of cases (8.9 + 1.0% SEM) than in relatives of controls (1.8 + 0.9% SEM). Among relatives of cases, cumulative risk was highest when the case had both early age at diagnosis and bone deformity (20.7 + 3.6% SEM) compared with risk when the case had early age at diagnosis but not bone deformity (10.8 + 3.2% SEM), bone deformity but not early age at diagnosis (5.8 + 1.3% SEM), or neither bone deformity nor early age at diagnosis (3.6 + 0.8% SEM). Risk in siblings of cases was higher when a parent was affected (22.1 + 8.0% SEM) than when both parents were unaffected (6.7 + 1.1% SEM).

These findings suggest that first-degree relatives of patients with Paget's disease have increased risk of developing the disorder, especially if the affected relatives have early age at diagnosis or deforming bone disease.
Estrogen binding and estrogenic responses in normal human osteoblast-like cells
Authors: Benz David J. , Dr. Haussler Mark R. , Komm Barry S.
Published: February 2010

A finite human cell line was established from trabecular bone explants obtained from a 48-year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using the following independent criteria: (1) the presence of histochemically detectable alkaline phosphatase (AP) activity; (2) response to the calciotropic hormone 1,25-(OH)2D3 as assessed by increased AP activity; (3) synthesis and secretion of the osteoblast-specific marker bone gla protein; and (4) expression of α1(I)-procollagen and α1(III)-procollagen mRNAs in a pattern similar to that of other osteoblast-like cell lines. In addition to these classic osteoblast markers, BG688 cells also possess approximately 2400 high-affinity (Kd = 0.45 nM) 17β-estradiol (E2) binding sites per cell. The binding of E2 to these sites is specific, and of the steroid hormone agonists tested, E2 and diethylstilbestrol elicited the greatest amount of competition with radiolabeled E2. BG688 cells were also shown to respond to a physiologic concentration (10 nM) of E2. In vitro translation products of poly(A)+ RNA obtained from control and hormone-treated cells revealed a pleiotropic influence of E2 on the relative abundance of several mRNAs as assessed by two-dimensional gel electrophoretic analysis of their corresponding peptides. E2 also elicits a twofold increase in the steady-state concentration of α1(I)-procollagen mRNA as demonstrated by northern blot hybridization. Thus, we here extend our previous data obtained in osteoblast-like osteosarcoma cells to indicate that a normal osteoblastic cell line is a target for the action of estrogen. The E2 responsiveness of these cells, coupled with their osteoblast-like phenotype, renders BG688 cells an attractive system in which to investigate the role of estrogens in the regulation of osteoblast function.
Genetic determinants of bone mass in adult women: A reevaluation of the twin model and the potential importance of gene interaction on heritability estimates
Authors: Dr. Slemenda Charles W. , Christian Joe C. , Williams Christopher J. , Norton James A. , Johnston C. Conrad
Published: February 2010

We estimated genetic effects on bone density in pre- and postmenopausal twins and critically considered the assumptions of the twin model. Bone mass in the radius, lumbar spine, and hip, anthropometric measurements, usual calcium and caffeine intake, tobacco and alcohol use, number of pregnancies and live births, menstrual history, usual physical activity, and medical history were measured in a volunteer sample of 171 twin pairs [124 monozygotic (MZ) and 47 dizygotic (DZ)], aged 25–80, free of diseases known to affect bone mass or mineral metabolism. At all skeletal sites, MZ intraclass correlations exceeded DZ correlations for both pre- and postmenopausal women, yielding highly significant estimates of heritability for bone mass. Adjustments for height, age, and environmental characteristics did not reduce the heritability estimates. However, many of these estimates were unrealistically high, suggesting some violation(s) of the assumptions of the twin model. Thus, the familial resemblance in bone mass is due primarily to genetic effects at all skeletal sites and at all ages, although the importance of genetic effects is diminished with aging, as evidenced by increasing within-MZ pair variability in older women. Because of failures in the assumptions of the twin model, however, particularly the greater MZ environmental similarity and the probability of gene interaction, heritability estimates are probably too high and require cautious interpretation.
Estrogen therapy and variable-resistance weight training increase bone mineral in surgically menopausal women
Authors: Notelovitz Morris , Martin Daniel , Tesar Rogene , Khan Farouk Y. , Probart Claudia , Fields Carol , McKenzie Lynda
Published: February 2010

This study was undertaken to examine the effect of estrogen replacement therapy alone and estrogen replacement therapy plus variable-resistance weight training on the bone mineral content of surgically menopausal women. A total of 20 surgically menopausal women were randomized and treated with either 0.625 mg conjugated estrogen daily or the same dose of estrogen plus a closely monitored exercise program involving the use of Nautilus muscle strengthening/endurance equipment. After 1 year's observation, the bone mineral density (BMD) of the spine determined by dual-photon absorptiometry increased in the exercising subjects by 8.3 ± 5.3% (p = 0.004), 95% confidence limits (CL) 3.9–12.8%; the group with estrogen replacement therapy alone maintained their BMD: 1.5 ± 12.4% (p = 0.36; 95% CL = −6.9–9.8%). The total body BMD of the exercising group increased by 2.1 ± 1.5% (p = 0.003; 95% CL = 0.8–3.3%); the nonexercising women had a nonsignificant 0.6 ± 2.9% change (p = 0.30; 95% CL = −1.4–2.5%). A significant increase of 4.1 ± 4.3% (p = 0.01; 95% CL = 0.8–7.4%) in the radial midshaft BMD of the exercising group was found; the estrogen alone group recorded a nonsignificant change of −0.3 ± 3.1% (p = 0.33; 95% CL = −1.7–2.4%). The results of this study suggest that variable-resistance training in estrogen-replete women adds bone to both the axial and appendicular skeleton.
Biphasic effect of calcitonin on tartrate-resistant acid phosphatase activity in isolated rat osteoclasts
Authors: Yumita S. , Professor Nicholson G.C. , Rowe D.J. , Kent G.N. , Martin T.J.
Published: February 2010

Tartrate-resistant acid phosphatase (TRAP) has been implicated as being involved in osteoclastic bone resorption, and calcitonin (CT) is known to inhibit the resorptive process. This study investigates the kinetics of CT action on TRAP activity in isolated rat osteoclasts using both biochemical and quantitative cytochemical methods. The latter technique has been developed to detect very small changes in intracellular TRAP activity at the single-cell level. The biochemical study showed that 10−9 M salmon CT (sCT) decreased TRAP activity in medium throughout the experimental period; TRAP activity in the cells was increased during the first 2 h but subsequently declined and was decreased to a significant level at 6 h. TRAP activity in sCT-treated osteoclasts measured by the cytochemical method showed significant increases within the first hour. This response was dose dependent between 1016 and 10−11 M sCT with EC50 at 8 × 10−14 M. After 1 h, the initial increase in intracellular TRAP activity in CT-treated osteoclasts was followed by a decline to below control levels, reaching statistical significance at 9 h. Treatment with forskolin (10−5 M) showed a similar trend, suggesting that this response is mediated by cyclic AMP-regulated phosphorylation events. From these results, we conclude that CT has two actions on TRAP in isolated rat osteoclasts: the first to inhibit its release, the second to inhibit its synthesis and/or increase its degradation.
Effects of guanine nucleotides and parathyroid hormone on inositol 1,4,5-trisphosphate metabolism in canine renal cortical tubular cell membranes
Authors: Coleman Daniel T. , Morrow Bruce S. , Bilezikian John P.
Published: February 2010

Parathyroid hormone (PTH) and guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) increase levels of the second messenger inositol 1,4,5-trisphosphate (IP3) and other inositol phosphates (IP) in several membrane preparations of PTH-responsive cells. We present evidence here indicating that in a membrane preparation of canine renal cortical tubular cells bPTH-(1–84), bPTH-(1–34), [N-Leu8,18Tyr34]bPTH-(3–34)NH2, and the human PTH related peptide fragment hPTHrP-(1–34)NH2 all increase levels of inositol phosphate (IP) but [Tyr34]-bPTH-(7–34)NH2 and hPTHrP-(7–34)NH2 have no significant effects on IP accumulation. Increases in IPs are generally attributed to increased formation of IPs and appear to be mediated by a G protein. However, increased levels of IPs may also result from inhibition of the phosphatases that are responsible for their metabolism. We investigated the effect of PTH and GTP-γS on the metabolism of IP3 in canine renal cortical tubular membranes. These membranes rapidly metabolize [3H]IP3 (47% at 15 s). Decreases in [3H]IP3 at all time points are accounted for quantitatively by increases in the sum of its breakdown products: [3H]IP2, [3H]IP1, and [3H]inositol. After 5 minutes of exposure to membranes, the vast majority of [3H]IP3 (84%) is converted to its terminal metabolite, [3H]inositol. GTP-γS (100 μM) inhibits the amount of [3H]IP3 metabolized in 15 s by 70% and reduces the amount of [3H]inositol ultimately formed in 5 minutes by 64%. ATP-γS, ATP, and 2,3-bisphosphoglycerate (100 μM) also inhibit [3H]IP3 hydrolysis in this preparation. The results demonstrating that nucleotides, such as GTP-γS, at concentrations used to demonstrate G protein-mediated increases in IP3 formation, can inhibit the metabolism of IP3 suggest that their effect to increase levels of IP3 may be due in part to inhibition of IP3 metabolism. In contrast, PTH-(1–34), PTH-(3–34), and hPTHrP-(1–34) (1 μM) do not reduce the amount of [3H]IP3 metabolized in 15 s, indicating that the site of action of PTH on IP3 levels is predominantly at steps before the hydrolysis of IP3.
Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: Substrate specificity and sensitivity to transport inhibitors
Authors: Dr. Dixon S. Jeffrey , Kulaga Andrew , Jaworski Ewa M. , Wilson John X.
Published: February 2010

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na+-dependent uptake system for L-ascorbate (J Membr Biol 111:83–91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37°C. Uptake of [L-14C]ascorbate (5 μM) was inhibited 98 ± 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 ± 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (< 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) = sulfinpyrazone > furosemide = 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.
Correlates of cortical bone mass among premenopausal and postmenopausal japanese women
Authors: Lacey Janet M. , Anderson John J.B. , Fujita Takuo , Yoshimoto Yoshio , Fukase Masaaki , Tsuchie Setsuko , Koch Gary G.
Published: February 2010

Few studies have examined the multifactorial risk factors of bone mass in Asian populations. This cross-sectional study was designed to explore relationships between bone mass and environmental variables, including dietary and life-style factors, in Japanese women living in Japan. A total of 178 Japanese women completed the study: 89 premenopausal, ages 35–40, and 89 postmenopausal, ages 55–60. Midradial bone mineral content (MBMC) and bone mineral content per unit area, referred to as bone density (MBMD), were measured using single-photon absorptiometry. The major results of this investigation were the following: (1) The postmenopausal women differed significantly from the premenopausal women in having lower radial bone parameters, lower mean height, later age of menarche, and higher dietary intakes of carbohydrates, vegetables, and milk with a lower intake of caffeine. (2) Current protein intake was a positive correlate of MBMC in both groups. (3) Intake of vegetables (leafy green, yellow, orange, and white) and current milk intake were positively associated with MBMC in the postmenopausal women. (4) For the premenopausal women, irregular menstrual cycles was a negative correlate of MBMC, and for the postmenopausal women, years of menopause was negatively associated with MBMC and MBMD. Longitudinal studies are needed to establish more conclusively associations among diet, life-style, and reproductive history and bone mass of Japanese women.
Demonstration that bone mass is greater in black than in white children
Authors: Bell Norman H. , Shary Judith , Stevens June , Garza Martha , Gordon Leonie , Edwards Jeanne
Published: February 2010

Osteoporosis and hip fractures are less common and bone mass is greater in black than in white women. To determine if bone mass is greater in black than in white children, bone mineral density (BMD) of the midradius by single-photon absorptiometry and BMD of the lumbar spine (L1-L4), trochanter, and femoral neck by dual-photon absorptiometry were measured in 20 black boys, 18 black girls, 33 white boys, and 35 white girls between the ages of 7 and 12 years. Mean age (10.4 ± 0.3 versus 10.2 ± 0.2 years) and body weight (39 ± 2 versus 38 ± 2 kg) in the blacks and whites, respectively, were not different in the two groups, and the ages and weights of the boys and girls were not different from each other. BMD were significantly greater in black than in white children at each site, in the black than in white boys at the trochanter and femoral neck, and in the black than in white girls at each site. In both races, BMD varied directly with age and body weight. Multivariate analysis showed that BMD were greater at the midradius, lumbar spine, trochanter, and femoral neck in the black than in the white children, that BMD of the lumbar spine was greater in the girls than in the boys, and that BMD of the trochanter and femoral neck were greater in the boys than in the girls. There were significant partial correlations between race and BMD and between BMD and body weight at each site, between sex and BMD at the lumbar spine, trochanter, and femoral neck, and between age and BMD at the midradius, trochanter, and femoral neck. Race, sex, age, and body weight together accounted for 49-66% of the variation in bone mass. Thus, BMD of the midradius, spine, and hip are greater in black than in white children, body weight and age are important determinants of bone mass, and some sex differences in bone mass are present at this age.
Calibration of dual-energy X-ray absorptiometry for bone density
Authors: Mazess Richard B. , Trempe Joel A. , Bisek Joseph P. , Hanson James A. , Hans Didier
Published: February 2010

Bone Mineral Content (Bmc, G) Using Dexa (Lunar Dpx) Was Measured On Known Hydroxyapatite Samples In A Water Bath In The Presence Of Uniform And Nonuniform Coverings Of Fat-Equivalent Materials. Selective Placement Of Paraffin Over Bone Had A Greater Effect Than Lard In Reducing Apparent Bmc, And Polycarbonate Plastic Had A Lesser Effect. Measured Bmc Was 100.1 ± 1.1% Of Actual Hydroxyapatite Weight When (1) Fat Over Bone Was About Twice The Mass Of Hydroxyapatite, And (2) The Surrounding Soft Tissue Was 15–30% Fat. There Was A Linear Relationship Between Observed And Expected Bmc, Area (Cm2), and bone mineral density (BMD, g/cm2) measured on an aluminum phantom using either the Lunar DPX or the Hologic QDR-1000. The measured area with the two densitometers was identical, but BMC differed. For both an anthropomorphic phantom and human subjects, use of a constant-threshold (0.2 g/cm2) edge-detection algorithm excluded less low-density bone from the transverse processes than the standard DPX edge-detection algorithm. Differences in edge detection could influence the results obtained with phantoms and in vivo and make system intercomparison difficult.
Kinetics of the osteoclast cytoskeleton during the resorption cycle in vitro
Authors: Lakkakorpi Paivi T. , Väänänen Kalervo H.
Published: February 2010

Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and Factin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With α-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.
Clodronate inhibits contraction and prevents the action of L-type calcium channel antagonists in vascular smooth muscle
Authors: Paspaliaris Vasilis , Leaver David D.
Published: February 2010

Clodronate (dichloromethylenebisphosphonate) decreased vasoconstriction of the isolated perfused rat tail artery mediated by norepinephrine and by Ca2+ in a K+-depolarizing solution. The norepinephrine contractile response was divided into two components by sequential manipulation of the composition of the perfusion fluid, where the first component is due to the release of Ca2+ from intracellular stores and the second to the influx of Ca2+ from extracellular fluid. Clodronate (20 μM) decreased only the first component of the response at a norepinephrine concentration of 50 nM, and both components of the response at a higher norepinephrine concentration (100 nM). The L-type Ca2+ channel blocking drugs, nicardipine (10 nM) and verapamil (1 μM), reduced the second component of the norepinephrine-mediated vasoconstriction, but in the presence of clodronate (20 μM) this blocking action was prevented. These results were confirmed by examining the interaction between clodronate and nicardipine on norepinephrine and K+-mediated lanthanum (La3+)-resistant unidirectional 45Ca uptake. Nicardipine (1–10 nM) decreased the norepinephrine (100 nM) and K+-induced (60 mM) La3+-resistant unidirection 45Ca uptake in a concentration-dependent manner, but in the presence of clodronate (20 μM) this concentration-dependent response was abolished. Thus, clodronate not only reduced agonist-induced Ca2+ release from intracellular stores and Ca2+ influx through L-type Ca2+ channels but also prevented L-type Ca2+ channel antagonists from exerting their effect. These results indicate clodronate has two sites of action during vascular smooth muscle contraction: the first on intracellular mobilization of Ca2+ and the second on L-type Ca2+ channels.
Interleukin-1α stimulates the release of prostaglandin E2 and phospholipase A2 from fetal rat calvarial cells in vitro: Relationship to bone nodule formation
Authors: Ellies Lesley G. , Heersche Johan N.M. , Vadas Peter , Pruzanski Waldemar , Stefanski Eva , Aubin Jane E.
Published: February 2010

We have shown previously that interleukin-1 (IL-1) has biphasic effects on the formation of bone nodules in long-term cultures of fetal rat calvarial (RC) cells (Ellies and Aubin, Cytokine 2:430–437, 1990). To determine the role of arachidonic acid metabolism in this process, we have examined the release of prostaglandin E2 (PGE2) and phospholipase A2 (PLA2) from RC cells under conditions that allowed concomitant analysis of the formation of bone nodules. Recombinant human IL-1α (rhIL-1α) stimulated PGE2 and PLA2 release in a time- and dose-dependent manner. PGE2 release was highest in preconfluent cultures (days 1–6) and was stimulated up to 8.5-fold in response to 50 U/ml of rhIL-1α. In contrast, extracellular PLA2 activity was maximal in postconfluent cultures, with 50 U/ml of rhIL-1α causing a 20-fold increase by day 15. PLA2 release by RC cells was not significantly affected by PGE2, the glucocorticoid dexamethasone, or the cyclooxygenase inhibitor indomethacin. Indomethacin partially blocked the inhibition of bone nodule formation caused by rhIL-1α, and exogenous PGE2 reversed this effect. Addition of group I PLA2 from Naja naja venom to RC cells had no effect on bone nodule development; however, group II PLA2 from Crotalus adamanteus venom inhibited the formation of bone nodules in a dose range similar to that induced by rhIL-1α. These results indicate that PGE2 release does not have a direct temporal correlation with increases in PLA2 activity. In addition, the data show that only part of the inhibition of bone formation seen with rhIL-1α is mediated by PGE2 and suggest that extracellular PLA2 also accounts for part of the inhibition.
Binding of insulin-like growth factors to cultured rat calvarial cells with differing biologic responses
Authors: Cantrell Paul R. , Frolik Charles A. , Ellis Lee F. , Dr. Williams Daniel C.
Published: February 2010

The insulin-like growth factors (IGFs) are considered important regulators of bone metabolism affecting a number of biologic responses in vitro. Primary fetal rat calvarial cells (PRC) and a cloned adult rat calvarial cell line (C3) both exhibit a concentration-dependent IGF stimulation of [3H]thymidine incorporation into DNA, but the C3 cells show a greater sensitivity and magnitude of response. IGF-I and IGF-II were nearly equipotent in PRC cultures, but IGF-I was more than twice as active as IGF-II in the C3 cultures. This effect of the IGFs on DNA synthesis in two bone cell cultures with different culture histories has been correlated with receptor and binding protein profiles. Specific high-affinity IGF binding sites were found in both cell types. In general, the sites present on PRC cells showed a preference for binding IGF-II over IGF-I, but C3 cells displayed two types of relatively specific binding sites. In both cell types [125I]IGF-I bound primarily to a protein with IGF type I receptor characteristics. However, in PRC cells, [125I]IGF-II cross-linked specifically with proteins that had IGF type II receptor characteristics plus several sites unique to these cells; in C3 cells, [125I]IGF-II bound to a 139 kD protein that could be displaced by either IGF-I or IGF-II. Finally, IGF-II-specific 85 and 67 kD proteins were common to both cell types. From these studies, it is apparent that the IGFs bind to a variety of high-affinity binding sites in bone cells and that these sites differ between a highly responsive and a less responsive bone cell population.