bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of parathyroid hormone-related peptide-responsive dual signal transduction systems in osteoblastic osteosarcoma cells: Role in PTHrP-induced homologous desensitization
Authors: Sugimoto Toshitsugu , Kano Junichi , Ikeda Kazuto , Fukase Masaaki , Chihara Kazuo
Published: February 2010

In Osteoblastic Umr-106 Cells, 10−7 M human (h) PTH-related peptide (PTHrP)-(1–34) significantly induced the formation of total inositol phosphates to the same degree as 10−7 M hPTH-(1–34), confirming that in addition to cAMP-dependent protein kinase (PKA), PTHrP possesses another signal transduction system, calcium/protein kinase C (Ca/PKC). Experiments were therefore performed to characterize the cross talk of these dual-signal transduction systems and its participation in the PTHrP-induced homologous desensitization of cAMP and cytosolic calcium (Cai) response in osteoblasts. Preincubation with 10−7 M hPTHrP-(1–34) caused homologous desensitization, resulting in a remarkable decrease in cAMP accumulation in response to further exposure to PTHrP. This effect was significant after 2 h pretreatment and reached a maximum at 6 h. Pretreatment with the PKC-activating phorbol ester phorbol 12-myristate-13-acetate (PMA, 10−6 M) for 30 minutes and 6 h caused a significant increase and decrease in cAMP responsiveness to PTHrP, respectively. Pretreatment with calcium ionophores (A23187 or ionomycin, 10−6 M), not for 30 minutes but for 6 h, caused a significant decrease in cAMP responsiveness to PTHrP. H-7 (an inhibitor of PKC, 50 μM) significantly blocked not only PMA- but also PTHrP-induced desensitization of the cAMP response. PTHrP caused the complete homologous desensitization of an increase in Cai within 30 minutes. Pretreatment with dibutyryl-cAMP (10−4 M) for 30 minutes caused significant inhibition of the PTHrP-induced increase in Cai, and pretreatment with Sp-cAMPS (10−4 M), a direct activator of PKA, for 30 minutes completely blocked the PTHrP-induced increase in Cai. Rp-cAMPS (10−4 M), an antagonist in the activation of PKA, slightly but significantly antagonized the PTHrP-induced homologous desensitization of the Cai response. The present study demonstrated the existence of cross-talk in PTHrP-responsive dual signal transduction systems and its participation in PTHrP-induced homologous desensitization.
CGRP Radioreceptor assay: A new diagnostic tool for medullary thyroid carcinoma
Authors: Wimalawansa Sunil J.
Published: February 2010

The Quantitative Analysis Of Immunoreactive Calcitonin (Ict) Is The Assay Of Choice For Diagnosis And Follow-Up Of Patients With Medullary Thryoid Carcinoma (Mtc). However, In A Small Percentage Of Patients With Mtc Or C Cell Hyperplasia (Cch), Basal And Stimulated Ict Levels May Not Be Significantly Elevated. In These Patients, Calcitonin Gene-Related Peptide (Cgrp) Radioreceptor Assay (Rra) Can Be Used Complementary To Immunoassay For Ct (Or Katacalcin) For Prompt Diagnosis Of Mtc And Cch. Cgrp Rra Is A Robust, Rapid, Sensitive, And Specific Determinant Of “Receptor-Recognized” Cgrp (Rr-Cgrp; Intact Molecule Of Cgrp) Either In Plasma Or In Tissue Extracts. Plasma Rr-Cgrp Levels Rose > 100% 2–5 Minutes After Stimulation With Intravenous Pentagastrin (Calcium Or Oral Alcohol) (P < 0.001), Whereas Icgrp Levels Were Raised To A Lesser Degree (P < 0.01). In Six Patients Who Had A False Positive Ict Response After Pentagastrin Or Had Raised Basal Ict Levels Measured With A Two-Site Immunoradiometric Assay, Rr-Cgrp Showed Only A Minimal Change. On The Other Hand, In Patients With Cch (True Positive, N = 8), Ict Was Increased By Only 40% After Pentagastrin But Rr-Cgrp Levels Rose By 140% (P < 0.001). No Change In Ict Or Rr-Cgrp Levels In Plasma Were Detected In Healthy Normal Volunteers After Administration Of Pentagastrin. Therefore, In Addition To The Plasma Ict Levels, Rr-Cgrp Would Resolve Some Of The Difficult Diagnostic Problems Associated With Mtc And Likely Improve The Specificity And Sensitivity Of Identifying Cch.
Molecular Cloning Of A Plasma Membrane Calcium Pump From Human Osteoblasts
Authors: Kumar Rajiv , Haugen James D. , Penniston John T.
Published: February 2010

The osteoblast plays a critical role in bone formation, bone remodeling, bone matrix formation, and matrix calcification. To better understand the process of osteoblast-controlled bone formation, we determined the structure and isoform types of the plasma membrane calcium pump from normal human osteoblasts. A complementary DNA library from normal human osteoblasts was screened for plasma membrane calcium pump clones. Sequencing and analysis of cDNA clones revealed the presence of a 3986 base pair cDNA that encoded a 1220 amino acid protein that was similar to the human plasma membrane calcium pump isoform 1. Polyadenylated RNA from human osteoblast cells contains bands of RNA approximately 5050 and 6750 bases long. Reverse transcription of polyadenylated RNA from human osteoblasts followed by amplification of the RNA-DNA duplex with calcium pump isoform-specific primers revealed the presence of isoforms 1 and 2 of the calcium pump. Isoform 4 was not detected. We conclude that normal adult human osteoblasts contain a plasma membrane calcium pump that is similar to the human plasma membrane calcium pump isoform 1. It is likely that this pump plays an important role in the cell biology of the human osteoblast.
Menatetrenone inhibits bone resorption partly through inhibition of PGE2 synthesis in vitro
Authors: Hara Kuniko , Akiyama Yasuhiro , Tajima Tetsuya , Shiraki Masataka
Published: February 2010

We studied the effect of menatetrenone, a vitamin K2 homolog, on bone resorption stimulated by interleukin-1α (IL-1α), prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone-resorbing activity was assessed by measurement of calcium and hydroxyproline in the media and calvariae. IL-1α (0.1–100 U/ml), 1,25-(OH)2D3 (1010-10−7 M), PGE2 (10−9-10−6 M), and PTH (3 × 10−8-3 × 10−7 M) dose dependently increased the levels of calcium and hydroxyproline in the medium. Indomethacin (10−6 M) completely inhibited bone resorption induced by IL-1α and partially inhibited bone resorption induced by 1,25-(OH)2D3. However, indomethacin did not affect the action of PGE2 or PTH. Menatetrenone (3 × 10−6-3 × 10−5 M) inhibited the bone resorption induced by IL-1α (2 U/ml), PGE2 (10−7 M), PTH (3 × 10−7 M), and 1,25-(OH)2D3 (3 × 10−10 M) in a dose-dependent manner. Menatetrenone also inhibited the PGE2 production stimulated by IL-1α. These results indicate that menatetrenone may inhibit bone resorption through at least two different mechanisms; one possibly is an inhibitory effect on prostaglandin production.
Pertussis toxin-sensitive activation of phospholipase A2 can be resolved from phosphoinositidase C in primary cultures of mouse osteoblasts using indomethacin
Authors: Sandy Jonathan R. , Meikle Murray C. , Farndale Richard W.
Published: February 2010

Recent work has established that various bone-resorbing hormones are able to activate phosphoinositide metabolism as well as eicosanoid production in osteoblast-like cells, although the relationship between these pathways is unclear. We used pertussis toxin and indomethacin to inhibit the stimulation of [3H]arachidonic acid release and [3H]phosphoinositide turnover caused by treating primary cultures of mouse osteoblasts with fetal calf serum. We found (1) that pertussis toxin and indomethacin each inhibited both pathways and (2) that although pertussis toxin inhibited [3H]arachidonic acid release to a greater extent than indomethacin, [3H]inositol phosphate accumulation was inhibited rather more effectively by indomethacin. These data suggest that whereas ligands in fetal calf serum activate [3H]arachidonic acid release largely directly via the action of a pertussis-sensitive G protein, activation of phosphoinositidase C is indirect, being substantially dependent upon eicosanoid production. These experiments suggest that serial activation of phospholipase A2 and phosphoinositidase C may occur in osteoblasts and that only the former enzyme is regulated by a pertussis toxin-sensitive G protein.
Secretion of osteocalcin and its propeptide from human osteoblastic cells: Dissociation of the secretory patterns of osteocalcin and its propeptide
Authors: Hosoda Kenji , Kanzaki Susumu , Eguchi Hiroshi , Kiyoki Mamoru , Yamaji Teizo , Koshihara Yasuko , Shiraki Masataka , Seino Yoshiki
Published: February 2010

Specific immunoassay systems for intact human osteocalcin (I-OC) and its 26-residue propeptide have been newly developed to assess their usefulness as biochemical markers of bone metabolism. Using human cultured osteoblastic periosteal cells, we monitored 24 h secretion of these molecules from the osteoblastic cells and also examined the deposition of Ca, P, and I-OC on the extracellular matrix. At day 5, both I-OC and its propeptide were secreted by osteoblastic cells in a concentration-dependent manner by treatment with 1,25-(OH)2D3. This propeptide was not detected in the serum of adult subjects but was detected in the serum of normal children, which confirmed this in vitro result of propeptide secretion. The secretion of I-OC into medium transiently decreased at day 11, when the rapid accumulation of I-OC, Ca, and P, namely mineralization, was observed on the extracellular matrix of osteoblastic cells, although secretion of the propeptide constantly increased throughout the culture period. Therefore, the ratio of the amount of propeptide to I-OC in the supernatant markedly increased when mineralization started. These data demonstrate the superior specificity of propeptide as a marker of osteoblastic function in vitro compared with I-OC and that monitoring the changes in propeptide to I-OC ratios in the culture supernatant may be useful for predicting the timing of mineralization on the extracellular matrix of osteoblastic cells.
Effects of weight and body mass index on bone mineral density in men and women: The framingham study
Authors: Felson David T. , Zhang Yuqing , Hannan Marian T. , Anderson Jennifer J.
Published: February 2010

We evaluated the association of weight and bone mass in elderly male and female subjects of the Framingham osteoporosis study, a subset of the Framingham study cohort. By examining the differences in the correlations of weight with bone mass among men and women in weight-bearing and non-weight-bearing sites and weight change since early adulthood, we attempted to understand different ways in which weight or body mass index affects bone mass. During biennial examination 20 of the Framingham cohort (1988–1989), 693 women and 439 men (mean age 76 years) had proximal femur bone mineral density assessed by dual-photon absorptiometry (DPA) and radius bone mass assessed by single-photon absorptiometry. The majority of these subjects also had spine measurements by DPA. Subjects had been weighed repeatedly over 40 years. After adjusting for other factors affecting bone density, we found that both recent weight and body mass index explained a substantial proportion of the variance in bone mineral density for all sites in women (8.9–19.8% of total variance, all p < 0.01) and for only weight-bearing sites (femur and spine) in men (2.8–6.9% of total variance, all p < 0.01). For bone mineral density at the proximal radius, weight and body mass index accounted for < 1% of variance in men (p NS). Weight change since biennial examination 1 (1948–1951) was the strongest explanatory factor for bone mineral density among women at all sites, but weight change did not affect radius bone mineral density in men. The effect of weight and of weight change on bone mineral density was in general much less in men than in women. Our results suggest that the strong effect of weight on bone mineral density is due to load on weight-bearing bones in both sexes. The sex difference is unexplained but may be due to adipose tissue production of estrogen in women after menopause.
Interleukin-1 receptor antagonist inhibits the hypercalcemia mediated by interleukin-1
Authors: Guise Theresa A. , Garrett I. Ross , Bonewald Lynda F. , Mundy Gregory R.
Published: February 2010

Recently, the effects of interleukin-1 (IL-1) on bone resorption in organ culture have been shown to be inhibited by an interleukin-1 receptor antagonist (IL-1RA), a novel monocyte cytokine in the IL-1 family. IL-1RA, which binds to IL-1 receptors and inhibits many of the effects of IL-1α and β, has been purified, cloned, and expressed. We used IL-1RA to investigate its effects on calcium homeostasis in vivo. After confirming that IL-1RA completely inhibited the effects of IL-1 on bone resorption in organ cultures, we tested the effects of IL-1RA on hypercalcemia mediated by IL-1 in normal mice and found that prolonged hypercalcemia provoked by IL-1 was completely inhibited by IL-1RA. The initial transient decrease in blood ionized calcium observed following an injection of IL-1 was also abrogated. IL-1RA had no effect alone on blood ionized calcium or on hypercalcemia mediated by parathyroid hormone (PTH) or PTH-related protein (PTHrP). These data suggest that antagonists to the IL-1 receptor may provide a useful therapeutic approach to osteoclastic bone resorption and hypercalcemia that is IL-1 dependent.
An uncoupling agent containing strontium prevents bone loss by depressing bone resorption and maintaining bone formation in estrogen-deficient rats
Authors: Marie Pierre J. , Hott Monique , Modrowski Dominique , De Pollak Cinderella , Guillemain Joel , Deloffre Pascale , Tsouderos Yannis
Published: February 2010

Trabecular bone loss in estrogen deficiency is associated with enhanced bone resorption with a smaller increase in bone formation. We previously reported that low doses of strontium can increase trabecular bone volume in rodents by affecting bone resorption and formation. In this study we determined the effect of a new divalent strontium salt (S12911) on bone loss induced by E2 deficiency. Sprague-Dawley female rats (230 g, n = 15–25 per group) were sham operated or ovariectomized (OVX) and treated with 17β-estradiol (E2, 10 μg/kg/day, sc) or S12911 by gavage at the dose of 77, 154, or 308 mg/kg/day or the vehicle. Treatment for 60 days with S12911 resulted in a dose-dependent increase in plasma, urine, and bone strontium concentrations without any deleterious effect on total or skeletal growth. OVX rats were osteopenic compared to sham rats as shown by decreased femoral dry bone weight and mineral content measured on bone ash and by DXA. Treatment of OVX rats with S12911 prevented bone loss as bone ash and bone mineral content were restored to the values in sham rats. Trabecular bone volume measured by histomorphometry on the tibial metaphysis was decreased by 46% in OVX rats and was corrected by E2. Treatment of OVX rats with S12911 increased the trabecular bone volume by 30–36%. Histomorphometric indices of bone resorption (osteoclast surface and number) were increased in OVX rats and were reduced by S12911 to the levels in sham rats. In contrast to this inhibitory effect on bone resorption, the osteoid surface, osteoblast surface, mineral apposition rate, and bone formation rate were as high in OVX rats treated with S12911 as in untreated OVX rats. In addition, plasma osteocalcin (OC) and alkaline phosphatase (ALP) levels remained elevated or were further increased in OVX rats treated with S12911. In contrast, treatment with E2 reduced both bone resorption and formation and plasma ALP and OC to the levels in sham rats. The data indicate that the divalent strontium salt S12911 is acting as an uncoupling agent that can prevent the femoral osteopenia and partially prevent the trabecular bone loss in E2-deficient rats by inhibiting bone resorption without reducing bone formation.
Production of leukemia inhibitory factor mRNA and protein by malignant and immortalized bone cells
Authors: Marušić Ana , Kalinowski Judith F. , Jastrzebski Sandra , Lorenzo Joseph A.
Published: February 2010

Leukemia inhibitory factor (LIF) is a recently characterized glycoprotein with complex biologic activities on bone cells. We tested various rodent and human immortalized and malignant bone cell lines and primary osteoblast-enriched cell cultures from fetal rat calvarial digests for expression of LIF mRNA and LIF protein. Both human and rodent immortalized and malignant cells expressed a single 4.4 kb mRNA transcript that hybridized to a human LIF cDNA probe in Northern blots. LIF mRNA was undetectable in unstimulated rodent osteoblast-like cells lines MC3T3-E1 and Pyla. However, treatment with LPS (10 μg/ml), TGF-β (1 ng/ml), TNF-α (100 ng/ml) or inhibitors of protein synthesis (cycloheximide, emetine, puromycin, and anisomycin) induced the expression of LIF message in these cells. In contrast, primary osteoblast-enriched cells did not express LIF mRNA in Northern blot assays either constitutively or after treatment with TNF-α or cycloheximide. The human osteosarcoma cells lines U-2 OS and Saos-2 constitutively expressed LIF mRNA and did not respond to LPS treatment. However, phorbol myristate acetate (PMA), an activator of protein kinase C, was a potent stimulator of LIF message in Saos-2 but not U-2 OS cells. The effects of PMA (0.5 ng/ml) on LIF mRNA in Saos-2 cells were detectable at 1 h and maximal at 6 h. TNF-α (100 ng/ml) and inhibitors of protein synthesis also increased LIF mRNA in both Saos-2 and U-2 OS cells. LIF protein was also detected constitutively in the conditioned medium from both Saos and U-2 OS cells. In addition, TNF (100 ng/ml) stimulated the release of LIF protein from both these cells and PMA (2.5 ng/ml) stimulated LIF protein in Saos-2 cells. These results show that several different human malignant and rodent immortalized clonal bone cell lines can express and regulate steady-state LIF mRNA levels and produce LIF protein but that primary cultures of fetal rat osteoblastic cells do not express this cytokine. Hence, LIF may regulate malignant osteogenic cell growth and function in bone but may not be an important regulator of normal bone metabolism.
Immunoassay for urinary pyridinoline: The new marker of bone resorption
Authors: Seyedin Saeid M. , Kung Viola T. , Daniloff Yuri N. , Hesley Robert P. , Gomez Baltazar , Nielsen Lori A. , Rosen Harold N. , Zuk Robert F.
Published: February 2010

Urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd) are markers of bone resorption that are elevated above normal in subjects with metabolic bone disease. Total Pyd and Dpd, both free and peptide-bound forms, can be measured by HPLC after hydrolysis and cellulose chromatography. Since free Pyd is the major component of total Pyd in urine, we developed an immunoassay using free Pyd as an immunogen. This assay is much easier to perform than HPLC, requires no sample preparation, and correlates well with total Pyd measurement by HPLC (r = 0.97) and with urinary hydroxyproline (r = 0.90). The antiserum reacts most strongly with free Pyd and Dpd and minimally with glycosylated and large peptide-bound forms. The sensitivity of the Pyd immunoassay is less than 25 nM. The intraassay CV is 5–10%; the interassay CV is 10–15%. Analytic recovery studies indicated negligible sample interference. Furthermore, measurement of the Pyd in the same individuals over a 30 day time period exhibited minimal day-to-day variation. Thus, the Pyd immunoassay provides a rapid and easy method for evaluation of Pyd in urine. Pyd immunoassay may serve as a practical method of screening for metabolic bone disease and for monitoring therapeutic treatment.
Mitogenic responsiveness of human bone cells in vitro to hormones and growth factors decreases with age
Authors: Pfeilschifter J. , Diel I. , Pilz U. , Brunotte K. , Naumann A. , Ziegler R.
Published: February 2010

Bone loss with aging may at least in part be due to inadequate bone formation. In this study, we examined whether the proliferation of osteoblast-like cells in vitro in response to local and systemic factors might be attenuated with age. A total of 36 cultures of osteoblast-like cells were obtained from outgrowths of human trabecular bone. Parathyroid hormone, growth hormone, calcitonin, transforming growth factor β, insulinlike growth factor I, and platelet-derived growth factor BB dose dependently increased DNA synthesis in all cultures. Increases in DNA synthesis with each of these factors were significantly negatively correlated with donor age in cultures obtained from the iliac crest bone of 50- to 70-year-old women. Cells from 61- to 70-year-old donors required approximately 10-fold higher concentrations of growth factors and hormones to yield comparable increases in DNA synthesis than cells from 51- to 60-year-old donors. A significant negative correlation between age and mitogenic responsiveness to platelet-derived growth factor and growth hormone, but not toward the other factors, was also observed in cultures from the femoral head trabecular bone of 60- to 90-year-old women. Our findings suggest that bone loss with aging may be partially due to a decreased capacity of osteoblasts to proliferate in response to systemic or locally released osteotropic factors.
Impact of anomalous vertebral segmentation on measurements of bone mineral density
Authors: Peel N.F.A. , Johnson A. , Barrington N.A. , Smith T.W.D. , Dr. Eastell R.
Published: February 2010

In anteroposterior (AP) bone mineral density (BMD) measurements of the lumbar spine (LS), the presence of ribs is used to identify vertebra T12. Similarly, in lateral LS-BMD measurements, the position of the iliac crest is used to identify the lumbar vertebrae. The aim of this study was to determine the impact of variations in spinal segmentation and iliac crest position on BMD measurements. In 375 women (ages 50–85 years) radiographs were taken of the thoracic and lumbar spine, as well as AP measurement of LS-BMD, by dual-energy x-ray absorptiometry (DXA). In 121 subjects lateral decubitus LS-BMD was also measured. Anomalous spinal segmentation was found in 16.5%, and L1 would have been incorrectly identified on the AP-DXA image in 13%. The change in BMC and BMD between adjacent vertebrae was greater in the upper than the lower lumbar spine. Misidentification of L1 for T12 resulted in underestimation of the bone mineral content in grams (BMC) of L1 by a mean of 11.5 ± 14.4% (SD; range -33.5 to 33.5%). For the usual region of interest, L2–4, the BMC (g) was underestimated by 8.4 ± 8.7% (range -1.5 to 29.2%), with the BMD (g/cm2) underestimated by 3.6 ± 4.8% (range -5.4 to 11.6%). The position of the iliac crest on the lateral decubitus DXA scans would have led to misidentification of either L2 or L4 for L3 in 15 cases (12%). This resulted in the BMD of L3 being underestimated by 2.7 ± 19.4% (range - 242.4 to 34.6%). We conclude that (1) anatomic variations in spinal segmentation are common; (2) the impact of mididentification of vertebra L1 on the AP scan is considerable if only one lumbar vertebra is studied but small if a larger area is analyzed and expressed as g/cm2; and (3) misidentification of L3 in the lateral projection can result in a large error in the BMD measurement of L3, although the mean error in terms of a population is small. Thus the impact of misidentification of lumbar vertebrae does not result in important overall changes when studying groups of subjects but on an individual basis could lead to incorrect diagnosis of osteopenia.
Isolation and complete amino acid sequence of osteocalcin from canine bone
Authors: Dr. Colombo Giovanna , Fanti Paolo , Yao Chinghua , Malluche Hartmut H.
Published: February 2010

Osteocalcin was purified in high yield and to homogeneity from the diaphysis of dog femora by the following steps: (1) acid demineralization of bone powder, (2) solid-phase extraction of acid-soluble proteins on Sep-Pak C18 cartridges, (3) gel filtration on Sephadex G-50, and (4) fast protein liquid chromatography on an Accell-QMA anion-exchange column. Starting from 30 g washed bone powder, approximately 7–10 mg pure protein was obtained in 2 days. The key step is the initial solid-phase extraction of osteocalcin from a large volume of a demineralized bone solution. The primary structure was established by automated sequence analyses of two tryptic peptides, of two endoproteinase Glu-C carboxy-termina) peptides, and of the first 30 amino acid residues of the intact protein. Dog osteocalcin contains 49 amino acids, has a molecular mass of 5654 daltons, contains no Thr, Met, Hyp, or Trp, has a disulfide bond between Cys 23 and 29, and is fully γ-carboxylated at residues 17, 21, and 24. Dog osteocalcin does not contain a pair of basic amino acids found at positions 43–44 in most other osteocalcins from mammals and birds. A computer search for homology indicated 88, 90, 84, 88, 66, and 57% sequence identity of dog osteocalcin with human, bovine, cat, monkey, chicken, and swordfish osteocalcin, respectively, and weaker homologies with the γ-carboxylated domains of blood-clotting proteins and the Pro-rich N-terminal extensions of myosin light-chain A1 and β-crystalline B1. The possible relevance of these homologies to the structure and potential functions of osteocalcin is discussed.
Dual-energy x-ray absorptiometry of the rat: Accuracy, precision, and measurement of bone loss
Authors: Griffin Michael G. , Kimble Robert , Hopfer Wendy , Pacifici Roberto
Published: February 2010

Dual-energy x-ray absorptiometry (DEXA) is now an established method of measuring bone mineral density (BMD) in humans. We have applied the technique to measuring BMD in the rat. The short-term precision of the technique was assessed by calculating the BMD coefficient of variation at the rat spine (1.20%), total femur (0.52%), proximal femur (1.16%), midfemur (1.00%), distal femur (0.96%), and proximal tibia (2.2%). The long-term precision of femoral measurements in rat cadavers over a 4 week period was 0.72% for the total femur and 1.2% for the distal femur. The accuracy of the technique was assessed by comparing DEXA-measured bone mineral content (BMC) in vitro and in vivo with ashed BMC. Results indicated a highly significant positive correlation between in vitro DEXA measurements and ash BMC (r = 0.99) and between in vivo DEXA measurements and ash BMC (r = 0.89). The ability of the technique to detect bone loss was assessed at the femoral site by comparing the BMD of ovariectomized (OVX) and sham-operated rats at baseline and at a 1 month follow-up. There was no significant difference in BMD between the groups at baseline. However, at 1 month follow-up the OVX group showed a significant (p < 0.001) decline in BMD at the distal femur (-8.6%) and in the total femur (-4.8%) compared with sham-operated rats. A comparison of retired breeder female rats with age-matched nulliparous rats indicated that the BMD of retired breeder rats was significantly lower than that of virgin females at all femoral sites (p < 0.01). The results suggest that the DEXA technique has the precision and accuracy necessary to study changes in rat BMD. Furthermore, the technique can be used quickly and noninvasively to detect a bone loss following ovariectomy in the rat, and the distal femur may be an optimal site for detecting such a bone loss.
Bone density and body composition on the pacific rim: A comparison between japan-born and u.s.-born japanese-American women
Authors: Kin Kokai , Lee Edmund J.H. , Kushida Kazuhiro , Sartoris David J. , Ohmura Akihiro , Clopton Paul L. , Inoue Tetsuo
Published: February 2010

Bone mineral density (BMD) of total body, spine, and proximal femur and the percentage of body fat in 151 U.S.-born Japanese-American women and 137 Japan-born immigrant Japanese-American women living in San Diego, California were measured using dual-energy x-ray absorptiometry. These data were compared with unpublished data from Japanese women obtained in previous studies in Hamamatsu, Japan. The age-adjusted BMD for the spinal level, femoral neck, Ward's triangle, trochanter, and total body, respectively, of U.S.-born Japanese-American women were 10.2, 9.8, 9.9, 9.2, and 2.7% higher than those of native Japanese women. The U.S.-born Japanese-American women had significantly higher body fat than immigrant Japanese-American women. Furthermore, the immigrant women had higher BMD and higher body fat than their native Japanese counterparts; however, no significant total-body BMD differences were found among the three groups after age, height, and weight were adjusted. The U.S.-born Japanese-American women had BMD values equivalent to those of white normals at the spine and femur. Significant life-style differences between U.S.-born and immigrant Japanese-American women were noted. Weight, exercise, early menarche, and years of lifetime estrogen exposure correlated positively with BMD. The significant negative correlates of BMD were age, smoking, and percentage of body fat. Our study presents data suggesting that immigration to the United States has produced a higher BMD in Japanese-American women that is attributable to changes in life-style and diet.
Reproductive correlates of bone mass in elderly women
Authors: Fox Kathleen M. , Magaziner Jay , Sherwin Roger , Scott Jean C. , Plato Chris C. , Nevitt Michael , Cummings Steven
Published: February 2010

Results from previous studies of reproductive factors and bone density have been conflicting; some demonstrate a beneficial effect, but others show a detrimental effect on bone density. The present study investigates the association of parity, lactation, and menstruation with radial bone density in 2230 white women, 65 years of age and older. Bone density was assessed by single-photon absorptiometry. Linear multiple regression was utilized to determine if reproductive factors were associated with radial bone density. The number of births, duration of menstrual bleeding, age at menarche, and years menstruating were significant independent predictors of postmenopausal bone density of the radius. A 1.4% increase in distal radius bone density was observed with each additional birth. Women who began menstruation at age 9 had 6.3% higher bone density than women who began at age 16. Women who menstruated for 3 days during each menstrual cycle had 2.8% less distal radius bone density than women who bled for 7 days. Each decade of menstruation was associated with a 2% greater distal radius bone density. No difference in bone density was demonstrated for women who breast-fed and women who did not. Length of the menstrual cycle, amount of menstrual flow, and irregularity of the menstrual cycle were not significantly associated with radial bone mineral density. In conclusion, pregnancy and menstruation are associated with postmenopausal bone density of the radius.
Effects of aerobic training on bone mineral density of postmenopausal women
Authors: Martin Daniel , Notelovitz Morris
Published: February 2010

A total of 76 women were enrolled and 55 naturally postmenopausal women completed a 12 month study investigating the effects of aerobic training plus calcium supplementation on lumbar (L2–4) bone mineral density (BMD) and forearm BMD. Training was conducted on treadmills at 70–85% of maximal heart rate for 30 or 45 minutes, three times per week for 12 months. Lumbar BMD was measured with dual-photon absorptiometry and forearm BMD with single-photon absorptiometry. Groups were similar with respect to age, weight, months since menopuase, height, maximal oxygen uptake (Vo2max), and lumbar and forearm BMD upon entering the study. Following training, percentage changes in Vo2max were significantly different between the control and exercise groups but not between exercise groups. ANOVA evaluating lumbar BMD revealed a nonsignificant interaction effect and no significant changes (p > 0.05) between groups or times. The control (N = 19), 30 minute (N = 20), and 45 minute (N = 16) groups percentage lumbar BMD changes (X ± SD) over 12 months were -0.61 ± 3.40, -0.48 ± 3.63, and 0.81 ± 4.53%, respectively. The 95% confidence limits for percentage changes in lumbar BMD for the control, 30 minute, and 45 minute groups were -2.25 to 1.02, -2.18 to 1.22, and -1.16 to 3.22%, respectively. Forearm BMD changes were also not significant. Improvement in lumbar BMD was weakly but positively correlated with improvements in VO2max, r = 0.28, p < 0.05. Women ≤ 6 years of the onset of menopause had an accelerated lumbar BMD loss compared to subjects who were >6 years postmenopausal, and this subset's BMD changes were examined. The recently postmenopausal control subjects' (N = 7) lumbar BMD mean percentage change was -3.36 ± 1.53 versus -1.67 ± 1.75% for the 11 recently postmenopausal exercise (30 + 45 minute groups) subjects, p < 0.05. None of the forearm BMD changes in the recently menopausal subset were significant. Thus, although 12 months of aerobic exercise training did not result in significant increases in forearm or lumbar BMD, training attenuated lumbar BMD loss in women who were ≤ 6 years of the onset of the menopause.
Effects of endurance exercise on bone mass and mechanical properties in intact and ovariectomized rats
Authors: Barengolts E.I. , Curry D.J. , Bapna M.S. , Kukreja S.C.
Published: February 2010

Exercise may play a role in the prevention of bone fractures in postmenopausal osteoporosis. The effects of endurance exercise on bone properties were assessed in 9-month-old sham-operated (SH) and ovariectomized (OVX) rats. The rats were either kept sedentary (SED) or were exercised (EX) on a rodent treadmill at 21 m/minute, 7% grade, 40 minutes/day, 4 days/week for 3 months. Bone mineral (by ash weight), morphometry, and biomechanical properties (by three-point bending) were evaluated after excision of bones at sacrifice. Ovariectomy resulted in a loss of bone mineral in femur, tibia, and fourth lumbar vertebra (L4), but biomechanical (force, deformation, stress, strain, and modulus of elasticity) and morphometric (length, cortial and medullary area, and moment of inertia) properties of femur were maintained. The ash weight of femur and tibia, but not L4, as well as femur yield and maximum force and moment of inertia, were improved in OVX-EX rats compared to OVX-SED animals. In SH rats exercise had no influence on ash weight of any of the three bones or femur morphometric properties, yet femur maximum force and plastic deformation were significantly enhanced compared to SH-SED rats. The results of the present study suggest that endurance exercise has beneficial effects on the bone mineral as well as biomechanical properties (femur yield and maximum force) during early stages after ovariectomy and improves the bending strength of the intact femur without an effect on bone mineral in sham-operated rats.
Endothelin-1 modulates calcium signaling by epidermal growth factor, α-thrombin, and prostaglandin E1 in UMR-106 osteoblastic cells
Authors: Tatrai Agnes , Stern Paula H.
Published: February 2010

Local factors play an important role in the regulation of bone metabolism. The homologous and heterologous desensitization of responses to these factors may be crucial in the modulation of bone cell signaling. In this study, the effects and interactions of endothelin-1 (25 nM), α-thrombin (0.9 μM), epidermal growth factor (40 nM), prostaglandin E1 (5 μM), and prostaglandin F1α (5 μM) were examined on calcium signaling in UMR-106 rat osteoblastic osteosarcoma cells. Intracellular calcium was measured using fluo-3 fluorescent dye. All agents elicited calcium transients at these concentrations and showed homologous desensitization to their repeated administration. Preincubation for 60 minutes with 500 μM monodansylcadaverine and 30 minutes or 24 h preincubation with 0.5 μM indomethacin did not affect homologous desensitization, suggesting that neither the internalization of receptors nor prostaglandins are involved in this event. Pretreatment for 3 minutes with 2 μM 4β-phorbol-12β,13α-dibutyrate significantly reduced the calcium elevations elicited by the first application of these compounds, whereas an inactive phorbol, 12,13-didecanoate, had no effect. Pretreatment for 4 minutes with 0.5 μM forskolin decreased the calcium signal response to PGE, only. Pretreatment with endothelin-1 for 3 minutes significantly decreased the calcium signals elicited by epidermal growth factor and α-thrombin. Prior administration of endothelin-1 significantly increased prostaglandin E1-stimulated calcium transients, whereas prostaglandin F1α responses were not affected. Preincubation with indomethacin did not alter any of the interactions. Responses to endothelin-1 were not significantly altered by 2–3 minutes pretreatment with the other factors, nor was there cross-desensitization among the other factors. The results could indicate that endothelin-1 has a unique and specific role in the modulation of bone cell signaling.
Role of the negative glucocorticoid regulatory element in glucocorticoid repression of the human osteocalcin promoter
Authors: Morrison Nigel , Eisman John
Published: February 2010

In osteoblast-like cells in culture, the human osteocalcin gene promoter basal activity is repressed by glucocorticoids, reflecting the repression of serum osteocalcin concentrations noted in syndromes of glucocorticoid excess. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active hormonal form of vitamin D, induces the osteocalcin promoter through a vitamin D response element (VDRE), and glucocorticoids also repress the vitamin D-induced promoter. This study addresses the role of a glucocorticoid receptor (GR) binding site overlapping the TATA box of the osteocalcin promoter, which had been proposed as a negative glucocorticoid response element (nGRE), invoking a steric interference mechanism of glucocorticoid repression. Confirmation of the role of the nGRE in regulating basal activity was obtained using promoter constructs containing a TATA box swap. However, a minor component of repression of 1,25-(OH)2D3 induced activity remained in the absence of the nGRE. In addition, glucocorticoid repression of the human osteocalcin promoter was shown to be cell line specific. This result is not compatible with a simple model of repression and suggests the existence of unidentified cell-specific factors that are involved in the repression event. Repression of the osteocalcin promoter was compatible with a composite model involving both the nGRE site and glucocorticoid regulation of factors that bind the vitamin D response element.
Detection and measurement of vitamins K1 and K2 in human cortical and trabecular bone
Authors: Hodges S.J. , Bejui J. , Leclercq M. , Delmas P.D.
Published: February 2010

Vitamin K plays an essential role in the gamma carboxylation of the gla-containing proteins of bone matrix, such as osteocalcin. We have measured the concentration of vitamin K1 (VK) and of the menaquinones MK-6, MK-7, and MK-8, the three major vitamin K2 moieties, in trabecular and cortical bone taken from the femoral neck of patients undergoing hip replacement. Both bone compartments were found to contain large amounts of VK, MK-6, MK-7, and MK-8. Concentrations were as high as those reported for the liver, the major storage organ for vitamin K. Further research is required to examine the association between vitamin K in bone and the gamma carboxylation of vitamin K-dependent bone proteins.
Oscillations in inositol 1,4,5-trisphosphate and diacyglycerol induced by vitamin D3 metabolites in confluent mouse osteoblasts
Authors: Grosse Brigitte , Bourdeau Agnès , Dr. Lieberherr Michèle
Published: February 2010

For the last 5 years, attention has focused on the nongenomic effects of 1,25-(OH)2D3, but considerably less is known about the mechanisms of the nonnuclear actions of 24,25-(OH)2D3. The present study examines and compares the rapid (5–90 s) effects of 100 pM to 10 nM 24,25-(OH)2D3, 10 pM to 1 nM 1,25-(OH)2D3, and 1–100 nM 25-OHD3 on the formation of inositol phosphates and lipids in confluent mouse osteoblasts. 24,25-(OH)2D3 and 25-OHD3 effects were dose dependent; those of 1,25-(OH)2D3, were dose dependent in a bell-shaped manner. The two dihydroxylated metabolites induced a multiphasic response in inositol 1,4,5-trisphosphate (IP3) formation with three stimulation peaks; the IP3 response to 25-OHD3 was monophasic. The amplitude of the IP3 response to 24,25-(OH)2D3 was greater and its oscillation period was slower than that induced by 1,25-(OH)2D3. The diacylglycerol (DAG) responses to secosteroids showed two stimulation peaks that appeared at different times depending on the secosteroid used. Pretreatment with neomycin totally inhibited the first DAG response; neomycin had no effect on the second peak of DAG induced by 25-OHD3, whereas it partially blocked the second response of DAG to 24,25-(OH)2D3 and 1,25-(OH)2D3. These data show for the first time that 24,25-(OH)2D3 can modulate phospholipid metabolism in confluent mouse osteoblasts as early as 5–10 s. The first pathway used by all three secosteroids is that of the hydrolysis of phosphatidylinositol 4,5-bisphosphate via phospholipase C activation, leading to the formation of the two second messengers, IP3 and DAG, since neomycin totally blocked the response. Thus, the action of these secosteroids on the osteoblast membrane may also implicate several steps of the phosphatidylcholine cycle, according to the metabolite tested. Finally, these data point to a direct interaction of vitamin D metabolites with specific membrane recognition moieties.
Human parathyroid hormone (1–34) and (1–84) increase the mechanical strength and thickness of cortical bone in rats
Authors: Dr. Ejersted Charlotte , Andreassen T.T. , Oxlund H. , Jørgensen P.H. , Bak B. , Häggblad J. , Tørring O. , Nilsson M.H.L.
Published: February 2010

An anabolic effect on bone of intermittent parathyroid hormone (PTH) treatment has been found in patients with osteoporosis and also in experimental animals. Controversies exist, however, about whether the positive effect on the trabecular bone balance occurs at the expense of the cortical bone. We examined the biomechanical quality of cortical bone after intermittent treatment with different doses of PTH and, furthermore, compared the effects of PTH-(1–34) and PTH-(1–84). Groups of rats were treated with biosynthetic human PTH-(1–34) or PTH-(1–84), 1.1, 3.3, 10, or 30 nmol/kg/day for 30 days. No changes in the body weights and no changes in the lengths of the femora were observed after the PTH treatments. The bio-mechanical properties were analyzed by means of a materials-testing machine. A dose-related increase in the bending strength and stiffness of the femora was found, and this increase in mechanical strength corresponds with a 9–12% increase in the cross-sectional area of the femoral diaphyses. The deflection capability and energy absorption were not influenced by any of the PTH treatments. No differences were found between the effects of PTH-(1–34) or PTH-(1–84) on the biomechanical properties of the femora. Consequently, intermittent treatment with biosynthetic PTH-(1–34) or PTH-(1–84) increased the formation of cortical bone, and the biomechanical competence of the femora was found to be preserved.
Influence of exercise on cancellous bone of the aged female rat
Authors: Yeh James K. , Aloia John F. , Chen Meng M. , Tierney Joan M. , Sprintz Sharon
Published: February 2010

Dual-energy x-ray absorptiometry and dynamic histomorphometry were used to examine the effect of treadmill exercise on the bone density and cancellous bone formation and resorption in the proximal tibia and fifth lumbar vertebra (L5) of the aged female rat. Female rats aged 14 months were divided into four groups: 8 controls and 10 exercised for a 9 week study and 8 controls and 9 exercised for a 16 week study. Exercise consisted of running on a flat-bed treadmill, 17 m/minute, 1 h/day, 5 days/week. Tibial metaphysis and L5 vertebral density of each rat were measured in the 16 week study by DXA at weeks 0, 9, and 16. Compared to the control group, a significant increase in bone density in both metaphyseal tibia and L5 vertebra was apparent at 16 weeks after exercise training (P = 0.046 and 0.025, respectively, by two-way ANOVA). Histomorphometric analysis showed that the trabecular bone eroded surface and the ratio of eroded to mineralizing surface in tibial metaphysis were significantly lower in the exercised than in the respective control group in both the 9 and 16 week studies. In L5 vertebra, these decreases by exercise were apparent only in the 16 week study. A significant increase in the bone formation rate was apparent in the cancellous bone of the tibia but not of the vertebra after 16 weeks of exercise (P < 0.05). The trabecular architecture (bone number and separation) of the L5 vertebra in the exercised rats did not differ from that of the controls in either study. The trabecular bone volume and trabecular number of the tibial metaphysis were higher and the separation was less in the exercised rats but not significantly different from the control at each time point. In conclusion, it was demonstrated that the tibial metaphyseal and L5 vertebral bone density were higher in the treadmill-exercised than in the nonexercised adult rat. Increased bone formation and suppressed resorption could account for the beneficial effect of exercise on cancellous weight-bearing bone. In cancellous non-weight-bearing bone, it could be primarily in suppressing bone resorption and inhibiting age-related loss.
Vertebral fracture assessment using a semiquantitative technique
Authors: Genant Harry K. , Wu Chun Y. , van Kuijk Cornelis , Nevitt Michael C.
Published: February 2010

The assessment of vertebral fracture by conventional radiography has been refined and improved using either semiquantitative or quantitative criteria. The inter- and intraobserver variability was determined for a semiquantitative visual approach that we routinely use in clinical studies for assessing prevalent and incident vertebral fractures. In addition, the semiquantitative approach was compared with a quantitative morpho-metric approach. The incidence and prevalence of vertebral fractures were determined in 57 postmenopausal women (age 65–75 years) by three independent observers. The radiographic basis for fracture definitions and the source of interpretative errors are illustrated. The results show excellent intraobserver agreement and good interobserver agreement for the semiquantitative technique. We conclude that the semiquantitative approach can be applied reliably in vertebral fracture assessment when performed using well-defined criteria.
Rapid publication: Flanking markers define the X-linked hypophosphatemic rickets gene locus
Authors: Econs Michael J. , Fain Pamela R. , Norman Michael , Speer Marcy C. , Pericak-Vance Margaret A. , Becker Peggy A. , Barker David F. , Taylor Arlene , Drezner Marc K.
Published: February 2010

X-linked hypophosphatemic rickets (HYP) is an X-linked dominant disorder characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. The defect in tubular phosphate reabsorption is probably secondary to an unidentified humoral factor. Identification of the humoral factor and a full understanding of the pathophysiology of the disease await the identification of the HYP gene. Previously we demonstrated that DXS257 and DXS41 are flanking markers for the HYP gene. Two markers, DXS365 and DXS274, are tightly linked to the HYP gene, but investigators have been unable to determine whether they are centromeric or telomeric to the disease gene. Since tightly linked flanking markers are necessary prerequisites to obtain the gene by positional cloning techniques, we sought to determine the relative positions of these markers to the HYP gene by expanding our data base for linkage studies. We also investigated a new polymorphic probe for linkage to HYP to construct a more detailed genetic map around the HYP locus. Our data indicate that the markers DXS365, DXS274, and DXS92 are tightly linked to the HYP locus and suggest a locus order of Xtel-(DXS444/DXS315)-DXS43-(DXS257/DXS365)-HYP-(DXS274/DXS41/DXS92)-DXS451-DXS319-Xcen. These results will facilitate attempts further to localize and clone the HYP gene.