bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Nitric Oxide Synthase Isoforms in Bone and Bone Cell Cultures
Authors: Helfrich Miep H. , Evans Deborah E. , Grabowski Peter S. , Pollock Jennifer S. , Ohshima Hiroshi , Ralston Stuart H. 
Published: April 2006

Abstract
Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine–NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.
Cyclization by a Specific Lactam Increases the Ability of Human Parathyroid Hormone (hPTH)-(1–31)NH2 to Stimulate Bone Growth in Ovariectomized Rats
Authors: Whitfield J. F. , Morley P. , Willick G. , Langille R. , Ross V. , Maclean S. , Barbier J.-R. 
Published: April 2006

Abstract
Human parathyroid hormone (hPTH)-(1–31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase-C as do hPTH(1–84) and hPTH(1–34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side-chain lactams have been introduced in the hydrophilic face of the receptor-binding region of the fragment's Ser17-Val31 amphiphilic α-helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this α-helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1–31)NH2, which had six times the AC-stimulating ability of hPTH(1–31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix-stabilizing lactam derivative [Leu27]-cyclo(Lys26-Arg30)-hPTH(1–31)NH2 did not have a greater AC-stimulating ability than hPTH(1–31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTH's anabolic action, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1–31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1–31)NH2 or [Leu27]-cyclo(Lys26-Arg30)-hPTH(1–31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1–31)NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1–31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.
Quantitative Ultrasound Techniques for the Assessment of Osteoporosis: Expert Agreement on Current Status
Authors: Glüer Claus-C. ,   FOR THE INTERNATIONAL QUANTITATIVE ULTRASOUND CONSENSUS GROUP
Published: April 2006

Abstract
Quantitative ultrasound (QUS) methods have been introduced in recent years for the assessment of skeletal status in osteoporosis. The performance of QUS techniques has been evaluated in a large number of studies. Reviewing existing knowledge, an international expert panel formulated the following consensus regarding the current status of this technology. To date, evidence supports the use of QUS techniques for the assessment of fracture risk in elderly women. This has been best established for water-based calcaneal QUS systems. Future studies should include the predictive validity of other QUS systems. Additional clinical applications of QUS, specifically the assessment of rates of change for monitoring disease progression or response to treatment, require further investigation. Its low cost and portability make QUS an attractive technology for assessing risk of fractures in larger populations than may be suitable or feasible for bone densitometry. Additional investigations that assess innovative QUS techniques in well defined research settings are important to determine and utilize the full potential of this technology for the benefit of early detection and monitoring of osteoporosis.
Binding and Cyclic AMP Stimulation by N-Terminally Deleted Human PTHs (3–84 and 4–84) in a Homologous Ligand Receptor System
Authors: Olstad Ole K. , Reppe Sjur , Loseth Ole P. , Jemtland Rune , Gautvik Kaare M. 
Published: April 2006

Abstract
We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3–84) and hPTH(4–84), employing the mating factor α (MFα) expression system. The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis. In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using [125I-Tyr36]chickenPTHrP(1–36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3–84) and hPTH(4–84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1–84) (Kd = 2.3 nM). In comparison, [Nle8,18,Tyr34]bovinePTH(3–34)NH2 and [Tyr36]cPTHrP(1–36)NH2 showed equal Kd's of 1.9 nM. Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM. At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1–84). hPTH(1–84) and [Tyr36]cPTHrP(1–36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively. The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or [Tyr36]cPTHrP(1–36)NH2. [Nle8,18,Tyr34]bPTH(3–34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited [Tyr36]cPTHrP(1–36)NH2-induced cAMP production by 40% when present at a 1000 M excess. These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.
Macrophage Colony Stimulating Factor Down-Regulates MCSF-Receptor Expression and Entry of Progenitors into the Osteoclast Lineage
Authors: Fan Xian , Biskobing Diane M. , Fan Dongjie , Hofstetter Willy , Rubin Janet 
Published: April 2006

Abstract
Macrophage colony-stimulating factor (MCSF), although necessary for entry of precursors into the early preosteoclast pathway, inhibits osteoclastogenesis at high doses. To clarify the relationship between MCSF and osteoclast formation, we investigated the effect of exogenous MCSF in murine bone marrow culture. Precursor proliferation and the expression of MCSF-receptor were examined after 4 days of culture in the presence or absence of accessory stromal cells. In both mixed marrow and destromalized cell cultures, exogenous MCSF dose-dependently decreased125I-MCSF binding (by 65 ± 5.0% at 3500 and 87 ± 16.7% at 7000 U/ml, respectively) while enhancing mononuclear cell proliferation after 3 days of exposure (by 2.8- and 6.3-fold, respectively). These effects were maintained 24 h after removal of exogenous MCSF and, as such, likely represented an MCSF-induced change in MCSF receptor-bearing cells. Exposure to exogenous MCSF (3500 U/ml) days 2–4 dose-dependently inhibited tartrate resistant acid phosphatase positive multinuclear cell (TRAP+ MNC) formation counted at the end of day 7, by 64.3 ± 4.1%. This inhibition of TRAP+ MNC formation was preceded by a 92 ± 9% decrease in the expression of carbonic anhydrase II mRNA measurable at 4 days. These results indicate that MCSF promotes proliferation of a population of cells expressing lower cognate receptor sites. Changes in MCSF-receptor expression appear to modulate the final lineage selection of the pluripotent monoblastic progenitor.
Aromatase in Human Bone Tissue
Authors: Sasano Hironobu , Uzuki Miwa , Sawai Takashi , Nagura Hiroshi , Matsunaga Gen , Kashimoto Osamu , Harada Nobuhiro 
Published: April 2006

Abstract
Peripheral aromatization of androgens exert estrogenic actions in many tissues. Recently in situ production of estrogens by aromatase was detected in human bone and cultured osteoblasts and has been proposed to participate in the maintenance of bone mass. We examined aromatase expression by immunohistochemistry and mRNA in situ hybridization in 16 cases of tibia (female 2 male, 14 female, 62 ± 5.2 years old) and quantified the level of aromatase mRNA in 28 cases of rib, femur, and lumbar vertebrae (16 male, 12 female, 58.0 ± 11.3 years old) by reverse transcriptase-polymerase chain reaction (RT-PCR) in order to study whether or not and in which cell types aromatase was expressed in human bone tissues. We also studied alternative use of multiple exons 1 of its gene and immunolocalization of type I 17β-hydroxysteroid dehydrogenase (HSD), which converts estrone produced by aromatase to estradiol. Strong aromatase immunoreactivity and mRNA hybridization as well as type I 17β-HSD immunoreactivity were detected in lining cells, osteoblasts, chondrocytes of articular cartilage, and adipocytes adjacent to bone trabeculae in all the cases examined. Amounts of aromatase mRNA varied greatly among the subjects (11.25 ± 9.77, 0.61–42.84 attomol/ng of total RNA). The amount of aromatase expression was not correlated with age or gender of the subjects but positively correlated with the degree of osteroporotic changes evaluated by radiological findings of lumbar vertebrae. Analysis of multiple exons 1 revealed that 1b or fibroblast type was predominantly (23/26) utilized as a promoter of aromatase gene expression. These results demonstrated that aromatase is expressed widely in human bone tissue and may play important roles in maintenance of human bone tissue.
Effects of Dihydrotestosterone on Bone Biochemical Markers in Sham and Oophorectomized Rats
Authors: Mason R. A. , Morris H. A. 
Published: April 2006

Abstract
Evidence exists to suggest that androgens stimulate bone formation in the estrogen-deficient state, however the mechanism of action is unclear. The following study investigates the effect of dihydrotestosterone (DHT) on biochemical markers of bone turnover and calcium homeostasis in sham and oophorectomized (oophx) rats when either vehicle, 40, 80, or 160 mg/kg body weight (bw) DHT were administered at the time of operation or at 15 weeks postoperation. Serum alkaline phosphatase (ALP) increased following DHT administration in sham and oophx rats in all groups (mean ALP ± SEM [U/l] week 8; sham vehicle, 40 ± 7; sham 160 mg DHT/kg bw, 72 ± 5; oophx vehicle, 60 ± 6; oophx 160 mg DHT/kg bw, 88 ± 11) (p &lt; 0.001). In contrast, serum osteocalcin was significantly suppressed in oophx rats administered DHT 15 weeks following operation (mean osteocalcin ± SEM [μg/l] week 8; oophx vehicle, 17.6 ± 3.5; oophx 160 mg DHT/kg bw, 10.5 ± 1) (p &lt; 0.01). Urine deoxypyridinoline was significantly decreased when DHT was administered 15 weeks postoophorectomy (p &lt; 0.001); however, urine hydroxyproline was not affected by DHT treatment in any group. Urine calcium was decreased by DHT treatment (mean Ca/Cr ± SEM week 8; sham vehicle, 0.87 ± 0.13; sham 160 mg DHT/kg bw, 0.24 ± 0.08; oophx vehicle, 0.68 ± 0.16; oophx 160 mg DHT/kg bw, 0.45 ± 0.1) (p &lt; 0.005) which was associated with an increase in the renal tubular reabsorption of calcium (p &lt; 0.05). This study demonstrates the direct effects of DHT on both bone cell activities and the renal handling of calcium.
Bone Elasticity and Ultrasound Velocity Are Affected by Subtle Changes in the Organic Matrix
Authors: Mehta Shreefal S. , Öz Orhan K. , Antich Peter P. 
Published: April 2006

Abstract
The mechanical competence of bone can be studied through the measurement of the components of its material elasticity, a property which can vary both in magnitude and in dependence upon orientation (anisotropy). While it is known that the elasticity is largely determined by the mineral constituents of the bone matrix, it is nonetheless clear that it must be also dependent upon the remaining constituents of bone material. In this work, the influence of organic components on the elasticity is explored by altering specific constituents of the bone matrix to varying degrees. This study addresses two questions: first, are the resulting changes in elasticity strongly or weakly dependent upon direction, and second, are they substantially dependent upon the nature and magnitude of the induced matrix alteration? To answer these questions, we performed different chemical manipulations of the bone matrix and measured the changes in elasticity and velocity using the technique of ultrasound critical angle reflectometry. Altering the properties of the organic matrix resulted in substantial and complex changes in the elasticity of bone. The observed changes were strongly dependent upon direction, could not be explained by changes in density alone, and varied strongly with the specific chemical treatment of the matrix. Immersion in urea selectively affected protein components of the organic matrix and resulted in reversible changes in velocity and elasticity, while removal of collagen caused anisotropic decreases and removal of all organic matter caused a collapse of all components of the elasticity. In conclusion, this study confirms that the organic matrix exerts a profound influence on the elasticity and indicates that the measurement of elastic properties at multiple directions is necessary in the assessment of bone mechanical competence.
Associations of Physical Activity and Calcium Intake with Bone Mass and Size in Healthy Women at Different Ages
Authors: Uusi-Rasi Kirsti , Sievänen Harri , Vuori Ilkka , Pasanen Matti , Heinonen Ari , Oja Pekka 
Published: April 2006

Abstract
Weight-bearing exercise and calcium intake are known to contribute to bone density. However, the relative significance of physical activity and calcium intake in the development of bone characteristics in functionally different weight-bearing and nonweight-bearing bone sites at different ages is poorly known. A total of 422 women in three age groups (25–30, 40–45, and 60–65 years) were screened from 1017 women and divided into four groups by their level of physical activity (high [PA+] and low [PA−]) and calcium intake (high [Ca+] and low [Ca−]). Total body bone mineral content (TBBMC), areal bone mineral density (BMD) of the femoral neck and distal radius, and selected dimensions and estimated strength variables (bone width, cortical wall thickness, cross-sectional moment of inertia, and section modulus of the femoral and radial shafts) were measured with dual-energy X-ray absorptiometry. Both high physical activity and high calcium intake were associated with a higher TBBMC when compared with low activity and calcium intake (1.8% and 4.6%, respectively). The BMD of the weight-bearing femoral neck was 5% higher in the PA+ groups than in the PA− groups, whereas calcium intake showed no such significant association. Neither physical activity nor calcium intake was associated with the BMD of the nonweight-bearing radius. However, both high physical activity and high calcium intake were related to larger and mechanically more competent bones in the femoral and radial shafts, the association for physical activity being stronger with increasing age. No significant interaction between physical activity and calcium intake was found with respect to any of the bone variables. These data from a cross-sectional study suggest that a moderate level of physical activity or a sufficient level of calcium intake, if maintained from childhood, can result in considerable long-term improvement in the mechanical competence of the skeleton. The clinical relevance of these findings is further emphasized by the fact that the observed patterns of physical activity and calcium intake pertain to customary lifestyle and are thus feasible targets for the primary prevention of osteoporosis.
Osteoprogenitor Cells as Targets for Ex Vivo Gene Transfer
Authors: Dr. Onyia J. E. , Clapp D. W. , Long H. , Hock J. M. 
Published: April 2006

Abstract
We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5–6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for β-galactosidase (β-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of β-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, β-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.
Colony Stimulating Factor-1–Induced Osteoclast Spreading Depends on Substrate and Requires the Vitronectin Receptor and the c-src Proto-Oncogene
Authors: Teti Anna , Taranta Anna , Migliaccio Silvia , Degiorgi Annamaria , Santandrea Elena , Villanova Ida , Faraggiana Tullio , Chellaiah Meena , Hruska Keith A. 
Published: April 2006

Abstract
The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1–induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1–induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the αvβ3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1–induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1–induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.
Effects of Estrogen Therapy of Postmenopausal Women on Cytokines Measured in Peripheral Blood
Authors: Rogers Angela , Eastell Richard 
Published: April 2006

Abstract
Estrogen replacement therapy (ERT) is known to prevent bone loss following the menopause, but the mechanism for this is unclear. Estrogen may suppress the secretion of certain bone-resorbing cytokines. The aim of this study was to assess the effect of ERT on the levels of cytokines measured in peripheral blood. We measured cytokines in 10 postmenopausal women (ages 56–59, 3–9 years since menopause) treated with ERT and 10 age-matched (54–59 years, 4–10 years since menopause) untreated women as controls. Samples of blood were taken and used for mononuclear cell cultures, whole blood (WB) cultures, and the separation of serum. The cultures were treated with lipopolysaccharide (LPS; 500 ng/ml) and hydrocortisone (10−6 M). The conditioned medium from cultures and the serum were then assayed for interleukin-6 (IL-6), IL-1α, IL-1β, IL-1 IL-1ra, tumor necrosis factor alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay. M-CSF and the soluble cytokine receptors soluble IL-6 receptor (sIL-6r) and soluble TNF receptor type 1 (sTNFr1) were also measured in serum and M-CSF in stimulated WB cultures. Measurements were corrected for mononuclear cell count. We also measured serum bone-specific alkaline phosphatase (ibAP) in all subjects. We found that LPS stimulated secretion of all cytokines both in WB and isolated cell cultures, and that this was attenuated by hydrocortisone. A significantly higher ratio of IL-1β/IL-1ra (p = 0.02) in LPS stimulated WB cultures was seen in the untreated women. Levels of IL-1β and IL-1α measured in WB cultures were lower and IL-1ra was higher in the ERT-treated group but these results were not significant. BAP was higher in the untreated group (p = 0.005) and correlated with IL-α/IL-1ra in the whole group (r = 0.49, p = 0.03). Results of other measurements showed no significant differences between groups. We conclude that estrogen may prevent bone loss following the menopause by altering the balance between IL-1β and IL-1ra.