bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone-selective analogs of human PTH(1–34) increase bone formation in an ovariectomized rat model
Authors: Lane Nancy E. , Kimmel Donald B. , Nilsson Magnus H.L. , Cohen Fred E. , Newton Suellen , Nissenson Robert A. , Strewler Gordon J.
Published: February 2010

Intermittent parathyroid hormone (PTH) therapy increases bone mass. The purpose of this study was to determine if analogs of human PTH(1–34) (hPTH[1–34]), which differ from the native sequence in their receptor-activating properties, could promote bone formation in an ovariectomized (OVX) osteopenic rat model. We synthesized two hPTH(1–34) analogs with single substitutions for serine in the 3-position that in vitro are partial agonists in kidney. In the renal cell line OK, maximal cyclic adenosine monophosphate (cAMP) activation by [His3]hPTH(1–34) was 50%, and maximal cAMP activation by [Leu3]hPTH(1–34) was 20% of that produced by hPTH(1–34). Both analogs were full agonists in UMR-106 rat osteosarcoma cells and other bone-derived systems, but both had reduced potency compared with hPTH(1–34). Six-month-old retired breeder Sprague-Dawley rats were ovariectomized, and five animals underwent sham operation. On day 56 post-OVX, five sham-operated and five pre-PTH treatment OVX animals were sacrificed, and the remaining animals were randomized into 10 groups of six animals each. All other animals were injected with one of the hPTH analogs or hPTH(1–34) at 0,4,40, or 400 μg/kg of body weight (BW)/day and were killed on day 84. Histomorphometry of the proximal tibia metaphysis and biochemical markers of bone turnover (osteocalcin and pyridinoline cross-links) were the primary endpoints. The cancellous bone volume was significantly lower at day 56 post-OVX (pretreatment) and at day 84 post-OVX (post-vehicle treatment) than at baseline. None of the compounds significantly increased the cancellous bone volume. Trabecular number declined after OVX and did not change with hPTH treatment. In contrast, the trabecular thickness declined after OVX but was higher after treatment with 40 μg/kg of BW/day or 400 μg/kg of BW/day of hPTH(1–34). In OVX rats, the mineralizing surface was higher than baseline at day 56 and fell toward control levels by day 84. All three peptides produced marked dose-related increases in the mineralizing surface and bone formation rates, but the two analogs were less potent than hPTH(1–34). Likewise, all peptides produced significant dose-related increases in the serum osteocalcin level. The osteoclast surface was not affected by OVX but was decreased with medium and high doses of hPTH(1–34). Pyridinoline cross-link excretion was not significantly affected by treatment with hPTH(1–34) but responded with a dose-dependent decrease to treatment with [His3]hPTH(1–34). These data suggest that bone selective analogs of hPTH(1–34) maintain the ability to induce bone formation but are less potent than hPTH(1–34).
In vivo demonstration that parathyroid hormone and parathyroid hormone–related protein stimulate expression by osteoblasts of interleukin-6 and leukemia inhibitory factor
Authors: Pollock Jennifer H. , Blaha Michelle J. , Lavish Sandra A. , Stevenson Sharon , Greenfield Edward M.
Published: February 2010

We have previously reported that parathyroid hormone (PTH) and PTH related protein (PTHrP) stimulate expression of interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) in osteoblasts in vitro. In the current study, we have developed a model of hormone injection into the subcutaneous space overlying mouse parietal bones to demonstrate that similar processes occur in osteoblasts in vivo. Specifically, PTH and PTHrP rapidly and transiently induce expression of the mRNAs encoding IL-6 and LIF. The effects are dose-dependent, with a maximal stimulation of approximately 50-fold for each cytokine. Although PTH and PTHrP activate both adenyl cyclase and phospholipase C-dependent signal transduction pathways, stimulation of IL-6 and LIF depends on adenyl cyclase since it is not reproduced by PTH(3–34), a partial agonist that only activates phospholipase C. These results confirm our previous in vitro studies and support the hypothesis that IL-6 and/or LIF are physiologically important mediators of at least some of the actions of PTH and PTHrP.
Long-term extracellular matrix metabolism by cultured human osteogenesis imperfecta osteoblasts
Authors: Fedarko Neal S. , Sponseller Paul D. , Shapiro Jay R.
Published: February 2010

Osteopenia due to deficient extracellular matrix synthesis is a hallmark of osteogenesis imperfecta (OI). Previous studies carried out within 72 h of osteoblast subculture, at an early stage of matrix synthesis, indicated that for osteoblasts derived from human OI patients the total amounts of collagen, osteonectin, and three proteoglycans were significantly reduced, while total amounts of thrombospondin, fibronectin, and matrix hyaluronan were elevated compared with age-matched controls. The current study was undertaken to follow OI osteoblast matrix metabolism as that matrix is synthesized, deposited, and matured. Steady-state metabolic radiolabeling was used to follow the metabolism of collagen, hyaluronan, and total proteoglycan by OI and normal osteoblasts for up to 5 weeks. Trabecular osteoblasts from non-OI controls showed an increase in total and matrix-associated collagen synthesis during the first and second week, reaching steady-state levels by week 4. In contrast, cultured OI osteoblasts did not increase either the total (medium + matrix-associated) or matrix-associated collagen during the entire 5-week period. Proteoglycan synthesis exhibited a pattern similar to that for collagen. OI-derived proteoglycans differed from controls in that levels in OI cultures did not reflect the normal time-dependent increase in total proteoglycan and proteoglycan matrix deposition. OI osteoblast hyaluronan synthesis was increased when compared with age-matched controls during 4 weeks of culture. In contrast, the ratios of calcium to phosphorus solublized from control and the OI extracellular matrix were not statistically different. Thus, with respect to the synthesis of collagen, proteoglycans, and hyaluronan, OI osteoblasts fail to parallel controls in depositing and elaborating extracellular matrix during 35 days in culture.
Total body calcium and bone mineral content: Comparison of dual-energy X-ray absorptiometry with neutron activation analysis
Authors: Ellis K.J. , Shypailo R.J. , Hergenroeder A. , Perez M. , Abrams S.
Published: February 2010

The aim of this study was to compare the dual-energy X-ray absorptiometry (DXA) measurement of bone mass with an independent measure of body calcium obtained by neutron activation analysis (NAA). Total body bone mineral content (BMC) was measured using DXA in 46 subjects in the age range 5–47 years (17 children, 28 young women, and 1 adult male). Total body calcium (TBCa) was measured in the same subjects by in vivo NAA. The correlation between the two measures of bone mass was highly significant (BMC[g] = 3.22 × TBCa[g] – 51.4, r > 0.98, p < 0.0001, SEEBMC = 122.7 g). When BMC was the independent parameter, the SEETBCa was 37.5 g. Bland-Altman analysis indicated a mean difference of 2.8 g with a standard error + 4.7 g for TBCaNAA versus TBCaDXA when the BMC values were converted to TBCa. The relative change in bone mass (ΔTBCa/ABMC) for DXA was higher than that reported for dual-photon absorptiometry versus NAA. The findings presented in this study provide translational equations among the DXA and NAA measurements and for the conversion of total body BMC to TBCa in children and young adults.
Regional bone mineral density interrelationships in normal and osteoporotic postmenopausal women
Authors: Prof. Nordin B.E. Christopher , Chatterton Barry E. , Schultz Christopher G. , Need Allan G. , Horowitz Michael
Published: February 2010

We describe a prospective study in which bone mineral density (BMD) was measured in total body and regions, proximal femur, lumbar spine, and forearm in 84 apparently normal postmenopausal women with normal spinal radiographs and in 47 women with 1–10 wedged or compressed vertebrae. There was a history of peripheral fracture in 35 of the 84 controls and 30 of the 47 osteoporotics (p < 0.02) but there was no association between vertebral fracture and wrist fracture. At all sites and regions, the differences in BMD between the “normal” and “osteoporotic” women was highly significant and all but “ribs” and “arms” remained significant after correction for menopausal age. In the whole set, and in both subgroups, the coefficients of correlation between sites and regions were all highly significant (p < 0.001). Nonetheless, some sites discriminated better between the two groups than others. Standardized odds ratios (OR) for vertebral fracture versus no-fracture were calculated by logistic regression and expressed as the rise in OR for each standard deviation (SD) fall in bone density. This ratio was greatest (3.4) in “pelvis” and weakest (1.7) in “ribs” but all were statistically significant. Geometric mean regression equations were calculated for all the 78 possible pairs of sites and regions in the 84 normal subjects and the deviations of the osteoporotic women from these normal slopes calculated. In most pairs of sites and regions, the vertebral fracture cases were scattered around the normal group's slope but fell lower down on both axes. The bone deficits in the osteoporotics compared with young normal women ranged from –14% in “head” to –40% in Ward's triangle and the T-scores ranged from –1.9 in “ribs” to –3.9 in the forearm. Sensitivity ranged from 17% in “ribs” to 36.2% in Ward's triangle. Specificity varied between 88 and 94% and the percent correctly classified ranged from 62.6% in “ribs” to 72.5% in Ward's triangle. We conclude that primary postmenopausal osteoporosis affects the entire skeleton but that some sites discriminate better between vertebral fracture and nonfracture cases regardless of whether they represent cortical or trabecular bone.
The prevalence of vertebral deformity in European men and women: The european vertebral osteoporosis study
Authors: O'Neill T. W. , Felsenberg D. , Varlow J. , Cooper C. , Kanis J. A. , Silman A. J. ,   The European Vertebral Osteoporosis Study Group
Published: February 2010

Our aim was to determine the prevalence of radiographically defined vertebral deformity, as a marker of vertebral osteoporosis, in different regions and populations within Europe. We used a cross-sectional population-based survey. Population-based sampling frames were obtained from 36 centers in 19 European countries. Stratified random sampling was used to recruit 15,570 males and females aged 50–79 years. Lateral spinal radiographs were taken according to a standardized protocol, and all X-rays were evaluated centrally. Vertebral deformity was morphometrically defined according to the published methods of McCloskey and Eastell. Based on the McCloskey method, the mean center prevalence of all deformities was 12% in females (range 6–21%) and 12% in males (range 8–20%). The prevalence increased with age in both sexes though the gradient was steeper in females. There was substantial geographical variation, with the highest rates in Scandinavian countries. Radiographically defined vertebral deformity is a common disorder and equally frequent in males and females. Using standardized methodology, there is important variation in occurrence across Europe, which might suggest clues to pathogenesis. (J Bone Miner Res 1996;11:1010–1018)
Analysis of signaling pathways used by parathyroid hormone to activate the c-fos gene in human SaOS2 osteoblast-like cells
Authors: Evans Dean B. , Hipskind Robert A. , Bilbe Graeme
Published: February 2010

We have evaluated the signaling pathways activated by parathyroid hormone (PTH) in SaOS2 human osteoblast-like cells correlating with induction of the c-fos proto-oncogene. Human PTH (1–34) (hPTH[1–34]) and hPTH(1–34) Nle8,18 Tyr34 induced the expression of c-fos mRNA in quiescent SaOS2 cells in a concentration-dependent manner. N-terminal truncations of hPTH(1–34) that fail to activate protein kinase A (PKA) also abolished c-fos mRNA induction. In gel retardation assays hPTH(1–34) led to a change in the mobility of specific, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB)-containing protein-DNA complexes identical to that caused by other activators of PKA. The appearance of this altered mobility complex correlated temporally with the induction of c-fos mRNA. Using a c-fos serum response element probe, a slowed protein DNA complex appeared upon serum, epidermal growth factor, and basic fibroblast growth factor treatment. This slowed complex reflects phosphorylation of the transcription factor ternary complex factor (TCF) mediated via activation of the mitogen-activated protein (MAP) kinase pathway. The MAP kinase cascade is also activated by protein kinase C (PKC), and treatment with phorbol ester led to the induced TCF shift. In contrast, PTH did not produce this shift, ruling out PTH activation of c-fos via PKC and the MAP kinase signaling cascade. Further evidence for this was the lack of effect of the highly selective PKC inhibitor CGP 41251 on c-fos induction by hPTH(1–34). The janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling cascade targets the v-sis-inducible element in the c-fos promoter via the induced binding of STATs. Interferon γ rapidly induced STAT binding in SaOS2 cells, unlike PTH. Thus, PTH induction of c-fos transcription appears to occur principally through activation of PKA that then targets CREB and the c-fos calcium/cAMP response element.
Osteoarthritis in cynomolgus macaques III: Effects of age, gender, and subchondral bone thickness on the severity of disease
Authors: Carlson Cathy S. , Loeser Richard F. , Purser Connie B. , Gardin Jean F. , Jerome Christopher P.
Published: February 2010

Knee joints from cynomolgus monkeys of both genders and a wide range of ages were examined to characterize further the natural history of osteoarthritis (OA) in these animals. The objectives of this study were to characterize better the subchondral bone changes previously noted in this disease, to determine whether the severity of OA in these animals is affected by age or weight, and to determine whether males and females are affected similarly. As had been seen in previous studies, the medial tibial plateau was the most severely affected site. The thickness of the subchondral plate in the medial tibial plateau increased with increasing severity of articular cartilage lesions in both males and females; however, in monkeys with subchondral plate thicknesses less than 400 μm, articular cartilage lesions were essentially absent. Subchondral plate thickness increased with increasing weight in both genders, but females had a higher subchondral plate thickness than males for a given body weight. There was no correlation between bone volume in the proximal tibial epiphysis and articular cartilage lesions of OA. The prevalence and severity of OA in the medial tibial plateau increased with increasing age, but were not affected by gender or weight. Although there was no correlation between articular cartilage lesions and body mass index or weight, the waist/hip circumference ratio and severity of articular cartilage lesions were correlated in the female monkeys. This work provides evidence that thickening of the subchondral bone plate may be more important than the volume of epiphyseal/metaphyseal cancellous bone in determining the biomechanical stresses in the joint and in influencing the development of articular cartilage lesions.
DNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling
Authors: Bronckers A. L. J. J. , Goei W. , Luo G. , Karsenty G. , D'Souza R. N. , Lyaruu D. M. , Burger E. H.
Published: February 2010

To study the fate of bone cells, we used the transferase-mediated, biotin-dUTP nick end-labeling (TUNEL) assay to detect DNA fragmentation during the formation of intramembranous and endochondral bone in newly born hamsters, mice, and rats. In alveolar bone forming around the developing tooth crowns, DNA fragmentation was found in three cell types: TRAP-negative mononuclear cells at the bone surface, osteocytes, and some but not all nuclei of TRAP-positive osteoclasts. Osteoblasts did not undergo DNA fragmentation. A strong positive correlation was found between contacts of TUNEL-positive osteocytes and osteoclasts. Extracellular bone matrix also stained occasionally for the presence of DNA fragments. During endochondral bone formation, TUNEL staining was detected in late hypertrophic chondrocytes of the epiphyseal growth plate. During rapid longitudinal growth of long bones, TUNEL-positive hypertrophic chondrocytes were found coincident with or slightly after invasion of blood vessels from the diaphysis. However, during slow longitudinal growth and in secondary ossification centers, DNA fragmentation was seen in hypertrophic chondrocytes still located within their lacunae. We conclude that some of the osteocytes in deeper layers of bone die within their lacuna and disperse nuclear fragments over the extracellular matrix, that a majority of the osteocytes are phagocytosed and degraded by osteoclasts at sites of intense bone resorption, and that during endochondral ossification, substantial numbers of late hypertrophic chondrocyte cells undergo cell death.
Insulin receptor expression in bone
Authors: Thomas D. M. , Hards D. K. , Rogers S. D. , Ng K. W. , Best J. D.
Published: February 2010

Insulin is known to be an important osteotropic hormone. To date, no study has specifically addressed the possibility that insulin receptor expression may be regulated by differentiation in bone. We report a heterogeneous distribution of insulin receptor (IR) within neonatal rat calvaria using a specific monoclonal antibody to the β-subunit of the rat insulin receptor (CT-1). Specific binding of CT-1 to mature osteoblasts was demonstrated, with little binding over periosteal tissues or osteocytes. Using enzymatically derived subpopulations of calvarial cells, we showed a correlation between alkaline phosphatase activity and insulin-stimulated 2-deoxyglucose (2-DOG) uptake and increased 125I-insulin binding. Since primary calvarial cultures contain many cell types, we compared 125I-insulin binding, insulin-stimulated 2-DOG uptake, and Northern blot analysis of IR mRNA in the clonal preosteoblast-like cell line UMR 201-10B and the mature osteoblast cell line UMR 106-01. It is shown that UMR 106-01 cells possess higher levels of IR mRNA, insulin binding, and insulin-stimulated glucose uptake, and that insulin up-regulated expression of mRNA of the glucose transporter GLUT1 by 3-fold. In contrast, insulin binding was negligible in UMR 201-10B cells, which expressed much lower levels of IR mRNA. UMR 201-10B cells did not possess an insulin-sensitive glucose uptake system, although they express GLUT1 mRNA. These data are consistent with the hypothesis that, as in muscle and fat, insulin receptor expression correlates with the stage of osteoblast differentiation in vivo and in vitro.
Effects of immobilization, three forms of remobilization, and subsequent deconditioning on bone mineral content and density in rat femora
Authors: Kannus Pekka , Järvinen Teppo L. N. , Sievänen Harri , Kvist Martti , Rauhaniemi Jyrki , Maunu Vesa-Matti , Hurme Timo , Jozsa Laszlo , Järvinen Markku
Published: February 2010

Disuse is associated with bone loss, which may not be recoverable. It is not known whether intensified remobilization is beneficial in restoring disuse-related bone loss nor if any such benefit would depend upon continuing mobilization for its maintenance. After an immobilization period of 3 weeks, the effects of free remobilization (11 weeks), and low- and high-intensity treadmill running (11 weeks) with and without subsequent deconditioning (18 weeks) on the bone mineral content (BMC) and density (BMD) of the hindlimb femora of Sprague-Dawley rats (n = 98) were studied using a dual-energy X-ray absorptiometric (DXA) scanner. Our hypothesis was that intensified remobilization is beneficial in restoring the BMC and BMD from disuse to normal while subsequent deconditioning is deleterious to these parameters. Immobilization for 3 weeks produced a significant BMC and BMD loss in the immobilized left femur (range −4.4 to −12.8%; p < 0.05-0.001). In the groups with free remobilization (free cage activity), the body weight-adjusted BMCs and BMDs always remained below those in the controls (range −2.3 to −12.1%; p values ranging from NS to <0.01). Both low- and high-intensity running restored BMC and BMD in the immobilized limb, the effect being better in the latter group. In both of these groups, the values of the immobilized left limbs and those of the free right limbs exclusively exceeded the corresponding values of the age-matched control rats (left limb values 3.0-21.1% higher with p values ranging from NS to <0.01; right limb values 7.9-21.4% higher with p < 0.05-0.01). However, after the deconditioning period of 18 weeks, the above described beneficial effects of low- and high-intensity running were lost, the left and right limb BMC and BMD values being lower than those in the age-matched controls (range −3.8 to −8.7%; p values ranging from NS to <0.05). In conclusion, this study clearly indicates the need for greater than normal activity to restore the BMC and BMD after disuse to normal levels. However, the benefits of intensified remobilization are lost if the activity is terminated, and therefore, after immobilization and disuse, bone loading activities should be continued, perhaps indefinitely.
Protein kinases of cultured osteoblasts: Selectivity for the extracellular matrix proteins of bone and their catalytic competence for osteopontin
Authors: Salih Erdjan , Ashkar Samy , Gerstenfeld Louis C. , Glimcher Melvin J.
Published: February 2010

The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with Mr's of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, Mr's 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.
Colony stimulating factor-1 plays a role in osteoclast formation and function in bone resorption induced by parathyroid hormone and parathyroid hormone-related protein
Authors: Dr. Weir Eleanor C. , Lowik Clemens W.G.M. , Paliwal Indu , Insogna Karl L.
Published: February 2010

Although colony stimulating factor-1 (CSF-1) plays a key role in osteoclast recruitment, studies examining the effect of CSF-1 on mature osteoclasts indicate that it may directly inhibit bone resorption by isolated rat osteoclasts. To define further CSF-1's role in bone remodeling, we examined the effect of neutralizing antisera to CSF-1 on basal and parathyroid hormone (PTH)-induced bone resorption using two organ culture assays designed to examine the recruitment of osteoclast precursors and the activation of mature osteoclasts, respectively. We first assessed whether PTH increases CSF-1 production from bone in organ culture by examining conditioned medium from 19-day-old fetal rat long bones in a mitogenesis assay employing a CSF-1-responsive cell line, CRX-1. Conditioned medium from untreated bones induced a titratable increase in CRX-1 cell proliferation, and treatment of bones with PTH for 72 h caused a significant increase in mitogenic activity. CSF-1 antiserum caused a significant decrease in mitogenic activity in conditioned medium, indicating that bone in organ culture produces CSF-1 constitutively and in response to PTH. To examine bone-derived CSF-1's role in bone resorption, we examined the effect of neutralizing antisera to CSF-1 on basal and PTH-induced bone resorption in the fetal rat long bone assay, which reflects activation of mature osteoclasts. Anti-CSF-1 caused a significant increase in unstimulated and PTH-induced bone resorption compared with control. By contrast, in the fetal mouse metacarpal assay, which examines proliferation and recruitment of osteoclast progenitors and precursors, anti-CSF-1 caused significant inhibition of PTH related protein (PTHrP)-induced bone resorption after 3 and 6 days of incubation. Consistent with these findings, histological examination of cultured 17-day-old fetal metacarpals demonstrated that anti-CSF-1 inhibits the formation of tartrate-resistant acid phosphatase-positive osteoclasts in PTHrP-treated explants, whereas it has no effect on unstimulated bones. We conclude that bone-derived CSF-1 may have a dual role in PTH/PTHrP-induced bone resorption by enhancing the appearance of osteoclast precursors while restraining the resorptive function of mature osteoclasts.
The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H+-ATPase
Authors: David Pe'Er , Nguyen Hieu , Barbier Alain , Baron Roland
Published: February 2010

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 ⩾ 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 μM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPases than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.
Immunolocalization of CD44 and the ERM family in bone cells of mouse tibiae
Authors: Nakamura Hiroaki , Ozawa Hidehiro
Published: February 2010

We studied the immunohistochemical localization of CD44, hyaluronate receptor, and the ezrin-radixin-moesin (ERM) family, actin binding proteins, in bone cells using confocal laser scanning microscopy and transmission electron microscopy to clarify the mechanism of the organization of their cytoskeletons. In osteoclasts, intense immunoreactivity to CD44 could be detected on their basolateral plasma membranes. There was less reactivity observed in the area of the plasma membrane in direct contact with the bone surface. The immunogold electron-microscopical method revealed that CD44 was mainly localized on the microvilli of the basolateral plasma membrane. The plasma membrane of the clear zone and the ruffled border were not immunolabeled with CD44. As for the ERM family, the basolateral plasma membrane of osteoclasts was stained with antimoesin monoclonal antibody, but not with ezrin or radixin. In osteoblasts attached to the bone surface, immunoreactivity to CD44 was restricted to their cytoplasmic processes. They showed immunoreactivities to radixin and moesin on the cytoplasmic side of their plasma membrane when in contact with each other. However, although osteocytes in the bone matrix demonstrate an intense immunolabeling with CD44 on their plasma membrane, they scarcely show immunoreactivity to the ERM family. These findings suggest that: (1) the CD44-moesin-actin filament system is involved in the organization of cytoskeletons in the basolateral plasma membrane of osteoclasts; and (2) other mechanisms, rather than the CD44 and the ERM family, may be involved in the cells of osteoblast lineage. (J Bone Miner Res 1996;11:1715-1722)
Development of mass, density, and estimated mechanical characteristics of bones in caucasian females
Authors: Haapasalo H. , Kannus P. , Sievänen H. , Pasanen M. , Uusi-Rasi K. , Heinonen A. , Oja P. , Vuori I.
Published: February 2010

Three hundred and thirty healthy Finnish girls and premenopausal women, aged 7–47 years, were examined to evaluate the natural development of bone mineral mass and density from early childhood to menopause. Bone mineral content (BMC, g) and areal density (BMD, g/cm2) were measured from the spine (L2–L4), femoral neck, trochanter region of the femur, and distal radius using dual-energy X-ray absorptiometry (DXA). In addition, the bone mineral apparent density (BMAD, g/cm3) was assessed from the above described skeletal sites, and the mechanical competence of the femoral neck was estimated. Special attention was paid to the timing of the peak values of these bone parameters as well as to the evidence of premenopausal bone loss. The BMC, BMD, and BMAD of the spine, femoral neck, and trochanter region of the femur achieved peak values around the age of 20, and the bone loss seemed to start soon thereafter. In contrast, the bone mass of the distal radius slightly increased between the ages of 20 and 47. In the femoral neck, the estimated bending strength achieved its peak value around the age of 20 and showed a slight decrease during the following decades. The highest body weight and neck-length adjusted strength values of the femoral neck were, however, found in early childhood, with the values decreasing linearly thereafter. In conclusion, this study supports previous findings of rapid bone mineral accumulation in late adolescence, and occurrence of the peak bone mass and density around the age of 20. Premenopausal bone loss seems to occur in the proximal femur and lumbar spine. Our observations of femur strength development imply that from childhood to menopause the mechanical strength of the femoral neck is well adjusted to the biomechanical loading requirements of the body. (J Bone Miner Res 1996;11:1751–1760)
Altered mineral metabolism and bone mass in children during treatment for acute lymphoblastic leukemia
Authors: Halton Jacqueline M. , Dr. Atkinson Stephanie A. , Fraher Laurence , Webber Colin , Gill Gerald J. , Dawson Susan , Barr Ronald D.
Published: February 2010

Children with acute lymphoblastic leukemia (ALL) often develop bone pain, abnormal gait, and unusual fractures while in remission and receiving continuing chemotherapy. A prospective longitudinal cohort study was undertaken of bone mass and biochemical mineral status in 40 consecutive children (27 male, 13 female, aged 0.3–17.0 years) receiving therapy on the Dana-Farber Cancer Institute protocol 87–01. Radiography, lumbar spine dual-photon absorptiometry, and biochemical measurements of mineral status were performed at diagnosis and at 6-month intervals throughout 24 months of chemotherapy. Eleven patients were not completely evaluated (4 deaths and 7 off study). Radiographic evidence of osteopenia was observed in 10, 64, and 76% at diagnosis, 12 and 24 months, respectively. Fractures occurred in 39% of children during treatment. Reduction in bone mineral content (BMC), as measured by Z scores, occurred in 64% of patients and was most severe in those greater than 11 years of age at diagnosis. Reduction in BMC during the first 6 months of therapy had a positive predictive value of 64%, while an increase in BMC had a negative predictive value of 82% for subsequent fracture. By 6 months of therapy, 31/37 (84%) children were hypomagnesemic, of whom 16 (52%) were hypermagnesuric. Plasma osteocalcin was subnormal at diagnosis in 29/40 (73%) but increased to normal by 6 months of treatment. Vitamin D status was normal throughout, but plasma 1,25-dihydroxyvitamin D remained subnormal in greater than 70% of children. Urinary cross-link N-telopeptide was normal at diagnosis and became elevated in 58% of children by the end of therapy. Suppressed bone mineralization is evident at diagnosis in a minority of children with ALL. Skeletal morbidity and a reduction in bone mineral mass become more prevalent during treatment, with increased bone resorption, perhaps mainly as a consequence of corticosteroid administration. (J Bone Miner Res 1996;11:1774–1783)
Bone density in type 1 gaucher disease
Authors: Pastores Gregory M. , Wallenstein Sylvan , Desnick Robert J. , Luckey Marjorie M.
Published: February 2010

Skeletal involvement is a major source of complications in patients with Type 1 Gaucher disease. To investigate the bone density and potential usefulness of bone densitometry in Gaucher disease, dual-energy X-ray absorptiometry was used to measure the density of the lumbar spine, femoral neck, trochanter, and distal radius in 61 adult patients ranging in age from 22 to 77 years. The mean bone density at each site was significantly lower than expected for age and sex. The severity of the osteopenia correlated significantly with other clinical indicators of disease severity, including the N370S/84GG genotype, prior splenectomy, and hepatomegaly. The bone density measurements also correlated significantly with the severity of skeletal disease as assessed by skeletal radiography. Vertebral density remained an independent predictor of the severity of bone involvement even after controlling for age, sex, weight, genotype, splenectomy, and hepatomegaly. These findings suggest that bone density measurements provide a quantitative assessment of bone involvement in Type 1 Gaucher disease, which may permit serial, noninvasive monitoring of bone changes in this progressive disorder. (J Bone Miner Res 1996;11:1801–1807)
Distinguishing affective and somatic dimensions of pain and depression: a confirmatory factor analytic study
Authors: Lerman Sheera F. , Rudich Zvia , Shahar Golan
Published: February 2010

In this study, we examined the overlap between pain and depression in a sample of 342 chronic pain patients treated at a specialty pain clinic. Confirmatory factor analysis was conducted to test the differentiation of pain and depression measured as latent factors derived from the subscales of the Short Form McGill Pain Questionnaire and the Center for Epidemiological Studies Depression Scale. The affective pain subscale did not load on latent depression and the somatic depression subscale loaded weakly on latent pain. Although pain and depression are linked, we found that affective pain is distinct from depression, and that somatic depression is distinct from pain. This finding justifies further examination of the casual relationship between pain and depression. © 2010 Wiley Periodicals, Inc. J Clin Psychol 66: 1–10, 2010.
Hybrid modeling of inulinase bio‐production process
Authors: Mazutti Marcio A. , Corazza Marcos L. , Maugeri Francisco , Rodrigues Maria I. , Oliveira J. Vladimir , Treichel Helen , Corazza Fernanda C.
Published: February 2010

BACKGROUND: A potential application of inulinase in the food industry is the production of fructooligosaccharides (FOS) through transfructosilation of sucrose. Besides their ability to increase the shelf‐life and flavor of many products, FOS have many interesting functional properties. The use of an industrial medium may represent a good, cost‐effective alternative to produce inulinase, since the activity of the enzyme produced may be improved or at least remain the same compared with that obtained using a synthetic medium. Thus, inulinase production for use in FOS synthesis is of considerable scientific and technological appeal, as is the development of a reliable mathematical model of the process. This paper describes a hybrid neural network approach to model inulinase production in a batch bioreactor using agroindustrial residues as substrate. The hybrid modeling makes use of a series artificial neural network to estimate the kinetic parameters of the process and the mass balance as constitutive equations.

RESULTS: The proposed model was shown to be capable of describing the complex behavior of inulinase production employing agroindustrial residues as substrate, so that the mathematical framework developed is a useful tool for simulation of this process.

CONCLUSION: The hybrid neural network model developed was shown to be an interesting alternative to estimate model parameters since complete elucidation of the phenomena and mechanisms involved in the fermentation is not required owing to the black‐box nature of the ANN used as parameter estimator. Copyright © 2010 Society of Chemical Industry
Tropical hornbills (Aceros cassidix, Aceros undulatus, and Buceros hydrocorax) use ballistic transport to feed with their large beaks
Authors: Baussart Sabine , Bels Vincent
Published: February 2010

The most common and plesiomorphic mechanism of food transport in tetrapods is lingual‐based. Neognathous birds use this mechanism for exploiting a large diversity of food resources, whereas paleognathous birds use cranioinertial mechanism with or without tongue involvement. Food transport in three hornbills' species (Aceros cassidix, A. undulatus, and Buceros hydrocorax) is defined by a ballistic transport mechanism. Only one transport cycle is used for moving the food from the tip of the beak to the pharynx. The tongue never makes contact with the food nor is it used to expand the buccal cavity. In hornbills, filmed through high‐speed video, time to food release occurred between 0.11 and 0.16 sec before time to maximum gape. The ballistic curves show similar patterns. Maximum gape angle is significantly different between the three species. Each species show a different kinematic and motor pattern of head movements associated with ballistic transport. In A. undulatus, head rotation follows a continuous pattern similar to that reported earlier in toucans. A. cassidix rotates head downward at the time of maximum gape to permit food to reach the pharynx without touching the mandible. B. hydrocorax elevates the head along the transport cycle to avoid contact with the food to the cavity of the upper beak. Selection of large food items in the diet may explain the evolutionary trend of using ballistic transport in the feeding behavior of hornbills, which play a key role in tropical forest ecology by dispersing seeds. J. Exp. Zool. 315:72–83, 2011. © 2010 Wiley‐Liss, Inc.
Administration of nonviral gene vector encoding rat β‐defensin‐2 ameliorates chronicPseudomonas aeruginosalung infection in rats
Authors: Hu Qiongjie , Zuo Peng , Shao Bing , Yang Shuo , Xu Guopeng , Lan Fen , Lu Xiaoxia , Xiong Weining , Xu Yongjian , Xiong Shengdao
Published: February 2010

Background

Beta‐defensin‐2 (BD‐2) plays an important role in host defense against pathogenic microbe challenge by its direct antimicrobial activity and immunomodulatory functions. The present study aimed to determine whether genetic up‐regulation of rat BD‐2 (rBD‐2) could ameliorate chronicPseudomonas aeruginosalung infection in rats.

Methods

Plasmid‐encoding rBD‐2 was delivered to lungsin vivousing linear polyethylenimine at 48 h before challenging with seaweed alginate beads containingP. aeruginosa. Macroscopic and histopathological changes of the lungs, bacterial loads, inflammatory infiltration, and the levels of cytokines/chemokines [interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, kertinocyte‐derived chemokine (KC), macrophage inflammatory protein‐2 (MIP‐2)] were measured at 3 and 7 days post‐infection (p.i.).

Results

The overexpression of rBD‐2 resulted in a significant increase in animal survival rate (at 3 days p.i.), a significant decrease in bacterial loads in the lungs (at 3 and 7 days p.i.), and significantly milder lung pathology. In addition, the overexpression of rBD‐2 led to increased infiltration of polymorphonuclear neutrophils (PMN), and elevated protein expression of cytokines/chemokines (IL‐1β, TNF‐α, KC and MIP‐2) at the early stage of infection (at 3 days p.i.), at the same time as being dramatically decreased at the later stage of infection (at 7 days p.i.).

Conclusions

Genetic up‐regulation of rBD‐2 increased animal survival rate, and reduced bacterial loads in lungs after bacterial infection. The overexpression of rBD‐2 also modulated the production of several cytokines/chemokines and increased PMN recruitment at the early stage of infection. Our findings indicate that the enhancement of BD‐2 may be an efficacious intervention for chronicP. aeruginosalung infection. Copyright © 2010 John Wiley & Sons, Ltd.
Failure of xenoimplantation using porcine synovium‐derived stem cell‐based cartilage tissue constructs for the repair of rabbit osteochondral defects
Authors: Pei Ming , Yan Zuoqin , Shoukry Mark , Boyce Brandon M.
Published: February 2010

The use of xenogeneic tissues offers many advantages with respect to availability, quality control, and timing of tissue harvest. Our previous study indicated that implantation of premature tissue constructs from allogeneic synovium‐derived stem cells (SDSCs) facilitated cartilage tissue regeneration. The present study investigated the feasibility of xenoimplantation of SDSC‐based premature tissue constructs for the repair of osteochondral defects. Porcine SDSCs were mixed with fibrin gel, seeded in polyglycolic acid (PGA) scaffolds, and cultured in a rotating bioreactor system supplemented for 1 month with growth factor cocktails. The engineered porcine premature tissues were implanted to repair surgically induced osteochondral defects in the medial femoral condyles of 12 rabbits. Three weeks after surgery, the xenoimplantation group exhibited a smooth, whitish surface while the untreated control remained empty. Surprisingly, 6 months after surgery, the xenoimplantation group displayed some tissue loss while the untreated control group was overgrown with fibrocartilage tissue. In the xenoimplantation group, chronic inflammation was observed in synovial tissue where porcine major histocompatibility complex (MHC) class II antigen positively stained in the engulfed foreign bodies. In addition, porcine source cells also migrated from the implantation site and may have been responsible for the observed loss of glycosaminoglycans (GAGs) underneath surrounding articular cartilage. The histological score was much worse in the xenoimplanted group than in the untreated control. Our study suggested that SDSC‐based xenogeneic tissue constructs might cause delayed immune rejection. Xenotransplantation may not be an appropriate approach to repair osteochondral defects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1064–1070, 2010
Cadmium concentration in durum wheat grain (Triticum turgidum) as influenced by nitrogen rate, seeding date and soil type
Authors: Perilli Patrizia , Mitchell Les G , Grant Cynthia A , Pisante Michele
Published: February 2010

BACKGROUND: Cadmium (Cd) is a trace element that has been associated with various human health problems. Cd enters plants, either by direct absorption through leaves or by uptake from soils, allowing Cd into the food chain. Nitrogen (N) fertilizer management is important in optimizing crop yield and protein content of durum wheat, but may influence Cd availability and hence Cd concentration in crops, with the effects being strongly influenced by environmental conditions and crop cultivar.

RESULTS: In field studies, Cd and protein concentration in durum wheat grain differed between cultivars and were strongly affected by N application, with only minor effects of N occurring on concentration and uptake of P and Zn. Protein content increased significantly with N application in five of six site‐years, with the response being generally independent of cultivar and seeding data. Cd concentration also increased with N application in five of six seeding dates, with the response being greater in AC Melita than Arcola in three of the six site‐years. There were large differences in Cd concentration from year to year and with seeding date, indicating a strong environmental influence.

CONCLUSIONS: This study shows that different cultivars accumulate different levels of Cd in the grain and that seeding date and nitrogen fertilizer management can influence grain Cd concentration, with the magnitude of effects varying with environmental factors. In the future we may be able to manipulate management practices to optimize protein concentration and minimize Cd concentration in durum wheat, which could help to address the health and safety concerns of consumers. © Society of Chemical Industry and Her Majesty the Queen in right of Canada