bluuebunny/crossref_metadata_2025 huggingface dataset:
Ability of four different techniques of measuring bone mass to diagnose vertebral fractures in postmenopausal women
Authors: Ott Susan M. , Kilcoyne R.F. , Chesnut Charles H.
Published: February 2010

Since bone mass has been shown to be an important determining factor of fractures in vitro, we undertook a study to evaluate whether bone mass measurements could separate postmenopausal women with vertebral compression fractures from women of a similar age without fractures. We also wanted to see if methods of measuring bone mass at the spine would be more sensitive or specific than methods that measured bone at the wrist or the entire skeleton. The techniques used were: total body calcium by neutron activation analysis (TBC), single photon absorptiometry (SPA), dual photon absorptiometry (DPA), and quantitative computed tomography (QCT). Normal women aged 20–85 were measured, but only those >50 yr were used in the analysis. Mean values for women with fractures were significantly lower than normals (p < .001): TBC 642 ± 103 g vs. 764 ± 114; SPA .658 ± .134 g/cm vs. .779 ± .142; DPA 3.75 ± .82 g/cm vs. 4.37 ± .86; QCT 59.0 ± 25.7 mg/cc vs. 92.6 ± 36.0. However, each of the methods showed considerable overlap between women with and without fractures. At 90% specificity the sensitivities of the tests were: TBC 34%; SPA 29%; DPA 33%; QCT 36%. When values were expressed as the % expected (based on age and height) then the sensitivities were: TBC 52%; SPA 36%; DPA 35%; QCT 44%. Using Bayes' theorem, we constructed curves showing the posttest probability of these tests at a prevalence of 20%. None of these bone mass measuring techniques showed complete separation between normal and osteoporotic women with fractures; about one-half of the women with fractures were below the normal range. The risk of having a fracture increases as bone mass declines, but our data suggest that bone mass is not the only factor leading to vertebral fractures in postmenopausal women.
Parathyroid hormone stimulation of mitosis in rat thymic lymphocytes is independent of cyclic AMP
Authors: Atkinson M.J. , Hesch R.-D. , Cade C. , Wadwah M. , Perris A.D.
Published: February 2010

The in vitro mitotic response of rat thymic lymphocytes to hPTH(1–34), hPTH (1–38), and 8,18 NIe hPTH(1–34) exhibits a dependency upon extracellular calcium. Removal of extracellular calcium or the addition of Verapamil (5 μg/ml) or trifluoroperazine (10 μM) abrogated the mitotic response. Mitogenic concentrations of 8,18 NIe hPTH(1–34) increased calcium 45 uptake from 4.49 ± 0.25 to 8.23 ± 0.75 pMol/106 cells/min. The intracellular calcium concentration, measured by Quin 2 fluorescence, also increased after addition of 8,18 NIe hPTH(1–34).

Parathyroid hormone-induced activation could not be demonstrated in an otherwise responsive thymocyte membrane adenylate cyclase. In intact cells mitogenic levels of 8,18 NIe hPTH(1–34) decreased intracellular cyclic AMP content. This response was blocked by both 3-isobutyl 1-methyl xanthine and trifluoroperazine, and may indicate activation of calcium-dependent phosphodiesterase.

We conclude that PTH stimulates thymic lymphocyte proliferation independently of cyclic AMP, and that changes in cellular calcium homeostasis are intimately involved in the action of PTH. In all of the assays employed, the hitherto antagonistic analogue 8,18 NIe 34 Tyr bPTH(3–34)amide proved to be an agonist. We postulate that the receptor utilized for this PTH action may not exhibit classical PTH structure-activity specificities.
Effects of extracellular calcium and magnesium on cytosolic calcium concentration in fura-2-loaded bovine parathyroid cells
Authors: Chen Chu J. , Anast Constantine S. , Posillico James T. , Brown Edward M.
Published: February 2010

A newly developed calcium-sensitive dye, Fura-2, was employed in dispersed bovine parathyroid cells to study the effects of extracellular calcium and magnesium on cytosolic calcium concentration and parathyroid hormone (PTH) release. In comparison with control cells, Fura-2-loaded parathyroid cells showed the same maximal rate of PTH release, set-point for extracellular Ca++ (the calcium concentration producing half of the maximal inhibition of PTH release), and maximal inhibition of PTH release (71.6%) by high extracellular Ca++. At an extracellular Mg++ concentration of 0.5 mM, raising extracellular Ca++ in a stepwise fashion from 0.5 mM to 2.0 mM produced a dose-dependent, statistically significant (p < 0.01) increase in cytosolic Ca++ from 198 ± 24 nM (0.5 mM Ca++) to 411 ± 21 nM (2.0 mM Ca++) which closely paralleled the concomitant decrease in PTH release. An elevation of extracellular Mg++ from 0.5 mM to 5 mM, at an extracellular Ca++ of 0.5 mM, resulted in a transient spike of cytosolic Ca++ which lasted for approximately 30 seconds, followed by a small but stable increase in the cytosolic Ca++ concentration (174 ± 7 nM vs. 237 ± 10 nM, n = 4, p < 0.01). Prior removal of extracellular calcium by addition of an excess of EGTA did not abolish the transient spike induced by high extracellular magnesium concentrations in Fura-2-loaded cells, suggesting that this rapid increase in cytosolic Ca++ arises, at least in part, from intracellular stores of Ca++. This is supported by the observation that pretreating cells with ionomycin resulted in disappearance of the magnesium-induced spike. In parallel experiments, the values for cytosolic calcium concentration at high and low extracellular calcium and magnesium concentrations in cells loaded with Fura-2 were comparable to those in cells loaded with Quin-2. These results show that Fura-2 may be employed to measure the cytosolic Ca++ concentration in dispersed bovine parathyroid cells and that extracellular Ca++ and Mg++ produced sustained increases in cytosolic Ca++ in cells loaded with this dye which are comparable to those seen with Quin-2-loaded cells. In addition, however, probably because of the lower loading concentrations possible with Fura-2, cells loaded with this dye show Mg++-induced spikes in intracellular Ca++, resulting, in part, from release of intracellular Ca++. These Ca++ transients support other data suggesting the possibility of an extracellular divalent cation receptor on parathyroid cells, which may produce some of its effects on parathyroid function by mobilizing intracellular Ca++.
Age-related rise in parathyroid hormone in man: The use of intact and midmoleeule antisera to distinguish hormone secretion from retention
Authors: Young Gloria , Marcus Robert , Minkoff Jerome R. , Kim Lance Y. , Segre Gino V.
Published: February 2010

Circulating levels of parathyroid hormone (PTH) rise with age in normal men and women. To resolve the basis for this observation we measured iPTH in 137 normal men and women, age 23 to 85 years, using two antisera which responded to different portions of the PTH molecule. A midmoleeule assay (Mid-PTH) employed antiserum NG-5, which recognizes mid- and carboxy-terminal portions of hPTH, whereas antiserum CK-67, which recognizes determinants in the 1–34 hPTH sequence, was used to measure intact PTH (NH2-PTH). Two-hour fasting urine was collected for measurement of creatinine clearance and excretion indices of phosphorus and cyclic AMP. Serum was analyzed for 25-hydroxyvitamin D (25-OHD) in addition to iPTH. Mid-PTH rose significantly with age in the 72 women (r = 0.38, p < 0.001) and in the 65 men (r = 0.40, p < 0.001). NH2-PTH rose with age in women (r = 0.23, p < 0.05), but a change in men was not significant (r = 0.19, n.s.). Cyclic AMP excretion rose significantly with age in both women (r = 0.42) and men (r = 0.41), whereas phosphorus excretion rose significantly in women only (r = 0.32, p < 0.01). 25-OHD levels were 27.5 ± 1.3 ng/ml for women and 26.1 ± 1.2 ng/ml for men. No change in 25-OHD was observed with age in women, and a significant decrease in men was due entirely to extremely high values in three young subjects. Endogenous creatinine clearance decreased with age in women (r = -0.65, p < 0.001) and men (r = -0.46, p < 0.001). In women, creatinine clearance correlated significantly with Mid-PTH (r = 0.21, p < 0.05), but the relationship of Mid-PTH to age remained significant when the effect of renal function was eliminated in the multiple regression. Creatinine clearance did not correlate significantly with NH2-PTH. Fourteen women with the highest values of NH2-PTH were compared with 12 women with the lowest NH2-PTH levels for multiple indices of PTH action and bone turnover, including urinary excretion indices for calcium, phosphorus, cyclic AMP, and hydroxyproline, and serum alkaline phosphatase activity and 25-OHD. In no case were significant differences observed between the two groups of elderly women. We conclude that increased iPTH with age in women reflects an increase in steady-state levels of intact hormone. Although a similar phenomenon probably occurs in men, results from men in this study did not reach significance. Although it is considered likely that elevated PTH levels may contribute to age-related bone loss, we found no correspondence of iPTH to several measures of PTH action and bone turnover. These results point out the need for a prospective evaluation of the relationship of iPTH to bone loss in elderly men and women.
Modifications of the mouse calvarial technique improve the responsiveness to stimulators of bone resorption
Authors: Lerner Ulf H.
Published: February 2010

The effect of a preincubation period, in basic medium or in medium with inhibitors of prostaglandin biosynthesis, on the response to different stimulators of bone resorption has been studied in an organ culture system using calvarial bones from neonatal mice. Bone resorption was assessed either by the release of 45Ca or by the release of 3H from [3H]-proline labeled bones. Preincubated bones were cultured for 18–24 hr in medium, with and without indomethacin, hydrocortisone, and dexamethasone, and then extensively washed before being transferred to culture medium containing different stimulators of bone resorption. Preincubation in medium containing indomethacin or corticosteroids resulted in an increased response to parathyroid hormone (PTH), prostaglandin E2 (PGE2), 1-alpha-hydroxyvitamin-D3 and thrombin as compared to the response in bones which were exposed to the stimulants directly after dissection. Preincubation in basic medium did not enhance the subsequent response to PTH. By using a preincubation period in indomethacin, the dose-response curves for the stimulatory effect of PTH and PGE2 on mineral mobilization could be sensitized as compared to the curves obtained with fresh bones. Thus, the concentration of agonists causing 50% stimulation of 45Ca release was decreased by a factor of 10. The threshold for actions of PTH and PGE2 on 45Ca release was 0.01–0.03 and 1–3 nmol/l, respectively.
Biphasic effects of epidermal growth factor on bone nodule formation by isolated rat calvaria cells in vitro
Authors: Dr. Antosz Mark E. , Bellows C.G. , Aubin J.E.
Published: February 2010

Osteoblast-like cells isolated from fetal rat calvaria (RC) form three-dimensional nodules in vitro having the morphological and immunohistochemical characteristics of bone. When administered continually over 21 days of culture, EGF caused a dose-related inhibition of nodule formation, as well as a reduction in the proportion of mineralized nodules, at concentrations between 10−12 to 10−7M. The same conditions caused an increase in both cell proliferation and saturation density in the cultures, suggesting that decreased nodule formation was not the result of general cell toxicity. Inhibitory concentrations of EGF caused a reversible alteration in cell shape, although changes in protein synthesis were not detectable. Indomethacin (5 × 10−7M) did not affect either nodule formation or its inhibition by EGF. When cultures were pretreated for various time periods with 10−8M EGF followed by its removal and continued culture in supplemented medium for 21 days, the number of bone nodules formed with brief exposures to EGF (4 hr up to 48 hr) was increased compared to the numbers formed in supplemented medium alone, whereas exposures to EGF of 4 days or longer decreased the number of nodules formed. These data indicate that EGF can either increase or decrease the osteogenic potential of RC cells in vitro depending on the duration of exposure to the factor.
The influence of sex steroid hormones on plasma calcitonin response to the calcium clamp in normal subjects
Authors: Tørring O. , Bucht E. , Sjöberg H.E.
Published: February 2010

The influence of sex steroid hormones on plasma calcitonin levels in healthy subjects was studied in 15 males and 10 premenopausal (PREMF) and 12 postmenopausal females (PMF). A standardized ionized calcium stimulus was achieved by means of the calcium clamp technique, and a sensitive RIA was used to determine immunoreactive CT (iCT) in plasma. Plasma iCT levels increased in response to the calcium clamp with an initial peak at 15 or 30 min. The iCT levels then declined but remained at an elevated level for the rest of the 180-min infusion period in all three groups. In males a positive correlation was found between the serum testosterone levels at 0 min and the estimates for initial iCT response, i.e., the change between 0 and 15 min (r = 0.80, n = 11, p < 0.01), between 0 and 30 min (r = 0.56, n = 15, p < 0.05) and between 0 and the maximum value (r = 0.68, n = 15, p < 0.01). In the two female groups no such correlation was found. Serum 17 β-estradiol or dehydroepiandrosterone sulfate concentrations were not correlated either to basal iCT levels or to the iCT response to the calcium clamp. PMF had lower levels of 17 β-estradiol than PREMF (p < 0.001), while the testosterone levels were similar. The iCT levels and response to the calcium clamp showed no significant difference in the two groups. The results indicate that sex difference in the plasma CT levels may be related to different testosterone levels. In addition, physiological levels of serum 17 β-estradiol seem to bear no direct relation to basal or Ca-stimulated iCT levels in pre- and postmenopausal females.
Biological activity of 19-nor, 10-keto, 25-hydroxyvitamin D3
Authors: Gray T.K. , Kleckner N.W. , Malloy P.J. , Feldman D. , Sivam G. , Dodd R.C. , Cohen M.S.
Published: February 2010

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10–25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10–25OHD3 induced differentiation of U937 cells. In these studies, 19/10–25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10–25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10–25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10–25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10–25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10–25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10–25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10–25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1: 100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently unexplained.
A simple spectrophotometric assay for micromolar amounts of lanthanum in the presence of calcium and phosphate
Authors: Fernandez-Gavarron Federico , Brand Joseph G. , Rabinowitz Joseph L.
Published: February 2010

A sensitive spectrophotometric assay for micromolar amounts of lanthanum in the presence of calcium and phosphate (as hydroxyapatite) was developed utilizing the change in absorption (at 652 nm) when the dye arsenazo III was complexed with lanthanum. Arsenazo III was used at a level of 25 μM and the solution pH was maintained at 3.1 with 0.2 M sodium acetate. Lanthanum concentrations down to 0.5 μM could be reliably assayed. Calcium ion did not complex well with arsenazo III at pH 3.1. With calcium present at 100 μM and lanthanum at 10 μM, the assay was 115 times more sensitive for lanthanum. The assay is simple, rapid, reproducible and, unlike the assay using radioactive lanthanum, can be performed at any time.
Biological activity of chicken calcitonin: Effects on neonatal rat and embryonic chick osteoclasts
Authors: Dempster D.W. , Murrills R.J. , Horbert W.R. , Arnett T.R.
Published: February 2010

Chicken calcitonin (cCT) has recently been synthesized according to nucleotide sequence data. We have investigated the in vitro effects of this hormone on the activity of disaggregated, neonatal rat and embryonic chick osteoclasts. While synthetic cCT inhibited bone resorption by neonatal rat osteoclasts at concentrations as low as 0.1 pg/ml, it failed to produce a significant reduction in bone resorption by embryonic chick osteoclasts, even at concentrations as high as 1 μg/ml. Similarly, cCT at 1 pg/ml reproducibly produced the rapid and dramatic inhibition of rat osteoclast motility characteristic of other calcitonins, as judged by time-lapse video recording, but did not impair the motile behavior of chick osteoclasts at concentrations up to a million-fold higher. Previous studies showing that embryonic chick osteoclasts were unresponsive to synthetic salmon calcitonin left open the possibility that the native hormone was required to produce an inhibitory effect. We conclude that the osteoclast is not a target cell for calcitonin in the embryonic chick; further studies will be required to define a role, if any, for this potent but enigmatic hormone in birds.
Bone histomorphometry of renal osteodystrophy in diabetic patients
Authors: Andress Dennis L. , Hercz Gavril , Kopp Jeffrey B. , Endres David B. , Norris Keith C. , Coburn Jack W. , Sherrard Donald J.
Published: February 2010

Bone biopsies and plasma parathyroid hormone (PTH) from 27 diabetic dialysis patients were compared to biopsies and PTH levels from matched patients without diabetes to determine if PTH has a role in preserving bone mass in diabetic renal osteodystrophy. Significantly lower values were present in the diabetic group for mineralized bone area (p < 0.003), osteoblastic osteoid (p < 0.01), resorptive surface (p < 0.001), fibrosis (p < 0.005), bone apposition rate (p < 0.01), bone formation rate (BMU level) (p < 0.04), and plasma PTH (p < 0.05). Bone-surface aluminum was higher in the diabetic group (44 ± 5% vs. 20 ± 5%, p < 0.005). Linear regression analysis revealed significant positive correlations of mineralized bone area with time on dialysis, bone formation rate, bone resorption, and PTH only in the group without diabetes. While both groups had significant positive correlations of PTH with osteoblastic osteoid and bone resorption, only in the nondiabetic group was there a positive correlation of PTH with bone apposition and bone formation rate (BMU level), observations suggesting that the lower bone formation in the diabetic patients may have arisen in part from a failure of PTH to promote bone mineralization. We conclude that relatively low PTH levels and high bone aluminum in diabetic patients with chronic renal failure may be responsible in part for low bone mass when compared to uremic patients without diabetes.
Effects of 1α,25- and 24R,25-dihydroxyvitamin D3 on aluminum-induced rickets in growing uremic rats
Authors: Vukičević Slobodan , Krempien Burkhard , Stavljenić Ana
Published: February 2010

Rats were subjected to a two-stage subtotal nephrectomy or sham operation, and treated with aluminum (Al) or both aluminum and vitamin D, metabolites for 5 weeks with a cumulative dose of 13.6 mg aluminum.

Animals were injected with 3H-thymidine and 3H-proline. The following analyses were performed: quantitative histology of tibial metaphyses and cytomorphometric electron microscopy of osteoclasts, quantitative (ICP-spectroscopy) and qualitative determination (histochemical staining) of aluminum within organs, and serum biochemistry (Ca, P, Mg, vitamin D3 metabolites, alkaline phosphatase, urea).

The following new facts of the aluminum-related bone disease became evident: (a) Application of aluminum to growing uremic rats induced rickets, whose major epiphyseal growth plate changes were 1α,25(OH)2D3-dependent. Addition of 1α,25(OH)2D3 prevented the formation of rachitic metaphysis, but failed to prevent osteoid accumulation on epiphyseal and metaphyseal trabecular surfaces. Moreover, calcitriol produced hyperosteoidosis and osteosclerosis in the same rats. Aluminum did not alter the function of osteoblasts, while osteoclasts seemed inactivated, (b) The development of rickets was associated with suppressed serum levels of 1,25(OH)2D3, reduced phosphorus level and the high content of aluminum in the bone, kidney, and liver. The addition of 24R,25(OH)2D3 markedly exaggerated the reduction of serum levels of calcitriol.

We suggested that aluminum induces rickets in growing uremic rats, which consists of two components: vitamin D refractory osteomalacia and 1α,25(OH)2D3-dependent epiphyseal growth plate changes.
An ultrastructural study on the multinucleation process of mouse alveolar macrophages induced by 1α,25-dihydroxyvitamin D3
Authors: Ejir Sadakazu , Segawa Akihisa , Miyaura Chisato , Abe Etsuko , Suda Tatsuo , Ozawa Hidehiro
Published: February 2010

The multinucleation process of isolated alveolar macrophages induced by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] was examined using a scanning electron microscope (SEM) and a transmission electron microscope (TEM).

At the beginning of culture, most of the macrophages were spherical in shape. During incubation with 1.2 × 10−8M 1α,25(OH)2D3, spreading macrophages appeared among the spherical macrophages, and they increased in number. Spreading macrophages extended many cytoplasmic processes toward adjacent macrophages, and interdigitations of these processes between those of neighboring cells were often seen. Two types of cell contact have been observed in the 1α,25(OH)2D3-treated cells. In some, cytoplasmic processes were put into the cytoplasm of the adjacent cells, where clathrinlike structures were observed at the inner membrane of the concave portion. In others, spreading macrophages occasionally came in contact with adjacent cells by a peripheral rim of their cytoplasm with gap junctions. Cytoplasmic continuity was rarely observed at the boundaries between the closely associated cells. The two types of cell contact were also found, though not frequently, in the untreated cells.

These results indicate that 1α,25(OH)2D3 promotes multinucleation of alveolar macrophages through spreading forms with the formation of gap junctions and the coated membrane invagination.
Actions of rat growth hormone-releasing factor and norepinephrine on cytosolic free calcium and inositol trisphosphate in rat C-cells
Authors: Dr. Fried Ronald M. , Tashjian Armen H.
Published: February 2010

Rat growth hormone-releasing factor (rGRF) and norepinephrine (NE) stimulate secretion of calcitonin (CT) and neurotensin (NT) from cultured C-cells. The mechanism by which these agents cause secretion has not been well studied. We have examined the actions of the CT and NT secretagogues rGRF and NE on cytosolic free calcium concentrations ([Ca2+]i) in the rat C-cell line rMTC 44-2. Because inositol trisphosphate (IP3) has been shown to cause release of intracellular calcium stores in several cell types, we have also examined the effects of rat GRF, NE, and increases in extracellular calcium on IP3 accumulation in rMTC 44-2 cells. Stimulation by 10−6M rGRF caused a biphasic response in [Ca2+]i consisting of a rapid spike to 136 ± 4% (mean ± SE) of basal [Ca2+]i. This increase in [Ca2+]i decayed to base line and then gradually increased to 173 ± 13% of basal [Ca2+]i. Stimulation by 10−6M NE gave a similar biphasic increase in [Ca2+]i. The increases in [Ca2+]i induced by both rGRF and NE were inhibited by pretreatment with EGTA or verapamil. rGRF, NE, and increasing concentrations of extracellular calcium, which all caused rapid increases in [Ca2+]i, failed to increase IP3 accumulation in rMTC 44-2 cells. These results suggest that rGRF- and NE-induced secretion in C-cells are mediated by changes in [Ca2+]i. These increases in [Ca2+]i appear to be generated by extracellular calcium influx rather than by release of intracellular calcium stores.
Detection of prefracture spinal osteoporosis using bone mineral absorptiometry
Authors: Ross Philip D. , Wasnich Richard D. , Vogel John M.
Published: February 2010

Bone mineral measurements have been criticized for their inability to clearly distinguish fracture and “non-fracture” populations. However, this failure is not unexpected, since some individuals in the “nonfracture” group have low bone mass and are at increased risk but have not yet experienced fractures. Although standard radiographs are not sensitive indicators of vertebral demineralization, they do identify some of the “prefracture” osteoporotic subpopulation within the nonfracture group. Prospective follow-up of 536 Japanese-American women demonstrated that 14 new spine fractures occurred in this prefracture osteoporosis group, whereas none occurred in the nonosteoporotic group (p ≤ 0.03). However, bone mineral content (BMC) measurements using photon absorptiometry were much more accurate than radiographs as indicators of spine fracture risk. BMC values were somewhat higher in the prefracture group than in those with existing fractures, but values for both groups were significantly lower than in nonosteoporotic patients even after adjusting for age, height, and weight (p < 0.0001). The magnitude of the difference was proportional to the trabecular bone content of the measurement site; the differences were greatest for the os calcis and lumbar spine, smaller for the distal radius, and least for the proximal radius. The prevalence of spinal osteoporosis (including both fracture and prefracture cases) was inversely proportional to BMC (p < 0.0001). Again, the relations were strongest for the os calcis and lumbar spine. These results indicate that BMC measurements are valid indicators of osteoporosis status, particularly when osteoporosis is defined to include both patients with existing fractures and those at increased risk for fractures. However, dual-photon spine BMC was adversely influenced by the presence of aortic calcification, arthritis, and other disease processes (p ≤ 0.0001).
Certain Vitamin D metabolites potentiate the expression of parathyroid hormone bioactivity
Authors: Bradbeer J.N. , Mehdizadeh S. , Fraher L.J. , Dr. Loveridge N.
Published: February 2010

With the development of a sensitive bioassay for the skeletal effects of parathyroid hormone (PTH), it has become possible to investigate the possible interaction between PTH and vitamin D3 metabolites. This assay is based on the stimulation of glucose-6-phosphate dehydrogenase (G6PD) activity in either the hypertrophic chondrocytes of the growth plate or the osteoblasts lining the metaphyseal trabeculae of rat metatarsals. The response to PTH is paralleled by the activity of dibutyryl cAMP. None of the vitamin D3 metabolites tested had any effect on enzyme activity when tested by themselves. However, both 1,25(OH)2D3 and 25(OH)D3 caused a dose-related potentiation of the response to PTH. Neither 1,24,25(OH)3D3 nor 1,25(OH)2D3 26,23-lactone potentiated the response to PTH. Because this potentiation of the response to PTH occurs after only 8 minutes, it is suggested that it represents a nongenomic response to the vitamin D3 metabolites.
Effect of prostaglandin F2alpha on human parathyroid adenomas: Evidence for uncoupling of parathyroid hormone secretion and cAMP accumulation
Authors: Fitzpatrick Lorraine A. , Martin Cathryn , Sharp Mark , Aurbach Gerald D. , Norton Jeffrey
Published: February 2010

Human parathyroid adenomas are aberrantly regulated by extracellular calcium. We tested pertussis toxin, which ADP-ribosylates and inactivates several guanine nucleotide regulatory proteins, to test the role of these proteins in the secretory control of adenomatous parathyroid tissue. Pertussis toxin did not affect basal cAMP accumulation in 12 adenomas and enhanced parathyroid hormone (PTH) release in 6 of 10 adenomas. Prostaglandin F2alpha (PGF2alpha) inhibited cAMP in three of six adenomas, and pertussis toxin pretreatment did not affect this result. PTH release in 7 of 10 adenomas was inhibited by PGF2alpha and pertussis toxin did not significantly alter PTH release. Pertussis toxin catalyzed ADP-ribosylation of a 40-kDa protein in all adenomas tested (n = 8). We conclude that cAMP accumulation was not affected by pertussis toxin but that in 6 of 10 adenomas, the toxin enhanced PTH release. We suggest that cAMP accumulation and PTH release may be uncoupled from negative control by inhibitory ligands in adenomatous tissue or that the G-proteins involved do not couple to regulatory receptors or to effector.
Osteoclasts in human osteopetrosis contain viral-nucleocapsid-like nuclear inclusions
Authors: Mills Barbara G. , Yabe Hiroo , Singer Frederick R.
Published: February 2010

We report the discovery of nuclear inclusions in the osteoclasts of three unrelated patients with benign osteopetrosis that resemble the osteoclast inclusions characteristic of Paget's disease of bone. These inclusions are morphologically and dimensionally identical to the nucleocapsids of a virus of the Paramyxoviridae family. Supporting a possible viral association with benign osteopetrosis is the observation of the presence of antigens of respiratory syncytial virus, measles virus, and/or mumps virus in the cells of all five patients whose paraffin-embedded bone specimens were tested. These included two patients whose osteoclasts contained nuclear inclusions. No patients with the malignant form of the disease have been studied. There is as yet no proof that a virus is causally related to human osteopetrosis even though a virus can produce an avian form of the disease.
Charged beads: Generation of bone and giant cells
Authors: Professor Krukowski Marilyn , Simmons David J. , Summerfield Anita , Osdoby Philip
Published: February 2010

Based on reports of electrically induced bone formation and findings that some materials used to promote bone ingrowth are stimulatory in bead form, the osteogenic potential of beads with different surface charges was examined. In this preliminary study, three types of Sephadex beads were injected into chick femora: type I, DEAE beads, positively charged; type II, CM beads, negatively charged; type III, G-25, uncharged. Beads were injected into the femoral midshaft, and after 3 days, 4 days, and 1 week, birds were sacrificed and femora were processed for histology. Type I beads: at 3 days, were surrounded by multinucleated giant cells; by 4 days, patches of bead-associated new bone were present along with giant cells; after 1 week, occasional bead-associated multinucleated cells were seen, but now most beads were surrounded by new intramedullary bone, forming an extensive bead-bone lattice. With bead types II and III, bead-associated new bone was seen at 3 days and 4 days only when beads lodged near the endosteum or in the metaphysis. At 7 days, no bone was seen with either of these two bead types. The response to the type I beads may be likened to a remodeling phenomenon with large numbers of giant cells at 3 days, new bone and giant cells at 4 days, and evidence only of bone formation at 7 days.
Sequential bone mineral content in small preterm infants with and without fractures and rickets
Authors: Dr. Koo Winston W.K. , Sherman Roberta , Succop Paul , Oestreich Alan E. , Tsang Reginald C. , Krug-Wispe Susan K. , Steichen Jean J.
Published: February 2010

Seventy-four infants with birth weights 1009 ± 28 grams and gestational age 28.6 ± 0.3 weeks (M ± SEM) were studied prospectively to test the hypotheses that bone mineral content (BMC) measured by photon absorptiometry, would be: (1) lower in very low birth weight (VLBW) infants with radiographic evidence of fractures and/or rickets (F/R), and (2) will continue to be lower over the first year when compared to VLBW infants without F/R. BMC and bone width (BW) of the distal one-third of left radius and ulna were measured at 5 weeks (n = 8), 14 weeks (n = 61), 26 weeks (n = 58), 40 weeks (n = 59), and 1 year (n = 52). Standardized radiographs of both forearms, and weight, length, and head circumferencce were also determined at each study age. Investigators and technicians involved in the photon absorptiometry measurements were unaware of the radiographic findings and vice versa. Twenty-three of 74 infants were found to have F/R. BMC of studied infants remained markedly below our previously determined range of “intrauterine bone mineralization,” even at 26 weeks after birth. There was no significant difference in BMC or BW between infants with and without F/R, either at the time of confirmation of F/R or during early follow-up; however, BMC was lower at ≥6 months and BW was lower at ≥9 months in infants with F/R. We suggest that the extremely low BMC measurements in early infancy predispose all VLBW infants to fractures and rickets.
Effect of parathyroid hormone on amino acid transport by cultured neonatal mouse calvarial bone cells
Authors: Yee John A.
Published: February 2010

The effect of synthetic bovine parathyroid hormone [bPTH-(1–34)] on amino acid uptake by confluent primary cultures of osteoblast-like cells isolated from neonatal mouse calvaria was studied. The uptake of proline and leucine by membrane transport Systems A, ASC, and L was discriminated on the basis of their sodium dependency and sensitivity to the system-specific amino acid analogs 2-(methylamino)-isobutyric acid (MeAIB) for System A and 2-amino-(2,2,1)-heptane-2-carboxylic acid (BCH) for System L. Treatment with 24 nM bPTH-(1–34) in serum-free EBSS for 4 hr increased the initial uptake rate of proline by 50–80% but had no effect on the uptake of leucine. Temporally, the increase in proline uptake was preceded by a 2-hr lag period and plateaued after 5–6 hr. A 5-min exposure to the hormone was sufficient to cause a significant increase in proline uptake measured 4 hr later. The magnitude of the increase was dose-related from 0.24 to 240 nM bPTH-(1–34), with the half-maximal effect occurring at 2.4 nM. Only the sodium-dependent, MeAIB-inhibitable component of proline uptake was elevated. Eadie-Hofstee analysis indicated that bPTH-(1–34) increased Vmax without changing the Km. Actinomycin D and cycloheximide prevented the hormone-stimulated increase, suggesting that RNA and protein synthesis were required. Treatment with either inhibitor alone caused a 30–35% decrease in proline transport that was not observed in the presence of bPTH-(1–34), indicating an effect not dependent on macromolecular synthesis. DBcAMP also increased proline uptake. Maximally effective concentrations of bPTH-(1–34) and DBcAMP did not increase transport above the level of bPTH-(1–34) alone. These results confirm that PTH and DBcAMP increase amino acid uptake in bone. In addition, they (1) identify osteoblast-like cells as targets for this action of PTH, (2) demonstrate that the effect is on amino acid transport System A, (3) suggest that the rise in proline uptake is a direct action of PTH that may involve effects on both synthesis and degradation of System A transport proteins, and (4) indicate that the effect may be mediated by cAMP.
The effects of transplantation of granulocyte-macrophage progenitors on bone resorption in osteopetrotic rats
Authors: Dr. Schneider G.B. , Relfson M.
Published: February 2010

Osteopetrosis in the ia (incisors absent) rat is the result of reduced bone resorption due to abnormal osteoclasts. This mutant and others have been used to determine the precursor(s) to osteoclasts. Hemopoietic stem cells, isolated from bone marrow of normal littermates, cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia rats. These studies were conducted to define further the precursor to the osteoclast by evaluating the effects of the transplantation of granulocyte-macrophage progenitors on bone resorption in the ia rat. Granulocyte-colony forming cells (G-CFC), granulocyte-macrophage-colony forming cells (GM-CFC), and macrophage-colony forming cells (M-CFC) were isolated from normal bone marrow using an FITC-labeled monoclonal antibody directed against rat Thy-1.1 and fluorescence-activated cell sorting. The isolates were evaluated in soft agar culture; granulocyte isolates generated 71% G-CFC of all colonies formed and were enhanced 27 times over unfractionated cells. Mixed isolates generated 57% GM-CFC of all colonies formed and were 15 times enhanced, while macrophage isolates were 75% M-CFC with an enhancement factor of 18. The isolated populations were injected into 3-week-old ia recipients and evaluated for the ability of these cellular isolates to correct the bone resorption defect by measuring the size of the tibial marrow cavity and by identifying morphologically normal osteoclasts. In addition, isolated populations of cells were labeled with FITC and injected into ia donors to determine if labeled osteoclasts developed. G-CFC isolates, which apparently contained osteoclast precursors that were co-purified with the G-CFC, were effective in correcting the skeletal defect and giving rise to FITC-labeled osteoclasts; GM-CFC were intermediate in their effect; and M-CFC were ineffective in both curing the osteopetrotic condition and giving rise to labeled osteoclasts.
Properties of parathyroid hormone receptors on circulating bovine lymphocytes
Authors: Yamamoto Itsuo , Bringhurst F. Richard , Potts John T. , Segre Gino V.
Published: February 2010

Binding of parathyroid hormone (PTH) to circulating bovine lymphocytes was studied using, as the radioligand, a synthetic sulfur-free analog of bovine PTH, [Nle8,Nle18,Tyr34]bPTH-(1–34)amide, which was labeled to high specific activity with 125I and was purified by reverse-phase high-performance liquid chromatography. Binding of PTH to lymphocytes satisfies several criteria indicative of a specific interaction between the hormone and its receptor. Specific binding is saturable at 3.3 fmoles of radioligand bound per 107 cells, occurs more rapidly at 37°C than at lower temperatures, and reaches equilibrium within 2 hr at 15°C. Inhibition of specific binding occurs with intact PTH, with biologically active PTH analog or fragment, and with synthetic PTH antagonists, but not with biologically inactive PTH fragments, or peptide hormones unrelated to PTH. There is an excellent correlation between the binding affinities of PTH agonists with receptors on lymphocytes and those previously reported with receptors in canine renal membranes, and on rat osteosarcoma cells. The dissociation constant (Kd) is approximately 10−9M, as calculated from the association and dissociation rate constants. This correlates very closely both with the apparent Kd, as estimated from Scatchard analysis of radioligand saturation and competition studies, and with previously reported Kd of PTH receptors in canine renal membranes and on intact rat osteosarcoma and opossum kidney cells. In addition, the relative binding affinity of intact hormone and a synthetic PTH agonist to receptors on lymphocytes correlates closely with the relative biologic potency of these peptides in stimulating adenylate cyclase in membranes from these cells. The presence of PTH specific receptors on lymphocytes is consistent with evidence suggesting that PTH-lymphocyte interactions may modulate bone resorption in vitro. Also, the ready accessibility of circulating lymphocytes may serve as a useful model system for examining PTH-receptor regulation in vivo.
Bone mineral content in the senescent rat femur: An assessment using single photon absorptiometry
Authors: Kiebzak Gary M. , Smith Ronald , Howe Juliette C. , Sacktor Bertram
Published: February 2010

The single photon absorptiometry technique was evaluated for measuring bone mineral content (BMC) of the excised femurs of the rat, and the system was used to examine the changes in cortical and trabecular bone from young adult (6 mo), mature adult (12 mo), and senescent (24 mo) male and female animals. BMC of the femur midshaft, representing cortical bone, apparently increased progressively with advancing age. The width of the femur at the scan site also increased with age. Normalizing the midshaft BMC by width partially compensated for the age-associated increase. However, when bone mineral values were normalized by the cortical area at the scan site, to take into account the geometric differences in the femurs of different aged animals, maximum bone densities were found in the mature adult and these values decreased slightly in the femurs from senescent rats. In contrast, the BMC of the femur distal metaphysis, representing trabecular bone, decreased markedly in the aged rat. The loss of trabecular bone was also evident from morphological examination of the distal metaphysis. These findings indicated that bone mineral loss with age was site specific in the rat femur. These studies provided additional evidence that the rat might serve as a useful animal model for specific experiments related to the pathogenesis of age-associated osteopenia.
Effect of glucocorticoid administration on intestinal, renal, and cerebellar calbindin-D28K in chicks
Authors: Dr. Tohmon Miyo , Fukase Masaaki , Kishihara Michizo , Kadowaki Seizo , Fujita Takuo
Published: February 2010

A radioimmunoassay for chick intestinal calcium-binding protein (calbindin-D28K, CaBP-28K) has been developed in our laboratory with a detection limit of 0.3 ng/ml. The values for CaBP-28K in vitamin D-deficient (-D) chicks ranged from a high value for the cerebellum (21,400 ± 580 ng/mg protein) to a scarcely detectable level in the liver (19.6 ± 2.2 ng/mg protein). After administration of vitamin D (vitamin D3 500 IU p.o. for 7 days) (+ D), the levels of CaBP-28K increased in the duodenum (52,300 ± 5,100 ng/mg protein), ileum (45,200 ± 740 ng/mg protein), cerebellum (22,000 ± 470 ng/mg protein), colon (15,200 ± 330 ng/mg protein), and kidney (13,460 ± 540 ng/mg protein). However, the increment in the level of CaBP-28K in each tissue after vitamin D administration was different; levels of CaBP-28K in the duodenum, ileum, and colon increased dramatically more than 200 times after vitamin D administration, whereas that in the kidney showed only a 2.5-fold increase and was unaltered in the cerebellum. On the other hand, glucocorticoid administration (dexamethasone, 1 mg/kg BW i.m. for 2 days) markedly inhibited the vitamin D-stimulated (vitamin D3 500 IU p.o. for 3 days) levels of CaBP-28K in the duodenum (from 23,373 ± 3,117 to 8,261 ± 968 ng/mg protein, p < 0.001), ileum (from 6,443 ± 1,342 to 3,712 ± 691 ng/mg protein, p < 0.01), and colon (from 4,335 ± 174 to 2,316 ± 590 ng/mg protein, p < 0.01. whereas glucocorticoid further increased the vitamin D-stimulated level of CaBP-28K in the kidney (from 3,922 ± 776 to 5,593 ± 450 ng/mg protein, p < 0.01). The level of CaBP-28K in the cerebellum was unaffected either in the presence or absence of vitamin D or glucocorticoid. Although the precise action of glucocorticoid on CaBP-28K is still unclear, these results indicate that glucocorticoid is inhibitory in the intestine and colon, but is augmentative in the kidney, promoting an increase in the vitamin D-stimulated CaBP-28K production. However, glucocorticoid did not affect the level of CaBP-28K in the cerebellum, irrespective of the presence or absence of vitamin D, suggesting a vitamin D- and glucocorticoid-independent control mechanism for CaBP-28K production in the cerebellum. As a final observation, bidirectional regulatory mechanisms of the vitamin D-dependent CaBP-28K may exist in chicks, one stimulatory, the other inhibitory.
Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass
Authors: Dr. Turksen Kursad , Kanehisa Junya , Opas Michal , Heersche Johan N.M. , Aubin Jane E.
Published: February 2010

The ability of osteoclasts (OC) to migrate and resorb bone is thought to be dependent on cytoskeletal function and adhesion. Therefore, we investigated the cytoskeleton and the adhesion patterns of rabbit OC on glass and on devitalized bone slices, using specific antibodies to cytoskeletal elements and fluorescence and interference reflection microscopy. Microtubules (MT) were similar in OC on both substrata, and appeared in a pattern typical of that described for many cells. Multiple centriolar complexes were observed in most OC, either as one large aggregate in the center of the cell or dispersed singly or in small aggregates close to individual nuclei. Staining of microfilaments (MF) was similar on both substrata and appeared primarily as an F-actin network. MF distribution was different in OC associated with resorption lacunae with intense staining over those regions. In the OC on glass, high F-actin staining was detectable at the periphery in dots and rosette-like structures, which also stained for vinculin. The adhesion patterns indicated that OC on glass do not make large focal contacts, but appear to make a few tiny focal contacts that are not associated with the rosette-like structures. Most of the undersurface of the OC appeared either to be involved in close contacts or to be separated by distances of >100 nm from the substratum. These studies indicate that the MF distribution and the adhesion patterns of rabbit OC are typical of motile cells, that the distribution of the cytoskeleton of rabbit OC on glass and on bone slices is similar, and that MF may be involved in the morphological changes associated with resorption.