bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and characterization of gap junctions in the osteoblastic MC3T3-E1 cell line
Authors: Yamaguchi Dean T. , Ma Defang , Lee Austin , Huang Jason , Gruber Helen E. 
Published: February 2010

Abstract
Gap junctions are channels connecting cells that function in cell-to-cell communication. Gap junctions are abundant in osteoblastic cells. Membranes enriched for gap junction plaques were obtained by differential centrifugation, followed by treatment of the membranes with potassium iodide and sarkosyl before sucrose density gradient centrifugation. Electron microscopy showed that the preparation was enriched for electrondense membranes consistent with gap junctions. Coomassie Blue staining of SDS-PAGE preparations revealed a prominent band at approximately 41 kD. Western analysis with a site-directed antibody, CT-360 (D. Laird, California Institute of Technology, Pasadena, CA), to the C-terminal portion of the rat heart connexin 43 molecule was positive in the MC3T3-E1 cell line, a phenotypic osteoblastic cell line derived from normal neonatal mouse calvariae. Western analysis using a monoclonal antibody, R5.21C, to rat liver connexin 32 was negative. Additionally, a prominent band at 59 kD was detected by CT-360 in both gap junction-enriched preparations and cell lysates. Treatment of diluted samples of gap junction-enriched preparations with sulfhydryl reducing agents in combination with detergents resulted in the enhancement and diminution of the 41 and 59 kD bands, respectively. Immunoprecipitation following [35S]methionine/[35S]cysteine labeling revealed a significant band detected at 122 kD in addition to the 41 kD band. To demonstrate functional gap junctions, transfer of lucifer yellow dye to surrounding cells was monitored after microinjection of a target cell. Between passages 10 and 25 in culture, functional cell coupling was found in approximately 70% of injected cells. Coupling was detected within 1–2 minutes after injection. Simultaneous microinjection of the CT-360 antibody with lucifer yellow resulted in the decoupling of cells. In conclusion, (1) MC3T3-E1 cells possess a 41 kD protein that is recognized by connexin 43 antibody to rat heart gap junction; (2) multimers of the MC3T3-E1 gap junctions occur in the preparation; and (3) functional coupling demonstrated by dye transfer may be regulated by region(s) in the C terminus of the connexin molecule.
Alcohol-induced bone disease in the absence of severe chronic liver damage
Authors: DR. Díez Adolfo , Puig Jordi , Serrano Sergi , Marin̄oso Maria-Lluisa , Bosch Jaume , Marrugat Jaume , Mellibovsky Leonardo , Nogués Xavier , Knobel Hernando , Aubía Jaume 
Published: February 2010

Abstract
To define and identify metabolic bone disease and mineral alterations induced by chronic heavy alcoholism in patients without severe liver damage, we studied a prospective series of unselected patients admitted to a 300-bed general hospital in Barcelona (Spain). A total of 26 chronic heavy drinkers of more than 150 g/day for at least 3 years were included. A general analytic and hormonal study, including liver biopsy in cases with any abnormality in liver function tests, and plasma and urine biochemistry with calcium regulating hormones and osteocalcin levels were determined. A transiliac bone biopsy after double-tetracycline labeling, with histomorphometric study of undecalcified bone, was performed. Statistical analysis was adjusted by age and sex by means of logistic regression. A total of 26 (20 men and 6 women) chronic alcohol abusers were studied. After adjustment for age and sex, alcoholic patients showed slight but significantly increased concentrations of plasma calcium (9.56 ± 0.56; OR = 17.93; 95% CI 3.17–101.48) and decreased cPTH (0.36 ± 0.11; OR = 0.097;95% CI 0.018–0.528) compared with controls. Osteocalcin values were low (1.49 ± 0.89, normal range 1.8–6.6). There was a significant decrease in bone volume, BV/TV (12.56 ± 5.29; OR = 0.06; 95% CI 0.01–0.34), with increased resorption surfaces, ES/BS (4.28 ± 2.43; OR = 9.86; 95% CI 2.16–45.07), and increased osteoclast number, N.Oc/TA (0.21 ± 0.37; OR = 6.41; 95% CI 1.27–32.25). Dynamic parameters were abnormal (Fisher's exact test), as follows: decreased BFR/BS (0.023 ± 0.028 versus 0.049 ± 0.020; p = 0.013), MAR (0.309 ± 0.269 versus 0.771 ± 0.529; p = 0.029), MS/BS (3.743 ± 4.433 versus 5.890 ± 2.882; p = 0.008, and OMR (2.402 ± 2.833 versus 3.057 ± 0.083; p = 0.011) and increased MLT (175.80 ± 405.49 versus 34.53 ± 8.117; p = 0.008). We conclude that chronic alcohol consumption induces osteopenia with low turnover and increased osteoclast number and resorption surfaces. The increase in plasma calcium levels and decreased PTH suggests a primary effect of alcohol on bone, resulting in bone loss and calcium infusion from bone into plasma. These effects were observed in the absence of significant liver damage.
Characterization of the osteogenic stromal cell line MN7: Identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing
Authors: Mathieu E. , Meheus L. , Raymackers J. , DR. Merregaert J. 
Published: February 2010

Abstract
Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the α2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.
Further definition of the protein kinase C activation domain of the parathyroid hormone
Authors: Jouishomme Hervé , Whitfield James F. , Gagnon Lyne , MacLean Susanne , Isaacs Richard , Chakravarthy Balu , Durkin Jon , Neugebauer Witold , Willick Gordon , Rixon Raymond H. 
Published: February 2010

Abstract
The protein kinase C (PKC) activation domain of the parathyroid hormone (PTH) was believed to be the 28–34 region of the molecule. We have now shown that PTH-(29–32) is the smallest PTH fragment that can stimulate significantly membrane-associated PKC activity in ROS 17/2 rat osteosarcoma cells. As was previously shown for full-length PTH-(1–84) and the fully bioactive PTH-(1–34) fragment, there were two peaks in the PKC response to PTH-(29–32): one peak was obtained with low picomolar concentrations and the other with much higher nanomolar concentrations of the fragment. The PKC-activating ability was unaffected by the loss of Asn33 and Phe34, but it was abolished by removing His32. Thus, the PTH-(28–31) and PTH-(29–31) fragments did not stimulate membrane-associated PKC activity. The much larger PTH-(1–31) fragment also did not stimulate membrane-associated PKC activity, although it stimulated adenylyl cyclase as strongly as PTH-(1–34). This functional sensitivity to the loss of the polar His32 was not caused by a specific need for His or another polar amino acid in this position because replacing it with the apolar Leu did not abolish adenylyl cyclase or PKC activation. It is concluded that the minimum, fully functional PKC activation domain of the PTH molecule is Gln29-Asp30-Val31-His32.
Quality control of bone densitometry in a national health survey (NHANES III) using three mobile examination centers
Authors: Wahner Heinz W. , Looker Anne , Dunn William L. , Walters Lana C. , Hauser Mary F. , Novak Catherine 
Published: February 2010

Abstract
A quality control (QC) program for bone mineral measurements at the proximal femur by dual-energy x-ray absorptiometry (DXA) was designed for the osteoporosis component of the Third National Health and Nutrition Examination Survey (NHANES III). Major elements of the QC program are (1) a QC center for review of all scans, (2) setup procedures for and continuous monitoring of daily QC procedures, (3) reference standards for cross-calibration, (4) longitudinal studies for assessment of instrument stability, (5) monitoring of technologist performance, and (6) training. This report describes the results of the QC program of the first half of this 6 year study, which began in 1988. Measurements were performed on 7376 subjects in three mobile examination centers, which traveled to a new location about every 3 months, a total of 44 locations. A small percentage (3.5%) of all scans were rejected, mostly because of patient motion during scanning, but 33% of the remaining scans required reanalysis at the QC center to refine the location of the regions of interest. Precision in spine and hip phantoms was below 1% at all ROIs. In 535 subjects with duplicate scans, age 20–91 of both sexes, examined in a blinded review, precision for BMD at the femur neck ROI was 3.2% (CV) and 5.1% for Ward's triangle BMD. The central review improved scan quality, increased the number of usable scans, and reduced significantly the range of the mean percentage difference in the duplicate scans. Minor alterations in machine function were observed and corrected. QC results on phantoms were similar to those obtained with stationary instruments in dedicated laboratories. Retrospective analysis of the regression slopes from QC records did not show a need for correction of the data base, indicating that the instruments were stable during the course of the study.
Long-term effect of calcium supplementation on bone loss in perimenopausal women
Authors: Elders Petra J. M. , Lips Paul , Netelenbos J. Coen , van Ginkel Floris C. , Khoe Edwin , van der Vijgh Wim J. F. , van der Stelt Paul F. 
Published: February 2010

Abstract
We observed in a controlled 2 year longitudinal trial in 248 perimenopausal women that a daily calcium supplement of either 1000 or 2000 mg Ca2+ significantly reduced lumbar bone loss and bone turnover in the first year of calcium supplementation. In the second supplementation year the rate of lumbar bone loss in the treated subjects was not significantly different from that in the control group, although two of the three biochemical parameters of bone turnover remained decreased throughout the study. To quantify further the long-term effect of calcium supplementation, we extended the study for another year in 214 women. In the women of the control group who were menstruating until the last year of the trial, the mean change in lumbar bone mineral density after 3 years was –3.2% of the initial value versus 1.6% in the calcium-supplemented groups (p &lt; 0.01). The decrease in lumbar bone loss in these supplemented premenopausal and early perimenopausal women remained statistically significant in the second and third years of supplementation. In the women who stopped menstruating before or during the study, the long-term reduction in lumbar bone loss was not significant (mean difference between control and treatment groups &lt;0.6% points after 3 years). The decrease in metacarpal cortical thickness (MCT) in the treated subjects during 3 years was on average –3.0% of the initial value in the control versus–2.0% in the supplemented subjects (P &lt; 0.01). The effect of calcium supplementation on MCT was not significantly related to the menopausal status of the subjects. Serum alkaline phosphatase, osteocalcin, and urinary hydroxyproline excretion decreased after calcium supplementation in all menopausal groups. These parameters remained decreased throughout the trial, with exception of alkaline phosphatase in the 1000 mg calcium group. We conclude that calcium supplementation substantially reduces cortical and trabecular bone loss in the years immediately preceding menopause. Although it reduces postmenopausal cortical bone loss to some extent, it does not prevent the menopause-related lumbar bone loss.
Paracrine interactions among parathyroid cells: Effect of cell density on cell secretion
Authors: Sun Feng , Maercklein Peter , Fitzpatrick Lorraine A. 
Published: February 2010

Abstract
Cell-cell interactions are important in the regulation of endocrine cell secretion. To investigate the possibility that cell communication may alter the regulation of parathyroid cell secretion, we utilized the reverse hemolytic plaque assay (RHPA) to measure parathyroid hormone (PTH) release from individual cells. Bovine parathyroid cells were dispersed and plated with protein A-conjugated erythrocytes at cell densities ranging from 0.9 to 36 X 102 cells/cm2 in 0.2 mM calcium. Cell populations were greater than 98% homogeneous as determined by immunocytochemistry and in situ hybridization for PTH mRNA. Plaques were developed and data analyzed for the amount of PTH per cell released (plaque area in μm2 X 104) and the determination of cell recruitment (% plaques formed). A positive correlation existed between parathyroid cell density and the amount of PTH released. As the distance between cells increased, the plaque area (amount of PTH released per cell) decreased (ranging from 1.0 X 1044 μm2 at 0.9 X 102 cells/cm2 versus 1.6 X 104 μm2 at 36 X 102 cells/cm2). The percentage of cells releasing PTH (recruitment) also decreased (16% at 0.9 X 102 cells/cm2 versus 47% at 36 X 102 cells/cm2). These data suggest that parathyroid cells in close proximity are stimulated to secrete more hormone than those at lesser densities. In addition, parathyroid cells are recruited to secrete PTH when plated at high density. Factor(s) released by the parathyroid cell may increase cell responsiveness and stimulate secretion in a paracrine fashion.
Correlation between low levels of estrogen receptors and estrogen responsiveness in two rat osteoblast-like cell lines
Authors: Davis Vicki L. , Couse John F. , Gray T. Kenney , Korach Kenneth S. 
Published: February 2010

Abstract
With the knowledge that estrogen replacement therapy can circumvent postmenopausal osteoporosis and with the discovery of estrogen receptors (ER) in cultures of normal osteoblast-like cells, extensive investigations have been directed toward understanding the role of the ER in normal bone homeostasis. ROS 17/2.8 and UMR-106-01, two established osteoblast-like cell lines derived from rat osteosarcomas, have been shown to have estrogen-regulated biologic responses. Only the ROS 17/2.8 cell line has been reported to contain ER. In this study, high-affinity, saturable binding sites characteristic of the ER were detected in UMR-106-01 cells by binding assays with the high-affinity ligand, [125I]17β-estradiol. An initial immunoconcentrtion step before western blot analysis also allowed detection of the full-length ER protein. In addition, northern blot analysis indicated that the entire ER transcript was expressed and that the half-life of the ER message was increased following cycloheximide treatment. Message levels were also regulated by removal of serum and treatment with estradiol. An estrogen-regulated reporter vector, ERET81CAT, was transfected into the UMR-106-01 cells to determine whether the detected level of ER was transcriptionally functional. Using this assay, estrogen responsiveness was evident; however, the response was inconsistent. Multiple factors, such as serum, estradiol, and cell density, influence the ER levels in these cells and probably cause fluctuations in the abundance of receptors available to induce the CAT response. When the cells are responsive, the ICI 164,384 antagonist could block the estrogen-induced activation of CAT.
The ROS 17/2.8 cells were also analyzed in parallel with the UMR-106-01 cells to allow comparisons between these two osteoblast-like cell lines because they exhibit phenotypes for two unique stages of differentiation. ROS 17/2.8 cells were found to contain more receptor sites/cell by the 125I-E2 (estradiol) binding assays, as well as higher levels of ER-specific transcripts, than UMR-106-01 cells (two- to threefold). This level of ER was consistently able to modulate estrogen-induced stimulation of the reporter CAT vector. Therefore, functional ER is expressed in both cell types, but the higher level of receptors found in the ROS 17/2.8 cell line improves the estrogen responsiveness of these osteoblast-like cells. These data also indicate that levels of ER that are low or undetectable by conventional methods are able to mediate biologic responses through direct interactions of the ER with the specific DNA response element.
Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime molddictyostelium discoideum
Authors: Dr. Rogers Michael J. , Watts Donald J. , Russell R. Graham G. , Ji Xiaohui , Xiong Xiaojuan , Blackburn G. Michael , Bayless Allan V. , Ebetino Frank H. 
Published: February 2010

AbstractBisphosphonates are inhibitors of bone resorption and are used increasingly as therapeutic agents for treating clinical disorders of skeletal metabolism. Their mode of action is still not fully understood. The demonstration that methylenebisphosphonate, a simple methylene analog of pyrophosphate, inhibits the axenic growth of amoebae of the slime mold Dictyostelium discoideum and is incorporated into adenine nucleotides suggested that this organism might be useful in elucidating the cellular effects of bisphosphonates. We examined 24 bisphosphonates, including all those of clinical interest as inhibitors of osteoclast-mediated bone resorption in vivo, for their effects on D. discoideum. All the geminal bisphosphonates inhibited growth of Dictyostelium, although the effectiveness of individual compounds varied widely. When the bisphosphonates were ranked there was a remarkable similarity between the order of potency as inhibitors of growth of Dictyostelium and the order of potency as inhibitors of bone resorption. Thus, bisphosphonates with more complex side-chain structures, especially those containing a nitrogen group, were more potent than simple substituted bisphosphonates, some inhibiting Dictyostelium growth even at concentrations below 10 μM. It therefore appears that the mechanism by which bisphosphonates prevent Dictyostelium growth could be similar to the mechanism by which these compounds affect the activity of osteoclasts. Because the mechanisms of action of bisphosphonates on osteoclasts remains unclear, Dictyostelium may provide an additional model for studying the biochemical mode of action of bisphosphonates. Furthermore, these studies suggest that Dictyostelium may also be a convenient organism for rapid evaluation of potentially active bisphosphonates.
Effect of parathyroid hormone-related peptide supplementation of soy protein formulas in the neonatal pig model
Authors: Hillman Laura S. , Forte Leonard R. , Veum Trygve , Ru Yuxia , Howard Barbara , Rowe Angela , Hillman Helena W. , Hillman Laura S. 
Published: February 2010

Abstract
PTH-related peptide (PTHrP) is found in all milks, including human and pig. To define a role for PTHrP in milk, 2-day-old piglets were randomized to receive soy formula devoid of PTHrP or supplemented with 1 nM synthetic PTHrP(1–86) (n = 8 per group). The number of serum samples with detectable PTHrP by immunoassay (Incstar) and radiometric assay (Nichols) was 9 of 33 and 3 of 13 in PTHrP− and 8 of 27 and 3 of 15 in PTHrP+ formula-fed piglets and 8 of 14 and 7 of 12 in naturally suckling piglets, respectively. Serum and urine concentrations of calcium and magnesium and total and bone alkaline phosphatase were similar in both groups at 3, 6, 10, and 17 days of age. No differences were seen in bone mineral content of the tibia measured by single-photon absorptiometry (BMC 0.22 + 0.06 and 0.22 + 0.10) or dual x-ray absorption (BMC 1.43 + 0.36 and 1.31 + 0.78) either in vivo or on excised bone or by measurement of Ca, Mg, or P content or total bone ash (1.26 + 0.26 and 1.38 + 0.28 mg). Intestinal histology, serum intestinal alkaline phosphatase, and net absorption and retention of Ca, Mg, and P in balances from age 11–17 days were all similar. As in humans, however, a developmental pattern was seen for phosphorus regulation in both groups. Serum phosphorus (mg/dl) increased between day 3 and days 6 and 10 of age and then fell to published values for 8-week-old suckling pigs at day 17 associated with an increase in urine phosphorus and cyclic AMP excretion. The rise in serum phosphorus from 3 to 6 days was suggestively attenuated in the piglets receiving PTHrP (4.6 + 1.5 in –PTHrP and 2.6 + 1.3 in +PTHrP; p = 0.02 by nonpaired t-test, ANOVA for treatment effect p = 0.14). Thus, although a suggestive early effect of milk PTHrP on phosphorus regulation may be present before the development of full responsiveness to endogenous PTH, no effects could be demonstrated on mineral absorption, renal regulation, or bone mineralization. Because the synthetic PTHrP studied was only 1–86 amino acids, an effect of more distal, carboxyl-terminal, peptides cannot be excluded.
Do variations in hip geometry explain differences in hip fracture risk between japanese and white americans?
Authors: Nakamura T. , Turner C.H. , Yoshikawa T. , Slemenda C.W. , Peacock M. , Burr D.B. , Mizuno Y. , Orimo H. , Ouchi Y. , Johnston C.C. 
Published: February 2010

Abstract
Despite lower femoral neck bone mass, Japanese women have a substantially lower incidence of hip fracture than North American whites. Reasons for this discrepancy were sought in a study of 57 Japanese and 119 white American women aged 50–79. All women were in good health. Bone mineral content (BMC) in the femoral neck, femoral neck length (NL), femoral neck angle (Ø), cross-sectional moment of inertia (CSMI), safety factor (SF), and fall index (FI) were calculated using dual x-ray absorptiometry. Height and weight were greater in Americans than in Japanese (1.62 versus 1.52 m; p &lt; 0.0001 and 66.0 versus 49.4 kg; p &lt; 0.0001, respectively). Mean BMC in the femoral neck and CSMI were greater in Americans than in Japanese (3.91 versus 3.02 g; p &lt; 0.0001 and 0.99 versus 0.57 cm4; p &lt; 0.0001, respectively). NL was longer in Americans (5.6 versus 4.4 cm; p &lt; 0.0001) and θ was larger in Americans (130 versus 128° p &lt; 0.01), whereas SF and FI were less in Americans than in Japanese (3.41 versus 5.12; p &lt; 0.0001 and 1.00 versus 1.40; p &lt; 0.0001, respectively). These results indicate that despite lower bone mass, Japanese women have lower risks of structural failure in the femoral neck, attributable primarily to shorter femoral necks and, to a lesser degree, a smaller femoral neck angle. Geometric characteristics of the femoral neck in Japanese women are associated with their lower hip fracture risk, and the measurement of proximal femoral geometry, combined with bone mass, may provide further clinical information about the risk of hip fracture.