bluuebunny/crossref_metadata_2025 huggingface dataset:
Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode
Authors: Zhang Keying , Zhang Yuzhong
Published: February 2010

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10−12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.
Quantitative Analyses and Transcriptomic Profiling of Circulating Messenger Rnas As Biomarkers of Rat Liver Injury
Authors: Wetmore Barbara A. , Brees Dominique J. , Singh Reetu , Watkins Paul B. , Andersen Melvin E. , Loy James , Thomas Russell S.
Published: February 2010

Serum aminotransferases have been the clinical standard for evaluating liver injury for the past 50-60 years. These tissue enzymes lack specificity, also tracking injury to other tissues. New technologies assessing tissue-specific messenger RNA (mRNA) release into blood should provide greater specificity and permit indirect assessment of gene expression status of injured tissue. To evaluate the potential of circulating mRNAs as biomarkers of liver injury, rats were treated either with hepatotoxic doses of D-(+)-galactosamine (DGAL) or acetaminophen (APAP) or a myotoxic dose of bupivacaine HCl (BPVC). Plasma, serum, and liver samples were obtained from each rat. Serum alanine aminotransferase and aspartate aminotransferase were increased by all three compounds, whereas circulating liver-specific mRNAs were only increased by the hepatotoxicants. With APAP, liver-specific mRNAs were significantly increased in plasma at doses that had no effect on serum aminotransferases or liver histopathology. Characterization of the circulating mRNAs by sucrose density gradient centrifugation revealed that the liver-specific mRNAs were associated with both necrotic debris and microvesicles. DGAL treatment also induced a shift in the size of plasma microvesicles, consistent with active release of microvesicles following liver injury. Finally, gene expression microarray analysis of the plasma following DGAL and APAP treatment revealed chemical-specific profiles.

Conclusion:

The comparative analysis of circulating liver mRNAs with traditional serum transaminases and histopathology indicated that the circulating liver mRNAs were more specific and more sensitive biomarkers of liver injury. Further, the possibility of identifying chemical-specific transcriptional profiles from circulating mRNAs could open a range of possibilities for identifying the etiology of drug/chemical-induced liver injury. Hepatology 2010
Combinatorial Effects of Interleukin 10 and Interleukin 4 Determine the Progression of Hepatic Inflammation Following Murine Enteric Parasitic Infection
Authors: Douglas Diana B. , Beiting Daniel P. , Loftus John P. , Appleton Judith A. , Bliss Susan K.
Published: February 2010

Mice lacking the immunoregulatory cytokine interleukin 10 (IL-10) develop necrotizing hepatitis after infection with

Trichinella spiralis

, and inflammation is dependent on the migration of intestinally activated CD4+ T cells into the liver. Hepatic production of IL-4 is elevated in these mice, and we hypothesized that it plays a role in the development of hepatic pathology. Wild-type (WT), IL-10 knockout (KO), IL-4 KO, and IL-10/IL-4 KO mice were orally infected, and disease progression was followed by histological examination, alanine aminotransferase assays, and flow cytometric analysis of hepatocellular content. Both IL-10 KO and IL-10/IL-4 KO mice experienced hepatocellular injury, but only IL-10 KO mice advanced to a necrotic phase. Hepatic CD4+ T cells were the major source of IL-4, and IL-10 regulated the number of intestinally-derived CD4+IL-4+ cells. Sequestration of activated neutrophils in the liver required IL-4, and neutrophil depletion prevented progression to overt necrosis. Adoptive transfer of intestinal WT CD4+ T cells inhibited neutrophil accumulation and inflammation, but their regulatory effects did not require IL-10 signaling.

Conclusion:

The absence of IL-10 led to hepatocyte injury during infection, but IL-4 was necessary for the development of neutrophil-dependent necrosis. These studies provide new insight into the combinatorial role of these cytokines and their targets in the generation and progression of hepatic inflammation. (Hepatology 2010;)
Impact of Epstein–Barr virus in the clinical evolution of patients with classical Hodgkin's lymphoma in Brazil
Authors: Souza Eni M. , Baiocchi Otávio C. G. , Zanichelli Maria A. , Alves Antonio C. , Assis Marianne G. , Eiras Daniel P. , Dobo Cristine , Oliveira José S. R.
Published: February 2010

Introduction

Classical Hodgkin's Lymphoma (cHL) has been frequently associated with Epstein–Barr virus (EBV), which can be found in a latent pattern in Reed‐Sternberg (RS) cells. However, the impact of the presence of EBV in RS cells and its prognosis are still controversial. We analysed the presence of EBV in RS cells and its influence in the clinical evolution of patients with cHL treated in two public hospitals in the city of São Paulo, Brazil.

Materials and Methods

We selected 97 patients with cHL from 1994 to 2004. Patients were only included in this study if they had (1) >18 years, (2) negative HIV serology, (3) undergone similar chemotherapy protocols, (4) paraffin blocks available with enough material for systematic review and histological reclassification and for detection of EBV in RS cells by in situ hybridization and immunohistochemistry and (5) clinical, epidemiological and laboratorial parameters available after a thorough chart review.

Results

EBV was identified in 52.5% of the cases. Mixed cellularity (MC) subtype was more common in EBV‐related tumours (25.5%) (p = 0.005). There was no difference on age, gender, stage and the presence of B symptoms between the two groups. The presence of EBV did not influence event free survival (EFS) (p = 0.38) or overall survival (OS) (p = 0.80) with a median follow‐up of 80 months.

Conclusion

We demonstrate that the prevalence of EBV‐related cHL in this Brazilian population is 52.5% and, that, the presence of EBV does not change the clinical evolution and OS of patients treated with similar chemotherapy protocols. Copyright © 2010 John Wiley & Sons, Ltd.
Pak1 and Pak2 are activated in recurrent respiratory papillomas, contributing to one pathway of Rac1‐mediated COX‐2 expression
Authors: Wu Rong , Abramson Allan L. , Symons Marc H. , Steinberg Bettie M.
Published: February 2010

Recurrent respiratory papillomas are premalignant tumors of the airway caused by human papillomaviruses (HPVs), primarily Types 6 and 11. We had reported that respiratory papillomas overexpress the epidermal growth factor receptor (EGFR), the small GTPase Rac1 and cyclooxygenase‐2 (COX‐2), and have enhanced nuclear factor‐κB (NFκB) activation with decreased levels of IκB‐β but not IκB‐α. We also showed that EGFR‐activated Rac1 mediates expression of COX‐2 through activation of p38 mitogen‐activated protein kinase. We have now asked whether the p21‐activated kinases Pak1 or Pak2 mediate activation of p38 by Rac1 in papilloma cells. Pak1 and Pak2 were constitutively activated in vivo in papilloma tissue compared with normal epithelium, and Rac1 siRNA reduced the level of both phospho‐Pak1 and phospho‐Pak2 in cultured papilloma cells. Reduction in Pak1 and Pak2 with siRNA decreased the COX‐2 expression in papilloma cells, increased the levels of IκB‐β and reduced the nuclear localization of NF‐κB, but had no effect on p38 phosphorylation. Our studies suggest that Rac1 → Pak1/Pak2 → NFκB is a separate pathway that contributes to the expression of COX‐2 in HPV‐induced papillomas, independently of the previously described Rac1 → p38 → COX‐2 pathway.
Formulation and characterization of radio‐opaque conjugated in situ gelling materials
Authors: Blakely Brandon , Hoon Lee Bae , Riley Celeste , McLemore Ryan , Pathak Chandrashekhar P. , Vernon Brent L.
Published: February 2010

X‐ray visibility is an integral design component of in situ gelling embolization systems for neurovascular treatment. The goals of this project included the synthesis and characterization of a unique intrinsically radio‐opaque in situ gelling material for neurovascular embolization. The gels formed using Michael‐Type Addition between pentaerythritol tetrakis 3‐mercaptopropionate (QT) thiols and poly(propylene glycol) diacrylate (PPODA) with the addition of the new material Iodobenzoyl poly(ethylene glycol) acrylate (IPEGA), a radio‐opaque agent, synthesized successfully as confirmed with 1H NMR. The PPODA and IPEGA were mixed using a syringe coupler with QT and buffer at pH 11 for 90 seconds. Gel mixes were weighed to provide equal molar thiols and acrylate groups, changing the present acrylate‐bearing compounds wt % ratios from 100 PPODA: 0 IPEGA, 90:10, 80:20, 70:30, 60:40, 50:50, and 0:100. Formulations with 10% and above of IPEGA were X‐ray visible. Rheology showed that increasing the amount of IPEGA decreased the storage. Kinetic FT‐IR studies indicate that the amphiphilic nature of the PEG backbone increased the reaction rate of the phase segregated reactants. Second order reaction constant modeling showed a change in initial reaction rate from 0.0029 to 0.0187 (M sec)−1 from the 10% to 50% IPEGA formulations respectively. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010
Vertebral morphology in aromatase inhibitor–treated males with idiopathic short stature or constitutional delay of puberty
Authors: Hero Matti , Toiviainen-Salo Sanna , Wickman Sanna , Mäkitie Outi , Dunkel Leo
Published: February 2010

Aromatase inhibitors (AIs), blockers of estrogen biosynthesis, delay bone maturation and therefore are used increasingly to promote growth in children and adolescents with growth disorders. The effects of treatment on skeletal health are largely unknown. Since estrogen deficiency is associated with various detrimental skeletal effects, we evaluated in this cross-sectional posttreatment study vertebral body morphology, dimensions and endplates, and intervertebral disks by the use of magnetic resonance imaging (MRI) in two cohorts of males previously treated with the AI letrozole or placebo. Males with idiopathic short stature received treatment with letrozole or placebo for 2 years during prepuberty or early puberty; males with constitutional delay of puberty received letrozole or placebo in combination with low-dose testosterone for 1 year during early or midpuberty. In males with idiopathic short stature, mild vertebral body deformities were found in 5 of 11 (45%) letrozole-treated subjects, whereas in the placebo group no deformities were detected (p = .01). In the cohort of males with constitutional delay of puberty, a high prevalence of endplate and intervertebral disk abnormalities was observed in both the letrozole- and the placebo-treated groups. We conclude that AI therapy during prepuberty or early puberty may predispose to vertebral deformities, which probably reflect impaired vertebral body growth rather than impaired bone quality and compression fractures. If AIs are used in growth indications, follow-up of vertebral morphology is indicated. © 2010 American Society for Bone and Mineral Research
Evidence that obesity does not influence the vitamin D-endocrine system in blacks
Authors: Epstein Sol , Dr. Bell Norman H. , Shary Judith , Shaw Sheryl , Greene Anne , Oexmann Mary Joan
Published: February 2010

As compared to nonobese white men and women, age-matched nonobese black subjects and obese white individuals show alterations in the vitamin D–endocrine system that are characterized by increases in mean serum immunoreactive parathyroid hormone (PTH), serum 1,25-dihydroxyvitamin D [1,25-(OH)2D], and urinary cyclic adenosine 3,5-monophosphate (cAMP) and by decreases in mean serum 25-hydroxyvitamin D (25 OHD) and in urinary calcium. Thus, both groups show secondary hyperparathyroidism which is associated with increased renal tubular reabsorption of calcium and increased renal synthesis of 1,25-(OH)2D. In view of these findings, studies were conducted in 10 obese black subjects (3 men and 7 women) and in 12 nonobese black individuals (7 men and 5 women), ranging in age from 20 to 35 yr, to determine whether obesity influences the vitamin D–endocrine system in blacks. Body weight averaged 99 ± 4 kg in the obese and 73 ± 3 kg in the nonobese subjects (p < .001). All of them were hospitalized on a metabolic ward and were given a constant daily diet containing 400 mg of calcium, 900 mg of phosphorus, 110 meq of sodium, 65 meq of potassium, and 18 meq of magnesium. Whereas mean serum Gla protein was significantly higher in the obese than in the nonobese black subjects (23 ± 4 vs. 14 ± 2 ng/ml, p < .05), mean serum total calcium, ionized calcium, phosphorus, magnesium, immunoreactive PTH by two different radioimmunoassays (399 ± 23 vs. 312 ± 26 pg/ml and 357 ± 26 vs. 392 ± 27 pg/ml), 25 OHD (5 ± 1 vs. 6 ± 1 ng/ml) and 1,25(OH)2D (43 ± 3 vs. 41 ± 3 pg/ml) were the same in the two groups. Also, mean urinary calcium (105 ± 13 vs. 101 ± 14 mg/d), phosphorus, sodium, potassium, magnesium and cAMP (3.12 ± 0.24 vs. 3.11 ± 0.47 nM/dl GF) and creatinine clearance were the same in the obese and nonobese black men and women. The results indicate that obesity does not produce alterations in the vitamin D–endocrine system that are over and above the changes present in nonobese black men and women.
The effect of ipriflavone (TC-80) on bone resorption in tissue culture
Authors: Tsuda Masao , Kitazaki Tadashi , Ito Toshio , Fujita Takuo
Published: February 2010

The fetal long bone culture system developed by Raisz for the assessment of bone resorption was modified to improve the sensitivity, by using radii and ulnae separately, based on the observation of the consistently higher release of 45Ca from the radii than ulnae. Effects of ipriflavone (TC-80), an isoflavonoid derivative currently under clinical trial for its effect on osteoporosis, on bone resorption were examined in this new system. Ipriflavone and its metabolites (5 out of 6) at 10 μg/ml or more inhibited basal 45Ca release from bones. The inhibitory effects were still demonstrated in the presence of submaximal concentration of parathyroid hormone (12.5 ng/ml). The effect of ipriflavone on bone resorption was apparently not due to its toxicity on bone cells, since the inhibition was reversible.
Clonal differences in prostaglandin synthesis among osteosarcoma cell lines
Authors: Dr. Rodan Sevgi B. , Wesolowski Gregg , Rodan Gideon A.
Published: February 2010

This study compares the metabolism of [14C]-arachidonic acid between PGE2 synthesizing (ROS 17/2.8) and nonsynthesizing (ROS 25/1) osteosarcoma cell lines. In both cell lines: (a) 90% of [14C]-arachidonic acid was taken up at 24 h. (b) More than 90% of the label was associated with phospholipids. (c) [14C]-arachidonic acid was rapidly taken up by phosphatidylcholine which reached the highest specific activity around 5 h while the labeling of other phospholipids was still increasing at 24 h. (d) Twenty-four hours after addition of [14C]-arachidonic acid only 4% of the label was associated with triacylglycerols in ROS 25/1 and 0.3% in ROS 17/2.8 cells. The calcium ionophore A23187 enhanced the release of [14C]-arachidonic acid from phospholipids in the PGE synthesizing osteoblastic cells (ROS 17/2.8 and 2/3) but had no effect in nonosteoblastic cells (ROS 24/1 and 25/1). ROS 17/2.8 and 2/3 cells converted the released arachidonic acid as well as exogeneously added arachidonic acid into PGE2. PGE2 synthesis depended on arachidonic acid concentration.

Among bone resorbing agents, parathyroid hormone and 1,25(OH)2D3 had no effect on PGE synthesis, whereas thrombin and rabbit serum stimulated PGE2 production. The effect of rabbit serum was abolished by heat inactivation.

The findings of this study indicate that the difference in PGE production between the osteoblastic and nonosteoblastic osteosarcoma cells are due mainly to differences in arachidonic acid conversion to PGE2.
Abnormal calcium metabolism caused by increased circulating 1,25-dihydroxyvitamin D in a patient with rheumatoid arthritis
Authors: Gates Sherman , Shary Judith , Turner Russell T. , Wallach Stanley , Bell Norman H.
Published: February 2010

A 35-year-old white male with rheumatoid arthritis who had developed hypercalcemia, hypercalciuria, and nephrolithiasis was found to be abnormally sensitive to vitamin D as a result of lack of regulation of circulating 1,25-dihydroxyvitamin D (1,25-(OH)2D). An increase in daily intake of vitamin D from 10 μg (400 units) per day to 50 μg (2000 units) per day produced an abnormal elevation in serum 1,25-(OH)2D, hypercalcemia, and hypercalciuria which were corrected by prednisone. Serum 25-hydroxyvitamin D initially was abnormally low, and increased with vitamin D to values which were in the low normal range. There were significant positive correlations between serum 1,25-(OH)2D (p < .05) and serum calcium and between serum 1,25-(OH)2D and urinary calcium (p < .05). Serum immunoreactive parathyroid hormone, initially in the lower range of normal, decreased further during hypercalcemia. A radiograph of the chest, gallium scan, and serum angiotensin-converting enzyme activity were normal. No granulomas or evidence of lymphoma were found in biopsies of the liver and of several lymph nodes. It is concluded that the abnormal calcium metabolism in this patient resulted from increased circulating 1,25-(OH)2D and that the defect in vitamin D metabolism was not related to sarcoidosis, other granulomatous disease, Hodgkin's disease, or lymphoma. The relationship, if any, of the abnormal metabolism of vitamin D and calcium to rheumatoid arthritis remains to be established.
Effects of human recombinant CSF-GM and highly purified CSF-1 on the formation of multinucleated cells with osteoclast characteristics in long-term bone marrow cultures
Authors: MacDonald B. R. , Mundy G. R. , Clark S. , Wang E. A. , Kuehl T. J. , Stanley E. R. , Roodman G. D.
Published: February 2010

Several studies have suggested that the osteoclast is derived from a mononuclear precursor which is found in bone marrow. We have developed a system for studying the formation of osteoclast-like multinucleated cells in long-term bone marrow culture of baboon cells. Recombinant human CSF-GM and highly purified CSF-1, both of which stimulate the proliferation of monocyte/macrophage precursors, were found to increase the number of osteoclast-like cells formed in long-term bone marrow culture. CSF-GM stimulated multinucleated cell formation more consistently than CSF-1. The subsequent addition of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to cultures initially treated with CSF-GM or CSF-1 further increased multinucleated cell formation. Autoradiographic studies indicate that CSF stimulated multinucleated cell formation by increasing the proliferation of the precursor cell, and that the potentiating effect of 1,25-(OH)2D3 was caused by fusion of the increased numbers of precursors. These studies suggest that the interaction of locally produced colony-stimulating factors with circulating calcium regulating hormones may be important in the control of osteoclast formation and bone resorption.
Characteristics of parathyroid hormone-specific cyclic changes of glucose-6-phosphate dehydrogenase activity in the distal convoluted tubule of the guinea pig
Authors: Sakaguchi Kazushige , Fukase Masaaki , Kobayashi Ikuo , Fujita Takuo
Published: February 2010

The effect of parathyroid hormone (PTH) on the time course of glucose-6-phosphate dehydrogenase (G6PD) activity in the distal convoluted tubule of a vitamin D-depleted guinea pig was determined using quantitative cytochemistry. G6PD activity decreased to the stable basal level 5 hrs after the initiation of the kidney segment maintenance cultures. The exposure of the tissues to 1 pg/ml of bovine PTH-(1–84) induced a cyclic change of G6PD activity, whereas neither carboxyl-terminal PTH nor other hormones tested showed such activity. After a 16-min exposure to bovine PTH-(1–84), the peak height of each cycle began to decrease until it disappeared at 34 min. The second exposure to this hormone at 46 min reinduced a similar cyclic change with a similar peak, indicating full viability of the cells. When bovine PTH-(1–84) was incubated with an excess amount of anti-bovine PTH antibody, the PTH-induced G6PD activity was completely abolished. Throughout a 14-min exposure to either human PTH-(1–84), human PTH-(1–34) or bovine PTH-(1–84), similar cyclic changes were observed with the constant peak height regardless of the dose (10−16-10−12M), although the cycle length shortened progressively as the dose was increased. They were equipotent on a molar basis between the concentrations of 10−16 and 10−13M at 6 min of hormone exposure. The present data demonstrate that the cytochemical bioassay of PTH in a vitamin D-depleted animal is based on a dose-dependent difference in the time course of G6PD activity.
Frequency and partial characterization of adenylate cyclase-stimulating activity in tumors associated with humoral hypercalcemia of malignancy
Authors: Stewart Andrew F. , Insogna Karl L. , Burtis William J. , Aminiafshar Ahmad , Wu Terrence , Weir Eleanor C. , Broadus Arthur E.
Published: February 2010

Parathyroid hormone-like adenylate cyclase-stimulating activity (ACSA) has previously been identified in small numbers of tumors or tumor-conditioned tissue culture medium derived from patients or animals with humoral hypercalcemia of malignancy (HHM). We examined the frequency with which this ACSA occurred in a large group of tumor extracts derived from patients with HHM (n = 20), and compared this to three control groups: normocalcemia-associated tumors (n = 20), hypercalcemic control tumors (n = 7), and normal, nonmalignant tissue samples (n = 10). Eighteen of 20 HHM-associated tumor extracts displayed ACSA whereas only 4 of 37 controls contained detectable ACSA.

ACSA in one tumor was partially purified, using sequential extraction steps and reverse-phase, high-performance liquid chromatography. Highly purified ACSA (4800-fold) also contained potent in vitro bone-resorbing activity. The molecular weight as assessed by gel filtration was approximately 40,000 D.

These findings provide strong support for the thesis that the humoral factor which is responsible for the syndrome of HHM is a parathyroid hormone-like adenylate cyclase-stimulating protein.
Kinetics of calcitonin receptor internalization in lung cancer (BEN) and osteogenic sarcoma (UMR 106–06) cells
Authors: Findlay David M. , Martin T. John
Published: February 2010

The fate of 125I-labeled calcitonin and calcitonin receptors in BEN and UMR 106–06 cells was studied after binding of the ligand. At 37°C but not at 4°C, 125I-labeled salmon calcitonin bound to both cell types and was internalized as evidenced by increasing resistance to removal by acid pH. This process was dependent on de novo protein synthesis. Cells were pretreated with salmon calcitonin and washed with acidified buffer to release cell-surface bound hormone and to allow assessment of cell-surface receptor concentration. It was found that initially there was a temperature- and time-dependent calcitonin-induced loss of binding capacity after exposure to salmon calcitonin, suggestive of endocytosis of the calcitonin-receptor complex. In the continued presence of calcitonin, receptors were lost only at the same rate as the normal turnover of calcitonin receptors as assessed in the presence of cycloheximide. These data are consistent with the presence of two functional populations of calcitonin receptors, only one of which is induced to internalize by ligand binding.
Identification of the calcitonin receptor by chemical cross-linking and photoaffinity labeling in human cancer cell lines
Authors: Moseley J. M. , Smith P. , Martin T. J.
Published: February 2010

Two methods have been used to covalently cross-link [125I]-salmon calcitonin to its receptor on a human lung carcinoma cell line, BEN, and the human breast cancer cell lines T47D and MCF 7. The first method was to use a specific photoaffinity derivative of salmon calcitonin and the second employed the chemical cross-linker, disuccinimidyl suberate. In both cases a cross-linked component of approximate molecular weight 80–90,000 on BEN cells was identified by polyacrylamide gel electrophoresis. This is consistent with the size of the cross-linked component found on T47D breast cancer cells using the photoactive salmon calcitonin as described in previous work. Disuccinimidyl suberate was unable to cross-link [125I]-salmon calcitonin either on T47D or MCF cells. However, photoactive salmon calcitonin cross-linked to a component of approximately 80–90,000 Mr on the MCF 7 cells. Thus, whereas the photoactive salmon calcitonin could cross-link a similar receptor component in all cell lines, the ability of disuccinimidyl suberate to do so was apparently cell specific. These data confirm that the calcitonin receptor comprises a component of approximately 85,000 Mr in cell lines examined thus far.
Vitamin D status and brush border membrane vesicles: 1,25-dihydroxyvitamin D3 induced destabilization
Authors: Putkey J. A. , Nemere I. , Dr. Norman A. W.
Published: February 2010

Purified chick duodenal brush border membrane vesicles (BBMV) were used to assess the effect of vitamin D on intestinal Ca2+ transport and membrane stability. BBMV preparations are right-side-out as judged by a nine-fold increase in accessibility of lactoperoxidase to core material actin in the presence of Triton X-100. Freshly prepared BBMV from vitamin D-deficient chicks support both sodium-dependent glucose transport and Ca2+ uptake. In vivo treatment with 1,25(OH)2D3 results in an 85% increase in the Vmax of Ca2+-uptake from 2.2 to 3.9 nmol/min/mg protein. The Km of Ca2+-uptake (0.9 mM) is independent of the vitamin D status of the chick. The majority of BBMV derived from vitamin D-replete chicks were destabilized and rendered incapable of supporting either sodium-dependent glucose uptake or Ca2+ uptake if they were held at 0–4°C for 2 to 24 h. In 40 separate experiments, 80% of membranes derived from vitamin D-replete chicks showed characteristics of destabilization, whereas only 24% of all control membranes exhibited a lack of viability.
Comparison of multinucleated cells elicited in rats by particulate bone, polyethylene, or polymethylmethacrylate
Authors: Dr. Glowacki J. , Jasty M. , Goldring S.
Published: February 2010

Osteoclasts, the multinucleated resorbing cells of bone, are identified by their characteristic morphology, unique cell membrane specializations, and more recently by the presence of cell surface antigens recognized by monoclonal antibodies. They are derived from mononuclear precursor cells of hematogenous origin. The precise relationship between osteoclasts and other types of tissue giant cells is unknown. This study was designed to examine factors involved in the recruitment and differentiation of multinucleated cells and to investigate the relationship between so-called foreign body giant cells and bone-resorbing osteoclasts.

Particles of various materials were implanted into subcutaneous pockets in rats. Histological, histochemical, and electron microscopic evaluations were made of specimens harvested 12 days after implantation. Large, foamy multinucleated cells were evident around particles of devitalized bone, polyethylene, and polymethylmethacrylate. Bone particles showed scalloped surfaces and were partially resorbed. Those cells adjacent to the bone particles stained positive for tartrate-resistant acid phosphatase, in contrast to the multinucleated cells adjacent to the other materials. All the cell types had extensive rough endoplasmic reticulum, abundant mitochondria, cytoplasmic vacuoles and dense bodies, giant centrospheres, and areas of fusion of plasma membranes. Cells in lacunae on the surface of the bone particles showed a clear zone of attachment to the bone substrate and ruffled borders, a feature characteristic of in osso osteoclasts. Although the giant cells occasionally displayed an extensive clear zone of attachment to the polyethylene and polymethylmethacrylate particles, no ruffled borders were detected.

The results of these studies show that the multinucleated cells elicited in response to different materials, although sharing many common features, do demonstrate certain features that are substrate specific. This model can be used to investigate the relationship between osteoclasts and other types of multinucleated giant cells and to help elucidate factors involved in osteoclast recruitment, differentiation, and regulation.
Isolation and characterization of highly serum-dependent cells released early from collagenase digested calvaria
Authors: Wong Glenda L. , Frantz K. , Lam C.
Published: February 2010

A subclass of highly serum-dependent bone cells has been identified among the cells released early from calvaria following digestion in collagenase. Partial purification for these cells has been carried out based on the observation that they require serum for attachment to polystyrene culture flasks. This subclass of bone cells differs from adherent cells and late released osteoblasts, in that they express almost no cAMP response to PTH, require high levels of serum (10%) for initial growth and proliferation, and do not increase DNA synthesis in response to PTH. In common with adherent cells and late released osteoblasts, their proliferation is decreased by 1,25(OH)2D3 at doses above 0.2 ng/ml and they respond to PGE2 with increased DNA synthesis. These similarities suggest an ontogenic relationship with osteoblasts. Based on their differences, however, provisional identification of these cells as relatively undifferentiated mesenchymal cells is suggested.
Redox studies of the epiphyseal growth cartilage: Pyridine nucleotide metabolism and the development of mineralization
Authors: Kakuta Saburo , Golub Ellis E. , Haselgrove John C. , Chance Britton , Frasca Peter , Dr. Shapiro Irving M.
Published: February 2010

The objective of this investigation was to examine the redox status of chondrocytes in normal and rachitic growth cartilages and to relate energy metabolism to cell maturation and the initiation of mineralization. The redox status was evaluated by chemical analysis and by microfluorimetric scanning of rapidly frozen, freeze-fractured tibial growth cartilages. In the normal epiphysis, the redox pattern of both avian and lagomorph cartilages were very similar. Thus, in the proliferative tissue zone the NAD/NADH ratio was high; in the hypertrophic zone, the cells appeared to be reduced. The sharp border between the two zones suggested that the redox shift may be associated with development of hypoxia. Induction of rickets resulted in a fivefold decrease in the total concentration of pyridine nucleotides in the proliferating and hypertrophic zones. Furthermore, the NAD/NADH ratio was profoundly disturbed. In the mineralizing zone, there was an accumulation of reduced pyridine nucleotide. Healing, initiated by administration of vitamin D to the rachitic birds, caused a rapid increase in NAD and NADH in all zones of the growth cartilage. It was concluded that vitamin D deficiency leads to changes in the energy metabolism of growth cartilage and that these changes were related to the defective mineralization of the rachitic tissue.
Effect of 1,25-dihydroxyvitamin D3 on mouse mammary tumor (GR) cells: Evidence for receptors, cellular uptake, inhibition of growth and alteration in morphology at physiologic concentrations of hormone
Authors: Gross Myron , Kost Susan Bollman , Ennis Bruce , Stumpf Walter , Kumar Rajiv
Published: February 2010

Mammary glands are target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We have examined a mouse mammary tumor cell line (GR) for receptors of 1,25(OH)2D3 and have examined alterations in the growth and morphology of these cells in response to 1,25(OH)2D3. GR cells contain a high affinity (Kd ∼ 10−11), lowcapacity receptor with a high specificity for 1,25(OH)2D3. The 1,25(OH)2D3 receptor in GR cells has a sedimentation coefficient of 3.5 and elutes from DEAE cellulose columns with ∼ 0.15 M KCI. These properties of the receptor are similar to those reported for other 1,25(OH)2D3 receptors. 1,25(OH)2D3 is internalized by GR cells in situ and specifically bound 1,25(OH)2D3 is found predominantly, if not entirely, in the nucleus as determined by cell fractionation and autoradiographic techniques. The incubation of GR cells in culture for 7 days with 1,25(OH)2D, markedly alters cell growth. Cell growth is retarded in a dose-dependent manner; physiologic concentrations (10−10M) of l,25(OH)2D3 retard cell growth by approximately 50%. In addition, GR cells incubated with 10−9 to 10−8M 1,25(OH)2D3 undergo marked morphological changes. The incubation of GR cells with other vitamin D metabolites such as 25-hydroxyvitamin D3 (25(OH)D3) at a concentration of 10−9M does not significantly alter cell growth or morphology. The presence of high affinity receptors for 1,25(OH)2D3, the specific internalization of 1,25(OH)2D3 predominantly into the nuclei, and the significant effects of physiological concentrations of 1,25(OH)2D3 on cell growth suggest a direct, specific, nuclear effect of 1,25(OH)2D3 on GR cells. The mouse mammary tumor model might be useful in examining the effect of 1,25(OH)2D3 on tumor formation.
Parathyroid cell surface autoantibodies that inhibit parathyroid hormone secretion from dispersed human parathyroid cells
Authors: Posillico James T. , Wortsman Jacobo , Srikanta Sath , Eisenbarth George S. , Mallette Lawrence E. , Brown Edward M.
Published: February 2010

Serum autoantibodies directed toward antigenic determinants on the surface of human parathyroid cells (PTAb-CS) have been demonstrated in a subset (8 of 23) of adult patients with idiopathic hypoparathyroidism (IHP). In sera from 3 of 8 patients with PTAb-CS, binding of these autoantibodies to their respective parathyroid cell surface antigen(s) resulted in marked inhibition of parathyroid hormone (PTH) secretion in an in vitro dispersed human parathyroid cell (dPTC) system. In 1 subject evaluated longitudinally, circulating levels of PTAb-CS, and the magnitude of the inhibitory effect on PTH secretion, temporally correlated with the clinical course of the hypoparathyroidism. These findings suggest a causative role for antibodies directed against cell surface antigens in parathyroid dysfunction in some cases of “autoimmune” hypoparathyroidism.
Effect of vitamin D metabolites on the expression of alkaline phosphatase activity by epiphyseal hypertrophic chondrocytes in primary cell culture
Authors: Hale Laura V. , Kemick Mary Lynn S. , Dr. Wuthier Roy E.
Published: February 2010

The effects of three vitamin D3 metabolites, 25-hydroxyvitamin D3 (25-(OH)D3), 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), and 24R,25-dihydroxyvitamin D3 (24R,25-(OH)2D3) on the activity of alkaline phosphatase (AP), a key enzyme involved in biomineralization, have been studied in primary cultures of chicken epiphyseal growth plate chondrocytes. Dosages of 1α,25-(OH)2D3 (10−12 to 10−7M) caused a progressive, dosage-and time-dependent decrease in cellular AP levels, IC50 occurring at approximately 10−12M. In contrast, 24R,25-(OH)2D3 at 10−13 to 10−10M stimulated cellular AP activity, half-maximal stimulation occurring at about 10−13M. At higher levels (10−10 to 10−7M), 24R,25-(OH)2D3 caused progressive reduction in AP activity. Maximal effects of 24R,25-(OH)2D3 were evident 48 h after administration of the metabolite. 25-(OH)D3 initially (24 h) caused a weak, dosage-dependent decrease in cellular AP activity, but after 48–72 h, low levels (10−13 to 10−11M) caused a dosage-dependent increase in AP activity. Higher levels of 25-(OH)D (> 10−10M) were clearly inhibitory to AP. These findings reveal that the AP activity of growth plate chondrocytes is exquisitely sensitive to both 1α,25- and 24R,25-(OH)2D3 but the response to each is in opposite directions. The paradoxical response of the cells to 25-(OH)D3 can be explained if the metabolite is slowly metabolized by a 24-hydroxylase to 24R,25-(OH)2D3 leading to stimulation of cellular AP. The inhibitory effects of all metabolites at higher levels (10−8 to 10−7M) may be explained if both 25-(OH)D3 and 24R,25-(OH)2D3 act as weak agonists for the 1α,25-(OH)2D3 receptor, mimicking its inhibitory action. The data show that vitamin D metabolites exert a direct effect on the growth plate chondrocytes. Studies are in progress to elucidate the mechanism of this AP response.
Differential effects of 1,25-dihydroxyvitamin D3 on cytosolic calcium in two human cell lines (HL-60 and U-937)
Authors: Desai Sanjay S. , Appel Michael C. , Dr. Baran Daniel T.
Published: February 2010

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to induce maturational changes in the human promyelocytic leukemia cell line HL-60 and in the human monocytic cell line U-937. Changes in cytosolic calcium have been reported to regulate cellular processes. We used the fluorescent dye Quin 2 to examine the effects of vitamin D metabolites on cytosolic calcium levels in HL-60 and U-937 cells. 1,25-(OH)2D3 (20 nM) increases cytosolic calcium by 24% over a 5-min period in HL-60 but not in U-937 cells. 1,25-(OH)2D3 (0.2 nM and 4 nM) has no effect on cytosolic calcium levels in either cell type. 24,25-(OH)2D3 (20 nM) has no effect on cytosolic calcium in HL-60 cells. Nifedipine (1 m M) has no effect on cytosolic calcium levels over 30 min and likewise does not block the 1,25-(OH)2D3-induced increase in cytosolic calcium in HL-60 cells. However, chelation of extracellular calcium with EGTA (10 m M) blocks the 1,25-(OH)2D3-induced increment in cytosolic calcium, but does not block the 1,25-(OH)2D3-induced maturational changes in HL-60 cells. The data suggest that 1,25-(OH)2D3 but not 24,25-(OH)2D3 increases cytosolic calcium in HL-60 cells within 5 min and the increment is due to increased influx of calcium. 1,25-(OH)2D3 modifies membrane permeability to calcium independent of calcium channels sensitive to nifedipine. Finally, 1,25-(OH)2D3-induced maturational changes in HL-60 cells can take place without an increase in cytosolic calcium.
Isolation of EGF-dependent transforming growth factor (TGFβ-like) activity from culture medium conditioned by fetal rat calvariae
Authors: Centrella Michael , Canalis Ernesto
Published: February 2010

A transforming growth factor of the beta class (TGF-β), defined by its ability to induce normal rat kidney cells (NRK, clone 49F) to form anchorage-independent large colonies in soft agar in the obligate presence of epidermal growth factor, has been prepared from culture medium conditioned by fetal rat calvariae. This activity was purified by acetic acid extraction, gel permeation chromatography, and two reversed-phase HPLC (rpHPLC) steps. Bone culture derived-TGFβ-like activity was soluble in 1.0 M acetic acid, eluted from Sephadex G-75 at relative molecular mass (Mr) 25,000, from μBondapak C18 rpHPLC at 63 ± 5% methanol in 0.1 M acetic acid, and from μBondapak CN rpHPLC at 36 ± 2% n-propanol in 0.1% trifluoroacetic acid. Based on specific activity estimations at each stage of purification, TGFβ-like activity was purified 2500-fold with a 14% recovery, and 1 I of conditioned medium yielded 1–2 μg of factor. Silver-stained polyacrylamide gels of this material after CN μBondapak rpHPLC revealed a predominant band of (Mr) 24,000.
The relative potency of a human tumor-derived PTH-like adenylate cyclase-stimulating preparation in three bioassays
Authors: Stewart Andrew F. , Wu Terence , Burtis William J. , Weir Eleanor C. , Broadus Arthur E. , Insogna Karl L.
Published: February 2010

Three bioassays are widely employed for the measurement of PTH-like adenylate cyclase-stimulating factors (ACSFs) drived from tumors associated with humoral hypercalcemia of malignancy. These include renal cortical adenylate cyclase (RAC) assays, rat osteosarcoma (ROS) adenylate cyclase assays, and fetal bone resorption (FBR) assays. A previous study has suggested that the potency of one human tumor-derived ACSF, expressed in PTH equivalents, was 30-fold higher in the ROS assay than in the RAC assay, but no study has directly compared all three bioassays using a single PTH standard and a single ACSF preparation.

We compared one partially purified ACSF preparation to a single lot of bPTH 1–34 in all three bioassays. The results indicate that the relative potency of this ACSF as compared to the PTH standard varied with the assay employed, with the ROS assay yielding a specific activity estimate 47.5-fold higher than the RAC, and the FBR 6.7-fold higher than the RAC but 7.1-fold lower than the ROS.

These findings support the possibility that distinct subpopulations of PTH receptors exist on different PTH target tissues. Further, they underscore the importance of bioassay choice when estimating the specific activity of tumor-derived ACSF preparations.
Evidence that low plasma 1,25-dihydroxyvitamin D causes intestinal malabsorption of calcium and phosphate in juvenile X-linked hypophosphatemic mice
Authors: Meyer Ralph A. , Meyer Martha H. , Gray Richard W. , Bruns M. Elizabeth
Published: February 2010

X-linked hypophosphatemic (Hyp) mice are a model for human sex-linked vitamin D-resistant rickets. We have reported intestinal malabsorption of calcium in young Hyp mice, and in this report we have explored the mechanism for it. To test for resistance of the intestine to 1,25(OH)2 vitamin D3, this hormone was continually infused via osmotic minipumps into 4-week-old normal and Hyp mice at 0,17, 50 or 150 ng/kg/day. After 3 days, 45Ca and inorganic 32P were administered by gavage, and the mice were sacrificed on the fifth day. The Hyp mice showed responses to the hormone equivalent to the normal mice in terms of increased intestinal absorption of both 45Ca and 32P, increased plasma isotope levels, increased femoral isotope content, and increased duodenal and renal 9 kD vitamin D-dependent calcium-binding protein (calbindin-D9K; CaBP). Plasma 1,25(OH)2D was measured in these mice. There were significant correlations of plasma 1,25(OH)2D to the intestinal absorption of 45Ca and 32P and to duodenal and renal CaBP. Plasma 1,25(OH)2D was also measured in stock normal and Hyp mice and was found to be lower in 4-week-old Hyp mice than in 4-week-old normal mice (113 ± 10 pM (n = 18) vs. 67 ± 10 (n = 20), normal vs. Hyp, p < .01), but unchanged at 13 weeks of age (77 ± 13 (n = 13) vs. 70 ± 15 (n = 15), NS). This observed difference in plasma 1,25(OH)2D between normal and Hyp mice at 4 weeks of age was sufficient to explain the observed normal-to-Hyp differences in intestinal absorption of 45Ca and duodenal and renal CaBP. It also explained 72 ± 18% of the observed difference in 32P absorption. We conclude that Hyp mouse intestine is not resistant to 1,25(OH)2D and that the lower plasma 1,25(OH)2D of 4-week-old Hyp mice causes intestinal malabsorption of calcium and phosphate.
The effects of ovariectomy and 17β-estradiol on cortical bone histomorphometry in growing rats
Authors: Turner Russell T. , Vandersteenhoven Jacob J. , Bell Norman H.
Published: February 2010

The effects of ovariectomy for four weeks and of 17β-estradiol for three weeks on histomorphometry of the tibial diaphysis were determined in young rats. The effects of ovariectomy on histomorphometry of subcutaneous implants of demineralized bone matrix were also examined. Groups of young female rats were either ovariectomized or sham operated. After surgery, the animals were weight matched and pair fed. Despite the same caloric intake, ovariectomized rats grew more rapidly than pair-fed, sham-operated controls but were significantly heavier at sacrifice in only one of three experiments. Ovariectomy did not change mean serum calcium, phosphate, 25-hydroxyvitamin D (25-OHD), or 1,25-dihydroxyvitamin D [1,25(OH)2D] but significantly lowered mean serum magnesium. Serum estradiol was not detectable in ovariectomized animals. 17β-Estradiol in ovariectomized animals significantly increased mean serum estradiol and lowered mean serum phosphate but did not change mean serum calcium, magnesium, 25-OHD, or 1,25(OH)2D, as compared to values in sham-operated controls. Bone formation rate was significantly enhanced in ovariectomized animals at both the endosteal and periosteal surfaces of the tibial diaphysis as compared to values in sham-operated controls. The increase in bone formation rate was reversed by 17β-estradiol at the periosteal but not endosteal surface. Ovariectomy increased the bone apposition rate, mineralization rate, and osteoid thickness of the tibial diaphysis. These increases were reversed by 17β-estradiol. In implants, ovariectomy increased the resorption of implant matrix and enhanced the formation of new matrix. Ovariectomy resulted in increases in forming surface and resorbing surface in the implants. The results of these short-term studies indicate (a) that ovariectomy in growing rats leads to increases in both bone formation and resorption, and (b) that these increases are reversed by treatment with 17β-estradiol.
Incidence of hip fractures in osteoporotic women treated with sodium fluoride
Authors: Riggs B. Lawrence , Baylink David J. , Kleerekoper Michael , Lane Joseph M. , Melton L. Joseph , Meunier Pierre J.
Published: February 2010

It has been suggested recently that, although fluoride therapy may decrease the occurrence of vertebral fractures, it could increase the risk of hip fractures. To evaluate this possibility, we combined retrospective data from five medical centers that have had a large experience with this therapeutic regimen. In 416 osteoporotic patients who were followed for more than 1,000 patient-years of fluoride treatment, there were 17 nontraumatic hip fractures. This incidence of 1.6% per year is similar to the incidence, 1.9% per year, for 120 of the patients in this series who had been followed prospectively for 3 years prior to initiation of fluoride therapy. The expected incidence for women of the same age in the general community is 0.5% per year. Thus, untreated osteoporotic women are at increased risk for hip fracture, but treatment with fluoride seems neither to decrease nor to increase the incidence of hip fracture substantially.
A tissue-specific protein in rat osteogenic tissues
Authors: Mardon Helen J. , Dr. Triffitt James T.
Published: February 2010

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and Polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.