bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal dynamics of myelination in the zebrafish spinal cord
Authors: Buckley Clare E. , Marguerie Anita , Alderton Wendy K. , Franklin Robin J. M.
Published: February 2010

Knowledge of the precise timing of myelination is critical to the success of zebrafish‐based in vivo screening strategies for potential remyelination therapies. This study provides a systematic review of the timing of myelination in the zebrafish spinal cord and a critique of techniques by which it may be accurately assessed. The onset of myelination was found to be 3 days postfertilization (d.p.f.); earlier than previously reported. This coincided with the dorsal migration and differentiation of oligodendrocytes and the expression of myelin basic protein (Mbp) transcripts and protein. Our data suggests that immunohistochemistry with zebrafish‐specific anti‐Mbp from 3 d.p.f. is the optimal histological method for myelin visualization, while quantification of myelination is more reliably achieved by quantitative polymerase chain reaction (qPCR) for mbp from 5 d.p.f.. Transgenic fluorescent lines such as olig2:EGFP can be used to assess oligodendrocyte cell number at 3 d.p.f. and the development of new, more specific lines may enable real time visualization of myelin itself. Quantitative ultrastructural analysis revealed that the myelination of zebrafish axons is regulated according to axonal growth and not absolute axonal size. This study confirms the use of the zebrafish larvae as a versatile and efficient in vivo model of myelination and provides a platform on which future myelination screening studies can be based. © 2010 Wiley‐Liss, Inc.
Causality Assessment in Drug-Induced Liver Injury Using A Structured Expert Opinion Process: Comparison to the Roussel-Uclaf Causality Assessment Method
Authors: Rockey Don C. , Seeff Leonard B. , Rochon James , Freston James , Chalasani Naga , Bonacini Maurizio , Fontana Robert J. , Hayashi Paul H.
Published: February 2010

Drug-induced liver injury (DILI) is largely a diagnosis of exclusion and is therefore challenging. The US Drug-Induced Liver Injury Network (DILIN) prospective study used two methods to assess DILI causality: a structured expert opinion process and the Roussel-Uclaf Causality Assessment Method (RUCAM). Causality assessment focused on detailed clinical and laboratory data from patients with suspected DILI. The adjudication process used standardized numerical and descriptive definitions and scored cases as definite, highly likely, probable, possible, or unlikely. Results of the structured expert opinion procedure were compared with those derived by the RUCAM approach. Among 250 patients with suspected DILI, the expert opinion adjudication process scored 78 patients (31%) as definite, 102 (41%) as highly likely, 37 (15%) as probable, 25 (10%) as possible, and 8 (3%) as unlikely. Among 187 enrollees who had received a single implicated drug, initial complete agreement was reached for 50 (27%) with the expert opinion process and for 34 (19%) with a five-category RUCAM scale (

P

= 0.08), and the two methods demonstrated a modest correlation with each other (Spearman's

r

= 0.42,

P

= 0.0001). Importantly, the RUCAM approach substantially shifted the causality likelihood toward lower probabilities in comparison with the DILIN expert opinion process.

Conclusion

: The structured DILIN expert opinion process produced higher agreement rates and likelihood scores than RUCAM in assessing causality, but there was still considerable interobserver variability in both. Accordingly, a more objective, reliable, and reproducible means of assessing DILI causality is still needed. Hepatology 2010
Novel Mechanism of Fetal Hepatocyte Injury in Congenital Alloimmune Hepatitis Involves the Terminal Complement Cascade
Authors: Pan Xiaomin , Kelly Susan , Melin-Aldana Hector , Malladi Padmini , Whitington Peter F.
Published: February 2010

Evidence suggests that most neonatal hemochromatosis (NH) is the phenotypic expression of gestational alloimmune fetal liver injury. Gestational alloimmune diseases are induced by the placental passage of specific reactive immunoglobulin G and often involve the activation of fetal complement by the classical pathway leading to the formation of membrane attack complex (MAC) as the effector of cell injury. We examined liver specimens from cases of NH, from cases of non-NH liver disease, and from infants without liver disease to determine if they would provide evidence that MAC is involved in hepatocyte injury. Sections were immunostained with anti-human C5b-9 complex, the terminal complement cascade (TCC) neoantigen formed in the assembly of MAC. Fetal liver injury in NH cases is associated with a severe loss of hepatocytes. In all NH cases examined, most remaining hepatocytes showed intense staining for TCC neoantigen, whereas hepatocytes in non-NH liver disease cases showed variable light staining. The percentage of hepatocytes containing TCC neoantigen in NH was much greater than that in non-NH liver disease, and there was no overlap between the groups. Findings in both groups suggest that hepatocytes have mechanisms to protect against MAC, including a biliary pathway for its excretion.

Conclusion:

The finding that all cases of proven NH contained TCC neoantigen far in excess of cases of other neonatal liver diseases suggests that a single process, namely congenital alloimmune hepatitis, is the principal cause of NH. MAC-mediated alloimmune injury in congenital alloimmune hepatitis is a novel mechanism of liver injury that results from an interplay of maternal adaptive immunity and fetal innate immunity. Hepatology 2010
The type 2 deiodinase Thr92Ala polymorphism is associated with increased bone turnover and decreased femoral neck bone mineral density
Authors: Heemstra Karen A , Hoftijzer Hendrieke , van der Deure Wendy M , Peeters Robin P , Hamdy Neveen A , Pereira Alberto , Corssmit Eleonora P , Romijn Johannes A , Visser Theo J , Smit Johannes W
Published: February 2010

The role of type 2 deiodinase (D2) in the human skeleton remains unclear. The D2 polymorphism Thr92Ala has been associated with lower enzymatic activity, which could result in lower local triiodothyronine (T3) availability in bone. We therefore hypothesized that the D2 Thr92Ala polymorphism may influence bone mineral density (BMD) and bone turnover. We studied 154 patients (29 men, 125 women: 79 estrogen-replete, 46 estrogen-deficient) with cured differentiated thyroid carcinoma. BMD and bone turnover markers [bone-specific alkaline phosphatase (BAP), cross-linking terminal C-telopeptide of type I collagen (CTX), procollagen type 1 amino-terminal propeptide (P1NP), and cross-linked N-telopeptide of type I collagen (NTX)] were measured. Effects of the D2 Thr92Ala polymorphism on BMD and bone turnover markers were assessed by a linear regression model, with age, gender, estrogen state, body mass index (BMI), serum calcium, 25-hydroxyvitamin D, parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), and free triiodothyroxine (T4) as covariables. Sixty patients were wild type (Thr/Thr), 66 were heterozygous (Thr/Ala), and 28 were homozygous (Ala/Ala) for the D2 polymorphism. There were no significant differences in any covariables between the three genotypes. Subjects carrying the D2 Thr92Ala polymorphism had consistently lower femoral neck and total hip densities than wild-type subjects (p = .028), and this was accompanied by significantly higher serum P1NP and CTX and urinary NTX/creatinine levels. We conclude that in patients with cured differentiated thyroid carcinoma, the D2 Thr92Ala polymorphism is associated with a decreased femoral neck BMD and higher bone turnover independent of serum thyroid hormone levels, which points to a potential functional role for D2 in bone. © 2010 American Society for Bone and Mineral Research
Enhancement of parathyroid hormone-responsive renal cortical adenylate cyclase activity by a cytosol protein activator from rat reticulocytes
Authors: Dr. Shane Elizabeth , Avioli Richard C. , Greene Virginia S. , Yeh Mary , Owens John M. , Bell Norman H. , Bilezikian John P.
Published: February 2010

The effects of the cytosol activator protein obtained from rat reticulocytes (RCAP) were investigated in a heterologous membrane system — partially purified cell membranes from dog renal cortex. RCAP enhanced the response of dog renal cortical adenylate cyclase to bovine parathyroid hormone (1–34) [bPTH (1–34)] from two- to three-fold. RCAP also enhanced the response to 5 μM arginine vasopressin, 10 μM glucagon, and 10 μM isoproterenol. Analysis of double-reciprocal plots of substrate concentration and enzyme activity indicated that bPTH (1–34) alone and together with RCAP increased the Vmax of the adenylate cyclase enzyme and did not alter the apparent Km of the enzyme for MgATP. Membranes from dog renal cortex contain 42K and 39K proteins that are ADP-ribosylated by cholera toxin and pertussis toxin, respectively, and appear to be the stimulatory (Ns) and inhibitory (Ni) guanine nucleotide binding proteins described in many other hormone-responsive membrane preparations. Similar to its effects in rat reticulocytes, RCAP inhibited ADP-ribosylation of Ns and enhanced ADP-ribosylation of Ni. The muscarinic agonist, carbachol, inhibited PTH-responsive adenylate cyclase activity in dog renal cortical membranes and this inhibition was reversed by RCAP. These results indicate that RCAP enhances stimulation of adenylate cyclase by a variety of hormones in a heterologous membrane preparation and supports the hypothesis that RCAP's site of action is common to all adenylate cyclase systems. RCAP may facilitate coupling between Ns and the catalytic unit of adenylate cyclase by a pertussis toxin-like effect to inactivate Ni. The dual effects of RCAP upon ADP-ribosylation of Ni and Ns α subunits suggest that a binding site for RCAP may exist at a site of homology between NSα and Niα.
Polyamines are involved in the 1-α,25-dihydroxyvitamin D3-induced fusion of mouse alveolar macrophages
Authors: Hayashi Takamune , Shinki Toshimasa , Tanaka Hirofumi , Abe Etsuko , Suda Tatsuo
Published: February 2010

We have reported that 1-α,25-dihydroxyvitamin D3 [1-α,25-(OH)2D3] directly induces fusion of mouse alveolar macrophages at a very high rate (circa 70–80%) by a mechanism involving protein synthesis (Proc. Natl. Acad. Sci. USA 80:5583, 1983; FEBS Letters 174:61, 1984). While examining further the mechanism of the 1-α,25-(OH)2D3-induced fusion of macrophages, we found that polyamines are involved in this mechanism. Mouse alveolar macrophages incubated with 12 nM 1-α,25-(OH)2D3 began to fuse at 36 h and the fusion rate increased linearly up to 60 h. Addition of as much as 0.05–5 mM α-difluoromethylornithine (α-DFMO), a specific inhibitor of ornithine decarboxylase, did not inhibit fusion appreciably, but addition of 0.05–5 μM methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, strikingly inhibited fusion. When macrophages were treated with both 12 nM 1-α,25-(OH)2D3 and 5 μM MGBG for the first 12 h and incubated further for 60 h in fresh medium containing 1-α,25-(OH)2D3, fusion was significantly inhibited, suggesting that the 1-α,25-(OH)2D3-induced synthesis of polyamines precedes fusion. The inhibition by MGBG of the 1-α,25-(OH)2D3-induced fusion was restored completely by adding 1 μM spermidine or spermine or 100 μM putrescine. None of the polyamines alone induced fusion. MGBG suppressed the 1-α,25-(OH)2D3-induced incorporation of [3H]-leucine into the trichloroacetic acid-insoluble fraction in macrophages, but its inhibitory effect was restored completely by adding 1 μM spermidine. When macrophages were incubated with [14C]-ornithine, the polyamine that accumulated most was [14C]-spermidine. 1-α,25-(OH)2D3 further enhanced the accumulation of [14C]-spermidine. The accumulation of [14C]-putrescine and spermine was not appreciably altered by the 1-α,25-(OH)2D3 treatment. Adding MGBG almost completely suppressed the accumulation of [14C]-spermidine and spermine, but it enhanced the accumulation of [14C]-putrescine considerably. These results indicate that spermidine or spermine is an important intracellular mediator of the 1-α,25-(OH)2D3 action in inducing protein synthesis, which in turn somehow induces fusion of alveolar macrophages.
Cyclosporine a inhibits calcemic hormone-induced bone resorption in vitro
Authors: Stewart Pamela J. , Green Orville C. , Stern Paula H.
Published: February 2010

We have investigated the in vitro effects of cyclosporine (CsA), a potent immunosuppressive agent, on bone resorption induced by calcemic hormones. CsA inhibited parathyroid hormone (PTH), prostaglandin E2, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), and osteoclast-activating factor induced resorption of fetal rat limb bones in a dose-dependent manner. Established ongoing resorptive activity in bone was also inhibited by CsA. The CsA inhibition of bone resorption could be partially surmounted by higher concentrations of PTH and 1,25(OH)2D3. The inhibitory effects of CsA on limb bone resorption were reversible. Neither protein nor DNA synthesis were inhibited by treatment of limb bones with CsA. Thus, the inhibitory effect of this agent on bone resorption is not a cytotoxic one. These data could suggest that the induction of bone resorption by the calcemic hormones involves an immune cell derived mediator such as a lymphokine.
Forskolin has both stimulatory and inhibitory effects on bone resorption in fetal rat long bone cultures
Authors: Dr. Lorenzo Joseph A. , Sousa Sandra , Quinton Joy
Published: February 2010

The diterpene forskolin which increases 3′,5′-cyclic adenosine monophosphate concentrations (cAMP) in intact cells by directly activating the enzyme adenyl cyclase, was examined for its ability to alter bone resorption in fetal rat long bone cultures. After 48 h, forskolin inhibited resorption at 1.0 and 10 μ However, after 120 h, it had a small stimulatory effect at 1.0 μM and no net effect on resorption at 10 μ Isobutylmethylxanthine (IBMX), which elevates cAMP levels in cells by inhibiting the enzyme 3′,5′-cyclic adenosine monophosphate phosphodiesterase, produced a resorptive response which was slightly different from that of forskolin. After both 48 and 120 h, IBMX at 0.1 mM stimulated resorption while at 1.0 mM, it had only inhibitory effects. In bones which were stimulated to resorb with either parathyroid hormone or 1,25(OH)2 vitamin D, forskolin inhibited resorption. The inhibitory effects of forskolin on hormonally stimulated resorption were transient in cultures treated with 1.0 μM but were sustained with 10 μM. Inhibitory responses to forskolin did not appear to result from toxicity since they were completely reversed when forskolin was removed from the media.

These results imply that agents which increases 3′,5′-cyclic adenosine monophosphate concentrations in bone activate two resorptive pathways: one which is inhibitory and another which is stimulatory.
Secretion and metabolism of monomeric human calcitonin: Effects of age, sex, and thyroid damage
Authors: Tiegs Robert D. , Body Jean Jacques , Barta Joyce M. , Dr. Heath Hunter
Published: February 2010

Some data suggest that human calcitonin (CT) secretion is lower in women than in men, decreases with age and the menopause, and is absent in thyroidectomized persons. To further explore CT secretory physiology, we have studied basal and calcium-stimulated plasma immunoreactive CT (iCT) and silica-extractable monomeric CT concentrations in 148 healthy volunteers and 33 patients with a history of thyroid damage (total or subtotal thyroidectomy, radioiodine treatment for thyrotoxicosis). Both whole-plasma iCT and extractable CT levels were lower basally and after calcium infusion in women than in men, basal levels being reduced about 50% and calcium-stimulated values about 75% from those of male subjects. There were no significant changes in basal iCT or extractable CT concentrations with age, and CT secretory capacity (CT response to calcium infusion) likewise did not change with age. Infusion of monomeric CT to constant concentration in 27 persons permitted estimates of CT metabolic clearance rates (MCRs) and secretion rates (SRs). Calculated MCRs of about 9 ml/min · kg−1 (persons aged 21–30 yr) and 6 ml/min · kg−1 (persons aged 54–70 yr) were in good agreement with published data, and did not differ between the sexes. SRs were dependent upon the assay method used to estimate basal plasma CT concentrations, being highest when whole-plasma iCT values were used. Based on estimates of plasma monomeric CT from the silica extraction procedure, the SR of CT was 59 ± 6 (SE) ng/d · kg−1 in men, and 22 ± 3 ng/d · kg−1 in women. Thyroid damage reduced, but did not abolish, apparent CT immunoreactivity, even in silica extracts of plasma. However, all subsets of thyroiddamaged patients had absent-to-markedly-impaired CT secretion in response to calcium infusion. We conclude that CT secretion is substantially lower both basally and after stimulation in women than in men, and that this difference in CT immunoreactivity probably reflects differences in circulating CT bioactivity. The sex difference in plasma CT concentrations probably results from lower rates of CT secretion in women, not increased MCR. There is no age-related decrease of plasma CT concentrations (or CT secretory reserve), calling into question the concept that a progressive deficiency of CT is partly resonsible for age-related (“senile”) osteoporosis. Surgical or radiation damage to the thyroid gland commonly abolishes C-cell response to calcium; such CT-deficient patients form a population suitable for determining whether or not reduced CT secretion can impair skeletal homeostasis.
Cytochemical bioassay of circulating concentrations of rat parathyroid hormone: Application to a study of age and sex
Authors: Dr. Peng Tai-Chan , Garner Sanford C. , Hirsch Philip F. , Posillico James T.
Published: February 2010

We have found that the cytochemical bioassay (CBA) method, originally developed to measure circulating levels of biologically active parathyroid hormone (bioPTH) in humans, also can measure endogenous concentrations of PTH in rats. Therefore, we have applied this assay method to examine the relationships between age and sex and the circulating levels of bioPTH, calcium, and phosphorus in Fischer rats. The concentration of bioPTH increased in both males and females from 5 to 15 months of age (p < .001) with no significant sex-related difference. Mean bioPTH values ranged from 2.3 to 3.6 pg (human PTH equivalents)/ml; these values were much lower (1 to 2 orders-of-magnitude) than those reported by others who used radioimmunoassays for PTH that do not discriminate between biologically active and inert PTH fragments. The CBA values for bioPTH were also 1/4–1/5 those obtained in rat serum by newer, more sensitive methods, the N-terminal specific PTH radioimmunoassay and the bioassay based on stimulation of chick renal adenylate cyclase.

Regression analysis did not reveal any significant correlation between serum calcium and age. However, serum inorganic phosphorus decreased significantly (p < .001) from 5 to 15 months of age in both male and female rats and was much lower in females than in males (p < .001). The fall in serum phosphorus was negatively correlated with the rise in bioPTH (p < .001).

This study, the first to our knowledge to use a CBA to detect physiological changes in the circulating levels of bioPTH in rats, demonstrates the usefulness of the assay in experiments in this species.
The effect of oral phosphate administration on major indices of skeletal metabolism in normal subjects
Authors: Silverberg Shonni J. , Shane Elizabeth , Clemens Thomas L. , Dempster David W. , Segre Gino V. , Lindsay Robert , Dr. Bilezikian John P.
Published: February 2010

The effect of brief periods of phosphate administration on indices of human skeletal metabolism was investigated. Thirteen subjects (8 women, 5 men; 19–36 years old) received 2 g of oral phosphate daily for 5 days. Serum phosphorus rose 26% (3.8 ± 0.1 mg/dl to 4.8 ± 0.1 mg/dl; p < .01) while total calcium fell (9.3 ± 0.1 mg/dl to 8.9 ± 0.1 mg/dl; p < .01). Parathyroid hormone levels increased by 50% (14.1 ± 2.0 pg/ml to 21.5 ± 1.7 pg/ml; p < .05) although values remained within the normal range. A persistent phosphaturia (0.64 ± 0.10 g/g Cr to 1.8 ± 0.4 g/g Cr; p < .05) and a 69% fall in urinary calcium (80.8 ± 10.0 mg/g Cr to 24.6 ± 6.0 mg/g Cr; p < .001) were observed. 1,25-dihydroxyvitamin D3 and urinary hydroxyproline concentrations did not change significantly but the bone γ-carboxyglutamic acid protein (BGP) concentration rose 41% by day 2 (9.6 ± 1.3 mg/ml to 13.5 ± 2.2 mg/ml; p < .005) and remained elevated throughout the study period. These results support the possibility that brief periods of phosphate administration may be useful in the therapy of disorders associated with low bone turnover, such as osteoporosis.
The solid, calcium-phosphate mineral phases in embryonic chick bone characterized by high-voltage electron diffraction
Authors: Dr. Lee Dosuk D. , Landis William J. , Glimcher Melvin J.
Published: February 2010

The solid mineral phases of calcium-phosphate (Ca-P) in the long bones from embryonic chicks between the ages of 9 and 13 days have been examined by high voltage (1.0 MV) electron microscopy and electron micro-diffraction. The study was undertaken to identify the chemical and crystallographic nature of the inorganic mineral phase(s) prepared under conditions which significantly reduce artifacts of specimen preparation and microscopic examination of the tissues.

Electron microdiffraction patterns of solid mineral phase particles in the osteoid matrices of the subperiosteal region of tibiae were principally those of poorly crystalline hydroxyapatite. In rare instances (less than 1% of the estimated volume of the mineral phase present in the zone of early mineralization), relatively large single crystals were observed within clusters of hydroxyapatite. From calculations of both interplanar spacings and measurements of angular displacement of diffraction reflections from single crystal microdiffraction patterns, two distinct phases other than hydroxyapatite were identified: brushite and β-tricalcium phosphate. A third phase, resembling an apatite, remains unidentified. The results suggest that very small amounts of nonapatitic phases of Ca-P exist in chicken bone tissue. No temporal relationship could be established between the nonapatitic and apatitic phases. There is at present no evidence from this study to support the concept that nonapatitic phases are precursors of a final apatitic phase in bone.
Production of and response to growth-stimulating activity in isolated bone cells
Authors: Wong Glenda L. , Roberts R. , Miller E.
Published: February 2010

Autologous growth-promoting activity has been shown to be secreted by bone organ cultures. To identify the cellular source of these growth factors, we have studied the activity present in cell extracts prepared from isolated bone cells released early or late from mouse calvariae following collagenase digestion. Previous studies have established that early released cell populations reside on the bone surface and consist of a heterogeneous mixture of cells that are less osteoblastic than late released cells. We find that soluble extracts of the latter cells contain more growth-promoting activity/mg of cellular protein than those of the former. By this criterion late released cells appear to be the major source of endogenous growth factors. On the other hand, upon exposure to bone cell-derived cell extracts, early released cells express a greater-fold increase in [3H]-thymidine incorporation into acid insoluble radioactivity than late released osteoblasts because of the high basal mitogenic activity of the latter which may be related to their production of autologous growth factor. These data suggest that both early and later released cells may be major targets for bone growth factors produced in situ by late released osteoblasts.
Chicken osteoclasts do not possess calcitonin receptors
Authors: Nicholson G.C. , Moseley J.M. , Sexton P.M. , Prof. Martin T.J.
Published: February 2010

Osteoclasts freshly isolated from embryonic chicks have been examined for calcitonin receptors using radio-iodine-labeled salmon calcitonin. Calcitonin binding to chick osteoclasts could not be shown by either autoradiography or biochemical binding studies. Furthermore, calcitonin did not stimulate cyclic AMP production. By contrast, rat osteoclasts have abundant calcitonin receptors, and a sensitive cyclic AMP response to calcitonin has been shown previously. It is concluded that chick osteoclasts do not possess calcitonin receptors, a finding which could explain the lack of calcitonin responsiveness observed in other avian osteoclast culture systems.
Demonstration that ethanol inhibits bone matrix synthesis and mineralization in the rat
Authors: Turner Russell T. , Greene Virginia S. , Bell Norman H.
Published: February 2010

The effects of ethanol on bone and mineral metabolism were investigated in 3 groups of male rats. The first group received ethanol administered as 36% of caloric content in a liquid diet for 3 weeks. A second group of pair-fed animals was given the same liquid diet, except that sucrose was substituted isocalorically for ethanol. A third group of rats was fed standard laboratory chow. The ethanol-treated rats gained significantly less weight than laboratory chow-fed controls but gained the same weight as the pair-fed animals. Ethanol-treated rats had a modest but significant decrease in mean serum calcium compared to pair-fed controls (10.3 ± 0.1 vs. 10.6 ± 0.1 mg/dl, p < .001). Mean serum phosphate, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone were the same in the 3 groups. The ethanol-treated animals showed significant decreases in mean tibial length (1.88 ± 0.01 vs. 1.98 ± 0.02 cm, p < .01), mean endosteal bone formation rate (0.0006 ± 0.0001 vs. 0.0026 ± 0.0003 mm3/day, p < .001) and mean periosteal bone formation rate (0.022 ± 0.001 vs. 0.026 ± 0.001 mm3/day, p < .01) compared to the pair-fed controls. The ethanol-treated rats demonstrated significant decreases in mean periosteal mineralization rate (7.5 ± 0.3 vs. 10.3 ± 0.6 μm/day, p < .01) and mean periosteal apposition rate (8.5 ± 0.5 vs. 11.0 ± 0.8 μm/day, p < .05) and a significant increase in mean periosteal osteoid thickness (15.5 ± 1.4 vs. 10.4 ± 0.8 μm, p < .01) compared to pair-fed controls. The laboratory chow-fed controls had higher mean endosteal bone formation rate (0.0040 ± 0.0004 mm3/day, p < .01), mean periosteal bone formation rate (0.034 ± 0.001 mm3/day, p < .01), mean periosteal mineralization rate (12.5 ± 0.7 μm/day, p < .01) and mean periosteal apposition rate (13.0 ± 0.7 μm/day, p < .01) than the pair-fed control animals. The results are interpreted to mean that in the rat ethanol alters mineral homeostasis and diminishes formation of bone matrix and mineralization of osseous tissue. These effects cannot be accounted for by alterations in the metabolism of vitamin D. Further, caloric restriction and inhibition of growth themselves diminish formation of bone matrix and mineralization of osseous tissue.
Effect of bisphosphonates on proliferation and viability of mouse bone marrow-derived macrophages
Authors: Dr. Cecchini Marco G. , Felix Rolf , Fleisch Herbert , Cooper Philip H.
Published: February 2010

Bisphosphonates (BP) are powerful inhibitors of bone resorption. Their mechanism of action, although still unclear, is now believed to be at the cellular level. In this study we investigated the effects of these compounds on proliferation, induced either by L-cell conditioned medium (CSF-1) or 4-phorbol 12-myristate 13-acetate (PMA) of bone marrow cells (BMC) and on CSF-1-induced proliferation and viability of bone marrow derived macrophages (BMDMΦ). BMC proliferation, measured by [3H]-TdR incorporation or by clonal assay in soft agar, was significantly inhibited by 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) and 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) at 2.5 × 10−7M and by dichloromethylenebisphosphonate (Cl2MBP) at 2.5 × 10−6M. This inhibitory effect was also confirmed on the proliferation, measured by [3H]-TdR incorporation, of BMDMΦ. In the absence of CSF-1, the viability of this latter cell population, estimated by DNA content per well and lactate dehydrogenase (LDH) released into the medium, was affected in the following order of concentrations: Cl2MBP, 1.0 × 10−4M; AHBuBP, 5.0 × 10−5M; and AHPrBP, 2.5 × 10−5M. Since osteoclasts and macrophages might share a common early progenitor cell, probably under the control of CSF-1, the effect exerted by BP on the proliferation of the macrophage precursors may also be extended to the osteoclast precursors.
1α,25-Dihydroxyvitamin D3 receptors in human thymic and tonsillar lymphocytes
Authors: Provvedini Diego M. , Rulot Christine M. , Sobol Robert E. , Tsoukas Constantine D. , Dr. Manolagas Stavros C.
Published: February 2010

In vitro activated human peripheral blood lymphocytes possess the receptor protein for 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the present study we have examined whether activated lymphocytes that occur in vivo in human thymuses and tonsils also possess receptors for 1,25(OH)2D3. Freshly isolated lymphocyte preparations, from five separate surgical specimens of thymus and tonsil, were depleted of monocytes and examined, before and after fractionation on a density gradient of Percoll, for [3H] 1,25(OH)2D3 binding by means of sucrose density gradient sedimentation, by saturation analysis of the binding, and by DNA-cellulose chromatography. The state of activation of the lymphocyte preparations was determined using [3H] thymidine incorporation, DNA and RNA quantitation (using acridine orange), and by determining the presence or absence of markers of activation (interleukin-2 receptor, transferrin receptor, and HLA-DR molecules). In both the thymic and the tonsillar lymphocyte preparations we detected a 1,25(OH)2D3-binding molecule possessing sedimentation coefficient of 3.3 S and dissociation constant of 10−10M as well as DNA binding capability. In thymic lymphocytes, the 1,25(OH)2D3 receptor concentration correlated positively with the number of lymphocytes expressing the transferrin receptor (r = 0.84; p < 0.05). In addition, in both thymic and tonsillar lymphocytes the concentration of 1,25(OH)2D3 receptors correlated positively with the number of cells in the G1a phase of the cell cycle (r = 0.79, p < 0.01, and r = 0.88, p < 0.001 for thymic and tonsillar lymphocytes, respectively). In contrast, the 1,25(OH)2D3 receptor concentration in these preparations did not correlate with the rate of cell proliferation. These results indicate that thymic and tonsillar lymphocytes in the early stages of activation possess in vivo the 1,25(OH)2D3 receptor protein, and suggest that 1,25(OH)2D3 may act in vivo on these cells.
Aminohexane diphosphonate in the treatment of paget' s disease of bone
Authors: Atkins Roger M. , Yates Ashley J.P. , Gray Richard E.S. , Urwin Graeme H. , Hamdy Neveen A.T. , Beneton Monique N.C. , Rosini Sergio , Dr. Kanis John A.
Published: February 2010

We studied the effects of the intravenous or oral administration of aminohexane diphosphonate (AHDP) in 42 patients with active Paget's disease of bone. Treatment of mouth (400 mg daily for 1 month) or intravenously (25 mg or 50 mg daily for 5 days) induced marked suppression of biochemical indices of disease activity. Urinary excretion of hydroxyproline fell to 39 and 42% of pretreatment values (oral and IV treatments respectively), and was followed by a similar decrease in the serum activity of alkaline phosphatase. In both groups of patients, disease activity remained suppressed for the 6 months of followup, and pain improved in 34 out of 37 patients who had bone pain attributed to Paget's disease. Both biopsies indicated that osteoblast and osteoclast numbers decreased with no adverse effects on mineralization. Neither regime was associated with significant side effects. We conclude that short courses of AHDP provide a promising treatment for the long-term control of Paget's disease.
Osteoclast-like cell formation in fetal and newborn long-term baboon marrow cultures is more sensitive to 1,25-dihydroxyvitamin D3 than adult long-term marrow cultures
Authors: Takahashi N. , Mundy G.R. , Kuehl T.J. , Roodman G.David
Published: February 2010

It is unknown if osteoclasts derived from animals at different developmental stages differ. To study this question, we used a long-term baboon marrow culture system in which osteoclast-like multinucleated cells (MNC) are formed. Fetal, newborn, or adult baboon marrow cultures were tested to determine if they differ in their responsiveness to osteotropic hormones. In fetal and newborn marrow cultures 1,25-dihydroxyvitamin D3 (1,25D3) at 10−10M significantly increased MNC formation with a maximal effect seen at 10−9M. Higher concentrations of 1,25D3 progressively decreased MNC formation. In contrast, in adult baboon marrow cultures, 10−9M 1,25D3 was required to significantly increase MNC formation, with a maximal affect at 10−8M 1,25D3. Calcitonin (25–200 ng/ml) inhibited MNC formation in fetal, newborn, or adult baboon marrow cultures treated with 1,25D3 in an identical manner. The effects of 1,25D3 on granulocyte-macrophage progenitor cells (CFU-GM), the probable precursors of MNC, were identical in fetal and adult baboon marrow cultures, with a significant inhibition of CFU-GM colony formation at 10−8M 1,25D3. These results suggest that 1) osteoclast precursors are more sensitive to some osteotropic hormones during the fetal and newborn periods, and 2) differences in the 1,25D3 sensitivity of osteoclast-like MNC formation in fetal, newborn, and adult baboon marrow cultures are not due to effects on early proliferating precursors but may result from effects of 1,25D3 on fusion of later precursors for MNC.
Relaxation of rat gastrointestinal smooth muscle by parathyroid hormone
Authors: Mok Lester L.S. , Cooper Cary W. , Thompson James C.
Published: February 2010

In this study we investigated whether parathyroid hormone (PTH) can produce relaxation of gastrointestinal (GI) smooth muscle as it has been reported to do for vascular and uterine smooth muscle. Muscle tissue preparations from rat stomach, duodenum, ileum, or colon were mounted in a 37°C tissue bath and perfused with oxygenated medium. Changes in isometric tension were recorded with a force-displacement transducer connected to a polygraph. Decreases in either resting tension or agonist-induced tension (0.5–1.0 μ M acetylcholine or carbachol) were observed within 1–2 min of PTH addition and were reversible upon removal of the peptide. All GI regions tested were responsive to PTH. Synthetic rPTH-(1–34) (0.1–100 nM) produced a dose-dependent relaxation of both fundic (ED50 = 5.2 nM) and colonic (ED50 = 2.5 nM) muscle strips. At 100 nM, a 90% decrease in fundic tension and a 70% decrease in colonic tension were seen. At 100 nM, bPTH-(1–34), but not bPTH-(7–34) or rat calcitonin gene-related peptide, also was effective in relaxing fundic or colonic muscle. Similarly, in the fundic muscle, 500 nM bPTH-(3–34) alone was ineffective, but it inhibited the effect of 5 nM rPTH-(1–34) when both peptides were tested in combination. Likewise, 100 nM bPTH-(3–34) also inhibited the relaxation induced by 5, 10, or 100 nM bPTH-(1–34). The results show PTH to be highly effective in nanomolar concentrations in causing relaxation of GI smooth muscle. The findings suggests that rat GI smooth muscle possesses receptors for PTH and indicate a potential role for PTH or PTH-related peptides in GI motility.
Evidence that pulsed electromagnetic fields inhibit coupling of adenylate cyclase by parathyroid hormone in bone cells
Authors: Cain Christopher D. , Adey W. Ross , Luben Richard A.
Published: February 2010

To investigate the biochemical effects of pulsed electromagnetic fields (PEMF) on bone in particular and on cell membrane-associated activity in general, we have studied the modification by PEMF of cAMP metabolism in primary calvarial bone cells. We report that PEMF inhibited cAMP accumulation stimulated by bovine PTH(1–34) peptide. After a 1-hr PEMF exposure, the cAMP response to PTH (2–7 min) was decreased in exposed cells to 48–70% (p < 0.05) of the response of unexposed cells; furthermore, this inhibition disappeared after 10–20 min with PTH. This inhibition occurred at submaximal PTH doses (2.4–7.3 nM) and no effect was observed at maximal PTH doses (24 nM). Thus with PEMF, the dose response curve for PTH became 0.5 log unit less sensitive. PEMF did not affect the cAMP response to cholera toxin and forskolin. However, when submaximal doses of both forskolin (0.5–1.0 μM) and PTH (0.24–2.4 nM) were used, forskolin prevented inhibition of cAMP production by PEMF in the range of fields and stimulus epochs which normally inhibit cAMP production.

It is proposed that PEMF inhibits PTH-stimulated coupling of the adenylate cyclase system and that this inhibition does not affect the intrinsic activity of the G-protein and the catalytic subunit.
Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone
Authors: Dr. Hefley Thomas J.
Published: February 2010

Crude bacterial collagenase is essential for the enzymatic isolation of cells from the membranous bone of neonatal mouse calvaria. We have employed the newly developed methodology of fast protein liquid chromatography (FPLC) to separate and quantify the isozymes of collagenase so that their role in the enzymatic isolation of cells might be determined. FPLC resolved as many as six protein peaks in less than 30 min using a single anion exchange column and separated collagenase isozymes into two classes. The Class I isozymes had a preference for the substrate Azocoll, a denatured collagen substrate, and the Class II isozymes had a preference for Hexapeptide, a synthetic substrate. Two preparations of chromatographically purified collagenase (CGN-A and CGN-B) were tested for their ability to release viable cells from bone. Both preparations of purified collagenase completely digested the calvaria in 120 min. The total cell yield obtained with CGN-A was 0.34 million cells per calvarium. The yield obtained with CGN-B was 1.01 million cells per calvarium. Each preparation of purified collagenase was analyzed using FPLC. CGN-A contained only Class I collagenase isozymes, whereas CGN-B contained a mixture of both Class I and Class II isozymes. The collagenase isozymes of CGN-B were separated by FPLC and then combined in a 4:1 ratio of Class II:Class I isozymes. Utilization of FPLC-separated collagenase isozymes for the cell isolation increased the total cell yield to 1.50 million cells/calvarium. We have concluded that there are many combinations of collagenase isozymes that will completely digest the extracellular matrix of bone. However, only a combination which favors the Class II isozymes will result in a low rate of cell destruction and high cell yields.
Retinole acid and glucocorticoids enhance the effect of 1,25-dihydroxyvitamin D3 on bone γ-carboxyglutamic acid protein synthesis by rat osteosarcoma cells
Authors: Nishimoto Satoru K. , Salka Carl , Nimni Marcel E.
Published: February 2010

Two 1,25-dihydroxyvitamin D3-controlled parameters in the osteoblastlike osteosarcoma cell line ROS 17/2, bone γ-carboxyglutamic acid-containing protein (BGP) and collagen synthesis, were measured after pretreatments with either retinoic acid (RA), or triamcinolone acetate (TRM). RA and TRM both caused double the expected increase in BGP secretion at 16 hr after treatment with 1,25-dihydroxy vitamin D3. Triamcinolone acetate concentrations of 10−8 and 10−9M or 10−6M retinoic acid were effective in enhancing the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion. Treatment with RA or TRM alone did not stimulate BGP secretion. RA alone had no effect on BGP secretion, while TRM inhibited BGP secretion. Collagen synthesis is inhibited by 1,25-dihydroxyvitamin D3. Neither retinoic acid nor triamcinolone acetate enhanced the 1,25-dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Retinoic acid by itself inhibited collagen synthesis but did not change the 1,25 dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Triamcinolone acetate by itself or together with 1,25-dihydroxy vitamin D3 increased collagen synthesis.

We conclude that, although both triamcinolone acetate and retinoic acid increase the 1,25-dihydroxy vitamin D3 stimulation of BGP secretion by ROS 17/2 cells, they have different effects on the regulation of collagen production. Thus, although both hormones increase the 1,25-dihydroxy vitamin D3 receptor concentration in these cells, their actions are not mediated solely by this mechanism.
Ultracytochemical investigation of calcium-activated adenosine triphosphatase (Ca++-ATPase) in chick tibia
Authors: Akisaka Toshitaka , Yamamoto Toshio , Gay Carol V.
Published: February 2010

The ultrastructural distribution of Ca++-ATPase in bone cells of growing chick tibia was investigated by a cytochemical method in order to gain insight into possible sites of calcium ion translocation. Both osteoclasts and osteoblasts showed a polar distribution of reaction product along the plasma membrane. In osteoclasts, enzymatic activity occurred along the portion of the plasma membrane facing the marrow but not along the ruffled border or clear zone. The reaction product in these cells was due solely to Ca++-ATPase action. In osteoblasts, the plasma membrane facing away from bone (apical and lateral membrane) was very intensely stained, whereas the basal membrane was unstained. The reaction product in these cells appeared to be the result of both Ca++-ATPase and Ca++, Mg++-ATPase. In osteocytes, no plasma membrane staining was detectable. Mitochondrial staining in all three types of cells was more sensitive to fixation than was the plasma membrane enzyme, suggesting that mitochondrial and plasma membrane Ca++-ATPases are chemically distinct, as biochemical studies have shown. In general, mitochondria in osteoclasts stained more intensely than those in osteoblasts or osteocytes. Mitochondrial and vesicular sites of activity may be related to intracellular calcium storage, whereas calcium ATPases of the plasma membrane are presumed to be involved in calcium efflux from the cells. Calcitonin treatment did not alter the enzymatic distribution or intensity in osteoclasts. The striking polar distribution of both osteoclast and osteoblast plasma-membrane activity suggests that directional calcium pumping by these cells may be of importance in bone-forming and bone-resorbing mechanisms.
Regulation of alkaline phosphatase expression in a neonatal rat clonal calvarial cell strain by retinoic acid
Authors: Ng Kong Wah , Gummer Paul R. , Michelangeli Valdo P. , Bateman John F. , Mascara Thomas , Cole William G. , Martin T. John
Published: February 2010

A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in α-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells. They will be useful models to study the regulation of ALP activity and type 1 collagen synthesis by various factors and the induction of expression of other phenotypic characteristics by RA.
Phorbol esters stimulate bone resorption in fetal rat long-bone cultures by mechanisms independent of prostaglandin synthesis
Authors: Lorenzo Joseph A. , Sousa Sandra
Published: February 2010

The phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate, which activate the enzyme protein kinase C, stimulated resorption in fetal rat long-bone cultures at concentrations of 1 and 10 μM. This effect appeared specific for active phorbol esters, since the inactive analogue 4-alpha-phorbol-12,13-didecanoate was without effect. The resorptive responses of fetal rat long-bone cultures to active phorbol esters differed from those previously described in newborn mouse calvaria cultures, since resorption stimulated by TPA in the rat long bones was not inhibited by either indomethacin (10 μM) or flufenamic acid (10 μM). However, calcitonin, an inhibitor of osteoclastic resorption, did decrease the response to TPA. There were some similarities between the response of fetal rat long-bone cultures to TPA and their response to epidermal growth factor (EGF). Like EGF, TPA stimulated DNA synthesis in the bones (measured as the incorporation of [3H]-thymidine) at concentrations below those necessary to stimulate resorption. TPA also did not stimulate resorption in the presence of aphidicolin (10 μM), an inhibitor of DNA synthesis that has been previously shown to block the resorptive response of these cultures to EGF. However, the responses of the cultures to TPA and EGF were not identical, since, unlike the effects of EGF, the stimulatory effects of TPA on DNA synthesis were biphasic. These results demonstrate that active phorbol esters stimulate bone resorption in fetal rat long-bone cultures through mechanisms that do not require prostaglandin synthesis but do appear to be mediated by osteoclasts. Hence, it is possible that one mechanism by which hormones stimulate resorption in bone is through activation of protein kinase C.
Growth and repair of cartilage: Organ culture system utilizing chondroprogenitor cells of condylar cartilage in newborn mice
Authors: Weiss Anna , Livne Erella , von der Mark Klaus , Heinegard Dick , Silbermann Michael
Published: February 2010

The zone of progenitor cells of mandibular condyles of neonatal mice was kept in an organ culture system for up to 8 days. Qualitative and quantitative determinations indicated a pronounced proliferative activity during the initial phases of the culture followed by a differentiation phase and the acquisition of typical hyaline cartilage. The mature hypertrophic chondrocytes were found to be surrounded by cartilage-specific macromolecules such as type II collagen, cartilage proteoglycans, and cartilage anchorin. The extracellular mineralization proceeded along matrix vesicles as is usually noted in vivo. A unique finding in this study was the observation that explants comprising cartilage progenitor cells and their adjacent extracellular matrix succeeded in repairing the damaged condylar in vitro.