Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although it is well recognized that lead accumulates in bone, skeletal tissue is considered primarily a sequestering compartment and not a site of toxic action for lead. However, exposure to lead is associated with impaired skeletal growth in children and reductions in indices of bone formation in laboratory animals. Osteoblastic ROS 17/2.8 cells were used in an effort to better understand the consequences of lead exposure on skeletal homeostasis. Studies on confluent cultures of ROS 17/2.8 cells revealed that lead (2-200 microM) had no effect on cell number or DNA and protein synthesis. However, alkaline phosphatase activity was reduced by lead in a dose- and time-dependent manner. Reductions in steady state alkaline phosphatase mRNA levels paralleled the lead-induced inhibition of enzyme activity. Moreover, lead exposure resulted in similar dose-dependent reductions in steady state type 1 procollagen and bone Gla protein mRNA levels. The effect of lead on osteoblastic gene expression in ROS 17/2.8 cultures, however, was selective in nature, as similar lead exposures resulted in no alterations in beta-actin or glyceraldehyde-3-phosphate dehydrogenase mRNA levels. These data demonstrate that lead, in the absence of over toxicity, specifically restricts the expression of certain aspects of the differentiated osteoblast phenotype. Such alterations in osteoblast function may contribute to the skeletal abnormalities observed in settings of lead intoxication.
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PMID:Regulation of osteoblastic gene expression by lead. 850 55

The adherence of osteoblast-like cells and the resorption activity of isolated osteoclasts on calcium sulphate hemihydrate (CSH) was investigated. After a 24 h incubation period, alkaline phosphatase staining showed that rat osteoblast-like cells ROS 17/2.8 attached on CSH. Neutral red (NR) and tartrate-resistant acid phosphatase (TRAP) staining revealed that osteoclasts attached on CSH. Furthermore, osteoclasts formed lacunae as revealed by scanning electron microscopy. Although the lacunae formed by osteoclasts on CSH were diverse in shape and form, the most common type had approximately a circular outline, with a well-defined margin similar to those formed on dentine. Less commonly, excavations appeared as a discontinuous area of resorbed CSH with the presence of a circular zone around the non-resorbed area. Finally, by using 10(-9) M calcitonin, evidence was obtained that NR-positive cells were osteoclasts (58.3% and 57.66% decrease of NR-positive mouse and rat cells detected on CSH after 24 h incubation). However, no inhibition was obtained with 10(-11) M calcitonin. The overall number of NR-positive osteoclasts adherent on 256 mm2 CSH was 43 +/- 14 and 42 +/- 3 for mice and rat, respectively. The overall number of TRAP-positive mouse osteoclasts was 67 +/- 12. Acetazolamide (10(-5) M), a carbonic anhydrase inhibitor, inhibited the number of adherent NR osteoclasts on CSH by 50.42% and 41.6% for mouse and rat, respectively. These results indicate that osteoblasts attach on CSH and osteoclasts resorb CSH in vitro.
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PMID:Osteoblast adherence and resorption activity of isolated osteoclasts on calcium sulphate hemihydrate. 857 71

KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose-dependently increased alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17/2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)(2)D3)-induced increase in osteocalcin synthesized by ROS 17/2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA-098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1 alpha,25(OH)(2)D(3), parathyroid hormone (PTH), and prostaglandin E2 (PGE2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclast-like cells. Coumestrol and 17beta-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17/2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17beta-estradiol on bone tissue is the inhibition of bone resorption.
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PMID:Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population. 861 81

The transforming growth factor-beta (TGF-beta) is a multifunctional homodimeric protein with an apparent molecular weight of 25 KDa. TGF-beta transduces signals by forming heteromeric complexes of their type-I (T beta R-I) and type-II (T beta R-II) serin/threonine kinase receptors. TGF-beta binds first to T beta R-II receptor, and then the ligand in this complex is recognized by T beta R-I, resulting in formation of a heteromeric receptor complex composed of T beta R-I and T beta R-II. Once received, T beta R-I becomes phosphorylated in the GS domain by the associated constitutively active T beta R-II and transmits the downstream signal. It has been reported that formation of the heteromeric complex is indispensible at least in epithelial cells for growth inhibition and extracellular matrix production induced by TGF-beta. In this study, the functional role of T beta R-II for the TGF-beta-induced signals in osteoblastic cells was investigated by using a dominant negative type of T beta R-II mutant receptors (T beta RIIDNR). ROS 17/2.8 and MG 63 cells were found to express T beta R-I, T beta R-II, and T beta R-III, and their cell growth was inhibited by TGF-beta, whereas alkaline phosphatase activity was stimulated. Cells that were stably transfected with the T beta RIIDNR plasmid showed decreased response to TGF-beta during growth and alkaline phosphatase activity. These results indicate that the intracellular serine/threonine kinase domain of T beta R-II is essential for signal transduction of the TGF-beta-induced alkaline phosphatase activity as well as growth inhibition.
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PMID:[Functional analysis of transforming growth factor-beta type II dominant negative receptor]. 874 21

The extracellular matrix protein tenascin is secreted by osteoblasts but absent from mineralized bone matrix. The current study was undertaken to test the hypothesis that tenascin regulates osteoblast behaviour. Three osteoblast-like cell lines UMR-106, ROS-17/2.8 (rat) and SAOS-2 (human) were used to investigate the role of tenascin in osteoblast morphology, differentiation and proliferation. Two of three cell lines adhered specifically to tenascin, remaining round and failing to spread. Tenascin as a substratum stimulated alkaline phosphatase activity (a marker of osteoblast differentiation) in two of three cell lines. Moreover, anti-tenascin in the medium caused a reduction in alkaline phosphatase levels in all three cell lines. Anti-tenascin also inhibited collagen synthesis, an important osteoblast function. Since it seemed possible that tenascin may exert its effects on cell function through its ability to cause cell rounding, the ability of cell shape change alone to influence alkaline phosphatase levels was investigated. Cells were incubated in the presence of cytochalasin D and alkaline phosphatase levels assayed. Alkaline phosphatase activity was not elevated by cytochalasin D treatment, indicating that cell rounding alone is insufficient to mimic the effect of tenascin. Anti-tenascin caused a slight increase in proliferation of SAOS-2 cells, indicating that tenascin is itself inhibitory. In ROS 17/2.8 and UMR-106 cells, in contrast, proliferation was inhibited by anti-tenascin. The results presented here indicate that tenascin is able to stimulate osteoblastic differentiation and that endogenous tenascin helps to maintain the functional state of cultured osteoblast-like cells.
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PMID:Modulation of osteoblast behaviour by tenascin. 879 46

Knowledge of the number and kinds of differentiation steps characterizing cells of the osteoblast lineage is inadequate. To analyze further osteoblast differentiation, a number of labs have generated monoclonal antibodies to osteogenic cells, derived from both normal bone and osteosarcomas. A variety of immunolabelling patterns on primary cell cultures, cell lines, and tissue sections has been reported, including cell surface, cytoplasmic, and extracellular matrix-associated patterns. Most of the antibodies selected recognize predominantly the mature osteoblast and osteocyte; in addition, however, antibodies have been generated that recognize pre-osteoblasts. Some recognize cells of both the osteoblast and chondroblast lineages and may contribute to a better understanding of the lineage and phenotypic relationships between these two cell types. In addition to recognition in vivo of cell subpopulations of discrete maturational stages, changes in the immunolabelling patterns in vitro have also documented a differentiation sequence in cells undergoing osteogenesis in cell and tissue cultures. In at least two cases, the antibodies have been used to isolate subpopulations of cells from bone, including relatively pure populations of osteocytes. With the exception of several antibodies that are against alkaline phosphatase or known matrix proteins including osteocalcin, the nature of the macromolecular species recognized by most of the antibodies generated to date are unknown. Recently, however, one antibody was used to clone the cDNA for the beta-galactoside-binding lectin, galectin 3 or epsilon binding protein (epsilon BP; IgE-binding protein; Mac-2), from a lambda gt11 osteoblast expression library; another was used to clone from an ROS 17/2.8-COS cell expression library the cDNA for OTS-8, a putative target gene of early response genes stimulated in response to phorbol esters in MC3T3-E1 cells. Neither of these macromolecules had previously been identified in bone cells, but the recent molecular and cellular analyses have shown them to be developmentally and/or hormonally regulated in osteoblastic cells. These antibodies extend the available markers and support earlier observations that a variety of molecules are differentially expressed by cells at different stages of the osteoblast lineage. This chapter will not be an exhaustive survey of all immunocytochemical and immunohistochemical analyses of osteogenic cells and tissues but will focus on the approach of eliciting novel monoclonal antibodies by the injection of osteogenic cells or crude bone extracts and its potential for establishing new markers of the osteoblast lineage. We have not included a large number of studies documenting the use of antibodies raised against several known bone matrix proteins; while these have been crucial in developing our current understanding of osteogenic differentiation, we sought rather to highlight the potential of the "random" injection approach.
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PMID:Monoclonal antibodies as tools for studying the osteoblast lineage. 884 13

A synthetic porous three-dimensional structure that can mimic the architecture of actual tissues, provide sustained release of nutrients or growth factors, and serve as a template for cell seeding would be an ideal substrate for tissue engineering. Poly(l-lactic acid) (PLLA) foams were fabricated for this purpose, based on the principle of phase separation from homogeneous naphthalene solutions. Complex shapes could be readily fabricated, and resulting foams had relatively uniform, open cells throughout the matrix. Densities and total pore-surface areas were in the range of 0.05-0.1 g/cm3 and 0.8-1.3 m2/g, respectively. The loss tangent of these foams ranged from 0.07 to 0.128, as measured by thermomechanical analysis. Naphthalene residue in the resulting foams went below 0.2 wt% after extensive vacuum sublimation. Feasibility of incorporating drugs or nutrients into such a highly porous structure was demonstrated by the dispersion of two model compounds, bromothymol blue (BTB) and sulforhodamine B (SD), in the matrix. Sustained release of BTB from the foam with a porosity as high as 87% was observed for more than 2 months. Alkaline phosphatase, as a model protein to be incorporated, lost approximately 30% of its bioactivity during the fabrication. As a cell-culture substrate, the PLLA foams performed as well as the flat PLLA surface in supporting the growth of rat osteosarcoma cells (ROS 17/2.8) and in maintaining their functions such as alkaline phosphatase activity and osteocalcin synthesis. UMR-106 cells cultured in the foam also expressed a higher degree of mineralization than those cultured on the flat PLLA substrate.
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PMID:Poly(L-lactic acid) foams with cell seeding and controlled-release capacity. 884 55

The actions of the hormonal form of vitamin D, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2 D3], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1 alpha, 25-(OH)2D3 in many cell types including osteoblastic cells. We have compared the effects of 1 alpha, 25-(OH)2D3 and a novel 1 alpha, 25-(OH)2D3 bromoester analog (1,25-(OH)2-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 x 10(-8) M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)2-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1 alpha, 25-(OH)2D3 and 1,25-(OH)2-BE intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 x 10(-8) M, both 1 alpha,25-(OH)2D3 and 1, 25-(OH)2-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)2-BE are similar to those of 1 alpha,25-(OH)2D3, and since 1,25-(OH)2-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors.
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PMID:Effects of the vitamin D3 analog 1 alpha, 25-dihydroxyvitamin D3-3 beta-bromoacetate on rat osteosarcoma cells: comparison with 1 alpha, 25-dihydroxyvitamin D3. 891 81

Galectin 3 is an endogenous soluble beta-galactoside-specific lectin originally identified and termed epsilon BP or IgE-binding protein in rat basophilic leukemia cells, but its wide tissue distribution and the multiple contexts in which it has been isolated have suggested that its function may not be limited to IgE binding but may include a role in cell growth regulation and differentiation, neoplastic transformation, and cell adhesion (Liu, 1990, Crit. Rev. Immunol., 10:289-306; Barondes et al., 1994, J. Biol. Chem., 269:20807-20810). After immunoscreening of a lambda gt11 cDNA expression library made from bone-nodule forming cultures of fetal rat calvaria (RC) cells with an antibody raised against osteoblastic cells (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), three cDNA clones were isolated and sequenced; the sequence matched that of rat galectin 3. Galectin 3 mRNA was detected in various fetal and adult rat tissues, including calvaria and cultured RC cells. In RC cells and the rat osteosarcoma cell line ROS 17/2.8, galectin 3 mRNA expression increased with time in culture, in contrast to its behavior in fetal rat skin fibroblasts (RSF) in which its expression decreased with time in culture. In a second rat osteosarcoma line, UMR 106.01, galectin 3 mRNA was almost nondetectable. The synthetic glucocorticoid dexamethasone (Dex) enhanced galectin 3 expression in RSF cell cultures, while 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) had no significant effect. In contrast, Dex downregulated and 1,25(OH)2D3 upregulated galectin 3 expression in RC and ROS 17/2.8 cells, especially at later time points in culture when expression of osteoblast-associated differentiation markers by these cell types is most marked. Immunolabeling with an antibody against rat galectin 3 to identify galectin 3 protein showed that cells labelled within both the ROS 17/2.8 and RC populations but with marked intercellular heterogeneity of intensity. Our data support the conclusion that galectin 3 is a previously unrecognized product of osteoblastic cells, that galectin 3 mRNA and protein expression increases with time in vitro concomitant with other markers of osteogenesis, including formation of bone nodules and expression of osteoblast-associated markers such as alkaline phosphatase, bone sialo-protein, and osteocalcin, and that its expression is regulated by hormones such as glucocorticoids and 1,25(OH)2D3 that modulate other aspects of the osteoblast phenotype.
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PMID:Expression and regulation of galectin 3 in rat osteoblastic cells. 895 96

The objective of this study was to evaluate the effects of metal ions, which may be released from orthopedic or dental implants, on osteoblast metabolism and differentiation. ROS 17/2.8 cells were cultured in F-12 medium for 7 days. Then Al+3, Co+2, Cr+3, Ni+2, Ti+4, and V+3 were added at concentrations less than their cytotoxic concentrations. After 3 days, DNA synthesis, succinate dehydrogenase activity, alkaline phosphatase (ALP) activity, and culture calcification were assessed. Northern blots were performed for ALP, osteocalcin (OCN), and osteopontin (OPN) mRNA transcription. The data indicated that Cr+3 and A1+3 had few inhibitory effects on ROS cell metabolism below their cytotoxic concentrations, Ni+2, Co+2, Ti+4, and V+3 affected all these parameters of ROS cell metabolism at concentrations below cytotoxic levels. For RNA analysis, A1+3 significantly suppressed the expression of ALP, OCN, and OPN at both cytotoxic and noncytoxic concentrations. Co+2 specifically suppressed ALP expression at cytotoxic concentrations. Cr+3 and Ni+2 inhibited OCN, OPN, and ALP gene expression only at cytotoxic concentrations. For Ti+4 and V+3 ions, gene expression at cytotoxic levels was not significantly affected as compared with the effects at noncytotoxic level. These results show that metal ions may alter osteoblast behavior even at subtoxic concentrations, but do not always affect the expression of all genes similarly.
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PMID:Effects of metal ions on osteoblast-like cell metabolism and differentiation. 897 50


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