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)

We have established two clonal cell lines, designated SM1/9 and SM25/3 from the mandibular condyles of newborn BALB/c mice by immortalization with the SV40 large T antigen. These cells have a high proliferative activity and have been maintained in culture for over 50 passages. They are polygonal in shape. Electron microscopic studies indicate an immature phenotype for both clones and a lack of prominent intracellular filaments typical of fibroblasts. SM25/3 demonstrates different biological properties as compared to SM1/9, it is tumourigenic in nude mice, has a faster growth rate and exhibits less differentiated features. Both cell lines have low constitutive levels of alkaline phosphatase, and the activity of this enzyme is increased significantly in a dose and confluency dependent manner by retinoic acid and 1,25 (OH)2 vitamin D3. The cells express transcripts for retinoic acid receptors mRAR-alpha and mRAR-gamma but not for mRAR-beta. They also express mRNA for the 1,25 (OH)2 vitamin D3 receptor. They co-express transcripts for collagen types I, II, III. Expression of mRNA for extracellular matrix proteins such as biglycan, osteopontin, PAI-1 is detected. Cultured cells do not express mRNA for osteocalcin and this transcript is not inducible with 1,25 (OH)2 vitamin D3 or retinoic acid. Chondrocyte markers such as link protein and aggrecan are not detected. In vitro assays indicate that the cell lines have a limited capacity for osteogenic or chondrogenic differentiation. Similarly agarose culture experiments and extended treatment with retinoic acid indicate that they do not resemble dedifferentiated chondrocytes. Both the cell lines appear to express a phenotype intermediate to osteoblasts and chondroblasts and possibly represent transitional differentiation stages of the progenitor cells of the mandibular condyle. These cells could serve as useful models in elucidating the pathways of early mesenchymal cell differentiation.
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PMID:Establishment and characterization of two clonal cell lines derived from murine mandibular condyles. 757 May 75

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue with 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion exchange chromatography. A monoclonal antibody C8F4 was developed to this core protein. The characteristics and specificity of the antibody were studied by an enzyme-linked immunosorbent assay (ELISA) using an alkaline phosphatase conjugated antibody (goat anti-mouse IgG). The antibody binding to the core protein was found specific and optimal at pH 7.0. The antibody recognizes either intact chondroitin sulfate-dermatan sulfate proteoglycan monomer, chondroitinase ABC digested monomer or chemically deglycosylated proteoglycan. Free chondroitin sulfates, keratan sulfate and hyaluronic acid did not compete for the antigenic sites in ELISA. Limited hydrolysis of the core protein by trypsin resulted in three peptides and only the peptide with a molecular weight M(r) = 40,000 was found capable of binding to hyaluronic acid. The antibody C8F4 recognized this hyaluronic acid binding peptide but did not recognize the other two peptides suggesting that the epitope(s) for this antibody is in the hyaluronic acid-binding region of the core protein. The antibody recognized the core proteins from bovine nasal cartilage proteoglycan and human aorta proteoglycan but did not recognize bovine aorta link protein, bovine serum albumin, human serum albumin, human transferrin, collagen Type I and fibronectin. The antibody was found useful to localize proteoglycans in atherosclerotic lesions in human aorta by immunohistochemical techniques.
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PMID:A monoclonal antibody that recognizes hyaluronic acid binding region of aorta proteoglycans. 768 Dec 90

We established a clonal chondrocyte-like cell line (TC6, TC stands for large T immortalized chondrocyte-like cell line) derived from articular cartilage of transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. TC6 cells exhibited spindle-like or polygonal morphology and grew well at 33 degrees C in alpha-minimal essential medium supplemented with 0.5% fetal bovine serum. After confluence, these cells formed nodules that were positive for staining with alcian blue. Northern blot analysis demonstrated that these cells expressed messenger RNAs (mRNA) of the genes encoding cartilage-specific proteins such as type II procollagen, link protein, and aggrecan. Furthermore, the expression of type II procollagen and link protein genes in TC6 cells was regulated by parathyroid hormone and basic fibroblast growth factor, suggesting the presence of the receptors for the hormone and cytokine. The expression of link protein mRNA in TC6 cells was regulated in a time-dependent manner and was enhanced in culture within a week and increased continuously up to 10-fold by the end of 4 weeks. Expression of mRNAs encoding type II procollagen and versican/PG-M also increased moderately during the culture period. TC6 cells expressed type I procollagen mRNA, however, its level declined along with time in culture in contrast to the enhancement of the genes encoding cartilage-specific molecules in these cells. Interestingly, alkaline phosphatase mRNA expression was barely detectable in the TC6 cells in their growing phase while it was enhanced dramatically more than 7-fold by day 14 in culture. These results indicate that the TC6 cells could serve as an excellent model for the studies on chondrocyte physiology.
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PMID:Establishment of a novel chondrocyte-like cell line derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. 891 72

The present study was performed to determine whether mammalian TGF-beta isoforms and Xenopus TGF-beta 5 elicit a differential chondrogenic response on mesenchymal cells during mouse limb development. Results showed that TGF-beta isoforms produced a distinct chondrogenic pattern depending on embryonic stage. When they were applied to 5 day micromass cultures of limb mesenchymal cells from embryonic stages 19, 20 and 21, a differential response to all four TGF-beta isoforms assayed was observed. By stage 19 the cells formed a uniform sheet of cartilage cells; by stage 20, mesenchymal cells were more responsive to TGF-beta 1 and TGF-beta 5 than at stages 19 and 21, showing an entire cell layer of chondrogenic cells with higher accumulation of extracellular matrix. The diminished effect of TGF-beta 2 and TGF-beta 3 at stages 20 and 21 was accompanied by a nodular pattern of chondrogenic cells rather than by a uniform sheet, as seen at stage 19. At stage 20 TGF-beta 1 and TGF-beta 5 enhanced the expression of sulfated proteoglycans, type II collagen, cartilage link protein and alkaline phosphatase activity. In contrast, TGF-beta 2 and TGF-beta 3 caused less expression in the same parameters. Only a transient exposure to TGF-beta isoforms at days 1 and 2 of culture stimulate chondrogenesis, indicating that TGF-beta isoforms could regulate chondrogenesis at early stages of chondrocyte differentiation. However, when TGF-beta isoforms were applied to low density cultures of mesenchymal cells, chondrogenesis was enhanced only by 25%, suggesting that TGF-beta isoforms enhanced cartilage differentiation to higher levels in micromass cultures than in situations in which little or no chondrogenic differentiation normally occurs.
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PMID:Differential effects of transforming growth factors beta 1, beta 2, beta 3 and beta 5 on chondrogenesis in mouse limb bud mesenchymal cells. 907 41

Proteoglycans (PGs) were extracted from a range of cartilaginous ovine connective tissues using 4 M GuHCl and separated by composite agarose polyacrylamide gel electrophoresis. Individual PG populations resolved by this electrophoretic system were identified in toluidine blue and Stains-All stained gel segments and also by conventional immunoblotting using a range of monoclonal antibodies to defined PG epitopes. These PG species were compared with aggregatable PG populations identified by affinity blotting using a biotinylated hyaluronan, and an avidin alkaline phosphatase/nitro blue tetrazolium 5-bromo-4-chloro indolyl phosphate detection system. Two major chondroitin sulfate- and keratan sulfate-substituted aggrecan populations were readily identified by affinity blotting in all of the connective tissue extracts. An additional slower migrating aggrecan species was also detected by affinity and immunoblotting in fetal disc extracts. This may represent an aggrecan species containing an intact carboxyl terminal G3 domain. Link protein was also detectable by affinity blotting; this was confirmed by immunoblotting using an anti-link protein monoclonal antibody (8-A-4). Fragments of aggrecan which contained a functional G1 domain were also detectable by affinity blotting. The biotinylated hyaluronan affinity blotting technique could detect as little as 100 ng (as hexuronic acid) of aggregatable PG. Affinity blotting therefore represents a useful new detection methodology which complements conventional immunoblotting protocols and yields information regarding the functional status of the G1 domain of individual PG populations.
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PMID:Detection of aggregatable proteoglycan populations by affinity blotting using biotinylated hyaluronan. 947 72

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.
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PMID:Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures. 1023 4

Biopsies removed from 57 patients considered for cartilage transplantation were grown at CTI Ltd. (47 biopsies) and at Tel Aviv University (10 biopsies). Tissue processing took place in dedicated laboratories. Explant cultures allowed cell number expansion. Fifty-four out of 57 biopsies grew cells. Fanning out of the cells began after 5-15 days in culture. Two passages later, cell numbers in the 10(7) range were achieved. Cells from all cultures expressed mRNA of aggrecan and link protein but not of alkaline phosphatase. Histochemical stains such as alcian blue pH 1 were negative in sparse monolayer cultures, but positive in pellet cultures. Immunohistochemistry demonstrated expression of collagen type I in monolayer cultures, switching to collagen type II in micromass cultures. Fibroblast growth factor receptor 3, a recently described characteristic receptor of precartilaginous cells, was expressed in monolayers and disappeared in micromass cultures. In conclusion, explants of articular chondrocytes cultured in vitro consistently yield monolayer cultures. The cells appear to revert to dedifferentiated chondrocytes, expressing a mesenchymal stem cell protein profile. Simultaneously, these cells regained their capacity to proliferate. Cultures held as micromass allowed reexpression of the differentiated phenotype traits.
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PMID:Characteristics of cartilage biopsies used for autologous chondrocytes transplantation. 1133 35

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.
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PMID:Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6. 1506 56

We examined the effect of the inflammatory mediator interleukin-1alpha (IL-1alpha) on cell proliferation, alkaline phosphatase (ALPase) activity, and the expressions of cartilage matrix proteins, bone morphogenetic protein-2 (BMP-2), and BMP-2 receptors in human chondrosarcoma cell line OUMS-27 (chondrocytes). The cells were cultured with Dulbecco's modified Eagle's medium containing 15% fetal bovine serum with 0, 1, 10, or 100 units/ml of IL-1alpha for up to 14 days. The expressions of cartilage matrix proteins, BMP-2, and BMP-2 receptors were estimated by determining mRNA levels using semiquantitative or real-time PCR and/or by determining protein levels using Enzyme-linked immunosorbent assay. Cell proliferation was decreased after 5 days in culture with IL-1alpha. The ALPase activity was decreased significantly in the presence of IL-1alpha until day 10 of culture. The expression of type II collagen was significantly decreased after 7 days in culture with IL-1alpha. The expressions of aggrecan and link protein were significantly decreased through day 14 of culture with IL-1alpha. The expression of BMP-2 was increased at days 3, 7, and 14 of culture with IL-1alpha, while the expression of type II receptor for BMP-2 was significantly decreased in the samples. These results suggest that IL-1alpha suppresses the expression of cartilage matrix proteins through a suppression of the autocrine action of BMP-2, brought about by the decrease in BMP-2 receptor expression in chondrocytes.
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PMID:Effect of IL-1alpha on the expression of cartilage matrix proteins in human chondrosarcoma cell line OUMS-27. 1548 96

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.
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PMID:Effects of IL-6 and soluble IL-6 receptor on the expression of cartilage matrix proteins in human chondrocytes. 1788 2


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