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)

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

The embryonic chick calvarium normally develops into an intramembranous bone without an intermediate cartilage stage, although cartilage-like calvarial cells have been observed in calcium-deficient chick embryos (Dev. Biol. 115, 215, 1986; Dev. Biol. 133, 221, 1989). To analyze the cellular basis of calvarial development, Incubation Day 14 embryonic chick calvarial cells were fractionated by Percoll gradient isopycnic centrifugation; after 12 days in monolayer culture, a subpopulation of cells (fraction F) was observed to differentiate into a rounded cellular morphology with refractile extracellular matrix. The cartilaginous nature of the extracellular matrix produced by fraction F cells was strongly suggested by the immunodetection of aggrecan and type II collagen, and Alcian blue staining. The other calvarial cell fractions (C, D, E) showed predominantly osteoblastic morphology, expressed alkaline phosphatase activity, and elaborated a collagen type I extracellular matrix. These findings suggest that a "chondrocyte-like" subpopulation of cells exist in the embryonic calvarium. To investigate how cellular interactions may influence the expression of osteogenic versus chondrogenic phenotypes by calvarial cells in vitro, the following cell type combinations were tested in high-density (20 x 10(6) cells/ml) micromass cultures: (1) total, unfractionated calvarial cells; (2) Percoll fractions (C to F) of calvarial cells; and (3) a highly chondrogenic cell type, Hamburger-Hamilton stage 23/24 chick limb bud mesenchymal cells. The micromass cocultures were set up either by mixing and plating two cell types to form the initial, single micromass or by plating the two cell types as separate, side-by-side cultures in the same culture well. The effects of interactions between cocultured calvarial and limb mesenchymal cells upon their respective differentiation fates were separately analyzed, on the basis of the number of Alcian blue-staining cartilage nodules in limb mesenchymal cells and [35S]-sulfate incorporation in both cell types. In cocultures with unfractionated or "osteoblast-like" fraction C and D calvarial cells, limb mesenchymal cells had decreased chondrogenesis. In separate cocultures with "chondrocyte-like" fraction F cells, limb mesenchymal cells exhibited enhanced chondrogenesis. Conditioned media from fraction C and D cells, and from fraction F cells, inhibited and enhanced limb mesenchymal cell chondrogenesis, respectively. Q35S]Sulfate incorporation was greater in (1) unfractionated and fractionated calvarial cells cocultured separately with limb mesenchymal cells, compared to calvarial cells cultured alone, and (2) fraction F cells, compared to other fractions or unfractionated calvarial cells. Interestingly, [35S]sulfate incorporation in fraction F cells was decreased when cocultured with fraction C, D, and E cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interactive cellular modulation of chondrogenic differentiation in vitro by subpopulations of chick embryonic calvarial cells. 785 37

Gene expression patterns have been investigated in prolonged cultures of rat mandibular condylar cartilage (MCC) cells to examine the possibility of culture-induced phenotypic changes. MCC cells were isolated from newborn rats and grown in the presence of 10% fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). MCC cells were passaged and cultured in the presence of 10% FBS and bFGF until confluent. After confluence, the medium was changed to that supplemented with 10% FBS, ascorbate, and beta-glycerophosphate (day 1). Mineralization and gene expression of MCC cells have been investigated. Mineralization, visualized by staining with Alizarin red, was observed to begin at day 13 in culture, and increased up to day 22 in culture, which was the length of this study. Type II collagen and aggrecan mRNAs were highly expressed at the start of culture (day 1-4) and decreased in a time-dependent manner. Type I collagen, alkaline phosphatase, and osteopontin mRNAs expressed biphasic patterns that peaked at the start of culture and the beginning of mineralization (day 13-16). Osteonectin mRNA was expressed throughout the culture period. Osteocalcin mRNA was expressed before the beginning of mineralization peaking at day 7. These observations suggest that the gene expression patterns of MCC cells can be categorized into two different periods in prolonged culture: maturation (day 1-10) and mineralization (day 13-22). The day culture system of MCC represents a new model system in which the differentiation of embryonic MCC cells can be examined.
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PMID:Changes in phenotypic gene expression in rat mandibular condylar cartilage cells during long-term culture. 859 45

The effects of ultrasound stimulation on various parameters of bone repair after diaphyseal injury were assessed in a standard rat femur fracture model. Bilateral closed femoral fractures were made in 79 skeletally mature male Long-Evans rats. An ultrasound signal consisting of a 200 microsecond burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 50 or 100 mW/cm2 spatial and temporal average, was applied to one fracture in each animal. The contralateral fracture was not exposed to ultrasound and served as a control. Mechanical testing of the healing fracture was performed 3 weeks after injury. In fractures treated with a 50 mW/cm2 ultrasound signal, the average maximum torque (223.5 +/- 50.5 Nmm compared with 172.6 +/- 54.9 Nmm, p = 0.022, paired t test) and average torsional stiffness (13.0 +/- 3.4 Nmm/degree compared with 9.5 +/- 2.9 Nmm/degree, p = 0.017) were significantly greater in treated than in control fractures. In animals treated with a 100 mW/cm2 ultrasound signal, the average maximum torque and torsional stiffness were greater in treated than in control fractures, but this trend did not reach statistical significance. Biochemical analysis of callus in ultrasound-treated and control fractures failed to demonstrate significant differences in cell number, collagen content, or calcium content. Evaluation of gene expression in fractures treated with 50 mW/cm2 ultrasound demonstrated a shift in the expression of genes associated with cartilage formation; aggrecan gene expression was significantly higher on day 7 after fracture and significantly lower on day 21 (p = 0.033 and 0.035, respectively). alpha 1(II) procollagen gene expression was similarly modified, but this trend did not reach statistical significance. Expression of genes coding for bone-related proteins, including alpha 1(I) procollagen, bone gamma-carboxyglutamic acid protein, alkaline phosphatase, and transforming growth factor-beta 1, did not differ between ultrasound-treated and control fractures. These data suggest that ultrasound stimulation increased the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation.
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PMID:Exposure to low-intensity ultrasound increases aggrecan gene expression in a rat femur fracture model. 889 75

Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins.
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PMID:Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure. 890 41

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

A clonal cell line named RMD-1 was established from the skeletal muscle of a 20-day fetal rat. RMD-1 represents a morphologically homogeneous population of undifferentiated mesenchymal cells, expressing alpha-smooth muscle actin and type I collagen, but no cartilage-associated genes. When cultured in agarose gel containing 100 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2; BMP-2), RMD-1 cells formed colonies and showed chondrocyte-like features as assessed by their ultrastructure, metachromatic staining with toluidine blue, and the production of large hydrodynamic-size proteoglycans. RMD-1 cells also differentiated into chondrocytes when the cells were plated at high density (over 2.5 x 10(5) cells/cm2) on type I collagen and incubated in medium containing 0.5% fetal bovine serum and 100 ng/ml of BMP-2. This chondrogenic differentiation was evidenced by a distinct morphological change into spherical cells, an increase in the levels of sulfated glycosaminoglycans, a decrease in type I collagen mRNA and the expression of cartilage-associated genes, including type II collagen, type IX collagen, aggrecan and alkaline phosphatase. In the presence of ascorbic acid and 10% serum, RMD-1 cells increased in size and expressed type X collagen as well as high alkaline phosphatase activity, then induced matrix mineralization. Thus, RMD-1 is a unique cell line that can differentiate from undifferentiated mesenchymal cells into hypertrophic chondrocytes.
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PMID:Establishment of bone morphogenetic protein 2 responsive chondrogenic cell line. 899 86

The pathogenesis of vitamin A-induced premature growth-plate closure was investigated in calves. A progressive increase in the severity of growth-plate lesions with time and a progressive increase in the extent of growth-plate involvement was observed. There was initial loss of metachromasia from the growth plate in a region that formed a narrow horizontal band of cartilage composed of the epiphyseal growth zone and a strip of reserve-zone cartilage. Immunostaining revealed there was loss of aggrecan, decorin, and biglycan from this region; however, it was doubtful that the regional loss of proteoglycan was a major contributing factor in the pathogenesis of premature growth-plate closure. This is because this region was the vestige of cartilage that remained when growth-plate closure was almost complete. The major alteration was premature mineralization of columnar cartilage and subsequent endochondral ossification. This caused the depth of the columnar zone to be reduced. Columnar-zone cartilage cells appeared immature where the matrix became mineralized and lacked the morphology of hypertrophic chondrocytes. The depth of the reserve-cartilage zone also was reduced as matrix mineralization of the columnar zone progressed, and further reduction in columnar cartilage depth occurred. Eventually, there was matrix mineralization within the adjacent reserve cartilage. The distribution of reaction product after immunostaining with antibodies to the following proteins was described during normal endochondral ossification: aggrecan, decorin, biglycan, versican, type I collagen propeptide, type I collagen, type II collagen, osteopontin, osteocalcin, osteonectin, bone sialoprotein, and alkaline phosphatase. Biglycan, type I collagen propeptide, type I collagen, osteopontin, osteocalcin, osteonectin, bone sialoprotein, and alkaline phosphatase were localized within the cytoplasm or surrounding matrix of hypertrophic chondrocytes. In vitamin-treated calves, these same proteins were found in regions undergoing premature matrix mineralization even though the chondrocytes did not have a hypertrophic morphology. Therefore, vitamin treatment did not cause just a selective expression, but it caused expression of a large number of matrix proteins normally associated with the hypertrophic chondrocyte phenotype. Finally, completely mineralized columnar and reserve cartilage were removed by a modeling/remodeling process similar to that seen in the metaphysis.
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PMID:Pathogenesis of vitamin (A and D)-induced premature growth-plate closure in calves. 926 93

Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of scleraxis, we overexpressed scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to in vitro translated scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing scleraxis and was supershifted by the antiserum raised against scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.
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PMID:Overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression in osteoblastic osteosarcoma ROS17/2.8 cells. 936 62

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II-expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II-expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II-expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.
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PMID:Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes. 944 16


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