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)

Bone morphogenetic protein-6 (BMP-6) is an osteoinductive factor that may have a regulatory role in the terminal differentiation of chondrocytes. We investigated the expression of BMP-6 messenger RNA in freshly isolated newborn calf rib chondrocytes separated by density gradient centrifugation into five highly enriched subpopulations at different stages of maturation as assessed by cell size and alkaline phosphatase activity. Expression of BMP-6 mRNA was compared with expression of type II collagen mRNA and type X collagen mRNA using Northern analysis. Type X collagen mRNA expression increased with increasing cell size whereas type II collagen mRNA varied little with cell size. BMP-6 mRNA expression was highest in small cells and lowest in the largest cells, which were maximally expressing type X collagen mRNA. This suggests that up-regulation of the BMP-6 gene may precede chondrocyte hypertrophy.
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PMID:Expression of bone morphogenetic protein-6 messenger RNA in bovine growth plate chondrocytes of different size. 778 61

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

During development and fracture repair, endochondral bone formation is preceded by an orderly process of chondrocyte hypertrophy and cartilage matrix calcification. Analysis of calcifying versus noncalcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel collagen, type X, and decreased synthesis of type II collagen, the major component of cartilage matrix. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo these changes in gene expression and to produce calcified matrix by exposure to ascorbic acid. The mechanism by which ascorbate produces these changes has been examined by analyzing the effect of the vitamin on prehypertrophic chick embryo sternal chondrocytes. Nuclear run-on assays demonstrated that ascorbate alters mRNA levels in chondrocytes by changing the transcription rates. The fact that marked changes in mRNA levels require 1-2 days of ascorbate exposure suggested that the effect of this vitamin on gene transcription may be secondary to other, earlier ascorbate-induced effects. Since cells cultured with ascorbate produce a collagen-enriched matrix, we examined the hypothesis that transcriptional changes were secondary to altered cell-matrix interactions. Chondrocytes were cultured after attachment to tissue culture plastic, in suspension, or on plates coated with collagen type I. Comparison of alkaline phosphatase activity with and without ascorbate addition demonstrated that under all of these conditions, induction of enzyme was dependent on the presence of ascorbate. When plates containing ascorbate-conditioned chondrocyte matrix were used as substrate for naive chondrocytes, the cells continued to require ascorbate for induction of high levels of alkaline phosphatase and type X collagen mRNA. Addition of the hydroxylation inhibitor, 3,4-dehydroproline, caused marked inhibition of collagen secretion as well as accumulation of underhydroxylated collagens within the cells. However, even in the presence of this inhibitor ascorbate was effective in inducing elevated alkaline phosphatase and type X collagen. These results indicate that the ability of ascorbate to induce chondrocyte hypertrophy does not depend on production of a collagen-rich matrix.
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PMID:Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion. 807 98

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.
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PMID:Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. 808 90

The rat tracheal cartilage was shown to calcify during development. The process of calcification was characterized in terms of distribution of alkaline phosphatase (ALP) activity and alterations to immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans during the development of the tracheal cartilage, in comparison with calcification of the epiphyseal growth plate cartilage. ALP activity was not identified in the tracheal cartilage in the course of calcification, which therefore differed from that in the growth plate. The tracheal cartilage matrix was not resorbed or invaded by type I collagen during calcification. This suggests that no osteogenesis is involved in calcification of the cartilage. Immunoreactivity for type II collagen became weaker in the central region of the tracheal cartilage during development. No net loss of proteoglycans was identified with Alcian blue staining after calcification of the tracheal cartilage. Immunoreactivity for chondroitin 4-sulphate increased in the calcified tracheal cartilage, while reactivity for chondroitin 6-sulphate was weaker in the calcified area than in the surrounding uncalcified region of the tracheal cartilage. The alteration of the extracellular matrices during development may be involved in the calcification of the rat tracheal cartilage.
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PMID:The process of calcification during development of the rat tracheal cartilage characterized by distribution of alkaline phosphatase activity and immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans. 821 23

The effect of IL-1 on expression of the mineralization-related phenotype by chondrocytes was examined. In cultures of rabbit growth plate chondrocytes, IL-1 beta at 0.1 ng/ml caused 95% decreases in alkaline phosphatase activity, alkaline phosphatase mRNA levels, the incorporation of 45Ca into insoluble material, and the calcium content during the hypertrophic stage. These effects of IL-1 beta were dose-dependent and were observed in 24-48 h. Furthermore, IL-1 beta suppressed increase in cell size and the syntheses of 1,25-dihydroxyvitamin D3 receptor and type X collagen, other markers of hypertrophy, but had little effect on the synthesis of total protein including type II collagen. The inhibition of calcification was observed only when chondrocytes were exposed to IL-1 before the onset of calcification: IL-1 treatment from the mineralization stage had a marginal effect on 45Ca incorporation into insoluble material. These results suggest that IL-1 inhibits chondrocyte hypertrophy and the onset of calcification in ossifying cartilage.
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PMID:Effects of interleukin-1 on syntheses of alkaline phosphatase, type X collagen, and 1,25-dihydroxyvitamin D3 receptor, and matrix calcification in rabbit chondrocyte cultures. 822 47

Neonatal pig bone marrow stromal cells (PBMSC) were tested in vivo and in vitro to establish their use as a large-animal model for the study of skeletogenesis. When implanted in diffusion chambers in athymic mice for 6-8 weeks, both freshly isolated pig bone marrow and passage 2 PBMSC formed partially mineralized cartilage, bone-like material, and fibrous tissue. The cartilage showed metachromatic, perilacunar staining with toluidine blue and safronin O, alcian blue staining for chondroitin and keratan sulfate, and intense immunostaining for type II collagen. Osteocalcin was immunolocalized to the mineralized regions, consistent with the formation of bone. Alkaline phosphatase was primarily observed in cell layers at boundaries between tissue types. Unstimulated monolayer cultures of PBMSC produced type I but not type II collagen, responded to dexamethasone (10(-8) M) with a 1.7-fold increase in alkaline phosphatase activity, and were stimulated to divide by basic fibroblast growth factor (1.5-fold; EC50 1 ng/ml). Transforming growth factor beta (TGF-beta) blocked both dexamethasone-induced alkaline phosphatase expression (EC50, 1 ng/ml of TGF-beta) and the mitogenic effects of bFGF (EC50 0.06 ng/ml of TGF-beta). When incubated for 10-14 days in medium containing dexamethasone, beta-glycerophosphate and ascorbate PBMSC formed mineralized nodules. Calcification occurred in the middle of the aggregates and was associated with intensely alkaline phosphatase positive cells and a dense type I collagen-rich matrix. PBMSC also displayed colony-forming unit-fibroblastic activity, with approximately 1 in 80 of the plated cells formed colonies > 128 cells over 14-21 days. PBMSC therefore mimic the known activities of stromal cells from other species, including the human, suggesting that they are a valid model for skeletal research.
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PMID:Preliminary characterization of porcine bone marrow stromal cells: skeletogenic potential, colony-forming activity, and response to dexamethasone, transforming growth factor beta, and basic fibroblast growth factor. 825 54

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGF beta were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.
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PMID:Inhibition of induced endochondral bone development in caffeine-treated rats. 836 35

The first human auricular (elastic cartilage) chondrocyte cell culture model is presented. These chondrocytes, harvested from fresh human cadaver auricular cartilage, are easily grown in culture. They produce type II collagen and abundant alkaline phosphatase. Like growth plate chondrocytes, human auricular chondrocytes respond mitogenically to both transforming growth factor-beta and basic fibroblast growth factor by increasing proliferation twofold. The growth factors exert a synergistic effect on thymidine uptake in this model, with maximal stimulation occurring at the dose combination of basic fibroblast growth factor, 10 ng/mL, and transforming growth factor-beta, 3 ng/mL. Human nasal septal (hyaline cartilage) chondrocytes do not increase proliferation in response to these two growth factors. Human auricular chondrocytes also increase matrix production in response to transforming growth factor-beta, as indicated by increased proteoglycan production and increased collagen synthesis. The increased matrix production combined with increased proliferative rate elicited with transforming growth factor-beta and basic fibroblast growth factor indicates that human elastic cartilage chondrocytes harvested from the external ear are better suited for in vitro cartilage implant growth than human hyaline cartilage chondrocytes. Further biochemical and molecular biological characterization of the human auricular chondrocyte cell culture model is currently under way. Additional work is also under way to generate multilayer culture growth on absorbable frameworks, in the pursuit of the long-term goal of in vitro production of autogenous cartilage implants.
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PMID:The human auricular chondrocyte. Responses to growth factors. 841 41

Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.
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PMID:Embryonic mouse pre-metatarsal development in organ culture. 843 20


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