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)

This study aimed to identify the behaviour of mandibular condylar chondrocytes in vitro. Cells were harvested from the mandibular condyles of 3 week-old S. D. rats. Cell structure, morphology and characteristics were assessed by phase-contrast microscopy, scanning electron microscopy, enzymohistochemistry and immunohistochemistry. The cultured condylar chondrocytes, stellated or spindle-shaped, could grow in several layers and form many cell colonies. They could secret proteoglycans, alkaline phosphatase, type II collagen, et al. The methods of isolation, culture and identification of condylar chondrocytes were presented and discussed, which can be adopted in probing further into the cellular mechanism of functional orthopedics.
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PMID:[The behaviour of rat mandibular condylar cartilage in cell culture]. 1220 13

The mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-beta or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-beta and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation.
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PMID:Basic helix-loop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. 1238 5

Deer antlers are the only mammalian organs that can be repeatedly regenerated; each year, these complex structures are shed and then regrow to be used for display and fighting. To date, the molecular mechanisms controlling antler regeneration are not well understood. Vitamin A and its derivatives, retinoic acids, play important roles in embryonic skeletal development. Here, we provide several lines of evidence consistent with retinoids playing a functional role in controlling cellular differentiation during bone formation in the regenerating antler. Three receptors (alpha, beta, gamma) for both the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families show distinct patterns of expression in the growing antler tip, the site of endochondral ossification. RAR alpha and RXR beta are expressed in skin ("velvet") and the underlying perichondrium. In cartilage, which is vascularised, RXR beta is specifically expressed in chondrocytes, which express type II collagen, and RAR alpha in perivascular cells, which also express type I collagen, a marker of the osteoblast phenotype. High-performance liquid chromatography analysis shows significant amounts of Vitamin A (retinol) in antler tissues at all stages of differentiation. The metabolites all-trans-RA and 4-oxo-RA are found in skin, perichondrium, cartilage, bone, and periosteum. The RXR ligand, 9-cis-RA, is found in perichondrium, mineralised cartilage, and bone. To further define sites of RA synthesis in antler, we immunolocalised retinaldehyde dehydrogenase type 2 (RALDH-2), a major retinoic acid-generating enzyme. RALDH-2 is expressed in the skin and perichondrium and in perivascular cells in cartilage, although chondroprogenitors and chondrocytes express very low levels. At sites of bone formation, differentiated osteoblasts which express the bone-specific protein osteocalcin express high levels of RALDH2. The effect of RA on antler cell differentiation was studied in vitro; all-trans-RA inhibits expression of the chondrocyte phenotype, an effect that is blocked by addition of the RAR antagonist Ro41-5253. In monolayer cultures of mesenchymal progenitor cells, all-trans-RA increases the expression of alkaline phosphatase, a marker of the osteoblastic phenotype. In summary, this study has shown that antler tissues contain endogenous retinoids, including 9-cis RA, and the enzyme RALDH2 that generates RA. Sites of RA synthesis in antler correspond closely with the localisation of cells which express receptors for these ligands and which respond to the effects of RA.
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PMID:A role for retinoic acid in regulating the regeneration of deer antlers. 1243 67

Epigallocatechin 3-gallate (EGCG), which is one of the components of green tea, was recently shown to inhibit endothelial cell growth in vitro and angiogenesis in vivo [5]. We have previously shown that bone and cartilage formation by bone morphogenetic protein (BMP) is highly dependent on the geometry of the carrier (vasculature-inducing or -inhibiting geometry [2]. To verify the function of angiogenesis in the BMP induction system, we examine in this article whether inhibition of angiogenesis enhances chondrogenesis and suppresses osteogenesis. Fibrous glass membrane used as a BMP carrier was mixed with 1.2 micrograms rhBMP-2 and 1-10 micrograms of EGCG and was implanted into rats subcutaneously. As the dose of EGCG increased, alkaline phosphatase activity and calcium content were decreased, whereas the type II collagen content was increased. The results clearly indicated that inhibition of vascularization enhanced chondrogenesis and suppressed osteogenesis.
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PMID:Inhibition of BMP-induced ectopic bone formation by an antiangiogenic agent (epigallocatechin 3-gallate). 1248 8

In order to analyze the phenotypic conversion of chondrocytes, mandibular condyles of mice and rabbits were cultured under cell and organ culture systems, and then examined by a combination of morphological and biochemical procedures. In organ culture, mandibular condylar cartilage (MCC) obtained from newborn mice began to mineralize from the central zone and then progressively widened towards the peripheral zone. Electron microscopic observations showed that with the increasing duration of the organ culture, chondrocytes at the central zone converted into spindle-shaped osteoblastic cells accompanying the formation of the bone type of thick-banded collagen fibrils. To obtain a better understanding of the chondrocytic conversion, immunolocalizations for type I and type X collagens and osteocalcin (OC) were examined in mouse MCC cells in cell culture. Type X collagen and OC were expressed almost simultaneously at the late stage of culture, and type I collagen was detected along the calcified nodules after the production of these proteins. Northern blot analysis in cell cultures of rabbit MCC indicated that type II collagen and alkaline phosphatase (ALPase) messenger ribonucleic acids (mRNAs) were highly expressed at day 7, but subsequently decreased. In contrast, mRNA for type I collagen was expressed at a low level on day 7 and peaked on day 12. The present results suggest that, morphologically and biochemically, cellular modification in MCC cells under culture conditions occurs at a cellular morphological level and also at marker-gene-expression level.
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PMID:Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization. 1255 19

The major aim of the current investigation was to evaluate the role of thiols during chondrocyte maturation and apoptosis. Using a thiol-sensitive fluorescent probe, we found that in chick growth plate chondrocytes, hypertrophy is accompanied by a decrease in the glutathione content. In this study, we show that the maturation-dependent loss of thiol, although not causing death of maturing chondrocytes, drastically increases susceptibility to apoptosis by oxidative and nitrosoactive stress. To investigate how the loss of thiol content in cultured chondrocytes affects the expression of the hypertrophic phenotype, we chemically manipulated intracellular thiol levels and analyzed the expression of important maturation markers. We found that thiol depletion causes a decrease in the expression of osteopontin, type X and type II collagen and a significant loss of alkaline phosphatase activity, suggesting that the expression of the hypertrophic phenotype is tightly regulated by redox levels in chondrocytes. Furthermore, severe thiol depletion profoundly affected cell survival under oxidative and nitrosoactive stress. It was concluded that the loss of thiol reserve is not only linked to the expression of the hypertrophic phenotype but also influenced chondrocyte survival, linking chondrocyte maturation and the activation of the apoptotic pathway.
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PMID:Maturation-dependent thiol loss increases chondrocyte susceptibility to apoptosis. 1267 27

One major problem of current cartilage repair techniques is that three-dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild-type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)-2 or -4 in alginate revealed that the formation of markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP-transfection. Cells were encapsulated in ultrahigh-viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously staining with haematoxylin-eosin or Alcian blue, immunohistochemical analysis, and quantitative RT-PCR confirmed the expression of chondrogenic markers (chondroitin-4- and -6-sulfate as well as type II collagen). Production of chondrogenic markers was particularly high in BMP-4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP-4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild-type cells and to some extent for BMP-2 transfected cells. The osteogenic markers, type I collagen, alkaline phosphatase, and Cbfa1 were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP-4 transfected cells as revealed quantitatively by real time RT-PCR. Thus, the in vitro results suggested that BMP-4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.
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PMID:Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate. 1455 23

Using a cell culture method, we histochemically and immunohistochemically investigated whether chondrocytes deriving from different origins, such as Meckel's or costal cartilages, express similar phenotypic characteristics. Chondrocytes isolated enzymatically from Meckel's and costal cartilages of 17-day embryonic mice both actively proliferated and formed cartilage nodules consisting of toluidine blue-positive proteoglycans and type II collagen. Both deposited calcified cartilaginous matrix as revealed by alkaline phosphatase (ALPase) activity and alizarin red staining throughout 3 weeks in culture. Immunostaining for osteopontin (OP), osteocalcin (OC), and osteonectin (ON) revealed that chondrocytes from both cartilages were positive for their proteins, but type I collagen was detected only in cells transforming from Meckel's chondrocytes late in the culture. Electron microscopy demonstrated that although costal and Meckel's chondrocytes had typical chondrocytic features during 2 weeks in culture, Meckel's chondrocytes transformed into osteocytic cells that produced thick, banded type I collagen fibrils. In contrast, costal chondrocytes maintained typical hypertrophic morphology throughout the final stage of culture. The present study suggests that Meckel's chondrocytes derived from neural crest-ectomesenchyme retain osteogenic potential, and differ from costal chondrocytes originating from mesoderm.
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PMID:Origin-associated features of chondrocytes in mouse Meckel's cartilage and costal cartilage: an in vitro study. 1457 66

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.
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PMID:Differential gene expression analysis using paraffin-embedded tissues after laser microdissection. 1462 59

Treatment with BMP-7 causes a shift in the differentiation pathway from myoblastic to osteoblastic in C2C12 mouse myoblast precursor cells in vitro. The underlying molecular mechanism is largely unknown. BMP-7 at 200 ng/ml completely inhibited myotube formation in C2C12 cells and dramatically induced alkaline phosphatase activity up to 20-fold when compared to untreated cells by day 12 in culture. The level of Runx2/Cbfa1 mRNA, a bone-specific transcription factor, was also stimulated up to 6-fold by BMP-7 with a peak at 24 h. In addition BMP-7 treatment stimulated a 55-fold increase in osteocalcin mRNA as early as 24 h after treatment. A novel finding was that the expression of the chondrocyte markers Sox9 and type II collagen was increased as well. Runx2/Cbfa1 is a molecular switch for osteoblast differentiation. To initiate the study of modulators of Runx2/Cbfa1, such as kinases and cofactors, during osteoblastic differentiation of C2C12 cells treated by BMP-7 in vitro, microarray analyses of gene expressions were performed. Microarray data suggested that a total of 882 transcripts were either up- or downregulated at least 2-fold. Cluster analyses revealed 76 genes (including ESTs) with expression patterns that paralleled Runx2/Cbfa1. Thirteen of these 76 genes were initially selected as potential transcription modulators for further study; including CCAAT/enhancer binding protein delta, distal- less homeobox 1, forkhead box F2, insulin-like growth factor binding protein 4, an ortholog of human osteoclast stimulating factor 1 and p300/CBP-associated factor. Some transcription modulators have been associated with osteoblastic differentiation or interacted with Runx2/Cbfa1. Most of them have not been extensively studied in osteoblastic differentiation and in relationship to Runx2/Cbfa1. Thus, these studies identify potential regulators for Runx2/Cbfa1 and osteoblast differentiation. In addition, our data revealed for the first time that BMP-7 not only induced the expression of osteoblastic differentiation markers but also stimulated the expression of chondroblastic markers in C2C12 cells.
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PMID:Identification of potential modifiers of Runx2/Cbfa1 activity in C2C12 cells in response to bone morphogenetic protein-7. 1474 33


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