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 compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of alpha 2(I)-collagen. The phenotype of the bone cell cultures was defined by a 3-4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of alpha 2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T----G point mutation in the second position of the 5' splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant alpha 2(I)-collagen allele compared to dermal fibroblasts.
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PMID:Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV. 164 48

A human malignant fibrous histiocytoma (MFH) cell line, designated as MFH-ino, was established from the maxillary tumor of a 45-year-old woman. Clinically, the original tumor was accompanied by extensive destruction of the surrounding tissues. Cells were obtained from the explant culture of tumor fragments. Both histiocytic and fibroblastic markers were observed in the histochemical and immunocytochemical studies of MFH-ino. The cells were positive for lysozyme, alpha-1-antichymotrypsin, and the collagen types I, III, IV, V, but were negative for alpha-1-antitrypsin, acetate esterase and type II collagen. As biochemical examinations of the culture cells, collagen synthesis was assayed by the measurement of hydroxyproline and the content increase in culture dishes with time after cell inoculation. Collagenase activity secreted in culture medium was also examined with FITC-labeled type I collagen as substrate, and high activity was detected at the late stage of the stationary phase. Further, the MFH-ino cells had high acid phosphatase activity while lacking alkaline phosphatase activity. These findings indicated that MFH-ino cells expressed the various properties of MFH, which will be of importance for understanding the biological behavior, and especially the collagen metabolism, of MFH.
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PMID:Establishment and characterization of a human neoplastic cell line (MFH-ino) derived from malignant fibrous histiocytoma of maxilla. 165 97

Samples of rat alveolar bone were first treated by collagenase digestion and then used as explants for cell culture. The cells obtained were subcultured and characterized by morphological and functional criteria. Their alkaline phosphatase activity was increased after incubation in 1,25-(OH)2 vitD3 10(-8) M whereas with gingival cells it did not change. The bone derived-cells organized nodular structures, synthesized type I collagen, Gla-protein, few type III collagen, and fibronectin. In the defined culture conditions no mineralization was observed. However, the method used allows to obtain cells from rat alveolar bone displaying some features of the osteoblastic phenotype.
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PMID:Isolation and characterization of rat alveolar bone cells. 165 92

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.
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PMID:Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. 169 81

The bone, liver, and kidney isozyme of alkaline phosphatase (ALP) has been measured in MG-63 human osteosarcoma cells after treatment with ascorbic acid (AA) and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both compounds were required to achieve maximum ALP activity. When grown in the absence of 1,25-(OH)2D3 cells had low basal ALP activity regardless of whether media contained AA. In AA-free medium, 1,25-(OH)2D3 (10 nM) increased ALP activity fourfold. Addition of AA further increased levels of ALP activity induced by 1,25-(OH)2D3 to 10-15 times those found in -AA controls. The earliest effects of 1,25-(OH)2D3 were seen after 24-48 h, and ALP activity continued to increase for 6-8 days. AA and 1,25-(OH)2D3 had similar effects on ALP activity in ROS 17/2.8 rat osteosarcoma cells. In MG-63 cells the effects of AA and 1,25-(OH)2D3 could not be simply explained by the ability of these compounds to inhibit cell growth because another mitotic inhibitor, hydroxyurea, had a minimal effect on ALP activity. 1,25-(OH)2D3-specific induction of ALP +/- AA was totally blocked by inhibitors of protein and RNA synthesis. Maximal ALP induction was obtained when cells were plated at low density. Consistent with our previous report (Franceschi et al. 1988 J Biol Chem 263:18938-18945), 1,25-(OH)2D3 rapidly stimulated type I collagen synthesis and acid-precipitable hydroxyproline production in MG-63 cells and this stimulation was further increased by AA. These results suggest that induction of the osteoblast marker, ALP, is directly or indirectly coupled to collagen matrix synthesis and/or accumulation.
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PMID:Regulation of alkaline phosphatase by 1,25-dihydroxyvitamin D3 and ascorbic acid in bone-derived cells. 170 22

Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast-like cells from spleen cells in mice. Osteoclasts were defined as tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP-positive MNCs) in which specific calcitonin receptors were identified by autoradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP-positive MNCs in response to 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] in co-cultures with spleen cells. A cell line, KS-4, was identified with the greatest potency in inducing osteoclast-like cell formation in co-culture with spleen cells. The capacity of KS-4 cells to produce this effect was much greater than that of two bone marrow-derived stromal cell lines (MC3T3-G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1 alpha, 25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805-1813, 1989). Parathyroid hormone (PTH) increased cAMP production in KS-4 cells, and PTH and interleukin-1 alpha also induced TRACP-positive MNCs in co-cultures with spleen cells. Contact between living KS-4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS-4 cells were killed by fixation. Separate cultures of either spleen cells or KS-4 cells formed no TRACP-positive MNCs. KS-4 cells synthesized predominantly type I collagen, formed bone nodules without added of beta-glycerophosphate in a long-term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS-4 cells have properties consistent with progression toward the osteoblast phenotype and represent a single cell line with the ability to promote osteoclast formation by a contact-requiring process.
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PMID:Cloning of an osteoblastic cell line involved in the formation of osteoclast-like cells. 170 73

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Gene expression in osteoblastic cells. 180 5

The non-invasive assessment of bone turnover has received increasing attention over the past few years because of the need for sensitive markers in the clinical investigation of osteoporosis. Markers of bone formation include serum total and bone-specific alkaline phosphatase, serum osteocalcin, and measurement of serum type I collagen extension peptides. Assessment of bone resorption can be achieved with measurement of urinary hydroxyproline, urinary excretion of the pyridinium crosslinks (Pyr and D-Pyr), and by measurement of plasma TRAP activity. For the screening of bone turnover in women at the menopause, and for the assessment of the level of bone turnover in elderly women with vertebral osteoporosis, serum osteocalcin and urinary Pyr and D-Pyr appear to be the most sensitive markers so far. Programmes combining bone mass measurement and assessment of bone turnover in women at the time of the menopause have been developed in an attempt to improve the assessment of the risk for osteoporosis. Efforts are being made to develop more convenient assays and to identify other markers of bone turnover. In the future a battery of various specific markers is likely to improve the assessment of the complex aspects of bone metabolism, especially in osteoporosis.
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PMID:What do we know about biochemical bone markers? 182 19

Recent evidence indicates that matrix vesicles (MV) interact with cartilage-specific collagens and other matrix proteins. Both type II and X collagens bind to and cosediment with MV. Our companion study shows that MV also are tightly coupled to proteoglycan link proteins (LP) and hyaluronic acid-binding region (HABR) in cartilage matrix. Here we sought to identify proteins responsible for the nexus between MV and matrix collagens using affinity chromatography with types I, II, and X collagen-Sepharose columns. Elution with NaCl step-gradients in the presence of nonionic detergent was used to assess the affinity between the MV proteins and the covalently attached collagens. Several MV proteins were found to bind to native type I, II, and X collagens but none bound to denatured type I collagen. Alkaline phosphatase, proteoglycan LP and HABR, and the 33- and 67-kDa annexins, bound with varying affinities to the native type I, II and X columns. In particular, LP and HABR, the 67-kDa annexin, and alkaline phosphatase bound with high affinity to the cartilage-specific collagens, although LP, HABR, and a 37-kDa protein also bound less tightly to native type I collagen. Thus, several MV proteins bind specifically to native type II and X collagens and should promote interaction between MV and the extracellular matrix. Such interactions may be important in MV formation, or in MV-mediated mineralization.
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PMID:Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen. 184 89

We present a new human osteosarcoma cell line designated OHS-4. These cells showed a high alkaline phosphatase activity that is not regulated by 1,25 dihydroxyvitamin D3. They exhibited a sensitive adenylate cyclase response to parathyroid hormone but not to prostaglandin E2 or human calcitonin. By Northern blot analysis we could detect type I collagen mRNA but none for type III collagen. The cells were able to produce human osteocalcin at a maximum level of 35 ng per million cells when exposed to 2.4 nM 1,25-dihydroxyvitamin D3 for 96 h. We purified this protein from conditioned media using successive chromatography and assessed its identity by partial amino acid sequencing. When injected into nude mice, the cells retained their osteogenic activity and developed calcified tumors. After Von Kossa staining, we observed nonmineralized osteoid deposits and mineralized deposits with a structure similar to that of trabecular bone by light microscopy. On the basis of its osteoblastic characteristics, this new osteosarcoma cell line may represent the human counterpart of the ROS 17/2 cell line. This cell line represents a valuable model for the isolation and characterization of human bone specific proteins.
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PMID:Characterization of a new human osteosarcoma cell line OHS-4. 186 Aug 86


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