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)

Cells of the clonal osteogenic cell line MC3T3-E1 were seeded onto a three-dimensional matrix of denatured collagen type 1 and cultured for a period of up to 8 weeks. Specimens were analyzed by histological, enzyme histochemical, immunocytochemical, and ultrastructural methods and by in situ hybridization between day 7 and day 56 after seeding. In 56-day cultures, the MC3T3-E1 cells were arranged in a three-dimensional network and formation of bone-like tissue was indicated by calcification of a newly synthesized collagen type I matrix resembling osteoid and surrounding osteocyte-like cells. The differentiating culture showed high expression of osteocalcin and alkaline phosphatase activity. NIH3T3 fibroblasts used as control cells passed through the network of the substrate forming a confluent monolayer underneath. This culture system offers a potentially powerful model for bone formation in vitro and for investigating the osteogenic potential of bone-derived cells.
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PMID:Bone formation by osteoblast-like cells in a three-dimensional cell culture. 229 23

The proliferation of human bone-derived cells (BDCs) was assessed in vitro, using [3H]thymidine incorporation and cell counting in a haemacytometer. The cells were cultured from human trabecular bone from 87 patients aged 2-88 years. The in vitro growth of these cells was unaffected by the chronological age of the donor. However, the cell number at confluence was shown to decrease with increasing donor age, this trend being most marked after 60 years of age. Other assays of the metabolic efficiency of the BDCs, namely, total protein, osteocalcin, and alkaline phosphatase synthesis, did not show any change with increasing donor age. These results suggest that while the ability of individual cells to divide and to perform specific synthetic activities is unimpaired with increasing age, other subtler changes may occur, leading to a decrease in the bone's osteogenic capacity.
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PMID:Effect of donor age on the growth in vitro of cells obtained from human trabecular bone. 230 56

The effect of donor age on the production of bone-like tissue and expression of cellular alkaline phosphatase was examined in cultures of cells obtained from rat bone marrow. Stromal cells were obtained from the bone marrow of young (5-6 weeks) and old (18 months) rats and cultured in vitro. After 28 days in first subculture, the following were quantified: (1) the total number of mineralised nodules and the size distribution of nodules and (2) the density of osteoblasts and osteocytes associated with nodules in histological sections. The doubling times of the cultures and the numbers of cells in cultures which expressed alkaline phosphatase activity were determined in separate experiments. Cells from young cultures produced three times more bone-like nodules than old cultures, although no differences were seen in the size distribution of nodules, or on osteoblast and osteocyte density. Doubling times for both groups were similar. The numbers of alkaline phosphatase (AP) positive cells was reduced by half in old cultures. These data show that this model may be useful for the study of the mechanisms of ageing on osteogenesis, and demonstrate a reduced osteogenic capacity in old cultures. The results suggest that this effect may be due to a reduction in the generation of cells of osteoblast lineage during ageing.
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PMID:Effects of donor age on osteogenic cells of rat bone marrow in vitro. 230 88

Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the osteoblastic lineage. One antibody, SB-1, reacts in frozen sections with a family of cells in bone, liver, kidney, and intestine which are identically stained by the histochemical substrate for alkaline phosphatase. In this report, biochemical and immunochemical evidence is presented to indicate that SB-1 is directed against an epitope on alkaline phosphatase which is shared by isoenzymes in a variety of chick tissues. In a solid-phase assay system, high dilutions (1:10(5] of ascites fluid were found to give significant binding of SB-1 to alkaline phosphatase extracted from chick limb or intestine. Partial purification of intestinal alkaline phosphatase on a Sepharose CL-6B column results in the co-elution of alkaline phosphatase enzyme activity and antibody-binding material; this indicates that SB-1 recognizes intestinal alkaline phosphatase rather than an impurity in the crude preparation. Furthermore, Western immunoblots of chick calvarial bone extract electrophoresed on a 5-20% SDS-polyacrylamide gel show that SB-1 reacts with a single 155 kD band which also is stained by the alkaline phosphatase histochemical substrate. In a similar set of experiments, SB-1 reacts with an intestinal alkaline phosphatase isoenzyme whose molecular weight is approximately 185 kD. From these studies, we conclude that SB-1 is specifically reactive with alkaline phosphatase isoenzymes present in bone, liver, kidney, cartilage, and intestine. The reactive epitope is stable to SDS denaturation, not associated with the active site of the enzyme, and dependent on disulfide bonds which impart secondary structure to the protein.
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PMID:A monoclonal antibody against the surface of osteoblasts recognizes alkaline phosphatase isoenzymes in bone, liver, kidney, and intestine. 235 24

Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with [3H]thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of [3H]thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of [3H]thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Clusters were predominantly AP-positive and located close to bone. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity [3H]thymidine at 1 and 2 days of culture completely blocked bone formation. These data indicate that a very small population of cycling osteoprogenitor cells is essential for bone formation in vitro and give rise to relatively small numbers of clonally distributed progenitors with limited proliferative capacity. The progeny of these clusters undergo restricted migration and differentiate into osteoblasts.
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PMID:Clonal distribution of osteoprogenitor cells in cultured chick periostea: functional relationship to bone formation. 237 58

The characteristics of a new osteosarcoma-associated cell surface antigen were studied by means of two murine monoclonal antibodies, TP-1 and TP-3, which were found to bind to two different epitopes on the same antigen, a monomeric polypeptide with a molecular weight of approximately 80,000. Immunohistochemical studies showed that the antigen was present in all osteogenic sarcomas tested, in most cases of malignant fibrous histiocytoma, in two malignant hemangiopericytomas and in a few synovial sarcomas, but not in other main groups of sarcomas and nonsarcomatous malignancies. In normal tissues it was detected only in clusters of cells in the adrenal medulla and in proximal kidney tubules. Also endothelial cells in proliferating capillaries in placenta and in most tumors were stained. The antigen was absent in resting but present in actively proliferating osteoblastic cells. The epitopes were resistant to proteolytic and sugar-cleaving enzymes but sensitive to high temperatures and could not be detected in paraffin-embedded specimens. The tissue distribution and properties of the antigen show that it is different from the sarcoma-associated antigens previously studied. In contrast to previous findings with three other anti-sarcoma monoclonal antibodies, no correlation was found between serum alkaline phosphatase activity and the amount of TP-binding substances in the same sera. Nevertheless, an apparently complex association between alkaline phosphatase and the TP-binding antigen seems to exist. Thus, the Mr 80,000 antigen extracted from an osteosarcoma cell line showed enzyme activity, whereas TP-binding molecules precipitated from patient sera contained alkaline phosphatase activity only in a few of the cases studied. Altogether our data suggest that the antigen defined by the TP antibodies may be a marker of osteoblastic differentiation. The pattern of antigen expression in malignant tumors is unique, inasmuch as the antigen is found selectively in sarcomas and in all 31 osteosarcomas tested.
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PMID:Expression and characteristics of a novel human osteosarcoma-associated cell surface antigen. 245 39

Detailed studies of the origin and differentiation of osteogenic cells can be facilitated by cell-specific markers. To this end, we immunized mice with a heterogeneous population of chick embryonic bone cells and subsequently generated and selected for monoclonal antibodies against cell surface determinants. Supernatants from growing hybridoma colonies were screened immunohistochemically against frozen sections of embryonic stage 35 (day 9.5) chick tibiae. Three cell lines, SB-1, SB-2, and SB-3, which each secrete a different antibody against osteogenic cells, have been successfully cloned, stabilized, and immortalized. Antibody SB-1 reacts with a family of cells in embryonic bone, liver, kidney, and intestine, which are identically stained by the histochemical stain for alkaline phosphatase. The SB-2 antigen is present only on osteoblasts, while the SB-3 antigen is expressed on the surface of osteoblasts, ependymal cells and ventricular myoblasts. Studies on the developmental progression of osteoblasts in the embryonic tibia indicate that the determinants recognized by SB-1, SB-2, and SB-3 are temporally coupled to the appearance of the pre-osteoblast marker alkaline phosphatase. Detailed morphologic analyses reveal that SB-1 reacts with a large family of osteogenic cells residing between the surface of newly formed bone and the overlying periosteal osteoprogenitor cells. By contrast, SB-2 and SB-3 appear to react with those mature osteoblasts involved in the secretion and mineralization of osteoid. Cells which are imprisoned within bone matrix (osteocytes) of the developing tibia are not recognized by these antibodies, but are immunostained by an osteocyte-specific monoclonal antibody which does not react with SB-1, SB-2 or SB-3 positive cells. The results reported here suggest the existence of an osteogenic cell lineage which is characterized by a series of discrete cell states prior to the overt expression of the Secretory Osteoblastic phenotype. We propose that the emergence and abatement of phenotypically distinct osteogenic cell surface antigens follows a precise spatial and temporal sequence which reflects the position of cells within the osteogenic lineage.
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PMID:First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts. 248 84

A series of four antibodies against rat osteoblasts have been produced using the hybridoma technique. After bone cells isolated from newborn rat calvariae by a sequential digestion procedure were cultured for 3 days, the cells were trypsinized and further maintained in rotation cultures overnight. Out of the cultured bone cells alkaline phosphatase-positive cells were sorted by flow cytometry and used as immunogens. The clones secreting the antibodies were selected on the basis of the abilities of these antibodies to bind to the bone cells but not to fibroblasts from neonatal rat head skins, in an enzyme-linked immunosorbent assay. Clones of two hybridomas, designated AOB-1 and AOB-2, were used to characterize the antigenic determinant(s) in osteogenic cells. The antibody showed the reactivity with isolated alkaline phosphatase-positive cells, osteogenic tissue cells in newborn rat calvaria, and mandibula, but not with the cells in head skin, lung, kidney, liver, or stomach as determined by immunofluorescence study. Western blot analysis has identified the antigenic determinants possessing apparent molecular weights of 210,000, 110,000, 65,000, 58,000, 40,000, 36,000, 32,000, 28,000, 25,000, 17,000, and 15,000 of osteoblast-rich monolayer cultured cells. According to the cell surface detection with biotin-avidin protein blotting technique, these fractions appear to be present as components of the cell surface of the osteoblast.
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PMID:Identification of osteoblast-specific monoclonal antibodies. 249 28

Previous in vitro studies have shown that salmon calcitonin had direct effects to increase parameters associated with embryonic chicken bone formation and to increase mouse and chicken osteoblast-line cell proliferation. The current studies demonstrate increased cell proliferation (i.e., [3H]-thymidine incorporation into DNA and tetrazolium salt reduction/deposition) in the osteoblastic murine cell line MC-3T3-E1 in response to salmon calcitonin (P less than 0.005) and to human calcitonin (P less than 0.005), but not to human calcitonin gene-related peptide. The current studies also show that salmon calcitonin increased several indices of murine bone formation. We found that 72 hours of exposure to salmon calcitonin [at 5 mU/ml-about 0.37 nM; mU/ml = milliunits of calcitonin activity/ml incubation medium (at 4,000 U/mg protein)] increased net 45Ca deposition (121% of control, P less than 0.05), net [3H]-proline incorporation 149% of control, P less than 0.001), and alkaline phosphatase activity (146% of control, P less than 0.01), in neonatal mouse half-calvaria. The calcitonin-dependent increase in alkaline phosphatase activity was not affected by co-incubation with 1 nM parathyroid hormone. Co-incubation with fluoride (which also increased net [3H]-proline incorporation and alkaline phosphatase activity in neonatal mouse half-calvaria, P less than 0.05, for each) enhanced the osteogenic response to low-dose calcitonin, (i.e., co-incubation with fluoride shifted the biphasic calcitonin dose-response curve to a range of lower calcitonin concentrations). The calcitonin-fluoride combinations had proportional effects on net [3H]-proline incorporation and alkaline phosphatase in the treated mouse calvaria (r = 0.78, P less than 0.005).
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PMID:Calcitonin (but not calcitonin gene-related peptide) increases mouse bone cell proliferation in a dose-dependent manner, and increases mouse bone formation, alone and in combination with fluoride. 250 8

Marrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA-15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]-proline-labeled cultures showed that MBA-15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50-fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70-fold. When cultures were grown to confluence and fed daily with ascorbic acid and beta-glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA-15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA-15 cell line possesses osteoblastic features in vitro and osteogenic capacity in vivo.
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PMID:Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo. 254 12


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