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)

The ability of noncollagenous bone proteins (NCBP) to inhibit calcium phosphate precipitation in vitro raises the question as to the nature and the relative efficiency of such proteins in vivo. To investigate this question NCBP from young adult sheep bones were fractionated using nondegradative techniques. Their relative activity was measured by their efficiency in preventing hydroxyapatite growth. On a weight (of protein recovered) basis, the activity is about equally divided between the Gla-containing protein, osteocalcin, and a phosphorylated protein essentially the same as osteonectin. On a molecular weight basis, the activity of the phosphorylated protein is almost three times higher than that of the Gla-containing protein. Finally, the inhibitory activity of the phosphorylated protein is alkaline phosphatase sensitive. The properties of this protein could provide a means of regulating the solubility of bone mineral and maintaining an equilibrium of calcium between bone and blood.
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PMID:A study of bone proteins which can prevent hydroxyapatite formation. 629 56

Estrogen deficiency is well recognized as a cause of bone loss in rats and humans. Likewise, treatment with estrogen results in prevention of this loss. Initially, this effect was thought to be indirectly mediated but, more recently, estrogen receptors (ER) have been reported in osteosarcoma cells and primary cultures originating from surgical waste, suggesting a direct effect of this steroid hormone. Detection of ER in skeletal tissues, however, has remained elusive. The purpose of this investigation was to establish the efficacy of the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique to detect ER in a well defined skeletal tissue (calvarial periosteum) that is responsive to the hormone. Primers were made specific to rat ER sequences. Total RNA was extracted from rat uterus, liver, spleen, and the periosteum using an organic solvent method. cDNA was synthesized from 2 micrograms total RNA. cDNA corresponding to 40 ng total RNA/sample produced intense PCR products for ER. In descending order of intensity were uterus, liver, bone, and spleen. Importantly, a similar time-course for estrogen-induced down-regulation of steady-state mRNA levels for alkaline phosphatase and osteonectin was observed in calvarial periosteum and tissues known to express estrogen receptors. These data provide in vivo evidence of ER mRNA in bone and suggest that at least some of estrogen's action on bone is directly modulated.
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PMID:Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum. 748 34

Water soluble derivatized dextran named E9 with a molecular weight of 45,000 g l-1 containing 58% methyl carboxylic acid unit, 19% benzylamide unit, and 26% sulfonate with a specific anticoagulant activity of 0.29 IU mg-1 was studied for its effects on human osteoblast growth and phenotype expression for short-term treatment. At concentrations between 1 ng ml-1 and 1 microgram ml-1 E9 has no effect on DNA synthesis whereas at higher concentrations DNA synthesis is inhibited in a dose related fashion (87% for 400 micrograms ml-1). For concentrations which do not modify osteoblast growth, E9 promotes alkaline phosphatase activity, type I collagen and osteocalcin synthesis with a maximum effect for 0.1-1 microgram ml-1. It has a synergistic effect with hPTH increasing AMPc. Moreover, osteonectin synthesis was enhanced in a dose-dependent manner between 0.1 and 5 micrograms ml-1. These results seem to indicate that E9 is able to stimulate human osteoblast phenotype expression and could be useful in clinical applications.
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PMID:Effect of a derivatized dextran on human osteoblast growth and phenotype expression. 752 43

The effects of retinoic acid (RA) on the expression of osteoblastic-related cell markers was examined. A marrow stromal osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constitutively express mRNA encoding for procollagen alpha 2 (I), osteonectin, osteopontin, biglycan, and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24 hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity displayed a different pattern. MBA-15.4, a preosteoblast cell line, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor beta (TGF beta). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor I (IGF-I) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic decrease in MBA-15.6 cell responses to PTH and PGE2, but no significant effects could be observed in other clonal lines.
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PMID:Differential effects of retinoic acid and growth factors on osteoblastic markers and CD10/NEP activity in stromal-derived osteoblasts. 752 53

Guided tissue regeneration (GTR) is a concept that evolved from the development of membrane barrier techniques which allow the repopulation of periodontal wounds by desirable cells, resulting in a so-called new attachment apparatus. To understand the biological mechanisms involved in membrane barrier-led periodontal healing, the histological localization of macromolecules phenotypical of bone and cementum formation was investigated in regenerating human periodontal tissues harvested after healing by placing barriers on teeth untreatable except by extraction. Using immunolocalization techniques, frozen sections of soft tissues and hard tissues under GTR barriers were stained with antibodies to osteonectin (LB-BON-II) and bone sialoprotein (BSP) (LF-6); alkaline phosphatase (AP) was detected histochemically. Frozen sections of regenerating periodontal tissue demonstrated the presence of spindle-shaped, fibroblast-like cells entrapped in a dense fibrillar extracellular matrix. Rounded cells aggregated to form nodules heavily stained by the Alcian blue method, indicating the presence of proteoglycans and strongly resembling those noted in hard-tissue sections. At the electron-microscopic level, the cytoplasm of the elongated cells had numerous cisternae of endoplasmic reticulum and Golgi saccules, indicating metabolic activity. Striated collagen fibres were scattered throughout the field of the sections. AP-stained soft-tissue sections demonstrated the presence cell-bound and extracellular AP. Osteonectin antibody staining confirmed the presence of this macromolecule in the extracellular matrix, particularly in the area of the cellular nodules. The dense network of connective tissue fibres was also stained.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunolocalization of bone matrix macromolecules in human tissues regenerated from periodontal defects treated with expanded polytetrafluoroethylene membranes. 757 38

Basic fibroblast growth factor (bFGF) may be involved in the development and repair of dentine and pulp because bFGF, its related peptides, and FGF receptors are expressed in dental mesenchymal cells. In this study, we examined the effects of bFGF on DNA synthesis, osteonectin/SPARC levels, alkaline phosphatase (ALPase) activity, their mRNA levels, and calcium levels in cultures of human pulp cells. Pulp cells were isolated from three healthy upper wisdom teeth of three patients and maintained separately. These cells produced SPARC, ALPase, and calcified nodules and there was a close correlation between the SPARC-synthetic activity of the cell lines and their levels of ALPase and calcification. The levels of SPARC, ALPase and calcium deposits in the three pulp cell cultures were 10-250 times those of human foreskin fibroblasts. Western blots showed that the pulp cells produced 38-kDa SPARC. Northern blots showed that the pulp cells expressed flg (FGF receptor type 1) transcripts throughout all culture stages, irrespective of the presence or absence of bFGF. The addition of bFGF to the pulp cultures suppressed the increases in ALPase activity, SPARC synthesis, and their mRNA levels, although it increased the incorporation of [3H]thymidine into DNA > 10-fold. The effects of bFGF on ALPase activity and SPARC synthesis were reversible. Furthermore, bFGF abolished the calcification of the extracellular matrix; the calcium content of bFGF-free cultures. These findings suggest that bFGF is a potent mitogen for human pulp cells and that it inhibits the expression of the odontoblast phenotype by the cells at least partly at pretranslational levels.
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PMID:Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells. 764 76

The spatial and temporal distribution of transcripts for the TRE/CRE-binding basic region-leucine zipper protein hXBP-1 was determined by in situ hybridization. Analysis of embryos from day 10.5 to 18.5 pc revealed high level expression of hXBP-1 RNA in two developing organ systems: 1) in bone and cartilage cells of the developing skeleton and toothbuds, and 2) in exocrine glands including the pancreas and the submandibular and salivary glands. High level expression was also found in whisker follicles and in selected cells in brown adipose tissue. In the developing skeleton, hXBP-1 RNA was expressed starting on day 11.5 pc in osteoblasts of newly formed intramembranous bone. Thereafter, hXBP-1 was expressed in both osteoblasts and preosteoblasts in bone formed directly by intramembranous formation as well as in bone formed during endochondral ossification. The most intense signal was observed in preosteoblasts and osteoblasts of newly forming bone. At day 11.5 pc low level hXBP-1 expression was also observed in matrix secreting chondroblasts of bones which are formed initially of cartilage, at the stage where they consist entirely of cartilage. Signal was also present in matrix producing chondroblasts of the mature zone of the growth region during endochondral ossification although at significantly lower level than in osteoblasts. hXBP-1 is thus the first transcription factor described, to our knowledge, whose level of expression is modulated during the osteoblast developmental sequence in vivo. The pattern of expression of hXBP-1 in the developing skeleton was found to be very similar to that of the genes encoding the tissue inhibitor of metalloproteinase and alkaline phosphatase throughout development. These observations suggest that hXBP-1 may play a role in regulating the expression of tissue specific genes (TIMP, osteonectin, osteopontin, osteocalcin) expressed in osteoblasts. It is intriguing that the promoter regions of several such genes contain potential hXBP-1 binding sites.
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PMID:In situ hybridization studies suggest a role for the basic region-leucine zipper protein hXBP-1 in exocrine gland and skeletal development during mouse embryogenesis. 769 55

We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.
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PMID:Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. 775 97

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca(2+)- and Pi-rich cell surface structures ranging from filamentous processes 0.14 +/- 0.02 microns by 0.5-2.0 microns, to spherical globules 0.70 +/- 0.27 microns in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 +/- 0.19 micron) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca(2+)-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.
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PMID:Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization. 775 59

Osteoblasts derived from Day 21 fetal rat calvaria grown on films of collagen type I exhibit an earlier and enhanced expression of the differentiated phenotype, compared to cells cultured on plastic. The temporal expression of genes characterizing three distinct periods of growth and differentiation are dramatically modified. During the initial proliferation period, expression of genes normally expressed at high levels on plastic (fibronectin, beta 1 integrin, and actin) was decreased from 50 to 70% in cells grown on collagen. Genes normally expressed at maximal levels in the postproliferative period (osteonectin, osteocalcin, and osteopontin) were up-regulated severalfold very early. Alkaline phosphatase enzyme activity was elevated 2- to 3-fold during the proliferation period, while mRNA levels remained low, suggesting post-transcriptional modifications. The most dramatic consequence of culture of cells on collagen is the accelerated and uniform mineralization of the matrix in contrast to the focal mineralization confined to bone nodules in cultures on plastic. Type I collagen supports maintenance of osteoblast phenotypic properties of passaged cells in the absence of glucocorticoid supplementation required for differentiation of osteoblasts subcultivated on plastic. Treatment of proliferating rat osteoblasts on plastic with 1,25(OH)2D3 blocks osteoblast differentiation and matrix mineralization. Although differentiation-related genes (alkaline phosphatase and osteocalcin) were up-regulated by vitamin D, culture on the collagen matrix could not overcome the inhibition of mineralization. Taken together, these studies define the critical role of type I collagen in mediating the signaling cascade for expression of a mature osteoblast phenotype and mineralization of the extracellular matrix in a physiological manner.
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PMID:The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization. 781 31


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