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)

Steel factor (SF) and LIF (leukemia inhibitory factor) synergistically promote the proliferation and survival of mouse primordial germ cells (PGCs), but only for a limited time period in culture. We show here that addition of bFGF to cultures in the presence of membrane-associated SF and LIF enhances the growth of PGCs and allows their continued proliferation beyond the time when they normally stop dividing in vivo. They form colonies of densely packed, alkaline phosphatase-positive, SSEA-1-positive cells resembling undifferentiated embryonic stem (ES) cells in morphology. These cultures can be maintained on feeder layers for at least 20 passages, and under appropriate conditions give rise to embryoid bodies and to multiple differentiated cell phenotypes in monolayer culture and in tumors in nude mice. PGC-derived ES cells can also contribute to chimeras when injected into host blastocysts. The long-term culture of PGCs and their reprogramming to pluripotential ES cells has important implications for germ cell biology and the induction of teratocarcinomas.
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PMID:Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. 138 Dec 89

Immuno-localization of BUdr was used to identify DNA synthesis in vitro in chicken embryonic bone cells stained positively or negatively for alkaline phosphatase activity. The results were similar to, but more sensitive than, our standard bioassay which assesses 3H-thymidine incorporation into DNA by liquid scintillation counting, and more rapid than autoradiographic localization of 3H-thymidine. SGF/IGF-II and bFGF stimulated cellular proliferation equally in ALP(+) and ALP(-) cells. In contrast, IGF-I and TGF-beta stimulate proliferation more in the ALP(-) than ALP(+) cells. The greatest increase in DNA replication of ALP(-) cells occurred following incubation with SGF/IGF-II or TGF-beta, and in the ALP(+) cells with SGF/IGF-II or bFGF. TGF-beta stimulated cellular proliferation at the lowest dose (1 ng/ml). The differential effect of the growth factors on each population of cells indicates that all these bone-matrix derived growth factors may play different roles in the local regulation of skeletal metabolism.
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PMID:Growth factor-induced proliferation of osteoblasts measured by bromodeoxyuridine immunocytochemistry. 176 62

Acidic (a) and basic (b) fibroblast growth factors (FGFs) are two related mitogenic and angiogenic factors. They are multifunctional in that they can affect proliferation and induce or delay differentiation. Both aFGF and bFGF were shown to stimulate proliferation of calvaria cells in situ as well as osteoblast-enriched calvaria-derived cells. bFGF was also found to suppress the expression of alkaline phosphatase, parathyroid hormone stimulatable adenylate cyclase, osteocalcin, and type I collagen in the osteoblastic ROS 17/2.8 cells. To explore a possible role for guanine nucleotide binding proteins we assessed the effects of pertussis toxin (PT) on FGF action. PT had opposite effects to those of bFGF on all parameters examined.
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PMID:Effects of acidic and basic fibroblast growth factors on osteoblastic cells. 261 59

The presence of many types of polypeptide growth factors in the mineralized extracellular matrix of bone is now well established. These factors are generally referred to as bone-derived growth factors (BDGFs), and are similar, or possibly identical, to the following species; platelet-derived growth factor (PDGF); acidic and basic forms of fibroblast growth factor (aFGF, bFGF); transforming growth factor beta (TGF-beta); and insulin-like growth factor 1 (IGF-1). Several osteoinductive factors, such as bone morphogenetic protein (BMP) and osteogenin, a skeletal growth factor (SGF), and osteoblast-derived BDGFs, have also been identified. Complete description of the biological functions of these BDGFs which are relevant to bone will ultimately require specific bioassays involving specific cell types in vitro, as well as in vivo animal implant models. Studies with primary rat osteoblast-like cells exposed either to mixed BDGFs, pure TGF-beta, or heparin-purified PDGF, aFGF, or bFGF from bovine bone have shown a general dose-dependent mitogenic effect. Phenotypic changes which accompany the BDGF-induced wave of proliferation include: decreased osteocalcin secretion and a reduction in 1,25-(OH)2 vitamin D3-stimulated osteocalcin synthesis; reduced alkaline phosphatase specific activity; decreased cyclic AMP responsiveness to parathyroid hormone (PTH); and increased collagen synthesis. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than that in serum. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for the unmasking or release of BDGFs from the mineralized matrix that result in local action on osteoblasts, endothelial cells, and other target cells are undoubtedly important for the development and maintenance of bone tissue.
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PMID:Polypeptide growth factors in bone matrix. 306 10

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.
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PMID:Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. 790 23

The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for vascular endothelial cells and a potent angiogenic factor. Expression of a chimeric receptor containing the extracellular domain of the flk-1 receptor fused to the transmembrane and intracellular domains of the human c-fms receptor in NIH-3T3 cells, resulted in the appearance of high affinity binding sites for 125I-VEGF165 on transfected cells. The binding of 125I-VEGF165 to the flk-1/fms chimeric receptor of the transfected cells as well as the VEGF165-induced autophosphorylation of the chimeric receptors were inhibited in the presence of low concentrations of heparin (1-10 micrograms/ml). In contrast, similar concentrations of heparin potentiated the binding of 125I-VEGF165 to the endogenous VEGF receptors of the transfected cells, indicating that to some extent, the effect of heparin on 125I-VEGF165 binding is receptor type-dependent. A soluble fusion protein containing the extracellular domain of flk-1 fused to alkaline phosphatase (flk-1/SEAP) was used to study the effects of heparin on the binding of 125I-VEGF165 to flk-1 in a cell-free environment. The fusion protein specifically inhibited VEGF165-induced proliferation of vascular endothelial cells, but bound 125I-VEGF165 inefficiently in the absence of heparin. Addition of low concentrations of heparin or heparan sulfate (0.1-1 microgram/ml) resulted in a strong potentiation of 125I-VEGF165 binding, whereas higher heparin or heparan sulfate concentrations inhibited the binding. The effect of heparin on the binding of 125I-VEGF165 to flk-1/SEAP could not be mimicked by desulfated heparin or by chondroitin sulfate. Both bFGF and aFGF inhibited the binding when low concentrations of heparin were added to the binding reaction. However, higher concentrations of heparin abolished the inhibition, indicating that the inhibition is probably caused by competition for available heparin. Taken as a whole, these results indicate that heparin-like molecules regulate the binding of VEGF165 to its receptors in complex ways which depend on the heparin binding properties of VEGF165, on the specific VEGF receptor type involved, and on the amount and composition of heparin-like molecules that are present on the cell surface of VEGF receptor containing cells.
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PMID:Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors. 817 51

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

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes.
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PMID:Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells. 865 99

The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1 beta (IL-1 beta) were not active. Furthermore, IL-1 beta but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurosporine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.
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PMID:Expression of the nucleoside triphosphate pyrophosphohydrolase PC-1 is induced by basic fibroblast growth factor (bFGF) and modulated by activation of the protein kinase A and C pathways in osteoblast-like osteosarcoma cells. 882 42

We previously reported that the total neurotrophic activity of hippocampal extracts was significantly (25-50%) reduced after 21-28 weeks of chronic ethanol treatment (CET) [23]. To test whether the level of a neurotrophic factor (i.e., ligand itself) is compromised, we measured nerve growth factor (NGF) protein and NGF mRNA contents using ELISA and Northern analysis. We reported that CET did not appear to reduce NGF protein, NGF mRNA or total neurotrophic activity when measured on sympathetic ganglia neurons [4]. We also observed that both NT-3 mRNA and bFGF mRNA levels were unaffected, but the BDNF mRNA levels was significantly reduced in CET rat hippocampus [18]. Neuronal degeneration and reduction of total neurotrophic activity after CET appear to be induced, at least partially, by compromised transcription of BDNF gene. CET may also induce functional changes in receptors for the neurotrophic factors. To investigate possible changes in neurotrophic factor-receptors, we examined Western blots (immunoblots) of rat cortex after 28 weeks of CET. After sonication and ultra-centrifugation, the supernatant of crude lysates of the cortex from individual animals was subjected to SDS-PAGE, electrotransfered to nitrocellulose membrane, incubated with anti-trk B antibody and secondary antibody conjugated to alkaline phosphatase, and reacted with chemiluminescent substrate. The membranes were then exposed to Kodak XAR film. Compared to controls (n = 6), CET rats (n = 6) appeared to have significantly higher band intensity (P < 0.01) of trk B-like protein at about 145 kDa, which suggests an up-regulation of trk B-like proteins to compensate the compromised level of certain subset (i.e., BDNF or NT-4/5, but not NGF) of neurotrophins in cortex.
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PMID:Up-regulation of high-affinity neurotrophin receptor, trk B-like protein on western blots of rat cortex after chronic ethanol treatment. 884 27


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