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)

To investigate the role and mechanism of action of epidermal growth factor (EGF) in the intestinal epithelium, we have studied its influence on proliferation and differentiation of Caco-2 cells, a human colon adenocarcinoma cell line exhibiting several characteristics of adult small intestinal enterocytes. A clone of Caco-2 cells synthesizing minimal amounts of transforming growth factor-alpha (TGF-alpha)/epidermal growth factor (EGF)-like activity was used in these studies. Cells grown in the presence of 20-200 ng EGF/ml exhibited increased DNA synthesis and proliferation; formation of morphologically poorly differentiated multilayers was observed at 200 ng EGF/ml. At all concentrations tested EGF produced a significant and marked reduction in sucrase activity, whereas other brush-border enzymes (aminopeptidase N, alkaline phosphatase, dipeptidylpeptidase IV) were only marginally affected. EGF influenced sucrase expression at two different levels. At 20 ng/ml, it affected primarily sucrase-isomaltase processing in the endoplasmic reticulum and/or increased its degradation. At 200 ng EGF/ml, a significant and marked reduction in sucrase-isomaltase mRNA levels and biosynthesis was observed. These results demonstrated that EGF has important and selective effects on Caco-2 cell proliferation and differentiation and may affect different cellular activities depending on its concentration.
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PMID:Inhibition of sucrose-isomaltase expression by EGF in the human colon adenocarcinoma cells Caco-2. 176 18

Two highly sensitive, nonradiolabeled assays for protein phosphotyrosine phosphatase (PTPase) have been developed. The first assay is based on the use of chemically synthesised phosphotyrosine-containing peptides that can be separated from the dephosphorylated peptide products by HPLC. In this assay, partially purified placental PTPase 1B dephosphorylated three dodecaphosphopeptides (corresponding to insulin receptor autophosphorylation sites at positions PY1146, PY1150, and PY1151) with approximately equal affinity (Km 1.3-2.5 microM), indicating that PTPase 1B shows no distinct preference for the site of dephosphorylation in these peptides. The second assay employs either a phosphopeptide or an autophosphorylated tyrosine kinase domain immobolized on microtiter plate wells. After reaction with PTPase, the remaining unconverted phosphosubstrate is detected in an ELISA using anti-phosphotyrosine antibodies. The latter assay was used to monitor PTPase activity during purification procedures and for characterizing PTPases. Modulation of PTPase activity by orthovanadate, heparin, Zn2+, and EDTA gave similar results in both assays. The immobilized autophosphorylated IR tyrosine kinase domain was a poor substrate for bovine liver alkaline phosphatase and seminal fluid acid phosphatase. The second assay also offers the potential for comparing PTPase activity toward several autophosphorylated tyrosine kinase domains, including those of the insulin, epidermal growth factor, and platelet-derived growth factor receptors.
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PMID:Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor. 181 86

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.
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PMID:Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells. 184 70

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.
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PMID:Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro. 193 45

We have investigated the individual and combined actions of epidermal growth factor (EGF), transferrin and hydrocortisone on the maturation of whole fetal mouse metanephroi maintained in serum-free conditions for up to 5 days. The presence of EGF (100 ng/ml) resulted in elevated levels of [3H]-thymidine incorporation when compared to controls; autoradiograms showed that the proliferation of mesenchymal cells in the nephrogenic zone is particularly enhanced as verified by cell counting. Brush border hydrolase activities (alkaline phosphatase and gamma-glutamyltransferase), on the other hand, were significantly diminished. Transferrin (5 micrograms/ml) slightly stimulated DNA synthesis and potentiated EGF mitogenic action. The activation of DNA replication by the growth factor seems to be mediated through the protein kinase C pathway. When added alone, hydrocortisone (10(-6) M) strongly inhibited DNA synthesis, stimulated hydrolase activities and exerted a positive effect on brush border differentiation. When combined with EGF or to EGF + transferrin, hydrocortisone counteracted the effects of these latter peptides on DNA synthesis and enzyme activities. Considering the earlier observation of a reciprocal relation between proliferation and differentiation during the neotubulogenic phase of kidney development, the results described in the current study suggest that synergistic and synarchic actions of these heterologous factors are involved in the regulation of tubulogenesis.
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PMID:Fetal mouse kidney maturation in vitro: coordinated influences of epidermal growth factor, transferrin and hydrocortisone. 195 5

Several clonal sublines of HCT-116 human colon adenocarcinoma cells were isolated and characterized on the basis of their growth characteristics, intrinsic enterocyte-like differentiation (as assessed by alkaline phosphatase and lactase activities), and responses to butyrate, an inducer of colon tumor cell maturation. The HCT-116 sublines were found to be heterogeneous and several phenotypically distinct clones were identified. Further characterization of these clones indicated that the effects of butyrate on cell growth, alkaline phosphatase activity, and lactase activity were distinct and separable. The growth of all of the clones were inhibited by butyrate (IC50 values varied from 0.44 to 1.5 mM), but the effects of this agent on alkaline phosphatase and lactase activities varied widely. In several sublines butyrate had no effect on either enzyme while in others one or both activities were induced. Additionally, the binding of 125I-epidermal growth factor (EGF) to cell surface receptors was found to be proportional to the expression of lactase activity in the cell. The D3 clone and other sublines with intrinsic lactase activities greater than 100 nmol/mg/min expressed a class of high-affinity EGF receptors (e.g., D3 cells had 3.48 X 10(4) EGF receptors/cell with a kd of 0.61 nM). Other clones with less lactase activity had undetectable levels of 125I-EGF binding. In clones which exhibited greater than twofold increases in lactase activity in response to butyrate, the expression of a large number of low-affinity EGF receptors was also induced. In one such clone, the P1 subline, lactase activity was increased from 70 nmol/mg/min to 230 nmol/mg/min after 96 h in 2 mM butyrate, and the expression of EGF receptors was increased from undetectable levels to 1.18 X 10(5) EGF receptors/cell (kd of 3.2 nM). Northern blot analysis indicated that the increased 125I-EGF binding after butyrate treatment may have been due, in part, to a greater than twofold accumulation of EGF receptor mRNA. In addition, the expression of the messages for transforming growth factor alpha (TGF-alpha) and transforming growth factor beta (TGF-beta) was examined in butyrate-treated cells. While TGF-alpha mRNA levels were found to correlate with EGF receptor message levels in the HCT-116 clones, TGF-beta mRNA expression was not found to correlate with the butyrate-induced growth inhibition or with increases in EGF receptor expression, alkaline phosphatase activity, or lactase activity in these cells.
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PMID:Increased cell surface EGF receptor expression during the butyrate-induced differentiation of human HCT-116 colon tumor cell clones. 197 61

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.
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PMID:Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor. 199 20

We have prepared plasma membranes from Balb/c 3T3 fibroblasts to study the transmodulation of the high affinity epidermal growth factor (EGF) receptor. Although phorbol esters do not transmodulate the high affinity EGF receptors on these membranes, the addition of platelet-derived growth factor (PDGF) or EGF to the membranes leads to the loss of high affinity EGF binding and to the phosphorylation of several membrane proteins, including the EGF receptor. The EGF receptor is phosphorylated at tyrosine residues although we have not yet established if this represents direct phosphorylation by the PDGF receptor kinase or is mediated by activation of other cell membrane-associated tyrosine kinases. Upon treatment of the membranes with PDGF, four major phosphoproteins (of apparent molecular masses of 69, 56, 38, and 28 kDa) are released from the membrane and can be retrieved from the supernatant fluid using a reversed-phase cartridge. As assessed by immunoprecipitation with an anti-phosphotyrosine antibody, all four proteins appear to be phosphorylated on tyrosine. The time course of dissociation of these proteins from the membranes closely parallels the loss of high affinity EGF receptors. The high affinity EGF receptor can be reconstituted on PDGF-transmodulated membranes by treating the supernatant fluid with alkaline phosphatase and adding the mixture to the membranes. It appears that dephosphorylation of the released proteins is sufficient to allow reassociation with the membranes and formation of the high affinity EGF receptor complex.
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PMID:Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor. 199 54

The concept of orthodontic tooth movement is based on the hypothesis that teeth move as a result of the biological response of periodontal tissues to the mechanical forces applied. There is a widely held hypothesis that mechanical stress generates an electrical signal which sets in motion the subsequent events, as in bone exposed to mechanical forces electrical currents are produced affect bone growth and remodeling. This implies a transduction mechanism which translates the electrical signal into a biochemical message, recognizable by the cellular machine. This study is aimed at the identification of the message and the investigation of its control. In fact, the effect of Pulsed Electromagnetic Fields (PEMF) on the intracellular second messenger, cytoplasmic Ca2+ in Human Periodontal Ligament Fibroblasts (HPLF) was investigated. The resting intracellular ionized calcium concentration ([Ca+2]i) of HPLF cells was 232.7 +/- 25.0 nM, and with PEMF [Ca2+]i increased from 12 hrs to 499.0 +/- 115.5 nM up to 12 hrs, then reached to a steady level through 24 hrs. The PEMF were also found to decrease the responses towards epidermal growth factor (EGF) and serum, when the degree of response was based on the intracellular Ca2+ transient. These effects of PEMF were mimicked by 12-0-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C. Some reports have suggested that fibroblasts of the periodontal ligament contain high alkaline phosphatase (ALPase) activity as much as osteoblast. Since similar results concerning the [Ca2+]i were obtained in osteoblast (OB)-like cells, this experiment also supports the hypothesis that fibroblasts of periodontal ligament have osteoblastic features.
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PMID:[Effect of pulsed electromagnetic fields (PEMF) on human periodontal ligament in vitro. Alterations of intracellular Ca2+]. 213 43

Effects of transforming growth factor (TGF)-beta, epidermal growth factor (EGF), insulin, 1, 25-dihydroxyvitamin D3 (1, 25 (OH)2D3), and parathyroid hormone (PTH) on the proliferation and differentiation of clonal dental pulp cells of rats were investigated. Interaction between growth factors (TGF-beta and EGF) and two hormones insulin and 1, 25 (OH)2D3, which have been noticed to accelerate the differentiation of the cells, were also studied, and the following results were obtained: 1) TGF-beta decreased alkaline phosphatase (ALPase) activity in a dose-dependent manner, and the inhibitory effect was not blocked by indomethacin, suggesting that the effect of TGF-beta on the cells may not be mediated by prostaglandins. Inhibitory effects of ALPase antagonists (L-phenylalanine, L-homoarginine, levamisole) on the activity were not affected by TGF-beta. TGF-beta showed no evident effect on the DNA synthesis (incorporation of [3H] thymidine) and collagen synthesis (incorporation of [2, 3-3H] proline into the collagenase-digestible protein) of the cells. 2) EGF stimulated the incorporation of [3H] thymidine and inhibited the ALPase activity. The inhibitory effect was not blocked by indomethacin, indicating that the EGF effect is not mediated by prostaglandins. Collagen synthesis was significantly inhibited by EGF. 3) Insulin showed a weak but significant inhibition of the DNA synthesis. Insulin increased the ALPase activity evidently, and accelerated the collagen synthesis significantly. 4) The vitamin 1, 25 (OH)2D3 significantly increased the ALPase activity though no significant changes were observed in the DNA synthesis and collagen synthesis. 5) PTH had no evident effect on the DNA synthesis and ALPase activity, but did tend to accelerate the collagen synthesis. 6) A study on the interaction between insulin and EGF or TGF-beta revealed that the acceleration of DNA synthesis induced by EGF was inhibited when the factor was combined with insulin, and the increase in ALPase activity elicited by insulin was inhibited by EGF and weakened by TGF-beta significantly when these factors were added simultaneously with the insulin. Or viewed another way, the inhibitory effect of EGF or TGF-beta on the ALPase activity was antagonized by insulin. The accelerative action of insulin on collagen synthesis was antagonized by EGF and potentiated by TGF-beta. 7) A study on the interaction between 1, 25 (OH)2D3 and EGF or TGF-beta revealed that 1, 25 (OH)2D3 inhibited the accelerating effect of EGF on the DNA synthesis and that the increasing effect of 1, 25 (OH)2D3 on ALPase activity was strongly inhibited by EGF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Effects of various growth factors and hormones on clonal rat pulp cells]. 213 79


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