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)

Osteoporosis is a known complication of diabetes mellitus, suggesting a role for insulin in bone homeostasis. We studied insulin receptors and insulin action in the osteoblast-like rat osteogenic sarcoma cell line ROS 17/2.8. These cells share many common features with the osteoblast, such as 1,25-dihydroxyvitamin D3 receptors, PTH receptors, and 1,25-dihydroxyvitamin D3-induced modulation of alkaline phosphatase activity and osteocalcin. Competition binding studies revealed high affinity insulin receptors, with an ED50 for insulin of 1 nM. The receptors were highly specific for insulin, with 60% inhibition of insulin binding by an antireceptor antibody, no competition by epidermal growth factor, and an ED50 of 300 nM for proinsulin. Steady state maximal insulin binding was obtained by 40 min at 37 C, and insulin degradation, as measured by trichloroacetic acid solubility, was 1%/h at 37 C. ROS cells readily internalized insulin, and under steady state binding conditions at 37 C, 56% of the cell-associated radioactivity consisted of intracellular material. Chloroquine (100 microM) inhibited intracellular processing of insulin, leading to a 300% increase in cell-associated insulin by 2 h (37 C). Photoaffinity labeling of the insulin receptor with the photosensitive analog of insulin, B2 (2-nitro-4-azidophenyl-acetyl)des-pheB1-insulin, followed by solubilization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed specific bands of 125K and 430K mol wt under reducing and nonreducing conditions, respectively. Thus, the structure of insulin receptors in ROS cells appears comparable to that of insulin receptors of known target tissues. Insulin action was also examined. Insulin did not stimulate [2-3H]deoxyglucose uptake or [1-14C]leucine incorporation into protein. In contrast, physiological concentrations of insulin inhibited alkaline phosphatase activity in nonconfluent cells. After exposure to insulin for 24 h, alkaline phosphatase activity was decreased compared to basal by 39.5% and 50% with 5 and 50 ng/ml insulin, respectively. In conclusion, ROS cells bind insulin to specific receptors that are similar to insulin receptors on other target tissues; receptors internalize insulin, which is then processed through a chloroquine-sensitive pathway; insulin does not affect membrane substrate transport; and insulin does inhibit the activity of an enzyme that is important in bone metabolism. ROS cells represent a model for studying insulin effects on bone.
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PMID:Demonstration of insulin receptors and modulation of alkaline phosphatase activity by insulin in rat osteoblastic cells. 353 Jul 24

Purified acidic fibroblast growth factor (aFGF) from bovine brain stimulates the proliferation of calvaria-derived osteoblastic cells. Maximum stimulation, relative to corresponding controls, was seen at 0.2% serum (2- to 3-fold), and no stimulation was seen in the absence of serum or under serum replete conditions. The effect was dose-dependent with an ED50 of around 750 pg/ml (47 pM). aFGF (5 ng/ml) sustained the growth of calvaria cells in culture during multiple passages (72 days) at 0.2% serum. In DNA synthesis assays aFGF produced 2- to 4-fold stimulation; insulin-like growth factor I had a slight effect on DNA synthesis on its own, but enhanced the effect of aFGF 2-fold. In cells fully stimulated by epidermal growth factor (5-fold), aFGF had no further effect. Stimulation of DNA synthesis peaked at 5 ng/ml, while higher concentrations were inhibitory. Recombinant aFGF (bovine sequence) also stimulated cell proliferation (1.5-fold), and its potency was augmented by heparin (50 micrograms/ml), about 2-fold. Using simultaneous histochemical staining for alkaline phosphatase activity and [3H]thymidine nuclear uptake we found that aFGF stimulates DNA synthesis to the same extent in alkaline phosphatase-rich (osteoblastic) and alkaline phosphatase-poor (nonosteoblastic) cells. However, after cell division there is a significant decrease in PTH-responsive adenylate cyclase (2- to 3-fold) and in alkaline phosphatase levels (4- to 8-fold). These findings indicate that aFGF is mitogenic to rat calvaria osteoblastic cells, its action requires additional factors, and its growth stimulation is associated with a reduction in phenotypic expression.
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PMID:Growth stimulation of rat calvaria osteoblastic cells by acidic fibroblast growth factor. 367 31

The purpose of this work was to study the direct influence of epidermal growth factor (EGF) on the maturation of the fetal mouse duodenum in organ culture. Duodenal explants, resected at 17 days of gestation, were cultured during 48 h at 37 degrees C in Leibovitz L-15 serum-free medium alone or supplemented with EGF (100 ng/ml). Differentiation of absorptive cells was evaluated by measuring brush border hydrolytic activities. After 48 h of culture with and without EGF, villous architecture and the fine structural characteristics of the tissues are preserved. In control explants, the level of alkaline phosphatase, maltase, trehalase, and sucrase activities as well as the protein and DNA contents remain comparable to the values measured in 17-day explants at the beginning of the culture period, while lactase activity falls drastically. In explants cultured with EGF, the level of alkaline phosphatase, maltase, and trehalase activities and the protein contents significantly increase while sucrase activity and DNA contents are unchanged, and lactase activity remains under the onset level. From these results, it was concluded that EGF influences directly the maturation of some brush border enzymes in the duodenum during the fetal period.
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PMID:Influence of epidermal growth factor on the maturation of the fetal mouse duodenum in organ culture. 387 51

A synthetic gene for human epidermal growth factor (hEGF) was joined to a sequence encoding the signal peptide of Escherichia coli alkaline phosphatase. This hybrid gene was placed under the control of the alkaline phosphatase gene (phoA) promoter in a recombinant plasmid, which was used to transfect E. coli. The hybrid protein that was expressed in host cells under conditions of phosphate limitation was processed accurately during the secretion process, and mature hEGF was recovered in the periplasmic fraction. On the other hand, no EGF was detected in the periplasmic space when the synthetic hEGF gene was not accompanied by the phoA signal sequence.
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PMID:Synthesis and secretion of human epidermal growth factor by Escherichia coli. 390 48

Rats were kept undernourished from birth to 24 days of age. At 17 days of age, the undernourished animals were divided into two groups and then injected with either saline or epidermal growth factor (EGF; 20 micrograms/kg) once a day for 7 days. They were killed 12-14 h after the last injection at which time the animals were 24 days old. During the experimental period the undernourished animals were prevented from weaning. A well-nourished group (weaned) which was injected with saline from 17 to 24 days of age, was also included. Undernutrition by itself significantly decreased body weight and the weight of the oxyntic gland area, antrum, and small intestine. This was also accompanied by a parallel reduction in DNA, RNA, and protein content in the oxyntic gland and small intestine. However, administration of EGF to undernourished rats resulted in a partial reversal of the situation. In undernourished rats, EGF caused significant enhancements in body weight as well as the weight of the gastrointestinal tissues and their protein and nucleic acid content when compared with the saline-treated undernourished controls. Furthermore, the magnitude of stimulation was found to be greater in the oxyntic gland than in the small intestine following EGF administration. The antral or serum gastrin levels were not affected by EGF. In both saline- and EGF-treated undernourished rats, lactase, sucrase, and alkaline phosphatase activities (expressed as total or specific activity) were found to be significantly higher than in the well-nourished animals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. 620 84

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.
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PMID:Characterization of cells isolated and cultured from human bone. 632 25

Although the primary cell type in human osteosarcoma is usually a neoplastic osteoblast, numerous other mesenchymal cell types may coexist in the same tumor. Previously described cloned, long-term osteosarcoma cell lines have had an osteoblastic phenotype. In this report, we describe a nonosteoblastic, long-term cell line derived from an osteosarcoma in a patient with Paget's disease. The cell line (FM-2) is nontransformed in having a low saturation density and anchorage-dependent growth, and it is nontumorigenic in nude mice. Important features of its fine structure include numerous elongated mitochondria, abundant Golgi and lysosomes, and a poorly developed rough endoplasmic reticulum. The line has high levels of lysosomal enzymes (acid phosphatase and N-acetylglucosaminidase) and low levels of alkaline phosphatase. It lacks numerous macrophage markers (lysozyme, C3, Fc receptors, and M1 antigen). The FM-2 line had a dose-dependent cyclic AMP response (7-fold increase) following treatment with calcitonin but not with parathormone. In 125I-calcitonin-binding experiments, we calculated approximately 5.3 +/- 0.2 X 10(3) receptor sites/cell with a kd of 1.8 +/- 0.1 X 10(-9) M. Conditioned medium from the FM-2 line was a potent stimulator of calcium release as assayed in a 45Ca-labeled fetal rat bone organ culture. This activity was not prostaglandin, vitamin D, parathormone, or epidermal growth factor, which are known stimulators of bone resorption. The FM-2 line does not appear to be derived from an osteoblast, macrophage, or fibroblast and may represent a calcitonin-responsive bone stem cell.
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PMID:Characteristics of a calcitonin-responsive cell line derived from a human osteosarcoma. 657 18

The effect of epidermal growth factor (EGF) on clone MC3T3-El cells that have osteoblastic activity was examined by phase-contrast microscopy and electron microscopy; hydroxyproline content, collagen synthesis, collagen pattern, and alkaline phosphatase (ALP) activity were also determined. We found that EGF (0.4 ng/ml) transformed the cells from their normal polygonal shape to a spindle-like morphology by 8 h. This hormone also caused dose-related suppression of hydroxyproline content and ALP activity which was detectable 2 days and 1 day, respectively, after EGF addition. Indomethacin did not affect hydroxyproline content and ALP activity, suggesting that the effect of EGF on the cells may not be mediated by prostaglandins. Epidermal growth factor at concentrations of 2 to 50 ng/ml significantly decreased collagen synthesis in the cells, whereas protein synthesis was stimulated. Electron microscopy demonstrated that collagen fiber formation was also reduced by EGF; an immature type of fibril was observed compared with the typical cross-striated one in the controls. Moreover, the hormone treatment also resulted in the appearance of type III collagen in addition to the type I already present in the cells. These suppressive effects of EGF on MC3T3-El cells in vitro suggest that this hormone may be involved in bone remodelling in vivo as well.
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PMID:Effects of epidermal growth factor on osteoblastic cells in vitro. 660 67

The FM-2 cell line is a cloned, immortalized cell line derived from a human osteosarcoma. Conditioned medium from FM-2 cultures contains a factor which stimulates calcium mobilization from fetal rat bone organ cultures. Treated bones contain increased numbers of osteoclasts and decreased bone matrix. This factor has a molecular weight of approximately 29,000 as determined by gel filtration. Its biological activity is dependent on a protein moiety and is completely inhibited by calcitonin. Its synthesis by the FM-2 line is dependent on cell density and replenishment of fresh medium. This factor is not parathyroid hormone, a vitamin D metabolite, prostaglandin E, epidermal growth factor, or osteoclast-activating factor, all of which have bone-resorbing activities. Also, FM-2-conditioned medium inhibits collagen synthesis in fetal rat calvaria cells and decreases alkaline phosphatase levels in an osteoblastic cell line, and these two properties coelute with the calcium-mobilizing factor from a hydroxylapatite column. These biological products, synthesized by a cell line derived from a tumor, may represent physiological factors normally synthesized by a subpopulation of bone cells.
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PMID:New bone resorption stimulation factor elaborated by a human osteosarcoma cell line. 660 87

Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10(-6) M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10(-8) M) and dbcAMP (10(-3) M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity.
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PMID:Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes. 674 50


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