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)

Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin, fibronectin; negative for factor VIII-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 +/- iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application.
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PMID:HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. 812 21

Osteoblasts from involved and noninvolved sutures and normal membranous bone in patients with premature unilateral coronal synostosis were harvested and grown in tissue culture. The cultures were characterized to establish specific basal metabolic parameters of cellular growth and the production of metabolites, including osteocalcin, alkaline phosphatase, platelet-derived growth factor, epidermal growth factor, tumor necrosis factor-alpha, and DNA. In addition, metabolic responses to two provocative bone trophic agents, parathyroid hormone and 1,25dihydroxyvitamin-D3 were ascertained. Osteoblasts from involved sutures (involved bone) exhibited altered indices of cellular metabolism when compared with osteoblasts derived from noninvolved sutures (noninvolved bone) in a basal state and when exposed to parathyroid hormone and vitamin D3 (p < or = 0.05). Involved osteoblasts produced significantly more osteocalcin and significantly less alkaline phosphatase than noninvolved osteoblast-derived cultures (p < or = 0.05). Secretion of platelet-derived growth factor and epidermal growth factor was also altered in the involved osteoblasts compared with the noninvolved osteoblasts (p < or = 0.05). Secretion of tumor necrosis factor-alpha was not significantly different between involved and noninvolved osteoblast-derived cultures.
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PMID:Plagiocephaly: premature unilateral closure of the coronal suture: a potentially localized disorder of cellular metabolism. 819 66

The ligand-stimulated tyrosine kinase activity of the normal human epidermal growth factor (EGF) receptor and a truncated EGF receptor lacking 164 carboxy-terminal (C-terminal) amino acids was examined in intact cells and after Triton X-100 extraction into Triton-soluble and -insoluble (cytoskeletal) preparations. Detergent extraction of the intact and truncated receptors appeared complete using 0.3% Triton as demonstrated by anti-EGF receptor immunoblots, tyrosine kinase assays, and marker enzyme (alkaline phosphatase) solubilization. Higher Triton concentrations yielded no additional EGF receptor extraction and began to inhibit EGF-stimulated kinase activity toward angiotensin II (AII). Furthermore, the tyrosine kinase activity of the truncated EGF receptor exhibited increased sensitivity to Triton extraction, suggesting a lower affinity or a more labile association of this receptor with the cytoskeleton. However, both EGF receptor forms had altered catalytic activity when associated with the cytoskeletal fraction, as evidenced by the increased phosphorylation of the exogenous substrates: AII, src-peptide, and [Val5]AII. Kinetic analyses of both receptor types revealed that the cytoskeletal fractions obtained using 0.3% Triton contain EGF receptor activity that exhibits a Michaelis-Menten constant (Km) for AII that is 2- to 3-fold more favorable than that calculated for the soluble receptor forms. EGF treatment of intact cells containing either the intact or truncated receptor revealed similar phosphorylated proteins in the soluble fraction of both cell types, although there was evidence for the enhanced phosphorylation of certain proteins (e.g. 115 and 50 kilodalton proteins) in cells containing the truncated receptor. There was also a greater number of tyrosine-phosphorylated proteins in the Triton-insoluble fraction of cells containing the truncated receptor, suggesting an altered specificity of this receptor toward selected cytoskeletal proteins. This work indicates that EGF receptor-cytoskeletal interaction may be an important consideration in the control of receptor-kinase activity and has examined the detergent sensitivity of this association. These studies also suggest that the C-terminal domain of the EGF receptor may affect cytoskeletal interaction in addition to influencing the receptor's catalytic capacity.
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PMID:Evidence for the potentiation of epidermal growth factor receptor tyrosine kinase activity by association with the detergent-insoluble cellular cytoskeleton: analysis of intact and carboxy-terminally truncated receptors. 824 11

We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.
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PMID:A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ. 841 66

Isolated proximal tubular (PT) and distal tubular (DT) cells from rat kidney were cultured for up to 9 days under serum-free, hormonally-defined conditions on 35-mm polystyrene culture dishes. Several hormonal and growth factor supplements were assessed for their ability to promote growth (increased protein and DNA content) and stability of differentiated phenotype (high activities of gamma-glutamyltransferase and alkaline phosphatase as brush-border membrane markers in PT cells; maintenance of high activities of glutamate dehydrogenase as a mitochondrial marker in both PT and DT cells; maintenance of low and high activities of lactate dehydrogenase in PT and DT cells, respectively; expression of cytokeratins). Basal supplemented media (DMEM/F12, 1:1 v/v) contained insulin, hydrocortisone, epidermal growth factor, sodium selenite and transferrin as supplements. Additionally, triiodothyronine selectively promoted growth and stability of differentiated phenotype in PT cells and thyrocalcitonin selectively promoted growth and stability of differentiated phenotype in DT cells. On Day 3 of primary culture, PT and DT cells were incubated for up to 8 h with either tert-butyl hydroperoxide (tBH; 0.5-10 mM), methyl vinyl ketone (MVK; 1-10 mM), or p-aminophenol (PAP; 1-10 mM) and cellular injury, as assessed by cellular release of lactate dehydrogenase, was determined. DT cells were significantly more susceptible to injury from both tBH and MVK, but the two cell populations were equally susceptible to injury from PAP, which is the same susceptibility pattern seen in freshly isolated cells. These results suggest that primary cultures of rat renal PT and DT cells reflect similar biochemical properties as freshly isolated cells and are, therefore, useful models for study of chemically induced injury.
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PMID:Susceptibility of primary cultures of proximal tubular and distal tubular cells from rat kidney to chemically induced toxicity. 854 48

The rat pituitary cell line GH4C1 secretes granins (chromogranin B and secretogranin II) and prolactin by the regulated secretory pathway. The intracellular storage of prolactin is preferentially induced by hormone treatment with estradiol, insulin, and epidermal growth factor. The goal of this study was to determine the effect of hormone treatment on storage of granins and constitutive secretory markers. The granins were efficiently stored in both hormone-treated and -untreated cells (17% of total secreted in 4 h). Secreted alkaline phosphatase (SEAP), a truncated membrane protein that would not be expected to enter secretory granules, and glycosaminoglycan, a marker for the constitutive secretory pathway, exhibited 70 80% secretion under both conditions. In comparison, the relative prolactin secretion was 31 and 68% from hormone-treated and -untreated cells, respectively. Phorbol ester and KCl stimulated prolactin secretion 2.3-fold from untreated cells and 5. 5-fold from hormone-treated cells. In contrast, SEAP secretion was stimulated 1.5-fold from both treated and untreated cells, consistent with secretion by the constitutive secretory pathway. Stimulated secretion of granins was detected from both hormone-treated and -untreated cells. These results suggest that granin and prolactin storage are differentially regulated and that the constitutive secretory pathway is not affected by hormone treatment.
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PMID:Differential storage of prolactin, granins (Chromogranin B and secretogranin II), and constitutive secretory markers in rat pituitary GH4C1 cells. 863 64

The effect of chicken growth hormone (cGH) on the proliferation and differentiation of avian growth-plate chondrocyte was evaluated in culture. In culture, addition of ascorbic acid to the culture media caused cell differentiation. Treatment of proliferating chondrocytes with cGH caused a time-dependent increase in collagen type II gene expression together with a decrease in the appearance of osteopontin (OPN) in the medium. In addition, the ascorbic acid-dependent increase in alkaline phosphatase (AP) activity was inhibited by cGH. IGF-I, on the other hand, caused an increase in AP activity in the ascorbic acid-treated chondrocytes. In the presence of ascorbic acid, cGH did not affect collagen type II gene expression or the appearance of OPN in the medium. Proliferation of avian growth-plate chondrocytes, in contrast to mammalian chondrocytes, was not stimulated by GH alone, although the presence of cGH was essential for chondrocyte survival in long-term culture. cGH in combination with epidermal growth factor (EGF) stimulated cell proliferation. These results suggest that GH inhibits differentiation in avian growth-plate chondrocytes, thereby sustaining their proliferative state and maintaining their sensitivity to growth factors such as EGF.
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PMID:Growth hormone inhibits differentiation of avian epiphyseal growth-plate chondrocytes. 867 49

An in vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzymes DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transferring the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7-10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and gamma-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.
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PMID:Normal rat kidney proximal tubule cells in primary and multiple subcultures. 879 58

We have investigated single and combined effects of calciotropic hormones and growth factors on the regulation of alkaline phosphatase (ALP) activity and calcium metabolism in an optimized serum-free bone organ culture system of embryonic chick tibiae. Parathyroid hormone PTH(1-34) alone mobilized calcium from bone tissue time- and dose-dependently and inhibited ALP activity. Both the bisphosphonate (BM 21.0955) and to a lesser extent salmon calcitonin alone slightly increased calcium uptake and inhibited the stimulation of bone resorption by PTH(1-34). 1,25(OH)2D3 mobilized calcium and inhibited ALP activity in contrast to 24,25(OH)2D3 which inhibited ALP activity but had no significant effect on calcium metabolism. Interestingly the combination of PTH(1-34) with 1,25(OH)2D3 but not 24,25(OH)2D3 reduced calcium mobilization. The combination of the midregional fragment PTH(28-48), which by itself has no effect on calcium metabolism, with 1,25(OH)2D3 reduced calcium mobilization more efficiently. Several PTH-regulated mediators have been assayed in this system. Of the tested growth factors, IGF-I at high concentrations caused bone resorption with no effect on ALP activity. TGF-beta 1 (transforming growth factor beta) and BMP-2 had no significant effect on calcium metabolism; however, ALP activity was inhibited by TGF-beta 1 and induced dose dependently by BMP-2. Of the other factors known to be present in bone, platelet-derived growth factor (PDGFA/B) and epidermal growth factor (EGF) had a small effect on calcium mobilization but had no effect on ALP activity. bFGF reduced ALP activity slightly without an effect on calcium metabolism. Our results show that this in vitro system can mimic some interactions of calciotropic hormones in vivo and allows the assaying of mediators in terms of regulation of ALP activity and of calcium metabolism.
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PMID:Individual and combined effects of calciotropic hormones and growth factors on mineral metabolism in embryonic chick tibiae. 920 16

An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein in E. coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E. coli using the PhoA signal sequence for protein export.
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PMID:Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence. 926 80


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