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)

A competitive enzyme immunoassay has been developed for the measurement of human interferon-gamma (IFN-gamma) in cell culture supernatants. The assay is based on the dose-dependent inhibitory effect of liquid phase IFN-gamma on the binding of a specific monoclonal antibody to recombinant IFN-gamma (rIFN-gamma) immobilized on microtitre plate wells. The extent of monoclonal anti-IFN-gamma inhibition was determined by the uptake of alkaline phosphatase-conjugated goat anti-mouse IgG and the subsequent development of enzyme substrate colour. Absorbance readings were taken and results for test samples were extrapolated from standard rIFN-gamma inhibition curves constructed as logit-log plots. Assay performance was assessed using three different monoclonal antibodies (clones 20G7, H-22 and GZ-4). Optimum sensitivity was achieved with the antibodies of higher affinity, 20G7 and H-22, which gave reliable quantification of IFN-gamma over a wide range of concentrations from 0.4 ng/ml (3.4 IU/ml), or less, to 250 ng/ml approximately 2000 IU/ml). The inhibition assay incorporates the advantages of specificity, reproducibility and convenience of performance which are the hallmarks of monoclonal antibody-based ELISAs. However, compared to the sandwich ELISAs previously described for human IFN-gamma, it is considerably more economical in its use of monoclonal anti-IFN-gamma, requiring < 50 ng of a single antibody per 96 well plate. It also uses relatively small volumes of test samples (50 microliters/well) which is particularly advantageous where limited amounts of cell culture supernatant are available for cytokine assays.
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PMID:A competitive inhibition ELISA for the quantification of human interferon-gamma. 831 91

We established bone marrow stromal cell lines from a transgenic mouse that harbors a temperature-sensitive mutant of the simian virus 40-derived large T-antigen under the control of a major histocompatibility complex (MHC) I promotor. These cell lines were screened for their ability to induce the formation of osteoclasts in a spleen cell/stromal cell coculture system. By means of this screen, five clones, referred to as marine bone marrow stromal clone 1 (mBMS-B1) mBMS-B2, mBMS-B14, mBMS-B18, and mBMS-B21, were selected for detailed characterization. Cell growth depends on culture conditions, i.e., cells grow at 33 degrees C in the presence of murine interferon-gamma, whereas cell proliferation ceases at 39 degrees C. The phenotype of the cells is also correlated with the culture conditions because the osteoclast inductive capacity is only seen at 39 degrees C, indicating that the cells undergo differentiation when the transforming agent is inactivated. These conditionally immortalized stromal cells can be induced to express a variety of markers that are typical for mature osteoblasts, e.g., alkaline phosphatase activity and expression of functional parathyroid hormone receptor after stimulation with soluble osteogenic protein 1 (sOP-1). mRNA analysis revealed the expression and regulation of osteopontin, osteonectin, and collagen alpha 1(I) as well as the inducibility of osteocalcin upon treatment with sOP-1. The cells have the potential to form mineralized nodules in supplemented medium. We observed expression of vascular cell adhesion molecule-1, which is stimulated upon treatment of the cells with 1 alpha,25-dihydrocholecalciferol after 4 days, indicating the presence of the receptor for this steroid. These cell lines represent a model to study mechanisms and factors involved in osteoblast differentiation.
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PMID:Establishment and characterization of conditionally immortalized stromal cell lines from a temperature-sensitive T-Ag transgenic mouse. 904 Oct 49

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-beta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-alpha, iNOS, IFN-gamma and c-fos, animals were injected IV with saline or 100 microg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-microm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-gamma, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-alpha, IFN-gamma, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.
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PMID:Enhanced immunohistochemical detection of autonomic nerve fibers, cytokines and inducible nitric oxide synthase by light and fluorescent microscopy in rat spleen. 911 Dec 38

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor kappaB (NF-kappaB) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IkappaBalpha and IkappaBbeta were analyzed during this period in an attempt to correlate NF-kappaB activity with IkappaB resynthesis. Degradation of IkappaBalpha was very rapid and was followed by recovery 1 h after LPS administration. IkappaBbeta degradation, which has been associated with persistent NF-kappaB activation, was complete at 1 h. However, a rapid recovery of IkappaBbeta in these tissues was observed at 3 h in parallel with the abrogation of NF-kappaB activity. Immunolocalization of newly synthesized IkappaBbeta by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IkappaBbeta by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IkappaBbeta with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IkappaBbeta. At the mRNA level, up-regulation of IkappaBbeta was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor alpha, but not interleukin-1 or interferon-gamma, promoted an important degradation of IkappaBbeta followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS- and tumor necrosis factor alpha- specific pathways involved in a rapid IkappaBbeta degradation and resynthesis and might explain the transient period of activation of NF-kappaB in these tissues upon stimulation with these factors. This rapid control of NF-kappaB function may contribute to the attenuation of the inflammatory response of these cells.
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PMID:Rapid Up-regulation of IkappaBbeta and abrogation of NF-kappaB activity in peritoneal macrophages stimulated with lipopolysaccharide. 928 99

Nitric oxide (NO) is known to be implicated in the metabolism of bone, especially as a mediator of cytokine effects on the remodelling of bone tissue. In this study we examine whether NO affects the osteoblast activation or the osteoclast differentiation of primary mouse osteoblast-like and osteosarcoma ROS 17/2.8 cell lines. Primary osteoblast and ROS 17/2.8 cells released NO upon stimulation of interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma. Sodium nitroprusside, a donor of nitric oxide, increased the activity of alkaline phosphatase in ROS 17/2.8 cells as well as the number of calcified nodule formations in primary mouse osteoblast-like cells. Sodium nitroprusside also completely inhibited 1 alpha, 25-(OH)2D3-induced osteoclast generation in a high concentration (100 microM). However, a low concentration of sodium nitroprusside (3-30 microM) significantly increased the generation of osteoclasts. These results indicated that NO appears to be an important regulatory molecule in the processes of bone formation and resorption. Hence, NO may be involved in the pathogenesis of bone loss in diseases associated with cytokine activation, such as periodontal disease and rheumatoid arthritis.
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PMID:Nitric oxide is a regulator of bone remodelling. 930 58

The derivatives of fumaric acid show antipsoriatic effects but details of the mechanism of action are largely unknown. The study focused on the effect of fumaric acid, dimethyl-fumarate, Zn-, Ca- and Mg-monoethyl-fumarate on the interferon-gamma (IFN-gamma)-induced expression of ICAM-1 and HLA-DR molecules on keratinocytes. Human hyperproliferative keratinocytes of the HaCaT cell line were exposed to IFN-gamma (10 U/ml) alone or in combination with fumaric acid and its derivatives for 48 hrs. The effect of fumarates was investigated semiquantitatively using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method. Subsequently, the effect of dimethyl-fumarate, the main component of "fumaric acid therapy", was evaluated quantitatively by means of an APAAP-ELISA technique. The semiquantitative evaluation revealed that in the micromolar dose range investigated only dimethyl-fumarate demonstrated substantial growth inhibition and down-regulation of the cell surface markers. In the quantitative evaluation, dimethyl-fumarate significantly (p</=0.05) suppressed the expression of ICAM-1 (84%) and HLA-DR (67%) on HaCaT keratinocytes at a subtoxic concentration of 4.0 microM as compared to untreated controls (100%). In contrast, concentrations of 4.0, 12 and 35 microM dimethyl-fumarate had no influence on the ICAM-1 and HLA-DR expression on IFN-gamma-exposed normal human epidermal keratinocytes in primary cultures. Thus, there is experimental evidence that dimethyl-fumarate may exert its antipsoriatic effect not only as an antiproliferative agent but also by down-regulation of ICAM-1 and HLA-DR molecules on hyperproliferative keratinocytes.
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PMID:The antipsoriatic dimethyl-fumarate suppresses interferon-gamma -induced ICAM-1 and HLA-DR expression on hyperproliferative keratinocytes. Quantification by a culture plate-directed APAAP-ELISA technique. 964 87

We have previously suggested that colorectal liver metastases might produce 'toxins' that reduce both quality of life (QoL) and survival. In this study we assessed whether QoL in patients with such metastases was related to immune activation, as determined by increased serum levels of interleukin 6 (IL6), soluble tumour necrosis factor receptor 1 (sTNFr1), soluble interleukin 2 receptor alpha (sIL2r alpha) or the interferon-gamma marker neopterin. Serum IL6, sTNFr1, sIL2r alpha, neopterin, alkaline phosphatase and carcinoembryonic antigen levels, liver metastasis volume, and QoL (Hospital Anxiety and Depression [HAD] scale, Rotterdam Symptom Checklist [RSC], and Sickness Impact Profile [SIP]) were measured in 43 patients. There were significant positive correlations between serum sIL2r alpha and HAD depression score (r = 0.66, P = 0.0001), RSC physical symptom score (r = 0.46, P < 0.01), and SIP score (r = 0.47, P = 0.009). Multiple regression analysis suggested that serum sIL2r alpha level was a significant independent predictor of HAD depression score. Although survival was shorter (logrank test P < 0.05) where sIL2r alpha, sTNFr1 and IL6 levels were higher, the ability of sIL2r alpha to predict HAD depression score was independent of survival.
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PMID:Relation between depression and circulating immune products in patients with advanced colorectal cancer. 981 54

We studied differences in ectopic osteoinduction in eight mouse inbred strains and an outbred strain. Antigen-extracted autolyzed rat bone gelatin was implanted under hind limb muscle fascia of 12-week-old males, and new bone formation was morphologically assessed on serial sections. Four weeks after implantation, less than half of the implants from CBA/J, A/J, BALB/cJ, and C3Hf/Bu mice showed induction of only cartilage. New cartilage was observed in all, and bone and bone marrow in 80% of the implants from AKR/J, C57BL/6J, DBA/2J, and RFM/Rij mice. Volume of the newly formed tissue ranged from 1.3% of the old matrix in A/J strain to 74.6% in DBA/2J strain. Outbred CD1 mice showed only weak cartilage induction. The "good" responders differed among themselves in the volume and type of newly induced tissue: DBA/2J, RFM/Rij, and AKR/J mice had a similar ratio of new bone and cartilage and abundant bone marrow, whereas the predominant newly induced tissue in C57Bl/6J mice was cartilage. The pattern of the expression of BMP-2, -4, and -7, alkaline phosphatase, osteocalcin, interferon-gamma, and granulocyte-macrophage colony-stimulating factor, measured by reverse transcriptase polymerase chain reaction, did not correlate with the type and the quantity of the newly induced tissue. Our results show that adult mice of inbred strains differ not only in the peak bone mass and morphology, but also ability to form new bone after an osteoinductive stimulus. Ectopic osteoinduction may be a useful in vivo model to investigate genetic determinants of endochondral osteogenesis, especially its immunological component.
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PMID:Genetic variability of new bone induction in mice. 1042 18

In this study we aimed to elucidate the physiological role of gammadelta intraepithelial lymphocytes (IEL) in the mouse intestine. For this purpose, we used T-cell receptor (TCR) Vgamma4/Vdelta5 transgenic mice (KN 6 Tg: BALB/c background, H-2d), and compared the immunological and physiological characteristics of the intestinal tracts of KN 6 Tg and non-transgenic (non-Tg) littermates. In KN 6 Tg littermates, 95% of small intestinal (SI) and large intestinal (LI) IEL expressed gammadelta TCR, and their TCR was replaced by Tg gammadelta TCR. In these mice, class II major histocompatibility complex (MHC) expression was up-regulated in the SI epithelium, compared with the non-Tg littermates, under specific pathogen-free (SPF) conditions. Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNAs of the I-Ealpha chain on the SI epithelial cells was higher in KN 6 Tg than in non-Tg littermates. However, in the LI, class II MHC molecules were not expressed in either KN 6 Tg or non-Tg littermates. The epithelial cell mitotic index in the SI, but not in the LI, was higher in KN 6 Tg than in non-Tg littermates under SPF conditions. However, differentiation markers for SI epithelial cells, such as alkaline phosphatase and disaccharidase (lactase, maltase and sucrase) activities, were similar in KN 6 Tg and non-Tg littermates. MHC class II molecule expression on the SI epithelium was absent in germ-free (GF) Tg mice, but was induced under SPF conditions, coinciding with the increase of interferon-gamma (IFN-gamma) mRNA in gammadelta TCR SI-IEL. These findings suggest that gammadelta TCR IEL regulate epithelial cell regeneration and class II MHC expression, but not cell differentiation in the SI. However, these functions were not observed in the gammadelta TCR IEL in the LI. In addition, the activation step in the gammadelta TCR SI-IEL is dependent on the presence of gut microflora.
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PMID:Physiological roles of gammadelta T-cell receptor intraepithelial lymphocytes in cytoproliferation and differentiation of mouse intestinal epithelial cells. 1044 10

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.
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PMID:Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts. 1058 92


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