Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.
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PMID:Production and characterisation of monoclonal antibodies recognising ovine IgE. 879 63

The complex temporal evolution of on-column exocytotic release of serotonin and proteins from individual rat peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 +/- 0.6 fmol; the average percentage of serotonin released was 28 +/- 14%. Events that are consistent with released serotonin from single submicrometer granules (250 aL each) were evident, each of which contained an average amount of 5.9 +/- 3 amol.
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PMID:Monitoring exocytosis and release from individual mast cells by capillary electrophoresis with laser-induced native fluorescence detection. 879 24

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep alpha1-proteinase inhibitor (alpha1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1. 1x10(3) M-1.s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2x10(6) M-1.s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between alpha1PI and alpha2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess alpha1PI and alpha2-macroglobulin, the alpha-chain being cleaved C-terminally and the beta-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.
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PMID:Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts. 916 5

Soybean Bowman-Birk protease inhibitor (BBI) is an inhibitor of serine proteases with two functional inhibitory domains of different specificities: one is specific for chymotrypsin-like proteases, the other for trypsin-like proteases. Chymase and tryptase are serine proteases which are stored in mast cell granules and released upon degranulation. This work investigated the inhibition of human chymase and tryptase by BBI. Active-site titration of human skin chymase by BBI demonstrated that BBI was a highly effective inhibitor of human chymase. Virtually stoichiometric inhibition of chymase by BBI was observed at 10 nM chymase. Kinetic studies of the inhibition reaction yielded an association rate constant of 4.0 x 10(5) M(-1) s(-1) and a dissociation rate constant of 1.7 x 10(-5) s(-1). From these two constants we estimate a K(i) of 50 pM. Chymase/BBI complexes did not dissociate in SDS-PAGE analyses under nonreducing conditions, consistent with the formation of a very tight complex with little tendency to dissociate. In contrast to chymase, human tryptase was not inhibited by BBI. These studies demonstrate that BBI is a good inhibitor of human chymase, exhibiting reaction properties better than physiological inhibitors described to date.
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PMID:Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase. 924 90

1. In vivo rectal distension (RD) induces a neurally mediated colonic net water hypersecretion in rats. Interleukin-1 beta (IL-1 beta) also induces neural colonic water hypersecretion involving the release of prostaglandins (PGs) and a mast cell degranulation in rats. This study investigated in vivo the role of IL-1, PGs and mast cells in RD-induced colonic hypersecretion. 2. Proximal colonic net water flux was determined using [14C]polyethylene glycol (PEG) 4000 (mol. wt. 4000) in anaesthetized rats. On strips taken from the distal colon: (i) a histological analysis was performed to determine the number of mucosal mast cells (MMC); and (ii) histamine levels were measured by radioimmunoassay after stimulation with compound 48/80. 3. RD induced a net colonic water secretion that was blocked by i.c.v. administration of IL-1ra (an IL-1 receptor antagonist) and indomethacin, and by systemic treatment with doxantrazole and indomethacin. RD decreased the number of resident mast cells and the release of histamine from the distal colonic strips. Moreover, using SDS-PAGE immunoblotting the expression of IL-1 beta was detected in the brain. 4. These results suggest that, in rats, RD induces colonic net water hypersecretion by the activation of a neuro-immunological reflex pathway, involving IL-1 beta, PG release and peripheral mast cell degranulation.
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PMID:Involvement of interleukin-1, prostaglandins and mast cells in rectal distension-induced colonic water secretion in rats. 948 85

To clarify the process of post-translational modification of L-histidine decarboxylase (HDC), we investigated the conversion of the 74-kDa form of HDC into the 53-kDa form in specialized organella of a rat basophilic/mast cell line (RBL-2H3). With treatment of streptolysin-O, RBL-2H3 cells released approximately 40% of HDC activity accompanied by over 90% of lactate dehydrogenase activity. Only the 74-kDa form of HDC was detected in the leaked fraction by SDS-polyacrylamide gel electrophoresis. The 74-kDa form in the homogenate of pulse-labeled cells was recovered in both the supernatant and particulate fractions, while the 53-kDa form was detected only in the particulate fraction containing marker proteins of microsomes, Golgi, and lysosomal granules. Confocal microscopic observation using double staining immunofluorescence with anti-GST fusion HDC antiserum showed that most of the HDC coexists with protein-disulfide isomerase, a typical marker of the luminal space of the ER. With treatment of digitonin, RBL-2H3 cells released only 74-kDa HDC. Trypsin digestion of digitonin-permeabilized cells resulted in the disappearance of the 74-kDa form but not the 53-kDa form. From these results, it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the ER, where it is converted to the 53-kDa form.
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PMID:Intracellular localization of the 74- and 53-kDa forms of L-histidine decarboxylase in a rat basophilic/mast cell line, RBL-2H3. 952 22

A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.
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PMID:Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle. 953 57

A mast cell granule protease has been isolated and purified from nematode-infected caprine jejunal homogenate by FPLC techniques and termed Goat Mast Cell Protease (GMCP). The purification steps were monitored for proteolytic activity against the synthetic substrate carboxybenzoyl-L-lysine thiobenzyl ester (BLT) and the presence of a homogenous protease preparation in the final sample was shown by SDS-PAGE electrophoresis. This protease was compared with enzymatic activity from isolated mucosal mast cells, which demonstrated the putative mast cell-derived source of the purified enzyme. Rabbit antiserum was raised against the protease and through the use of immunohistochemistry and Western blotting techniques the mast cell origin of the protease was confirmed. NH2-Terminal amino acid sequence analysis demonstrated a high degree of homology between GMCP and other previously isolated mast cell proteases including sheep mast cell protease (SMCP). Substrate analysis showed that GMCP also had an unusual dual chymotrypsin-like and trypsin-like activity similar to SMCP and bovine duodenase.
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PMID:The isolation and purification of a dual specific mast cell-derived protease from parasitised caprine jejunal tissue. 955

Chymase, a major product of mast cell activation, is secreted as a fully active enzyme. We have prepared recombinant human prochymase and have investigated the conditions under which it may be activated by dipeptidyl peptidase I (DPP I). The gene for human chymase was cloned in a baculovirus vector and expressed in High Five insect cells, and the recombinant protein purified by heparin-agarose and gel-filtration chromatography. The purified prochymase was homogeneous by SDS/PAGE with the same molecular mass as native human chymase, and its identity confirmed by N-terminal sequence analysis and Western blotting with chymase-specific antibodies. Treatment with DPP I to remove the N-terminal dipeptide prosequence resulted in enzymatically active chymase, with substrate and inhibitor profiles very similar to those of the native human enzyme. The activation of prochymase by DPP I was strongly inhibited by heparin (IC50 = 0.5 microg ml[-1]) and histamine (IC50 = 2 mM), though these mast cell products had little effect on the action of DPP I towards a low molecular-mass substrate. The pH optimum of DPP I was also higher and narrower with prochymase. The inhibitory action of heparin was lost at NaCl or KCl concentrations sufficient to elute prochymase from a heparin agarose column. Dextran sulphate was as inhibitory as heparin, whereas chondroitin sulphate C was more than 10-fold less effective. Our findings suggest that the activation of prochymase might be restricted to the early stages of vesicle maturation, when the pH is close to neutrality and the histamine and heparin concentrations are low.
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PMID:The conversion of recombinant human mast cell prochymase to enzymatically active chymase by dipeptidyl peptidase I is inhibited by heparin and histamine. 957 89

The murine T1 gene encodes a membrane-bound glycoprotein (T1M) and a soluble variant (T1S) which represents the ectodomain of the receptor-type form. T1 is an orphan receptor belonging to the interleukin-1 receptor family. Its biological function is currently unknown. We analyze the expression of the two T1 proteins in mast cells and fibroblasts by using a set of monoclonal antibodies (MAb) that specifically recognize the extracellular portion of the T1 receptor. To generate anti-T1 MAbs, we immunized Lewis rats with a eukaryotically expressed chimeric protein consisting of the T1-receptor ectodomain fused to a human immunoglobulin domain. The two MAbs DJ4 and DJ8 were shown to specifically detect the murine T1M protein on the surface of primary IL-3-dependent bone marrow-derived mast cells as shown by flow cytometry and immunohistochemistry. Both antibodies were also capable of immunoprecipitating the membrane-associated 110-120 kDa T1M protein from mast cell lysates. In serum-stimulated but not in quiescent NIH3T3 fibroblasts, DJ4 and DJ8 MAbs detected both the soluble T1S protein as a 45-65 kDa band on SDS polyacrylamide gels as well as the membrane-bound 95 kDa T1M protein. The T1M protein in fibroblasts was less abundantly expressed and exhibited a lower molecular weight than the mast cell-produced T1M, probably as a consequence of different protein glycosylation. The MAbs described here represent highly specific reagents and valuable tools that should facilitate the establishment of the murine T1 protein expression pattern thus contributing to the solution of the question of its function.
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PMID:Expression analysis of the soluble and membrane-associated forms of the interleukin-1 receptor-related T1 protein in primary mast cells and fibroblasts. 962 50


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