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)

Whole-cell patch-clamp recordings of membrane currents were performed in combination with measurements of mediator secretion from mucosal-type mast cells (rat basophilic leukemia cells, subline 2H3), to determine the involvement of membrane conductances induced upon depletion of intracellular Ca2+ stores. In patch-clamp experiments, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-induced depletion of internal Ca2+ stores led to activation of two distinct membrane conductances, a Ca2+ current and a Cl- current. The Ca2+ current was blocked by 100 microM La3+, which did not affect the Cl- current. In contrast, 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid produced selective blocked of the Cl- current. Remarkably, the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid, and N-phenylanthranilic acid (NPAA) inhibited not only the Cl- current but also the Ca2+ current. IC50 values for the blockade of the Ca2+ inward current by NPPB, niflumic acid, and NPAA were determined to be 23, 150, and 190 microM, respectively. In secretion experiments, thapsigargin-induced depletion of internal Ca2+ stores stimulated serotonin release, which was found to be strictly dependent on extracellular Ca2+. In the presence of 100 microM La3+ secretion was almost completely inhibited. In contrast, only 50% of secretion was suppressed by 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid, which fully blocked the Cl- current without affecting Ca2+ influx, as monitored by electrophysiological experiments. The other Cl- channel blockers produced a very different pattern for the inhibitory dose dependence of secretion, with IC50 values for NPPB, niflumic acid, and NPAA of 23, 60, and 180 microM, respectively. Taken together, these findings suggest that Ca2+ store depletion leads to concomitant activation of Cl- and Ca2+ currents. Blockade of the latter is apparently an additional mode of action for diarylaminocarboxylate-type Cl- channel blockers inhibiting mast cell secretory responses.
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PMID:Blockade of capacitive Ca2+ influx by Cl- channel blockers inhibits secretion from rat mucosal-type mast cells. 774 67

In order to investigate the functional similarities of the high affinity receptor for IgE (Fc epsilon RI) and the T cell receptor for antigen, we have developed a high efficiency polyethylene glycol-mediated fusion method to make somatic hybrids between cells from a mast cell line (RBL-2H3) and cells from T lymphoma cell lines (Jurkat and HPB-ALL). Using flow cytometry to select for the heterologously fused cells, we demonstrated that aggregation of the T cell receptor results in the efficient secretion of [3H]5-hydroxytryptamine from RBL cell-derived granules. In addition, both receptors mediate Ca2+ mobilization in the hybrid cells that is insensitive to inhibition by the protein kinase C activator phorbol-12-myristoyl-13-acetate (PMA). In contrast, Ca2+ mobilization caused by aggregation of Fc epsilon RI in the parent RBL cells is completely inhibited by PMA. The results indicate that these two different receptors for foreign antigen can substitute for each other to trigger responses in the hybrid cells that are unique to each cell type. The methodology employed has general utility for studying signal transduction mediated by mammalian cell surface receptors.
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PMID:Fc epsilon RI and the T cell receptor for antigen activate similar signalling pathways in T cell-RBL cell hybrids. 849 25

The role of leukotrienes on bone resorption was tested in a well-standardized model of bone remodeling by inhibiting their biosynthesis with BWA4C, a specific inhibitor of 5-lipoxygenase. After extraction of their upper molars unilaterally, 30 Wistar rats were divided into three groups; the first remained untreated (control group), the second received 80 mg/kg/day of BWA4C dissolved in polyethylene glycol 300 (experimental group), and the third received only the vehicle (sham-treated group). After four days of experiment, the animals were killed and the resorption profile was assessed along the antagonist mandibular buccal cortex. The main result was a dramatic decrease in the number of TRAP-positive mononucleated preosteoclasts in the experimental group (-69%, p < 0.0005 and p < 0.003 vs. the control and sham-treated groups, respectively). This drop was related to a significant decrease in the number of osteoclasts. Neither the activation of the differentiated osteoclasts nor their mean interface with the bone surface were affected by BWA4C. Concomitantly, the mast cell population residing near the vascular network limiting the periosteum was markedly and significantly increased by the treatment. These mast cells were mostly degranulating, i.e., were in a state of activation that we previously found to related to resorption. These data suggest (1) that the leukotrienes are involved in the recruitment of osteoclast progenitors, and/or their differentiation into preosteoclasts, and (2) that mast cells responded to leukotriene inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The 5-lipoxygenase inhibitor BWA4C impairs osteoclastic resorption in a synchronized model of bone remodeling. 855 28

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.
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PMID:Activation of methotrexate-phenylalanine by monoclonal antibody--carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro. 856 31

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

The aim of this study was to measure the effects of food restriction on antigen-induced bronchoconstriction and inflammatory cell influx in guinea pigs and to determine the role of plasma cortisol and catecholamine concentrations. Ovalbumin (OA; 0.3 mg/kg, i.v.) was administered to OA-sensitized, anesthetized guinea pigs which had been allowed free access to food or had been food restricted for 18 h prior to OA challenge. In addition to higher plasma levels of epinephrine (30% increase) and cortisol (33% increase), fasted guinea pigs had significantly lower (60% decreased) maximal bronchoconstrictor responses to OA than nonfasted, sensitized litter mates. Additionally, groups of fasted or fed animals were subdivided into two additional treatment groups: (1) saline-pretreated or (2) polyethylene glycol 400 (PEG)-pretreated (1 ml/kg, p.o., 1 h prior to antigen challenge). In saline-treated, fasted animals, bronchoconstrictor responses to antigen were significantly diminished (67% decreased) and epinephrine and cortisol levels were increased (64 and 34%, respectively) compared to the corresponding fed group. In both fasted and fed groups, the PEG-treated guinea pigs had higher plasma epinephrine and cortisol levels than animals which received saline, but no significant differences were detected within the PEG-treated group. Plasma norepinephrine concentrations were lower in all fasted groups. In a separate model in conscious guinea pigs, there were no differences in aerosol OA-induced bronchoconstriction and eosinophil influx between fasted and fed groups. However, compared to the saline pretreatment group, PEG administration reduced the antigen-induced bronchoconstriction and eosinophilia in both fed and fasted guinea pigs. We speculate that the reduced responsiveness to antigen in fasted versus fed animals may result from food-restriction-induced, stress-related release of epinephrine and cortisol from the adrenal glands, thereby suppressing mast cell degranulation or reducing responsiveness to spasmogenic and chemotactic mediators. In addition, the results suggest that oral dosing with 100% PEG may enhance this phenomenon.
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PMID:Food restriction-mediated adrenal influences on antigen-induced bronchoconstriction and airway eosinophil influx in the guinea pig. 975 48

Diquat, a nonselective desiccant herbicide, induces a significant secretion of fluid into the lumen of the gastrointestinal tract of rats at sublethal doses (from 0.5 to 50 mg/kg). This study investigated the effect of an acute low-level exposure to diquat (0. 1, 0.5, and 1 mg/kg) on intestinal net water flux and the mechanisms involved. In anesthetized rats, an intestinal loop (7 cm) was infused with Ringer's buffer containing [14C]-polyethylene glycol 4000. After equilibration, diquat (0.1, 0.5, and 1 mg/kg) was added to Ringer's buffer during 60 min. Net water flux was calculated according to [14C] activity determined in the effluent collected at 15-min intervals. Infused in the intestinal loop for 60 min at doses of 0.5 and 1 mg/kg but not at 0.1 mg/kg, diquat induced an intestinal net water secretion during 180 min with a maximal effect at the highest dose used and during the first hour following the end of diquat infusion. Diquat-induced (1 mg/kg) intestinal net water secretion was blocked by a neurotoxin, tetrodotoxin (5 micrograms/kg iv), doxantrazole (5 mg/kg ip), a mast cell stabilizer, and two inhibitors of NO synthases: l-NAME (25 mg/kg ip) and aminoguanidine (2 mg/kg ip). It is concluded that a single low-level (0.5 and 1 mg/kg) intrajejunal administration of diquat induces a net water intestinal secretion and that this secretory effect is nerve-mediated, implying mast cell degranulation and NO release.
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PMID:Low-level exposure to diquat induces a neurally mediated intestinal hypersecretion in rats: involvement of nitric oxide and mast cells. 977 2

Architectural features of synthetic ligands were systematically varied to optimize inhibition of mast cell degranulation initiated by multivalent crossing of IgE-receptor complexes. A series of ligands were generated by end-capping poly(ethylene glycol) (PEG) polymers and amine-based dendrimers with the hapten 2,4-dinitrophenyl (DNP). These were used to explore the influence of polymeric backbone length, valency, and hapten presentation on binding to anti-DNP IgE and inhibition of stimulated activation of RBL cells. Monovalent MPEG(5000)-DNP (IC(50) = 50 nM), bivalent DNP-PEG(3350)-DNP (IC(50) = 8 nM), bismonovalent MPEG(5000)-DNP(2) (IC(50) = 20 nM), bisbivalent DNP(2)-PEG(3350)-DNP(2) (IC(50) = 3nM) and DNP(4)-dendrimer ligands (IC(50) = 50 nM) all effectively inhibit cellular activation caused by multivalent antigen, DNP-bovine serum albumin. For different DNP ligands, we provide evidence for more effective inhibition due to (i) preferential formation of intra-IgE cross-links by bivalent ligands of sufficient length, (ii) self-association of monovalent ligands with longer tails, and (iii) higher probability of binding for bisvalent ligands. We also show that larger DNP(16)-dendrimers of higher valency trigger degranulation by cross-linking IgE-receptor complexes, whereas smaller DNP-dendrimers are inhibitory. Thus, features of synthetic ligands can be manipulated to control receptor occupation, aggregation, and inhibition of the cellular response.
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PMID:Highly effective poly(ethylene glycol) architectures for specific inhibition of immune receptor activation. 1459 88

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.
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PMID:Affinity chromatography of tryptases: design, synthesis and characterization of a novel matrix-bound bivalent inhibitor. 1559 13

In vivo efficacy of novel anticancer agents has been hindered by the inability to deliver effective concentrations of drugs to tumors. The use of macromolecules such as antibodies and polymers for enzyme delivery to tumors has revealed that catalyzing the conversion of a nontoxic prodrug into its cytotoxic form can generate an effective level of cytotoxic agents at tumor sites. This study primarily focuses on the synthesis and characterization of methoxypoly(ethylene glycol)-modified carboxypeptidase A (CPA) for solid tumor targeting. The molecular weight of CPA has been successfully altered from 35 to 40-50 kDa via attachment of a defined number of mPEG moieties. Relatively pure mPEG-CPA conjugates containing one, two, and three mPEG chains were obtained at preparative scale quantities through controlled PEGylation followed by fractionation that involved size-exclusion chromatography. An enhancement in kinetic properties including k(cat) and k(cat)/K(m) towards hippuryl-L-phenylalanine (hipp-L-phe) was observed in mPEG-CPA conjugates. An increase in the V(m) appeared to be responsible for this enhancement. The attachment of mPEG to CPA substantially improved the stability of the enzyme with respect to the specific peptidase activity toward the model substrate. This finding is particularly important in the development of a novel CPA/methotrexate-alpha-peptide system in solid tumor chemotherapy.
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PMID:Methoxypoly(ethylene glycol)-conjugated carboxypeptidase A for solid tumor targeting: part I: synthesis and characterization. 1586 40


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