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)

In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3'-terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal-chelate complex with ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra-acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal-binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.
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PMID:Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails. 190 25

1. Stimulation of mast cells by externally applied secretagogues activated a slowly developing membrane current. With high external and low internal chloride (Cl-) concentrations, the current reversed at about -40 mV, but when external Cl- was made equal to internal Cl-, the reversal potential shifted to about 0 mV, demonstrating that the current carrier was Cl-. 2. In addition to external agonists, internally applied cyclic AMP and high concentrations of intracellular calcium [Ca2+]i could also activate the Cl- current. However, elevated [Ca2+]i produced only slow and incomplete activation. This suggests that the Cl- current is not directly Ca2+ activated. Also, activation of Cl- current by external agonists and by cyclic AMP was unimpaired when [Ca2+]i was clamped to low levels with internal ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA), indicating that elevated [Ca2+]i is not necessary for activation of the Cl- current. Although activation by cyclic AMP was faster than that produced by elevated [Ca2+]i, it still required tens of seconds; thus the effect of cyclic AMP was also likely to be indirect. 3. Internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) could also activate the Cl- current, suggesting the involvement of a G protein in the control of the current. 4. The variance associated with the Cl- current was small, and noise analysis gave a lower limit of about 1-2 pS for the single-channel conductance. The Cl- current was reduced by 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), and during DIDS blockade, the variance of the current increased. This suggests that DIDS enters and blocks the open channel. 5. Activation of the Cl- current would make the membrane potential negative following stimulation of a mast cell, thus providing a driving force for entry of external calcium via the stimulation-induced influx pathways described in the preceding paper (Matthews, Neher & Penner, 1989).
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PMID:Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells. 255 69

The presynaptically active snake venom neurotoxin beta-bungarotoxin (beta-Butx) is known to affect neurotransmitter release by binding to a subtype of voltage-activated K+ channels. Here we show that mast cell degranulating (MCD) peptide from bee venom inhibits the binding of 125I-labeled beta-Butx to chick and rat brain membranes with apparent Ki values of 180 nM and 1100 nM, respectively. The mechanism of inhibition by MCD peptide is noncompetitive, as is inhibition of 125I-beta-Butx binding by the protease inhibitor homologue from mamba venom, toxin I. Beta-Butx and its binding antagonists thus bind to different sites of the same membrane protein. Removal of Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid inhibits the binding of 125I-beta-Butx by lowering its affinity to brain membranes.
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PMID:Inhibition of beta-bungarotoxin binding to brain membranes by mast cell degranulating peptide, toxin I, and ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. 313 91

1 Treatment of purified rat peritoneal mast cells at 37 degrees C with concentrations of the non-ionic detergent nonidet P40 (NP40) up to 0.005% (v/v) failed to reduce their viability. 2 There was a marked reduction in the histamine releasing capacity of NP40-treated mast cells upon challenge with a variety of selective (adrenocorticotrophic hormone 1-24 (Synacthen), rabbit anti-rat IgE antiserum, adenosine triphosphate (ATP) and the calcium ionophore, A 23187) and non-selective (rabbit anti-rat mast cell antiserum plus complement) histamine liberators. 3 Nonidet P40 (0.005%) was found to reduce the activity of a mast cell membrane 'ecto-enzyme', calcium-activated ATPase, by about 45% when presented at the time of its assay.
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PMID:The effects of nonidet P40 on the function of rat peritoneal mast cells in vitro. 616 28

The specific IgE response that appears in subjects immunized with tetanus toxoid does not induce hypersensitivity reactions at subsequent immunizations. The type I immune response, therefore, was studied both in vivo and in vitro, in 11 subjects who had specific IgE antibodies for tetanus toxoid. The results showed that: 1. the specific IgE antibodies are heterogeneous regarding their affinity for the mast cell and basophil receptors; 2. the specific IgG antibodies for tetanus toxoid, at serum concentrations, are not able to interfere with the in vitro specific basophil degranulation; 3. in the PEG precipitate there are aggregates of specific IgE antibodies for tetanus toxoid. In vitro, these molecular aggregates are not able to sensitize the basophil cells.
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PMID:Functional characterization of specific IgE antibodies for tetanus toxoid. 662 28

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

Polyethylene glycol (PEG)-conjugated Hb (PEG-Hb) is being considered as a blood substitute. Previously, we showed that PEG-Hb extravasates rapidly from the intestinal mucosa and causes transient epithelial sloughing, resulting in temporary unimpeded passage of material between the intestinal lumen and the microcirculation. The present study quantifies the time course of factors related to this disturbance. Anesthetized Sprague-Dawley rats (350-450 g) were injected with a bolus of PEG-Hb (10 mg/ml) in saline. Control animals received saline, alone or with Dextran 70 (5 mg/ml). After 2, 8, 15, 60, or 90 min, the small intestine was perfusion fixed for microscopy (4 animals for each time point). Epithelial cell detachment and mucosal mast cell degranulation peaked at 2 and 8-15 min, respectively, but by 90 min were back to normal. Goblet cell secretion increased with time up to 8-15 min, after which it leveled off. Mean interstitial width was significantly greater 8 min after injection than for controls and continued to increase with time. In capillaries, endothelial fenestral diaphragms were replaced by thick, amorphous structures. Mesenteric mast cell degranulation was significantly greater 60-90 min after injection compared with controls. We propose that these results are consistent with intravascular injection of PEG-Hb invoking a transient inflammatory response in the intestine.
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PMID:Ultrastructural effects of intravascularly injected polyethylene glycol-hemoglobin in intestinal mucosa. 968 51

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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