The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Angela Santoni - One of the best experts on this subject based on the ideXlab platform.
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activation of syk tyrosine kinase is required for c cbl mediated ubiquitination of fceri and syk in RBL Cells
Journal of Biological Chemistry, 2002Co-Authors: Rossella Paolini, Rosa Molfetta, Laurie O. Beitz, Juan Zhang, Andrew M. Scharenberg, Mario Piccoli, Luigi Frati, Reuben Siraganian, Angela SantoniAbstract:Engagement of the high affinity receptor for IgE (FceRI) on mast Cells and basophils results in FceRI β and γ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FceRI engagement on RBL-2H3 Cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FceRI β and γ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL Cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FceRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FceRI and activated Syk to the proteasome for degradation.
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Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of FcεRI and Syk in RBL Cells
The Journal of biological chemistry, 2002Co-Authors: Rossella Paolini, Rosa Molfetta, Laurie O. Beitz, Juan Zhang, Andrew M. Scharenberg, Mario Piccoli, Luigi Frati, Reuben Siraganian, Angela SantoniAbstract:Abstract Engagement of the high affinity receptor for IgE (FceRI) on mast Cells and basophils results in FceRI β and γ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FceRI engagement on RBL-2H3 Cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FceRI β and γ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL Cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FceRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FceRI and activated Syk to the proteasome for degradation.
Klaus Aktories - One of the best experts on this subject based on the ideXlab platform.
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Pleiotropic role of Rac in mast cell activation revealed by a cell permeable Bordetella dermonecrotic fusion toxin.
Cellular signalling, 2010Co-Authors: Heidi Stratmann, Carsten Schwan, Joachim H. C. Orth, Gudula Schmidt, Klaus AktoriesAbstract:To activate the GTPase Rac in rat basophilic leukemia (RBL) Cells and mouse bone marrow-derived mast Cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL Cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast Cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell activation.
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Inhibition of Calcium Release-activated Calcium Current by Rac/Cdc42-inactivating Clostridial Cytotoxins in RBL Cells
The Journal of biological chemistry, 2000Co-Authors: Nabil Djouder, Ulrike Prepens, Klaus Aktories, Adolfo CavaliéAbstract:Abstract Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 Cells was studied.Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL Cells mediated by the high affinity antigen receptor (FceRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca2+ mobilization induced by FceRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca2+. In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca2+ influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca2+ mobilization most likely by regulating the Ca2+ influx through calcium release-activated calcium channels activated via FceRI receptor in RBL Cells.
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Influence of Clostridium botulinum C2 toxin on FcɛRI-mediated secretion and tyrosine phosphorylation in RBL Cells
Naunyn-Schmiedeberg's archives of pharmacology, 1998Co-Authors: Ulrike Prepens, Holger Barth, Jörg Wilting, Klaus AktoriesAbstract:We studied the effects of the binary Clostridium botulinum C2 toxin on stimulated [3H]serotonin release and protein tyrosine phosphorylation in RBL 2H3 hm1 Cells. Actin was specifically ADP-ribosylated by C2 toxin in intact Cells resulting in a 2–3 fold increase in antigen- or calcium ionophore (A23187)-induced degranulation. The effects of C2 toxin were time- and concentration-dependent. Toxin treatment, which dramatically changes the morphology of RBL Cells, was not sufficient to induce mediator release in the absence of activators of secretion. Antigen- and A23187-stimulated tyrosine phosphorylation of 60–80kDa and 110–120kDa proteins was reduced or blocked after C2 toxin incubation. Treatment of RBL Cells with the tyrosine phosphatase inhibitor pervanadate reversed the inhibitory effect of C2 toxin on stimulated protein tyrosine phosphorylation indicating activation of phosphatases by C2 toxin. The data indicate that disassembly of the actin cytoskeleton by C2 toxin facilitates FcɛRI-mediated signal-secretion coupling and suggest a role of the actin cytoskeleton in phosphatase regulation in RBL Cells.
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ADP-ribosylating and glucosylating toxins as tools to study secretion in RBL Cells.
Advances in experimental medicine and biology, 1997Co-Authors: Ulrike Prepens, Ingo Just, Fred Hofmann, Klaus AktoriesAbstract:The influence of different ADP-ribosylating and glucosylating cytotoxins on stimulated protein tyrosine phosphorylation and secretion in rat basophilic leukemia (RBL) Cells was studied. Treatment of RBL Cells with Clostridium botulinum C2 toxin, which specifically ADP-ribosylated monomeric G-actin and caused complete depolymerization of the actin cytoskeleton in intact Cells, inhibited Fc epsilon RI receptor-mediated tyrosine phosphorylation of various proteins in a time- and concentration-dependent manner with maximal effects at 100 ng/ml C2I and 200 ng/ml C2I. C2 toxin (10 ng/ml C2I and 20 ng/ml C2II) increased antigen- or calcium ionophore (A23187)-stimulated [3H]serotonin release maximally by about 3 fold. Clostridium botulinum C3, which ADP-ribosylated Rho in intact RBL Cells, had no effect on protein tyrosine phosphorylation and stimulated secretion. In contrast, the cytotoxic Clostridium difficile toxin B (ToxB), which glucosylated the Rho-subtype family members RhoA and Cdc42, blocked or reduced antigen- or calcium ionophore-mediated [3H]serotonin release, respectively, and decreased tyrosine phosphorylation of a 110 kDa protein. The data indicate that different actin pools control tyrosine phosphorylation and secretion in RBL Cells and suggest that Rho subfamily proteins regulate secretion independently of the actin cytoskeleton.
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Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia Cells by Clostridium difficile toxin B (monoglucosyltransferase)
The Journal of biological chemistry, 1996Co-Authors: Ulrike Prepens, Ingo Just, Von Eichel-streiber C, Klaus AktoriesAbstract:Abstract Treatment of rat basophilic leukemia (RBL) 2H3-hm1 Cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL Cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 μg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL Cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL Cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.
I S Ambudkar - One of the best experts on this subject based on the ideXlab platform.
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distinct ca2 permeable cation currents are activated by internal ca2 store depletion in RBL 2h3 Cells and human salivary gland Cells hsg and hsy
The Journal of Membrane Biology, 2004Co-Authors: X Liu, Klaus Groschner, I S AmbudkarAbstract:Store-operated Ca2+ influx, suggested to be mediated via store-operated cation channel (SOC), is present in all Cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents (I SOC) in human submandibular glands Cells (HSG) and human parotid gland Cells (HSY) with I CRAC (Ca2+ release-activated Ca2+ current) in RBL Cells. Internal Ca2+ store-depletion with IP3 or thapsigargin activated cation channels in all three cell types. 1 μM Gd3+ blocked channel activity in all Cells. Washout of Gd3+ induced partial recovery in HSY and HSG but not RBL Cells. 2-APB reversibly inhibited the channels in all Cells. I CRAC in RBL Cells displayed strong inward rectification with E rev(Ca) = >+90 mV and E rev (Na) = +60 mV. I SOC in HSG Cells showed weaker rectification with E rev(Ca) = +25 mV and E rev(Na) = +10 mV. HSY Cells displayed a linear current with E rev = +5 mV, which was similar in Ca2+- or Na+-containing medium. pCa/pNa was >500, 40, and 4.6 while pCs /pNa was 0.1,1, and 1.3, for RBL, HSG, and HSY Cells, respectively. Evidence for anomalous mole fraction behavior of Ca2+/Na+ permeation was obtained with RBL and HSG Cells but not HSY Cells. Additionally, channel inactivation with Ca2+ + Na+ or Na+ in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY Cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.
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Distinct Ca^2+-permeable Cation Currents Are Activated by Internal Ca^2+-Store Depletion in RBL-2H3 Cells and Human Salivary Gland Cells, HSG and HSY
The Journal of Membrane Biology, 2004Co-Authors: X Liu, Klaus Groschner, I S AmbudkarAbstract:Store-operated Ca^2+ influx, suggested to be mediated via store-operated cation channel (SOC), is present in all Cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents ( I _SOC) in human submandibular glands Cells (HSG) and human parotid gland Cells (HSY) with I _CRAC (Ca^2+ release-activated Ca^2+ current) in RBL Cells. Internal Ca^2+ store-depletion with IP_3 or thapsigargin activated cation channels in all three cell types. 1 μM Gd^3+ blocked channel activity in all Cells. Washout of Gd^3+ induced partial recovery in HSY and HSG but not RBL Cells. 2-APB reversibly inhibited the channels in all Cells. I _ CRAC in RBL Cells displayed strong inward rectification with E _rev(Ca) = >+90 mV and E _rev (Na) = +60 mV. I _SOC in HSG Cells showed weaker rectification with E _rev(Ca) = +25 mV and E _rev(Na) = +10 mV. HSY Cells displayed a linear current with E _rev = +5 mV, which was similar in Ca^2+- or Na^+-containing medium. p Ca/ p Na was >500, 40, and 4.6 while p Cs / p Na was 0.1,1, and 1.3, for RBL, HSG, and HSY Cells, respectively. Evidence for anomalous mole fraction behavior of Ca^2+/Na^+ permeation was obtained with RBL and HSG Cells but not HSY Cells. Additionally, channel inactivation with Ca^2+ + Na^+ or Na^+ in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY Cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.
Theoharis C. Theoharides - One of the best experts on this subject based on the ideXlab platform.
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Differential effect of flavonoids on inhibition of secretion and accumulation of secretory granules in rat basophilic leukemia Cells.
International journal of immunopharmacology, 1999Co-Authors: Michael G. Alexandrakis, Richard Letourneau, William Boucher, Leena K. Singh, Panagiotis Theofilopoulos, Theoharis C. TheoharidesAbstract:Abstract Rat basophilic leukemia (RBL) Cells resemble mucosal mast Cells (MMC) and develop few secretory granules under normal culture conditions. RBL Cells have been used for the study of secretion and for the possible involvement of MMC in food allergies and irritable bowel syndrome (IBS). The flavonoid quercetin is one of very few molecules that inhibit RBL cell proliferation and constitutive histamine release; it also induces synthesis of rat mast cell protease (RMCP) II and accumulation of secretory granules. Even though quercetin is available as a food supplement over the counter, some early studies had indicated it may be carcinogenic. We, therefore, compared the effect of quercetin to that of other flavonoids with similar structure. Flavone, kaempferol, myricetin and morin were investigated for their action on RBL cell secretion of β -hexosaminidase stimulated by anti-DNP serum and DNP-BSA, as well as on secretory granule development. Quercetin, myricetin and kaempferol inhibited RBL cell secretion significantly only at 10 −4 M. Flavone inhibited secretion at 10 −4 , 10 −5 and 10 −6 M; it also maximally induced secretory granule accumulation as evidenced by light and electron microscopy. In contrast, morin which differs structurally only by one extra hydroxyl group had minimal effect. These results indicate that flavone is capable of inhibiting stimulated secretion and inducing secretory granule development at reasonable concentrations.
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Quercetin-induced expression of rat mast cell protease II and accumulation of secretory granules in rat basophilic leukemia Cells
Biochemical pharmacology, 1993Co-Authors: Jan Trnovsky, Richard Letourneau, Eman G. Haggag, William Boucher, Theoharis C. TheoharidesAbstract:Abstract Rat basophilic leukemia (RBL) Cells are considered to be similar to bone-marrow derived mast Cells and to mucosal mast Cells (MMC), the latter of which may be involved in inflammatory bowel diseases. RBL Cells are not able to accumulate histamine and secretory granules under regular growing conditions. Here we show that the flavonoid quercetin, which inhibits mast cell secretion of histamine, also inhibited RBL cell proliferation and constitutive histamine release while it induced synthesis of rat mast cell protease (RMCP) II and triggered processes leading to accumulation of secretory granules. Cell viability was also retained in the presence of quercetin, whereas untreated Cells did not survive past 6 days of growth. Quercetin did not affect the expression of mRNA for α-subunit of immunoglobulin E (IgE) receptor, but led to increased expression of mRNA for, and synthesis of RMCP II, which is a marker protein for MMC. Many of these granules showed metachromasia with toluidine blue after 3 days of growth, stained red with alcian blue counterstained with safranin after 8 days of growth, and contained electron dense material. Our results suggest that RBL Cells have the capacity to progress to a more mature state and may lend themselves to further analysis of a growth regulator(s) with action similar to that of quercetin.
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Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.
The Journal of clinical investigation, 1991Co-Authors: Charalabos Pothoulakis, Theoharis C. Theoharides, J T Lamont, R. Eglow, Ning Gao, J. B. Rubins, Burton F. DickeyAbstract:The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) Cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL Cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL Cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.
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Two-dimensional gel electrophoretic profile of rat basophilic leukemia (RBL) and mast Cells.
Life sciences, 1991Co-Authors: Demetrios Kouretas, J. Trnovsky, M. Holdridge, Theoharis C. TheoharidesAbstract:RBL Cells are not differentiated, but resemble mucosal mast Cells (MMC). Two-dimensional (2-D) gel electrophoresis following isoelectric focusing (IEF) was performed using purified rat peritoneal mast Cells and RBL Cells. Certain similarities were identified with silver staining between mast Cells and stationary phase (72 hr) RBL Cells. RBL Cells were also labelled with [35S]-cysteine in order to study the specific expression of proteins during logarithmic or stationary growth phases. Only stationary phase RBL Cells appeared to specifically express three proteins of 42, 55 and 93 kD and were still capable of secreting histamine in response to immunoglobulin E (IgE) and specific antigen. These results suggest that specific RBL cell proteins may be used as markers for further analysis of their maturation/differentiation.
Rossella Paolini - One of the best experts on this subject based on the ideXlab platform.
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activation of syk tyrosine kinase is required for c cbl mediated ubiquitination of fceri and syk in RBL Cells
Journal of Biological Chemistry, 2002Co-Authors: Rossella Paolini, Rosa Molfetta, Laurie O. Beitz, Juan Zhang, Andrew M. Scharenberg, Mario Piccoli, Luigi Frati, Reuben Siraganian, Angela SantoniAbstract:Engagement of the high affinity receptor for IgE (FceRI) on mast Cells and basophils results in FceRI β and γ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FceRI engagement on RBL-2H3 Cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FceRI β and γ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL Cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FceRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FceRI and activated Syk to the proteasome for degradation.
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Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of FcεRI and Syk in RBL Cells
The Journal of biological chemistry, 2002Co-Authors: Rossella Paolini, Rosa Molfetta, Laurie O. Beitz, Juan Zhang, Andrew M. Scharenberg, Mario Piccoli, Luigi Frati, Reuben Siraganian, Angela SantoniAbstract:Abstract Engagement of the high affinity receptor for IgE (FceRI) on mast Cells and basophils results in FceRI β and γ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FceRI engagement on RBL-2H3 Cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FceRI β and γ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL Cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FceRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FceRI and activated Syk to the proteasome for degradation.
