Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

• Klíčová slova
• FRAP, FcεRI signaling, STIM1-ORAI1 coupling, alkanol, flow-FRET, heat shock response, membrane fluidizer, store-operated calcium entry,
• MeSH
• cholesterol MeSH
• cytokiny MeSH
• heptanol MeSH
• mastocyty * MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• cytokiny MeSH
• heptanol MeSH

Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)-mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E2 and stem cell factor. Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.

• Klíčová slova
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• antigeny metabolismus   MeSH
• degranulace buněk MeSH
• kyselina ursolová MeSH
• lipidy farmakologie   MeSH
• mastocyty metabolismus   MeSH
• myši MeSH
• receptory IgE * metabolismus   MeSH
• triterpeny * farmakologie   metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• lipidy MeSH
• receptory IgE * MeSH
• triterpeny * MeSH
• vápník MeSH

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.

• MeSH
• benzothiazoly MeSH
• chinoliny MeSH
• diaminy MeSH
• fluorescenční barviva analýza   MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• organické látky analýza   MeSH
• polymerázová řetězová reakce metody   MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• benzothiazoly MeSH
• chinoliny MeSH
• diaminy MeSH
• fluorescenční barviva MeSH
• kvartérní amoniové sloučeniny MeSH
• organické látky MeSH
• SYBR Green I MeSH Prohlížeč

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.

• MeSH
• aktivace enzymů MeSH
• antigeny metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• časové faktory MeSH
• cholesterol metabolismus   MeSH
• detergenty farmakologie   MeSH
• DNA metabolismus   MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• fragmentace DNA MeSH
• imunoblotting MeSH
• konfokální mikroskopie MeSH
• konformace proteinů MeSH
• krysa rodu Rattus MeSH
• kyselina myristová metabolismus   MeSH
• kyselina palmitová metabolismus   MeSH
• luminescentní proteiny metabolismus   MeSH
• membránové mikrodomény metabolismus   MeSH
• metabolismus lipidů MeSH
• myši MeSH
• oktoxynol farmakologie   MeSH
• precipitinové testy MeSH
• receptory IgE metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• sfingolipidy metabolismus   MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu chemie   metabolismus   fyziologie   MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• tyrosin metabolismus   MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zelené fluorescenční proteiny MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• cholesterol MeSH
• detergenty MeSH
• DNA MeSH
• fosfotyrosin MeSH
• kyselina myristová MeSH
• kyselina palmitová MeSH
• luminescentní proteiny MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• oktoxynol MeSH
• receptory IgE MeSH
• rekombinantní proteiny MeSH
• sfingolipidy MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosin MeSH
• vápník MeSH
• zelené fluorescenční proteiny MeSH

The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.

• MeSH
• antigeny Thy-1 imunologie   MeSH
• buněčná membrána imunologie   MeSH
• cholesterol fyziologie   MeSH
• detergenty MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fluorescenční protilátková technika MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mastocyty imunologie   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• precipitinové testy MeSH
• sekvence nukleotidů MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny Thy-1 MeSH
• cholesterol MeSH
• detergenty MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• skupina kinas odvozených od src-genu MeSH

Rat peritoneal and pleural mast cells and rat basophilic leukemia cells, RBL-2H3, have been previously shown to be activated by Thy-1-specific monoclonal antibodies (mAb). In the present study we investigated the mechanism of Thy-1-mediated activation and compared it with activation induced by cross-linking of the high-affinity IgE receptor. Binding of an IgG Thy-1 x 1-specific mAb, MRCOX7 (OX7), to RBL-2H3 cells and mast cells, and activation of RBL-2H3 by the OX7 were abrogated by pretreatment of the cells with phosphatidyl inositol-specific phospholipase C (PI-PLC). The F(ab')2 fragment of OX7, in contrast to the Fab' fragment, induced cell activation as well as intact OX7 mAb. Cells sensitized with IgE exhibited an increased responsiveness to anti-Thy-1 antibodies suggesting formation of functional complexes of IgE receptor/IgE/Thy-1/anti-Thy-1. Pretreatment of RBL-2H3 cells with cholera toxin potentiated activation induced by IgE+antigen (Ag) and IgE+OX7, but had no effect on activation induced by OX7 antibody alone. Similarly, dexamethasone had no effect on OX7-induced activation but inhibited IgE+Ag- and IgE+OX7-induced activation. Analysis of phosphotyrosine-containing proteins in RBL-2H3 cell lysates revealed that IgE+Ag and IgE+OX7 induced a marked increase in tyrosine phosphorylation of several proteins that were not tyrosine phosphorylated in cells exposed to OX7 mAb alone. Similar results were obtained when RBL-2H3-derived cells, expressing transfected mouse Thy-1.2, were activated with Thy-1.2-specific IgM antibody. The combined data suggest that Thy-1-specific antibodies activate cells by a mechanism that is different from activation induced by cross-linking of high-affinity IgE receptor.

• MeSH
• antigeny povrchové imunologie   MeSH
• antigeny Thy-1 MeSH
• cholerový toxin imunologie   MeSH
• dexamethason imunologie   MeSH
• fosfodiesterasy imunologie   MeSH
• fosfolipasa C fosfoinositidové signalizace MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• mastocyty imunologie   MeSH
• membránové glykoproteiny imunologie   MeSH
• monoklonální protilátky imunologie   MeSH
• reagencia zkříženě vázaná MeSH
• receptory Fc imunologie   MeSH
• receptory IgE imunologie   MeSH
• tyrosin imunologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• antigeny Thy-1 MeSH
• cholerový toxin MeSH
• dexamethason MeSH
• fosfodiesterasy MeSH
• fosfolipasa C fosfoinositidové signalizace MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• membránové glykoproteiny MeSH
• monoklonální protilátky MeSH
• reagencia zkříženě vázaná MeSH
• receptory Fc MeSH
• receptory IgE MeSH
• tyrosin MeSH

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cells (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with src-related protein-tyrosine kinase p53/p56lyn and other protein-tyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large ( > 10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.

• MeSH
• akutní bazofilní leukemie MeSH
• antigeny povrchové izolace a purifikace   metabolismus   MeSH
• antigeny Thy-1 MeSH
• C-terminální Src kinasa MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfodiesterasy MeSH
• gelová chromatografie MeSH
• imunoblotting MeSH
• krysa rodu Rattus MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• makromolekulární látky MeSH
• membránové glykoproteiny izolace a purifikace   metabolismus   MeSH
• molekulová hmotnost MeSH
• monoklonální protilátky MeSH
• nádorové buňky kultivované MeSH
• protoonkogenní proteiny pp60(c-src) izolace a purifikace   metabolismus   MeSH
• receptory IgE metabolismus   MeSH
• skupina kinas odvozených od src-genu * MeSH
• tyrosinkinasy izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• antigeny Thy-1 MeSH
• C-terminální Src kinasa MeSH
• fosfodiesterasy MeSH
• lyasa fosfatidylinositoldiacylglycerolu MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• makromolekulární látky MeSH
• membránové glykoproteiny MeSH
• monoklonální protilátky MeSH
• protoonkogenní proteiny pp60(c-src) MeSH
• receptory IgE MeSH
• skupina kinas odvozených od src-genu * MeSH
• tyrosinkinasy MeSH