11 |
L'activation continuelle de SHP-1 dans les radeaux lipidiques des neutrophiles humains suite à une stimulation au GM-CSF contribue à l'altération de leurs fonctions effectrices observées avec le vieillissementFortin, Carl January 2006 (has links)
Il a été montré que les fonctions et la prolongation de la survie cellulaire des neutrophiles humains par les médiateurs de l'inflammation tendent à diminuer avec le vieillissement. Les protéines tyrosines phosphatase (PTPs), comme SHP-1, sont un des mécanismes qui permettent de moduler à la baisse et de terminer ces fonctions inflammatoires qui sont modulées par l'action des cytokines. Nous avons étudié le rôle des PTPs dans l'altération due au vieillissement des fonctions des neutrophiles humains. L'utilisation d'un inhibiteur pharmacologique des PTPs a suggéré une dérégulation de l'activité phosphatasique avec le vieillissement. Cette dérégulation était confirmée aussi dans le cas de l'apoptose mesurée après 18 heures d'incubation. L'activité phosphatasique de SHP-1 purifiée par immunoprécipitation de neutrophiles de sujets jeunes ou âgés stimulés par le GM-CSF est altérée de façon significative après une minute de stimulation chez les sujets âgés. Dans les sujets jeunes SHP-1 est déplacée des radeaux lipidiques après 1 minute de stimulation par le GM-CSF alors que chez les sujets âgés, SHP-1 est présente à tous les temps de stimulation utilisés. Des immunoblots faits avec des anticorps anti-phosphotyrosine et anti-phosphosérine ont montré une augmentation de la phosphorylation en sérine dans les neutrophiles des sujets jeunes après une stimulation au GM-CSF alors que ce n'était pas le cas chez les sujets âgés. Nous avons aussi trouvé des altérations dans l'activation et le recrutement aux radeaux lipidiques de la Src kinase Lyn chez les neutrophiles des sujets âgés. De plus, nous avons démontré que SHP-1 est continuellement recrutée à Lyn chez les sujets âgés alors que cette interaction, qui est observée dans des cellules non stimulées chez les sujets jeunes, est défaite par la stimulation au GM-CSF. En conclusion, les altérations observées dans la modulation de l'activité de SHP-1 par le GM-CSF dans les radeaux lipidiques sont un des facteurs qui contribuent à la diminution des effets du GM-CSF sur les neutrophiles humains avec le vieillissement.
|
12 |
Analýza vlivu inhibitorů Src kináz na adhezní signalizaci v lidských hematopoietických buňkách / Analysis of the effects of Src kinase inhibitors on adhesion signaling in human hematopoietic cellsObr, Adam January 2012 (has links)
Adhesion of hematopoietic cells to the bone marrow microenvironment is important for their proper development. It is proven that Src-family kinases (SFK) regulate cell adhesion, although their exact role in the regulation of adhesion signaling remains unclear. Since adhesion processes are investigated mainly in adherent cell types, far less is known about hematopoietic cells. However, defects in the cell adhesion accompany a number of hematological diseases, like chronic myeloid leukaemia (CML). SFK overexpression is one of the proposed mechanisms of resistance to the first-line CML treatment, imatinib mesylate. Second generation drugs (e. g. dasatinib) inhibit SFK together with Bcr-Abl. Additionally, SFK-specific inhibitors (PP2, Src inhibitor-1) are also available, but there are no studies about effects of these drugs on cellular adhesivity of hematopoietic precursors. To explore the dynamics of hematopoietic cell adhesion to the extracellular matrix, we introduced a new approach using the RTCA xCELLigence DP system along with the well-established method of fluorimetric detection of adherent cell fraction. Our general observation is that various drugs (dasatinib, imatinib, PP2, Src inhibitor-1) induce pro-adhesive effects in several leukemic cell lines. Direct comparison of the kinetics of...
|
13 |
Biologický význam fosforylace tyrosinu 90 v SH3 doméně kinázy Src / Biological relevance of the tyrosine 90 phosphorylation in SH3 domain Src kinaseKoudelková, Lenka January 2013 (has links)
Kinase Src plays an essential role in signal transduction from activated surface receptors. Src is involved in signal pathways that participate in the control of cell proliferation, differentiation or motility. That is why Src activation undergoes strict and complex regulation. Inactive conformation is maintained by intramolecular inhibitory interactions. SH3 domain associates with a polyprolin helix in CD linker whereas SH2 domain binds phosphorylated C-terminal tyrosine 527. Both regulatory domains maintain contacts with the lobes of a kinase domain thereby stabilizing an inactive conformation of the catalytic domain. Transition to an active state is accompanied by a disruption of these inhibitory interactions. Conformation changes are substantially influenced by the phosphorylation status of key tyrosines 416 and 527. Phosphoproteomic analysis revealed new Src tyrosine residue, which can be phosphorylated in vivo. It has been found, that tyrosine works as an additional regulator of Src activity. This is Tyr 90, which forms one of the hydrophobic pockets in the binding surface of Src SH3 domain. Based on the expression of phosphomimic mutant Src 90E in S. pombe or in SYF lineage, it has been observed, that Tyr 90 phosphorylation elevates Src kinase activity. The reason is that the phosphate...
|
14 |
Fluoreszenz-mikroskopische Untersuchung der Inaktivierung der Tyrosinkinase SRC im Integrin alphaIIb-beta3 -Signalweg / Studies on the inactivation process of the tyrosine kinase Src in the integrin alphaIIb-beta3 signaling pathway by fluorescence microscopyVielreicher, Martin Christian January 2008 (has links) (PDF)
Essentiell für die Blutstillung (Haemostase) ist die Thrombozyten- oder Blutplaettchen-Adhaesion und die Thrombus-Bildung. Beide Vorgaenge werden hauptsaechlich durch den Thrombozyten-Rezeptor Integrin alphaIIb-beta3 vermittelt. Nach Bindung des Liganden Fibrinogen aendert sich die Rezeptor-Konformation, Integrine assoziieren und ein intrazellulaeres Signalnetzwerk wird aktiviert, welches die Organisation des Aktin-Zytoskeletts steuert. Diese Zytoskelett-Reorganisationen sind Grundlage für zellulaere Adhaesions- und Aggregations-Prozesse. Die Signalvermittlung vom Integrin zum Zytoskelett wird durch die Protein-Tyrosinkinase Src eingeleitet, deren Aktivitaetszustand den Signalweg reguliert. Bei der Src-Aktivierung wird Tyrosin 418 durch Autokatalyse phosphoryliert. Die Kinase muss jedoch wieder inaktiviert werden. Dies übernimmt in Plaettchen ausschliesslich die Tyrosinkinase Csk (C-terminale Src Kinase) durch Phosphorylierung von Tyrosin 529 im C-terminalen Ende des Proteins. Die Csk-vermittelte Inaktivierung von Src stellt den entscheidenden Kontrollschritt des alphaIIb-beta3-vermittelten Signalwegs dar. Obwohl bekannt ist, dass die Src-Aktivierung bei der Zelladhaesion an den Zellraendern der Lamellipodien geschieht und man den Mechanismus und die Kinetik der Src-Csk Interaktion genauer versteht, ist bislang immer noch unbekannt, wo und wie Src inaktiviert wird bzw. welche Rolle der Src-Inaktivierung genau zukommt. FRET (Fluoreszenz-Resonanz-Energie-Transfer) ist ein physikalischer Effekt, mit dem Interaktionen beliebiger fluoreszenzmarkierter Proteine mikroskopisch detektiert werden koennen. Diese Technik wurde genutzt, um die Src-Csk-Interaktion waehrend der alphaIIb-beta3-vermittelten Fibrinogen-Adhaesion in einer etablierten Thrombozyten-Modellzelllinie (A5-CHO) direkt visualisierbar zu machen. Es zeigten sich starke Src-Csk Interaktionen (FRET-Signale) an den Zellraendern aktiver Lamellipodien und zusaetzlich in Fokalkontakten, wo beide Proteine mit Vinculin, einem Fokalkontakte-Marker, co-lokalisierten. Die Proteininteraktionen folgten einem hochdynamischen Ablauf. Nach der Akkumulation der Src-Csk Komplexe an den Zellraendern wanderten sie in Abstaenden von 2-3 Minuten nach innen, fragmentierten und bildeten schliesslich stabile Fokal-Adhaesionen. FRET-Signale an den Zellraendern fanden sich vor allem in ruhenden Lamellipodien bzw., waehrend des Lamellipodien-Rückzugs, in wachsenden Lamellipodien traten die FRET-Signale dort dagegen nicht auf. In unabhaengigen biochemischen Tests im Zeitfenster der FRET-Beobachtungen wurde ein spezifischer Anstieg der Src-Tyr529-Phosphorylierung (Inaktivierung) und eine parallele Abnahme der Src-Tyr418-Phosphorylierung (Aktivierung) gemessen. Weiterführende Ergebnisse lieferten Versuche mit Src- und Csk-Mutanten. Die Co-Expression von Wildtyp-Src mit Kinase-inaktivem CskK222R hatte weder einen Effekt auf die Adhaesion und Ausbreitung der Zellen noch auf die Praesenz von FRET, es aenderte sich jedoch drastisch die zellulaere Verteilung der FRET-Signale sowie das Wachstum und die Form der Lamellipodien. Die Co-Expression von Wildtyp-Csk mit konstitutiv aktivem SrcY529F verursachte dagegen eine stark verringerte Adhaesionsfaehigkeit und Hemmung der Lamellipodien-Bildung. Die Fokal-Adhaesionspunkte in diesen Zellen waren sehr schwach und ueberdimensioniert und lagen ungeordnet verteilt in der Adhaesionsebene. Zusaetzlich verursachte SrcY529F eine starke Ueberaktivierung des Zytoskeletts und das fast vollstaendige Verschwinden der FRET-Signale. Die ermittelten Daten zeigen, dass die enge Kontrolle der Src-Aktivitaet durch Csk eine bedeutende Rolle für die funktionelle Zell-Adhaesion and -Ausbreitung spielt. Co-Immunpraezipitations-Resultate und Messungen der Menge an markiertem Protein in Zellen, in welchen FRET detektierbar war, untermauern zusaetzlich unsere These, zum ersten Mal die Src-Regulation durch Csk in lebenden Zellen direkt beobachtbar gemacht zu haben. Dieser neue FRET-Ansatz kann auch als Reporter-System für Prozesse der Src-Inaktivierung in anderen Signalwegen und Zellen angewendet werden. Das Messprinzip kann weiterhin auf das Studium der Inaktivierung weiterer Mitglieder der Familie der Src-Kinasen (in verschiedensten Signalwegen) erweitert werden. / Platelet adhesion and thrombus formation required for functional hemostasis depends on integrin receptor mediated “outside-in” signaling to the cytoskeleton. Integrin alphaIIb-beta3 is the major integrin on the platelet surface and acts as a specific receptor for the plasma protein fibrinogen. Fibrinogen binding causes clustering of integrins within the plasma membrane activating the protein tyrosine kinase Src (signal initiation) by phosphorylation of tyrosine 418. Src, however, is negatively regulated by another tyrosine kinase, Csk (C-terminal Src kinase), which phosphorylates tyrosine 529. Although, in adhering cells, it is believed that Src is getting activated at lamellipodia leading edges, neither the cellular location nor the dynamics and exact role of Src inactivation is known to date. Here, we studied Src inactivation during alphaIIb-beta3-dependent adhesion to fibrinogen in the established platelet model cell line A5-CHO. Using a live cell FRET (fluorescence resonance energy transfer) microscopy technique with CFP and YFP label molecules (cyan and yellow fluorescent protein), we were able to image highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. Every 2-3 minutes, signals detecting Src-Csk interactions (complexes) appeared at the cell periphery before they begin to move inward in the cell and reorganize while lamellipodia start to protrude (grow). FRET signals were also found in small accumulations at the fringe and also further to the centre of the adhesion plane (focal complexes and adhesions). Src and Csk co-localize with vinculin (a focal adhesion marker) within these regions. During the runtime of FRET observation a specific increase in Src-Tyr529 phosphorylation with a parallel decrease in Src-Tyr418 phosphorylation was observed supporting the idea that Src inactivation occurs within the cells. The role of Src-Csk interaction was studied in further detail using Src and Csk mutants. The data revealed that co-expression of inactive CskK222R did not alter the presence of FRET signals, but fundamentally changed its distribution within the cell. Furthermore it caused lamellipodia shape changes and a tendency of constant lamellipodia protrusion. Co-expression of constitutively active SrcY529F in turn caused a severe adhesion and spreading dysfunction. Adherent cells showed very weak, disorganized and oversized focal adhesions, a hyper-activated cytoskeleton (visible in fast-changing membrane blebs) and absence of FRET signals. Results from immunoprecipitation analyses and protein level determination within cells, in which FRET was detectable, further supported that we were able, for the first time, to directly visualize Src (and integrin) regulation by Csk control in live cells. The results show that Src control by Csk is ultimately required for lamellipodia and focal adhesion function and thus for cell anchorage and spreading. The novel FRET-approach reported here can be readily applied to other integrin and signaling pathways including the study of closely related Src family kinases (SFKs). Results may also contribute to a better understanding of the processes of tumor formation.
|
15 |
The role of interleukin-1 receptors in brain cell signallingNguyen, Loan January 2010 (has links)
IL-1α and IL-1β are two IL-1 agonists which signals at the same receptor complex composed of IL-1R1/IL-1RAcP. However, IL-1α and IL-1β exert differential actions. A recent CNS-specific IL-1 receptor accessory protein, called IL-1RAcPb, has been characterised but its actions are unknown. In T cell line, over expression of IL-1RAcPb negatively regulate IL-1 action (Smith et al, 2009), but over-expression of IL-1RAcPb in HEK cell line induces IL-1 signaling (Lu et al, 2008). The role of IL-1RAcPb has not been studied in primary cells. The aim of this project was to investigate the role of IL-1RAcPb in IL-1-induced actions in neurones and glia, and to determine IL-1α and IL-1β differential actions in these two cell types. The role of IL-1RAcPb in IL-1-induced protein expression and IL 1α and IL-1β differential effects were investigated by treating WT and IL 1RAcPb-/- neurones and glia with IL-1α or IL-1β in the presence or absence of IL-1RA for 24 h followed by assessment of IL-6 induction by ELISA. The mechanism of IL-1RAcPb actions were studied by examining the effects of IL-1α or IL-1β on p38, ERK1/2 and Src kinase activation in neurones and glia by Western blot analysis. SB203580 (p38 inhibitor), UO126 (ERK1/2 inhibitor), and PP2 (Src kinase inhibitor) were used to determine the contribution of p38, ERK1/2 and Src kinase activation to IL-1-induced IL-6 synthesis in neuronal cultures. In WT neurones, IL-1α and IL-1β were equipotent at inducing IL-6 synthesis and p38 activation, whilst both ligands failed to induce ERK1/2 or Src kinase activation. In IL-1RAcPb-/- neurones, IL-1α and IL-1β induced similar levels of IL-6, but IL-1β was more potent than IL-1α at inducing p38 activation. IL-1α-induced p38 activation was reduced in IL-1RAcPb-/- neurones compared to WT neurones. In contrast to WT neurones, ERK1/2 was activated in IL-1RAcPb-/- neurones in response to IL-1α, whilst Src kinase was not activated by IL-1α or IL 1β. IL-1-induced IL-6 synthesis was abolished by IL-1RA, SB203580, UO126 and PP2. Interestingly PP2, a specific Src kinase inhibitor also partially inhibited basal ERK1/2 activity. In WT glial cells, IL-1α was more potent than IL-1β at inducing IL-6 synthesis but both cytokines induced ERK1/2 activation with equal potency. In IL-1RAcPb-/- glia, IL-1α and IL-1β were equally potent at inducing IL-6 synthesis and ERK1/2 activation. However, IL-α-induced-IL-6 synthesis was reduced in IL 1RAcPb-/- glia compared to WT glia. In both WT and IL-1RAcPb-/- glia, IL-1α and IL-1β induced p38 activation but not Src kinase activation . In conclusion, this study showed that in neurones, IL-1RAcPb may contribute to IL-1α-induced p38 activation but negatively regulates IL-1-induced ERK1/2 activation, therefore IL-1RAcPb may have specific effects on different signalling pathways. The effect of IL-1RAcPb could also be cell specific, as IL 1RAcPb contributed to IL-1α-induced p38 signalling in neurones but IL-6 production in glia. The role of IL-1RAcPb remains largely unknown and more investigations are required to elucidate its role in IL-1 signalling in the brain.
|
16 |
Sequence Specificity of Src Homology-2 DomainsTan, Pauline H. 06 January 2012 (has links)
No description available.
|
17 |
Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina : Focus on α2-Adrenergic and Endothelin Receptor Signaling SystemsHarun-Or-Rashid, Mohammad January 2016 (has links)
Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury. The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2-ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells. The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury.
|
18 |
Differentiation and malignant transformation of epithelial cells:3D cell culture modelsCapra, J. (Janne) 06 March 2018 (has links)
Abstract
The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis. / Tiivistelmä
Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta.
|
19 |
Regulační úlohy proteinů PAG a CSK v FcɛRI signalizaci žírných buněk / Regulatory roles of PAG and CSK in FcɛRI signaling of mast cellsPotůčková, Lucie January 2017 (has links)
8 1 ABSTRACT (EN) This thesis is focused mainly on understanding mechanisms of regulatory roles of C-terminal Src kinase (CSK) and phosphoprotein associated with glycosphingolipid- enriched microdomains (PAG) in the high-affinity IgE receptor (FcɛRI)-mediated signaling of murine mast cells. FcɛRI activation is initiated by aggregation of the receptor by complexes of multivalent antigen with IgE, followed by activation and enhanced activities of protein tyrosine kinases, phosphatases, adaptor proteins and number of other signal transduction molecules. The signaling events result in mast cell degranulation and release of variety of proinflammatory mediators, responsible for initiation of allergy and other inflammatory diseases. Understanding the function of key regulatory molecules controlling FcεRI-mediated mast cell activation, degranulation, and cytokines production could have therapeutic impact. CSK is a major negative regulator of Src family tyrosine kinases (SFKs) that play a critical role in various immunoreceptor signaling events. However, its function in mast cell activation has not been completely understood. Because of its cytoplasmic localization, CSK was assumed to be brought to the vicinity of the plasma membrane- bound SFKs via binding to membrane-bound adaptors and PAG was a major candidate....
|
20 |
INVOLVEMENT OF SRC TYROSINE KINASE AND CALCIUM-HANDLING IN AIRWAY SMOOTH MUSCLE EXCITATION-CONTRACTION COUPLINGHumber, Brent T. 04 1900 (has links)
<p><strong>Introduction</strong></p> <p>Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA<sub>2</sub>b and RyRs play a role in ASM excitation-contraction coupling.</p> <p><strong>Aim</strong></p> <p>We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.</p> <p><strong>Methods</strong></p> <p>Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10<sup>-4 </sup>M), src kinase family inhibitors PP1 (10<sup>-5 </sup>M) and PP2 (10<sup>-5 </sup>M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.</p> <p><strong> </strong></p> <p><strong>Results</strong></p> <p>Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA<sub>2</sub>b, RyR1 and RyR2 were unchanged by the house dust mite treatment.</p> <p><strong>Conclusion</strong></p> <p>These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca<sup>2+</sup>-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.</p> / Master of Science (MSc)
|
Page generated in 0.0718 seconds