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Studies into the mechanism of action of murine interleukin-3Sorensen, Poul Henrik Bredahl January 1990 (has links)
The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hemopoietic cell line, B6SUtA. These cells were first used to identify the mIL-3 surface receptor as a monomeric
67 kDa protein with a pI of approximately 6.2. Further studies suggested the presence of an additional mIL-3 binding protein with an apparent molecular mass of 140 kDa. Then, in an attempt to gain some insights into the mechanism of action of mIL-3, molecules other than mIL-3 were tested to determine their effects on cell proliferation. Murine granulocyte -macrophage colony-stimulating factor (mGM-CSF) was found to be as potent as mIL-3 in stimulating B6SUtA cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, both stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA cells exposed for brief periods to mIL-3, mGM-CSF or TPA were analyzed for changes in phosphorylation patterns using metabolic [superscript]32p-labeling. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a
68 kDa cytosolic protein, while all three agents stimulated the serine-specific phosphorylation of a 67 kDa membrane protein. Furthermore, using antibodies to phosphotyrosine, it appeared that mIL-3 stimulated tyrosine phosphorylation of 67 kDa and 140 kDa membrane proteins, as well as of 40, 55 and 90 kDa cytosolic proteins. The 90 kDa protein was also tyrosine phosphorylated in response to mGM-CSF, suggesting that this phosphorylation results from a common step in mIL-3 and mGM-CSF-stimulated signaling pathways. These phosphotyrosine containing proteins were not detected in TPA-treated cells. Moreover, evidence from a variety of studies is presented that the 140 kDa but not the 67 kDa mIL-3 receptor becomes phosphorylated on tyrosine residues when B6SUtA cells bind mIL-3. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate
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Die Rolle von NF-kappaB und MEK in der Apoptosesuppression durch Raf / The role of NF-kappaB and MEK in suppression of apoptosis by activated RafTränkenschuh, Wolfgang January 2009 (has links) (PDF)
Aus genetischen und zellbiologischen Untersuchungen ist bekannt, dass die Proteinkinase Raf Apoptose unterdrücken kann, die Effektoren sind jedoch im Detail nicht klar. Am Modell der IL-3 abhängigen Zellinie 32D wurde die Rolle des Transkriptionsfaktors NF-kappaB in der Apoptosesuppression durch aktiviertes Raf untersucht. Unter Verwendung zweier aktiver Raf-Mutanten ergaben sich Hinweise auf eine proapoptotische Funktion, passend zu den NF-kappaB zugeschriebenen proaptotischen Zielgenen. Für den Raf-Effektor MEK ist eine antiapoptotische Funktion bekannt. Hier gelang es mit einer hyperaktiven Mutante (deltaStu-MEK-LIDEMANE) aus IL-3 abhängigen 32D Zellen einen Faktor-unabhängigen Zellpool (FID) zu generieren. Das von den bekannten Vorstellungen über Zytokin-abhängige Zellinien abweichende Verhalten, nach dem erst die Mutation von mehr als einem Onkogen zu einer Faktorunabhängigkeit führt, wurde genauer charakterisiert. Die FID-Zellen sind weiter von den Signalmolekülen MEK und PI3-Kinase abhängig und ein autokriner Mechanismus konnte ebenso wie die Expression von Signalmolekülen auf der Zelloberfläche ausgeschlossen werden. / The protein kinase Raf is able to suppress apoptosis, although its effectors are not known in detail. The IL-3-dependent cell line 32D served as a model for the suppression of apoptosis by activated Raf in order to elucidate the role of the transcription factor NF-kappaB. We used two different active Raf mutants in our experimental approach and found evidence of a proapoptotic role for NF-kappaB. This stresses the significance of the proapoptotic target genes of NF-kappaB. The Raf effector MEK has an established antiapoptotic role. Our hyperactive MEK-mutant (deltaStu-MEK-LIDEMANE) turned IL-3-dependent 32D cells into cytokine-independent cells contrary to the traditional model of cytokine dependence. The cytokine-independent cells still employed the MEK and PI3-Kinase pathways. An autocrine mechanism could be excluded as well as the presence of surface signalling molecules.
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Effects of interleukin-3 and c-kit ligand on the in vitro survival of human hematopoietic progenitor cells and stem cellsBrandt, John E. January 1993 (has links)
This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).
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Interleukin -3 receptor expression and function in now-hemopoietic cells / Eija Korpelainen.Korpelainen, Eija January 1995 (has links)
Errata inserted on back end papers. / Includes bibliographical references. / 99 leaves, [9] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Identifies a novel site of action for IL-3, and suggests that it can influence immune and inflammatory responses and hemopoiesis by acting not only on hemopoietic cells but also on vascular endothelium. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996?
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Regulation of myeloid progenitor cell proliferation: the effects of steel factor on a human factor-dependent cell lineHendrie, Paul Curtis January 1993 (has links)
This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).
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Suppression der Apoptose durch C-Raf erfordert MEK1 und Phosphatidylinositol 3-Kinase abhängige Signale / Apoptosis Suppression by C-Raf Requires MEK1- and Phosphatidylinositol 3-Kinase-Dependent Signals.Gise, Alexander von January 2007 (has links) (PDF)
Unterhalb des Interleukin 3 (Il-3) Rezeptors sind zwei Ras-abhängige Signalwege beschrieben, die entweder zur Aktivierung von C-Raf oder von PI3-Kinase (PI3K)/Proteinkinase B (PKB, AKT) führen und Wachstum und Überleben vermitteln. Frühere Untersuchungen des Mechanismus, über den C-Raf Apoptose unterdrückt, zeigten die Notwendigkeit einer Anwesenheit der zytoplasmatischen Kinase an den Mitochondrien. Diese Translokation konnte entweder durch Überexpression des antiapoptotischen Proteins Bcl-2 oder aber durch Fusion der Kinase mit dem mitochondriellen Protein Mas p70 erreicht werden. Aktiviertes mitochondriell gebundenes C-Raf ist nicht in der Lage ERK1 und ERK2 zu aktivieren, vermag aber durch Inaktivierung des proapoptotischen Bcl-2 Familienmitgliedes BAD Apoptose zu unterdrücken. Ungeachtet dieser Ergebnisse deuteten andere genetische und biochemische Untersuchungen auch auf eine Bedeutung der Raf Effektoren MEK und ERK in der Unterdrückung des programmierten Zelltodes hin. Im Rahmen dieser Arbeit wurde daher die Bedeutung von MEK und MEK-abhängigen Signalwegen für das zelluläres Überleben untersucht. Wir nutzten für diese Untersuchungen überwiegend die Il-3 abhängige Zelllinie 23D. MEK war essentiell für das zelluläre Überleben und Wachstum nach Stimulation durch Il-3. Eine konstitutiv aktive MEK1 Mutante verzögerte signifikant das Einsetzen der Apoptose nach Entzug des Wachstumsfaktors, während eine dominant negative Mutante den Zelltod akzelerierte. In der Fibroblastenzelllinie NIH 3T3 unterdrückte eine konstitutiv aktive Mutante von ERK2, ähnlich effektiv wie onkogenes MEK, durch Doxorubicin induzierten Zelltod. Diese Beobachtung lässt auf einen, das Überleben der Zelle vermittelnden, Signalweg von MEK schließen, der zur Aktivierung von ERK führt. Der protektive Effekt von aktiviertem MEK in 32D Zellen wurde durch MEK- und PI3K-abhängige Mechanismen vermittelt. Die dabei beobachtete Aktivierung von PI3K führt zur Phosphorylierung und Aktivierung von AKT. Die Abhängigkeit von MEK und PI3K Signalwegen konnte auch für den Schutz von 32D Zellen vor Apoptose durch onkogenes C-Raf gezeigt werden. Diese Befunde ließen sich ebenso in der Il-3 abhängigen pro-B Zelllinie BaF3 verifizieren, was darauf schließen lässt, dass die Rekrutierung von MEK/ERK im antiapoptotischen Signalweg von aktiviertem Raf ein allgemeingültiger Mechanismus ist. Dass in diesem antiapoptotischen Signalweg von C-Raf auch der PI3K Effektor AKT notwendig ist zeigten weitere Untersuchungen, in denen eine dominant negative Mutante von AKT den protektiven Effekt von aktiviertem C-Raf inhibierte, während eine konstitutiv aktive Form von AKT einen synergistischen Effekt mit C-Raf in der Unterdrückung der Apoptose hatte. Diese Daten zeigen einen, zelluläres Überleben vermittelnden Effekt von Raf, der durch MEK und AKT vermittelt wird. / Two Ras effector pathways leading to the activation of C-Raf and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by C-Raf demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting C-Raf to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active C-Raf is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the C-Raf effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic C-Raf. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.
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Mass spectrometric studies on glycoprotein oligosaccharides : a modified procedure for the liquid secondary ion mass spectrometric analysis of glycoprotein oligosaccharides. Studies on the nature of glycosylation on baculovirus-expressed mouse interleukin-3Hogeland, Kenneth Eden 23 April 1993 (has links)
Graduation date: 1993
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Transcriptional regulation of the GM-CSF gene in T lymphocytes / Cameron Stuart Osborne.Osborne, Cameron Stuart January 1996 (has links)
Addendum pasted on front end papers. / Includes bibliographies. / 109, [99] leaves, [5] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the investigation as to whether the mouse granulocyte-macrophage colony-stimulating factor and interleukin-3 genes are regulated in a similar manner as those of the human, focussing on regulation through an enhancer. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1996
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The molecular basis of IL-3, Il-5 and GM-CSF receptor activation / Frank Charles Stomski.Stomski, Frank Charles January 1997 (has links)
Copies of author's previous publications inserted. / Bibliography: leaves 153-182. / xv, 183, [10] leaves, [27] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / From experimental data presented, combined with molecular modelling, proposes a hexameric model of active IL-3, IL-5 and GM-CSF receptor complexes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998?
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Microfluidic blood fractionation and lysis towards analysis of cytokine levels in red blood cellsBarrett, Laura January 2019 (has links)
In the process of blood analysis, for biomarker detection, blood plasma or serumis analyzed, while Red blood cells (RBCs) are usually considered waste. A recentstudy has found that cytokine concentrations in RBC lysate are on average 12-fold higher than in plasma. Microfluidic devices for the extraction of plasma fromwhole blood and containers of dried blood spots have already been developed.They o↵er a small tool size, low required sample volume, and low productioncost, which makes them suitable for point-of-care applications.In this thesis, an approach of isolating and lysing RBCs in a commercial bloodfilter using a microfluidic device was investigated. The design is based on a previouslydeveloped microfluidic device for plasma extraction. It was used for plasmaextraction, washing, and RBC lysing with RBC lysis bu↵er. The device’s functionalitywas examined, and the output lysate analyzed through measurements ofhemoglobin concentration, optical microscopy and an Elisa test of the cytokineIL-3.The results show that 48% of the devices were subject to one of two problems.One was slow filling of the capillary channel, and the other was the lysate beingvisibly clear. These problems were attributed to blood cells obstructing the filtersand increasing flow resistance. The device lysate viewed through an optical microscopewas shown to contain a significant amount of blood cells in some cases,suggesting that cells were passing through the filters. The mean lysis efficiency ofthe devices was determined to be 20%, which with the Elisa test results, suggeststhat there is low rates of mixing between the RBCs and the lysate within the filtermatrix. In conclusion, the tested method of isolating and lysing RBCs needs tobe improved in terms of reliability and efficiency. It was shown to work in someof the cases, and so shows promise for future development. / Röda blodkroppar (RB) ses vanligtvis som avfall vid blodmatningar av biomarköreri plasma eller serum. Men en ny studie har funnit att koncentrationerna av cytokiner i lyserade RB är i snitt 12 gånger högre än i plasma. Mikrofluidiska apparater som extraherar plasma från helblod har redan utvecklats. Deras fördelarär att de är små, använder små provvolymer och är billiga att producera. Detta gör dem lampliga for patientnara analyser, eller s.k. point-of-care-användning.I det här arbetet prövas en metod för att isolera och lysera RB i ett kommersiellt blodfilter, med hjälp av en mikrofluidisk apparat. Designen av apparaten är baserad på en mikrofluidisk apparat som utvecklats för plasmaextrahering. Den används för plasmaextraktion, tvätt och lysering med lyseringsbu↵ert. Appa-ratens funktionalitet undersöktes, och RB-lysatet analyserades med mätningar avhemoglobinvärden, ljusmikroskopi och en Elisa-mätning av cytokinet IL-3.Resultaten visar att 48 % av apparaterna hade något av två problem. Det ena problemet var att den kapillära kanalen fylldes långsamt, och det andra var att lysatet var till synes ofärgat. Dessa problem tillskrivs att blodceller blockerar filtren och ökar flödesmotståndet. I ljusmikroskop visade sig lysatet från apparaten i vissa fall innehålla stora mängder blodceller. Detta tyder på att celler passerat igenom filtret. Medelvärdet av lyseringse↵ektiviteten i apparaterna visades vara 20 %. Tillsammans med Elisa-resultaten tyder detta på att vätskan i filtret blandas i otillräcklig utsträckning. Sammanfattningsvis behöver metoden för att isolera och lysera RB förbättras gällande tillförlitlighet och e↵ektivitet. Den visadesig fungera i vissa fall och är därför lovande för framtida utveckling.
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