1,488 research outputs found
High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry.
Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. (c) 2013 International Society for Advancement of Cytometry
ErbB receptorok szerepe epitél eredetű tumorokban: receptor tirozinkinázok, mint a tumor ellenes terápiák lehetséges target molekulái. = Role of ErbB receptors in epithelial tumors: receptor tyrosine kinases as targets for anticancer therapy.
Az erbB2-erbB2 Ă©s erbB2-erbB1 molekulák homo- Ă©s heteroasszociáciĂłja - akárcsak más erbB hetero-oligomerekĂ© - fontos szerepet játszhatnak a jelátvitelben Ăgy az immunoterápiában is. KĂsĂ©rleteket vĂ©geztĂĽnk annak felderĂtĂ©sĂ©re, hogyan modulálja ezt a folyamatot más kölcsönhatĂł molekulák, pl. integrinek, CD44 (hialuronsav receptor) sejtfelszĂni jelenlĂ©te. Az ErbB2 ellenes immunterápia (trastuzumab kezelĂ©s) során kialakulĂł trastuzumab rezisztencia lehetsĂ©ges okait is vizsgáltuk. FRET eredmĂ©nyeink alapján elkĂ©szĂtettĂĽk ErbB2 dimer molekuláris modelljĂ©t. A trastuzumab rezisztens JIMT-1 ill. MKN-7 valamint a szenzitĂv SKBR-3 Ă©s N87 tumor sejtvonalakon vĂ©gzett összehasonlĂtĂł kĂsĂ©rleteink alapján megállapĂtottuk, hogy a trastuzumab kezelĂ©s korai elkezdĂ©sĂ©vel mĂ©g a rezisztens sejtek esetĂ©n is rĂ©szleges válasz Ă©rhetĹ‘ el. A xenograft kĂsĂ©rletek rámutattak arra, hogy az antitest által közvetĂtett citotoxicitás (ADCC) a trastuzumab kezelĂ©s kedvezĹ‘ hatásának elsĹ‘dleges oka. A geldanamycin ill. 17AAG sejtosztĂłdást gátlĂł hatása hatĂ©konyabbnak bizonyult trastuzumab rezisztens sejtekben. Ugyanakkor a CD44 leszabályozása, valamint a hialuronsav szintĂ©zis gátlása növelte a trastuzumab kezelĂ©s hatĂ©konyságát. Mindezek az eredmĂ©nyek arra utalnak, hogy cĂ©lzott adjuváns kezelĂ©s nagymĂ©rtĂ©kben fokozhatják a trastuzumab terápia hatĂ©konyságát. | Homo- and heteroassociations of erbB2-erbB2 and erbB2-erbB1 molecules ? similarly to that of other ErbB hetero-oligomers ? play an important role in signal transduction and immunotherapy. Our experiments aimed to reveal whether the cell surface presence of interacting molecules such as integrin and CD44 (receptor for hyaluronic acid) molecules can modulate these processes. We have also studied the possible causes of trastuzumab resistance developing during the ErbB2 targeted immunotherapy. We have prepared a useful molecular model of ErbB2 dimer on he basis or our FRET results. By comparing the behavior of trastuzumab resistant (JIMT-1 and MKN-7) and sensitive (SKBR-3 and N87) tumor cell lines we found that early trastuzumab treatment can be partially beneficial even in resistant cell lines. It was also revealed that the primary reason of the efficacy of trastuzumab treatment in xenograft models is the antibody dependent cell citotoxicity (ADCC). The treatment using geldanamycin or its derivative 17AAG was more efficient in trastuzumab resistant cells than in sensitive cells. Downregulation of CD44 and inhibition of synthesis of hyaluronic acid increased the efficiency of the trastuzumab treatment. These results suggest that targeted adjuvant therapy can significantly enhance the efficacy of the trastuzumab therapy
Az MHC I. sejtfelszĂni szervezĹ‘dĂ©se = Cell surface organization of MHC I.
A pályázat keretĂ©ben tanulmányoztuk a T limfociták sejtfelszĂni fehĂ©rjĂ©inek eloszlását. MegállapĂtottuk, hogy a Kv1.3 K+ csatornák Ă©s a T sejt receptor integráns rĂ©szĂ©t kĂ©pzĹ‘ CD3 molekulák átfedĹ‘ membrán domĂ©nekben helyezkednek el. Konfokális mikroszkĂłpos eredmĂ©nyeinket áramlási citometriás energia transzfer mĂ©rĂ©sekkel is megerĹ‘sĂtettĂĽk. FelismertĂĽk, hogy a CTL-target sejt kölcsönhatás során a Kv1.3 K+ csatornák az immunolĂłgiai szinapszisban feldĂşsulnak. Megalkottuk az IL-2/IL-15R rendszer dinamikus heterotetramer modelljĂ©t: a citokin kötĹ‘dĂ©se az alegysĂ©gek konformáciĂłjának/közelsĂ©gĂ©nek megváltoztatásával kialakĂtja a megfelelĹ‘ nagy affinitásĂş αβγc heterotrimert. Fluoreszcenciás festĂ©kek Ă©s arany nanorĂ©szecskĂ©k közti energia transzferen alapulĂł távolságmĂ©rĹ‘ mĂłdszert dolgoztunk ki. Energia transzfer mĂ©rĂ©sek hatĂ©kony vĂ©gzĂ©sĂ©re számĂtĂłgĂ©pes programokat hoztunk lĂ©tre, melyek működĂ©sĂ©t számos rendszeren teszteltĂĽk. | In the frame of the project we studied the distribution of cell surface proteins of T lymphocytes. We proved that the Kv1.3 K+ channels are located in the same membrane domains as the CD3 molecules which are part of the T cell receptor. Confocal microscopic data were in good agreement with those of the flow cytometric energy transfer measurements. We showed that the Kv1.3 K+ channels are expressed at higher concentrations in the immunological synapse during CTL-target cell interaction. We described the dynamic heterotetramer model of the IL-2/IL-15R system: the binding of the appropriate cytokine results in the formation of high-affinity αβγc heterotrimeric receptors. We introduced a novel energy transfer method for measuring distances on the cell surfaces using fluorescent dyes and nanoparticles. We elaborated computer programs for determining energy transfer efficiencies and tested them on several biological systems
Ioncsatornák szerepe az immunrendszer sejtjeinek differenciálódásában és proliferációjában = The role of ion channels in differentiation and proliferation of immune cells.
ElektrofiziolĂłgiai Ă©s biokĂ©miai mĂłdszerek kombinálásával számos Ăşj, potenciálisan immunszuppresszĂv hatásĂş Kv1.3 ioncsatorna blokkolĂł toxint fedeztĂĽnk fel, melyeket a az Anurotonus phaiodactylus, a Centruroides elegans Thorell, a Centruroides suffusus suffusus Ă©s a Tityus stigmurus skorpiĂłk mĂ©rgĂ©bĹ‘l izoláltunk. Kimutattuk, hogy a Kv1.3 ioncsatorna inaktiváciĂł lassulásában döntĹ‘ szerepet játszik a 399-es pozĂciĂłban találhatĂł hisztidin protonálĂłdása, mely elektrosztatikus kölcsönhatáson keresztĂĽl fokozza az inaktiváciĂł sebessĂ©gĂ©t meghatározĂł K+ kötĹ‘hely betöltöttsĂ©gĂ©t. ValĂłszĂnűsĂtettĂĽk, hogy a Shaker K+ csatorna esetĂ©ben az aktiváciĂłs Ă©s inaktiváciĂłs kapu közötti csatolás alapvetĹ‘ tĂ©nyezĹ‘ a sejtmembránon átáramlĂł ionfluxus szabályozásában. KĂsĂ©rletes eredmĂ©nyeink szerint a Kv1.3 ioncsatornák eloszlása a plazmamembránban nem vĂ©letlenszerű, karakterisztikus mintázatot mutat. Citotoxikus Ă©s target sejt konjugátumok vizsgálatakor azt tapasztaltuk, hogy a Kv1.3 csatornák a kĂ©t sejt által kĂ©pzett immunolĂłgiai szinapszisba (IS) rendezĹ‘dnek át, a csatornák polarizált expressziĂłt mutatnak. A sejtfelszĂni molekulák eloszlásának Ă©s távolságának kvantitatĂv jellemzĂ©sĂ©re Ăşj biofizikai Ă©s számĂtĂłgĂ©pes eljárásokat dolgoztunk ki. FarmakolĂłgiai Ă©s biofizikai mĂłdszerekkel sikerĂĽlt alátámasztanunk, hogy az Ă©retlen dendritikus sejtek NaV1.7 nátrium, mĂg az Ă©rettek Kv1.3 kálium ioncsatornát fejeznek ki. | Combination of electrophysiological and biochemical approaches resulted in the identification and characterization of several new Kv1.3 blocker peptide toxins with potential immunesuppressive effect from the venoms of Anurotonus phaiodactylus, Centruroides elegans Thorell, Centruroides suffusus suffusus and Tityus stigmurus scorpions. It has been shown that protonation of His399 plays a dominant role in the regulation of the kinetics of slow inactivation of Kv1.3, suggesting a model in which the protonated histidines influence the occupancy of the K+ binding site determining the rate of inactivation. We suggest that coupling between the activation and inactivation gates of Shaker K+ channels is a fundamental factor in the regulation of the transmembrane ion flux. We showed that the surface distribution of Kv1.3 ion channels in the plasma membrane of T cells is nonrandom.. Kv1.3 channels were recruited into the immunological synapse formed between cytotoxic and target cells thereby showing a polarized channel expression. For the numerical characterization of membrane protein distributions and distances we developed new biophysical and computer methods. A combination of pharmacological, biophysical and molecular biological methods showed NaV1.7 sodium and Kv1.3 ion channel expression in the immature and mature dendritic cells, respectively
TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs
TCR gene-modified T cells for adoptive therapy simultaneously express the transgenic (tg) TCR and the endogenous TCR which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer (FRET) we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization)
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