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001-es BibID:	BIBFORM004705
035-os BibID:	(scopus)18544371851 (wos)000173421300040
Első szerző:	Dornan, Saffron
Cím:	Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction / Dornan, S., Sebestyen, Z., Gamble, J., Nagy, P., Bodnar, A., Alldridge, L., Doe, S., Holmes, N., Goff, L. K., Beverley, P., Szollosi, J., Alexander, D. R.
Dátum:	2002
Megjegyzések:	An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk analysis Antigens,CD4 Antigens,CD45 Antigens,CD8 Energy Transfer Fluorescence Human immunology Lymphocyte Specific Protein Tyrosine Kinase p56(lck) metabolism Phosphorylation Receptors,Antigen,T-Cell Signal Transduction Support,Non-U.S.Gov't
Megjelenés:	The Journal of Biological Chemistry. - 277 : 3 (2002), p. 1912-1918. -
További szerzők:	Sebestyén Zsolt Gamble, John Nagy Péter (1971-) (biofizikus) Dóczy-Bodnár Andrea (1970-) (biofizikus) Alldridge, Lou Doe, Senam Holmes, Nick Goff, Lindsey K. Beverley, Peter Szöllősi János (1953-) (biofizikus) Alexander, Denis R.
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001-es BibID:	BIBFORM004665
035-os BibID:	(scopus)0034033048 (wos)000086686100012
Első szerző:	Hederer, Rosemarie A.
Cím:	The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells / Hederer, R. A., Guntermann, C., Miller, N., Nagy, P., Szollosi, J., Damjanovich, S., Hale, G., Alexander, D. R.
Dátum:	2000
Megjegyzések:	Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo.The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Antibodies, Monoclonal Antibodies, Neoplasm Antigens, CD Antigens, CD45 Calcium Calcium Signaling Cells, Cultured Comparative Study Energy Transfer enzymology Fluorescence genetics Glycoproteins Human immunology In Vitro Inositol 1,4,5-Trisphosphate Isoenzymes Jurkat Cells metabolism Phospholipase C physiology Receptors, Antigen, T-Cell Signal Transduction Support, Non-U.S.Gov't T-Lymphocytes Transfection
Megjelenés:	International Immunology. - 12 : 4 (2000), p. 505-516. -
További szerzők:	Guntermann, Christine Miller, Nigel Nagy Péter (1971-) (biofizikus) Szöllősi János (1953-) (biofizikus) Damjanovich Sándor (1936-2017) (biofizikus) Hale, Geoffrey Alexander, Denis R.
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001-es BibID:	BIBFORM039658
035-os BibID:	(scopus)0036628631 (wos)000176656500002
Első szerző:	Sebestyén Zsolt
Cím:	Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer / Zsolt Sebestyén, Péter Nagy, Gábor Horváth, György Vámosi, Reno Debets, Jan W. Gratama, Denis R. Alexander, János Szöllősi
Dátum:	2002
ISSN:	0196-4763
Megjegyzések:	Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1-10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method.METHODS:Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.ResultsTo increase the accuracy of FCET measurements, we applied a long wavelength donor-acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency.CONCLUSIONS:We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Algorithms analysis Antibodies Biophysics Calibration Cell Line, Transformed Cells Comparative Study Dyes Energy Transfer Flow Cytometry Fluorescein Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Dyes Humans Hungary instrumentation Lasers Membrane Proteins methods Proteins Reproducibility of Results Research Spectrometry, Fluorescence Subtraction Technique Support Tumor Cells, Cultured egyetemen (Magyarországon) készült közlemény
Megjelenés:	Cytometry. - 48 : 3 (2002), p. 124-135. -
További szerzők:	Nagy Péter (1971-) (biofizikus) Horváth Gábor (1974-) (biofizikus) Vámosi György (1967-) (biofizikus) Debets, Reno Gratama, Jan Willem Alexander, Denis R. Szöllősi János (1953-) (biofizikus)
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001-es BibID:	BIBFORM004741
035-os BibID:	(scopus)0345528144
Első szerző:	Szöllősi János (biofizikus)
Cím:	The application of fluorescence resonance energy transfer to the investigation of phosphatases / Szollosi, J., Alexander, D. R.
Dátum:	2003
ISSN:	076-6879 (Print)
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk analysis Animals Biophysics chemistry Energy Transfer Flow Cytometry Fluorescence Fluorescence Resonance Energy Transfer Hungary Kinetics metabolism methods Phosphoprotein Phosphatase Research Support
Megjelenés:	Methods in Enzymology. - 366 (2003), p. 203-224. -
További szerzők:	Alexander, Denis R.
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