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001-es BibID:BIBFORM023492
Első szerző:Matkó János (biológus)
Cím:Analysis of cell surface molecular distributions and cellular signaling by flow cytometry / J. Matkó, L. Mátyus, J. Szöllősi, L. Bene, A. Jenei, P. Nagy, A. Bodnár, S. Damjanovich
Dátum:1994
ISSN:1053-0509
Megjegyzések:Flow cytometry is a fast analysis and separation method for large cell populations, based on collection and processing of optical signals gained on a cell-by-cell basis. These optical signals are scattered light and fluorescence. Owing to its unique potential ofStatistical data analysis and sensitive monitoring of (micro)heterogeneities in large cell populations, flow cytometry?in combination with microscopic imaging techniques?is a powerful tool to study molecular details of cellular signal transduction processes as well. The method also has a widespread clinical application, mostly in analysis of lymphocyte subpopulations for diagnostic (or research) purposes in diseases related to the immune system. A special application of flow cytometry is the mapping of molecular interactions (proximity relationships between membrane proteins) at the cell surface, on a cell-by-cell basis. We developed two approaches to study such questions; both are based ondistance-dependent quenching of excited state fluorophores (donors) by fluorescent or dark (nitroxide radical) acceptors via Förstertype dipole-dipole resonance energy transfer (FRET) and long-range electron transfer (LRET) mechanisms, respectively. A critical evaluation of these methods using donor- or acceptor-conjugated monoclonal antibodies (or their Fab fragments) to select the appropriate cell surface receptor or antigen will be presented in comparison with other approaches for similar purposes. The applicability of FRET and LRET for two-dimensional antigen mapping as well as for detection of conformational changes in extracellular domains of membrane-bound proteins is discussed and illustrated by examples of several lymphoma cell lines. Another special application area of flow cytometry is the analysis of different aspects of cellular signal transduction, e.g., changes of intracellular ion (Ca2+, H+, Na+) concentrations, regulation of ion channel activities, or more complex physiological responses of cell to external stimuli via correlated fluorescence and scatter signal analysis, on a cell-by-cell basis. This way different signaling events such as changes in membrane permeability, membrane potential, cell size and shape, ion distribution, cell density, chromatin structure, etc., can be easily and quickly monitored over large cell populations with the advantage of revealing microheterogeneities in the cellular responses. Flow cytometry also offers the possibility to follow the kinetics of slow (minute- and hour-scale) biological processes in cell populations. These applications are illustrated by the example of complex flow cytometric analysis of signaling in extracellular ATP-triggered apoptosis (programmed cell death) of murine thymic lymphocytes.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
fluorescence
flow cytometry
energy transfer
electron transfer
protein-protein interaction
signal transduction
egyetemen (Magyarországon) készült közlemény
Megjelenés:Journal Of Fluorescence 4 : 4 (1994), p. 303-314. -
További szerzők:Mátyus László (1956-) (biofizikus) Szöllősi János (1953-) (biofizikus) Bene László (1963-) (biofizikus) Jenei Attila (1966-) (biofizikus) Nagy Péter (1971-) (biofizikus) Dóczy-Bodnár Andrea (1970-) (biofizikus) Damjanovich Sándor (1936-2017) (biofizikus)
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2.

001-es BibID:BIBFORM006044
Első szerző:Matkó János (biológus)
Cím:Biphasic effect of extracellular ATP on the membrane potential of mouse thymocytes / Matko J., Nagy P., Panyi G., Vereb G. Jr., Bene L., Matyus L., Damjanovich S.
Dátum:1993
Megjegyzések:Extracellular ATP induced changes in the membrane potential of thymocytes from BALB/c mice were analyzed. At concentrations below 0.1 mM, ATP hyperpolarizes the cell membrane on the time scale of development of the Ca(2+)-signal. After a longer time hyperpolarization turns to depolarization. ATP concentrations higher than 0.5 mM caused rapid depolarization without previous hyperpolarization. Verapamil, quinine or the absence of extracellular Ca2+ blocked the hyperpolarization by ATP. In Na(+)-free medium the magnitude of depolarization decreased. Our data suggest a contribution of Ca(2+)-activated K+ channels to the hyperpolarizing effect of ATP at lower concentrations. The direction of membrane potential changes is determined presumably by a sensitive balance of ATP-receptor mediated Ca(2+)- and Na(+)-influx and the Ca(2+)-activated K(+)-channel activity.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Adenosine Triphosphate
Animal
Biophysics
Cell Membrane
cytology
drug effects
Hungary
Kinetics
Membrane Potentials
Mice
Mice,Inbred BALB C
pharmacology
physiology
Quinine
Support,Non-U.S.Gov't
Thymus Gland
Verapamil
Megjelenés:Biochemical and Biophysical Research Communications. - 191 : 2 (1993), p. 378-384. -
További szerzők:Nagy Péter (1971-) (biofizikus) Panyi György (1966-) (biofizikus) Vereb György (1965-) (biofizikus, orvos) Bene László (1963-) (biofizikus) Mátyus László (1956-) (biofizikus) Damjanovich Sándor (1936-2017) (biofizikus)
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3.

001-es BibID:BIBFORM046282
Első szerző:Nagy Péter (biofizikus)
Cím:Ion-channel activities regulate transmembrane signaling in thymocyte apoptosis and T-cell activation / Nagy Péter, Panyi György, Jenei Attila, Bene László, Gáspár Rezső, Matkó János, Damjanovich Sándor
Dátum:1995
ISSN:0165-2478
Megjegyzések:Several examples have shown that plasma membrane ion channels (e.g., Ca2+ and K+ channels) make an important contribution to lymphocyte activation or thymocyte apoptosis. Here we report on the importance of these ion channels in the sensitivity or resistance of lymphoid cells to extracellular ATP-induced apoptosis. Thymocytes of Balb/c mice responded to extracellular ATP (ATPex) sensitively, with an immediate increase in the intracellular calcium level and later with an increased membrane permeability to low MW markers. Mature (medullary) thymocytes showed a higher sensitivity than did cortical thymocytes. Three human lymphoma cell lines, including SUPT13, a cell line reported to be sensitive to TcR/CD3 activation-induced apoptosis, showed a high resistance to ATPex action. These observations suggest that maturation/differentiation state-dependent activity or disappearance of early ATP-receptor operated signaling systems (including ion channels) are critical for the cells in developing towards apoptosis. Using the patch-clamp technique we demonstrated that bretylium tosylate (a particular K(+)-channel blocker) known as inhibitor of T-lymphocyte proliferation also influences the single-channel properties of voltage-gated K+ channels through depressing whole-cell K+ currents. This finding is yet another example underlying the importance of K+ channel activity in T-lymphocyte proliferation.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Immunology Letters. - 44 : 2-3 (1995), p. 91-95. -
További szerzők:Panyi György (1966-) (biofizikus) Jenei Attila (1966-) (biofizikus) Bene László (1963-) (biofizikus) Gáspár Rezső (1921-2001) (fizikus) Matkó János (1952-) (biológus) Damjanovich Sándor (1936-2017) (biofizikus)
Pályázati támogatás:T14655
OTKA
F13335
OTKA
T6163
OTKA
T6221
OTKA
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4.

001-es BibID:BIBFORM004878
035-os BibID:(scopus)26444586247 (wos)000232299300007
Első szerző:Nagy Péter (biofizikus)
Cím:Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins / Nagy, P., Bene, L., Hyun, W. C., Vereb, G., Braun, M., Antz, C., Paysan, J., Damjanovich, S., Park, J. W., Szollosi, J.
Dátum:2005
ISSN:1552-4922
Megjegyzések:The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
analysis
Biophysics
Calibration
Cells
chemistry
Cyclin-Dependent Kinase 2
Cyclin-Dependent Kinase Inhibitor p27
Energy Transfer
Flow Cytometry
Fluorescence
Fluorescence Resonance Energy Transfer
Hungary
metabolism
methods
Protein Binding
Proteins
Research
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
Support
Megjelenés:Cytometry. Part A. - 67 : 2 (2005), p. 86-96. -
További szerzők:Bene László (1963-) (biofizikus) Hyun, William C. Vereb György (1965-) (biofizikus, orvos) Braun, Manuel Antz, Christof Paysan, Jacques Damjanovich Sándor (1936-2017) (biofizikus) Park, John W. Szöllősi János (1953-) (biofizikus)
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5.

001-es BibID:BIBFORM004634
035-os BibID:(scopus)0032101894 (wos)000073937700007
Első szerző:Nagy Péter (biofizikus)
Cím:EGF-induced redistribution of erbB2 on breast tumor cells : flow and image cytometric energy transfer measurements / Nagy, P., Bene, L., Balazs, M., Hyun, W. C., Lockett, S. J., Chiang, N. Y., Waldman, F., Feuerstein, B. G., Damjanovich, S., Szollosi, J.
Dátum:1998
Megjegyzések:erbB2, a member of the epidermal growth factor (EGF) receptor-type tyrosine kinase receptor family, is overexpressed in breast carcinomas with poor prognosis. We examined the cell surface association of this receptor with itself and with other cell surface proteins by the Forster-type fluorescence resonance energy transfer using whole antibodies and Fab fragments. We found that erbB2 molecules homoassociate in unstimulated SK-BR-3, BT474 and BT474-M (a metastatic version of the parent BT474 line) breast tumor cells, and that the interaction was enhanced by EGF treatment in suspensions of SK-BR-3 and BT474-M cells. BT474 cells (with low EGF receptor expression) and attached SK-BR-3 cells do not respond to EGF. Image microscopic energy transfer measurements found considerable pixel-by-pixel heterogeneity in the homoassociation state of erbB2. In accordance with the EGF-induced redistribution of erbB2, EGF receptor was found to be in close proximity to erbB2 in FRET measurements. By labeling different epitopes on erbB2 and the lipid bilayer, we were able to prepare an epitope map of erbB2 molecule. Our data suggest the existence of dynamic cell surface patterns of erbB2 and point to functions fulfilled by these molecular complexes.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
analysis
biosynthesis
Breast Neoplasms
Carcinoma
Cell Membrane
chemistry
drug effects
Energy Transfer
Epidermal Growth Factor
Female
Flow Cytometry
Fluorescence
Human
Hungary
metabolism
methods
Models
Molecular
pharmacology
Receptor
erbB-2
Receptors
Transferrin
Support
Non-U.S.Gov't
Tumor Cells,Cultured
Squamous Cell
ultrastructure
Megjelenés:Cytometry. - 32 : 2 (1998), p. 120-131. -
További szerzők:Bene László (1963-) (biofizikus) Balázs Margit (1952-) (sejtbiológus, molekuláris genetikus) Hyun, William C. Lockett, Steven J. Chiang, Nancy Y. Waldman, Frederick Feuerstein, Burt G. Damjanovich Sándor (1936-2017) (biofizikus) Szöllősi János (1953-) (biofizikus)
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