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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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001-es BibID:	BIBFORM006077
Első szerző:	Vereb György (biofizikus, orvos)
Cím:	Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells / Vereb, G., Jr., Szollosi, J., Matyus, L., Balazs, M., Hyun, W. C., Feuerstein, B. G.
Dátum:	1996
Megjegyzések:	Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk antagonists and inhibitors Biophysics Bradykinin Ca(2+)-Transporting ATPase Calcium Calcium Channel Blockers Calcium Channels Calcium Signaling Dose-Response Relationship,Drug drug effects Fluorescence Glioblastoma Human Hungary Lanthanum Manganese metabolism Neuroglia pharmacology Platelet-Derived Growth Factor Signal Transduction Support, Non-U.S. Gov't Support, U.S.Gov't, Non-P.H.S. Support, U.S.Gov't, P.H.S. Terpenes Thapsigargin Tumor Cells,Cultured
Megjelenés:	Cytometry. - 24 : 1 (1996), p. 64-73. -
További szerzők:	Szöllősi János (1953-) (biofizikus) Mátyus László (1956-) (biofizikus) Balázs Margit (1952-) (sejtbiológus, molekuláris genetikus) Hyun, William C. Feuerstein, Burt G.
Internet cím:	Intézményi repozitóriumban (DEA) tárolt változa
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001-es BibID:	BIBFORM004890
035-os BibID:	(scopus)26444597596 (wos)000232299300017
Első szerző:	Vereb György (biofizikus, orvos)
Cím:	Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence / Vereb, G., Feuerstein, B. G., Hyun, W. C., Fulwyler, M. J., Balazs, M., Szollosi, J.
Dátum:	2005
ISSN:	1552-4922
Megjegyzések:	Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk analysis Biophysics Bromodeoxyuridine Calcium Calcium Signaling Cell Adhesion Cell Culture Cell Cycle Cells Cells,Cultured Dna drug effects genetics Glioblastoma Hungary metabolism methods pathology pharmacology Platelet-Derived Growth Factor Research Spectrometry,Fluorescence Support
Megjelenés:	Cytometry. Part A. - 67 : 2 (2005), p. 172-179. -
További szerzők:	Feuerstein, Burt G. Hyun, William C. Fulwyler, Mack J. Balázs Margit (1952-) (sejtbiológus, molekuláris genetikus) Szöllősi János (1953-) (biofizikus)
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