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001-es BibID:	BIBFORM046090
Első szerző:	Bacsó Zsolt (biofizikus)
Cím:	Raft and cytoskeleton associations of an ABC transporter : P-glycoprotein / Zsolt Bacso, Henrietta Nagy, Katalin Goda, László Bene, Ferenc Fenyvesi, János Matkó, Gábor Szabó
Dátum:	2004
ISSN:	0196-4763
Megjegyzések:	A novel flow cytometric assay has been described in an accompanying report (Gombos et al., METHODS: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents. RESULTS: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping. CONCLUSIONS: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levelsújratöltve - BIBFORM004828
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Cytometry. - 61 : 2 (2004), p. 105-116. -
További szerzők:	Nagy Henrietta Goda Katalin (1969-) (biofizikus) Bene László (1963-) (biofizikus) Fenyvesi Ferenc (1977-) (gyógyszerész, gyógyszertechnológus) Matkó János (1952-) (biológus) Szabó Gábor (1953-) (biofizikus)
Pályázati támogatás:	T032563 OTKA 034393 OTKA T046945 OTKA
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001-es BibID:	BIBFORM002233
035-os BibID:	WOS:000242995600010
Első szerző:	Goda Katalin (biofizikus)
Cím:	Complete inhibition of P-glycoprotein by simultaneous treatment with a distinct class of modulators and the UIC2 monoclonal antibody / Katalin Goda, Ferenc Fenyvesi, Zsolt Bacsó, Henrietta Nagy, Teréz Márián, Attila Megyeri, Zoltán Krasznai, István Juhász, Miklós Vecsernyés, Gábor Szabó
Dátum:	2007
Megjegyzések:	P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99mTc-hexakis-2- methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban UIC2 monoclonal antibody P-glycoprotein
Megjelenés:	The Journal of Pharmacology and Experimental Therapeutics 320 : 1 (2007), p. 81-88. -
További szerzők:	Fenyvesi Ferenc (1977-) (gyógyszerész, gyógyszertechnológus) Bacsó Zsolt (1963-) (biofizikus) Nagy Henrietta Márián Teréz (1950-) (radiobiológus) Megyeri Attila (1968-) (orvos) Krasznai Zoltán (1950-) (biofizikus) Juhász István (1956-) (bőrgyógyász, bőrsebész, kozmetológus, klinikai onkológus) Vecsernyés Miklós (1959-) (gyógyszertechnológus, endokrinológus) Szabó Gábor (1980-) (orvos) jr
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001-es BibID:	BIBFORM046091
Első szerző:	Gombos Imre
Cím:	Cholesterol sensitivity of detergent resistance : a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins / Imre Gombos, Zsolt Bacsó, Cynthia Detre, Henrietta Nagy, Katalin Goda, Márton Andrásfalvy, Gábor Szabó, János Matkó
Dátum:	2004
ISSN:	0196-4763
Megjegyzések:	Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method. METHODS: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents. RESULTS: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach. CONCLUSIONS: A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Cytometry. Part A. - 61A : 2 (2004), p. 117-126. -
További szerzők:	Bacsó Zsolt (1963-) (biofizikus) Detre, Cynthia Nagy Henrietta Goda Katalin (1969-) (biofizikus) Andrásfalvy Márton Szabó Gábor (1953-) (biofizikus) Matkó János (1952-) (biológus)
Pályázati támogatás:	T034393 OTKA TS044711 OTKA T032563 OTKA T046945 OTKA
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001-es BibID:	BIBFORM004839
035-os BibID:	(WOS)000220105600025 (scopus)1342345242
Első szerző:	Nagy Henrietta
Cím:	Distinct groups of multidrug resistance modulating agents are distinguished by competition of P-glycoprotein-specific antibodies / Nagy, H., Goda, K., Fenyvesi, F., Bacso, Z., Szilasi, M., Kappelmayer, J., Lustyik, G., Cianfriglia, M., Szabo, G.
Dátum:	2004
Megjegyzések:	P-glycoprotein (Pgp) is one of the ABC transporters responsible for the multidrug resistance of cancer cells. The conformational changes of Pgp that occur in the presence of substrates/modulators or ATP depletion are accompanied by the up-shift of UIC2 monoclonal antibody (mAb) binding. In the case of cyclosporin A, vinblastine or valinomycin, this up-shift was found to be concomitant with the near-complete suppression of labeling with other mAbs specific for Pgp epitopes overlapping with UIC2, while pre-treatment with verapamil or Tween 80 brings about a modest suppression. Here we have extended these observations to 44 Pgp interacting agents, and found that only 8 fall into the cyclosporin-like category, inducing a conformational state characterized by the complete UIC2 dominance. The rest of the drugs either did not affect antibody competition or had a modest effect. Thus, Pgp substrates/modulators can be classified into distinct modalities based on the conformational change they elicit.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Adenosine Adenosine Triphosphatases Animals Anti-Bacterial Agents Antibodies Antibodies,Monoclonal Antineoplastic Agents Binding,Competitive Biophysics Calcium Calcium Channel Blockers Cells Cyclosporine Detergents Drug Resistance,Multiple Drug Resistance,Neoplasm Epitopes Flow Cytometry Fluorescein Fluoresceins genetics Humans Hungary immunology Ivermectin metabolism Mice Nih 3T3 Cells P-Glycoprotein pharmacology physiology Research Substrate Specificity Support Valinomycin Verapamil Vinblastine egyetemen (Magyarországon) készült közlemény
Megjelenés:	Biochemical and Biophysical Research Communications. - 315 : 4 (2004), p. 942-949. -
További szerzők:	Goda Katalin (1969-) (biofizikus) Fenyvesi Ferenc (1977-) (gyógyszerész, gyógyszertechnológus) Bacsó Zsolt (1963-) (biofizikus) Szilasi Mária (1953-) (tüdőgyógyász, klinikai immunológus, allergológus, belgyógyász) Kappelmayer János (1960-) (laboratóriumi szakorvos) Lustyik György Cianfriglia, Maurizio Szabó Gábor (1953-) (biofizikus)
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