45 results on '"Attila Jenei"'
Search Results
2. Understanding FRET as a Research Tool for Cellular Studies
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János Szöllősi, György Vereb, Dilip Shrestha, Attila Jenei, and Péter Nagy
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Proteomics ,ErbB ,Green Fluorescent Proteins ,Review ,Computational biology ,Living cell ,CD1d ,Molecular heterogeneity ,Catalysis ,Immune synapse ,Inorganic Chemistry ,lcsh:Chemistry ,Fluorescence lifetime ,Fluorescence Resonance Energy Transfer ,Elméleti orvostudományok ,Physical and Theoretical Chemistry ,Molecular Biology ,Biological sciences ,lcsh:QH301-705.5 ,Spectroscopy ,Physics ,Molecular interactions ,IL2 ,IL15 ,Organic Chemistry ,Fluorescence intensity ,Orvostudományok ,General Medicine ,Flow Cytometry ,Computer Science Applications ,Cell biology ,Förster resonance energy transfer ,Microscopy, Fluorescence ,lcsh:Biology (General) ,lcsh:QD1-999 ,FRET ,Functional significance ,Anisotropy ,Methods for measuring FRET ,Major Histocompatibility Complex (MHC) ,Signal Transduction - Abstract
Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1-10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.
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- 2015
3. A fogászatban alkalmazható keresztkötött hialuronsav alapú hidrogélrendszerek szintézise és hatóanyag leadásának vizsgálata
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Tünde Rente, József Bakó, Kinga Bágyi, Attila Jenei, and Csaba Hegedűs
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General Medicine - Abstract
A hialuronsav biológiai lebonthatóságát, biokompatibilitását kiaknázva a hialuronsav alapú rendszerek alkalmazása egyre nagyobb teret kap a fogászatban is. A célzott hatóanyagszállítás megvalósítására az egyik legelterjedtebb eljárása biopolimerek keresztkötése. Jelen munka célja olyan hialuronsavbázisú rendszerek előállítása és vizsgálata, amelyek a fogászatban is alkalmazható hatóanyag-leadó rendszerek alapjául szolgálhatnak. A különböző arányban (25, 50, 75, 100%) térhálósított szerkezetek kialakítása a hialuronsav és a 2,2’ (etiléndioxi)bisz(etilamin) kondenzációs reakciójával, karbodiimid felhasználásával történt. A részecskék méretének meghatározásakor a dinamikus lézer fényszórás fotometriai (DLS) mérések eredményeként megállapítható, hogy mind a négy módosított polimer trimodális méreteloszlást mutatott, továbbá, hogy a módosítás arányának növekedésével a részecskék mérete csökkent. A transzmissziós elektronmikroszkópos (TEM) felvételek szintén alátámasztják ezen eredményeket. A keresztkötött származékok hatóanyagleadó tulajdonságának jellemzése fotometriás méréstechnikával történt, modellvegyületként metilénkéket alkalmazva. Eredményeink azt bizonyították, hogy a keresztkötő ágens arányának növelésével a hatóanyag kioldódásának sebessége növekszik, amely a további kísérletekben egy új injektálható hatóanyag-kibocsátó rendszer fejlesztésének alapját képezheti.
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- 2017
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4. Preparation and application of highly porous aerogel-based bioactive materials in dentistry
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István Lázár, Andrea Kuttor, József Bakó, Attila Jenei, Csaba Hegedus, Tünde Rente, István Fábián, Farkas Kerényi, and Melinda Szalóki
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Materials science ,Pore diameter ,Scanning electron microscope ,Műszaki tudományok ,Aerogel ,Biological effect ,Anyagtudományok és technológiák ,Chemical engineering ,Highly porous ,Confocal laser scanning microscopy ,General Materials Science ,Composite material ,Porosity ,Mesoporous material - Abstract
In this study, the possibility of preparation and application of highly porous silica aerogel-based bioactive materials are presented. The aerogel was combined with hydroxyapatite and β-tricalcium phosphate as bioactive and osteoinductive agents. The porosity of aerogels was in the mesoporous region with a maximum pore diameter of 7.4 and 12.7 nm for the composite materials. The newly developed bioactive materials were characterized by scanning electron microscopy. The in vitro biological effect of these modified surfaces was also tested on SAOS-2 osteogenic sarcoma cells by confocal laser scanning microscopy.
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- 2014
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5. [Effect of BMP-2 treatment on the morphology and proliferation of human embryonic palatal derived mesenchymal preosteoblast cells]
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Edit, Hrubi, László, Imre, Zsolt, Bacsó, Sándor, Bíró, Attila, Jenei, and Hegedűs Csaba
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Osteoblasts ,Palate ,Bone Morphogenetic Protein 2 ,Humans ,Cell Differentiation ,Mesenchymal Stem Cells ,Embryonic Stem Cells ,Cell Line ,Cell Proliferation - Abstract
In dental implantation missing tooth or teeth are replaced by artificial root. To reduce the time required for the integration newest trends are the enhancement of bone formation around the implant by bioactive molecules, growth factors. Such a molecule is bone morphogenetic protein-2 (BMP-2) accepted by US Food and Drug Administration (FDA). In these kind of applications effect of BMP-2 is tested in vitro on appropriate cell lines. One of these cell lines is the osteoblast like human embrionic palatal mesenchymal cell line (HEPM). In our experiments the effect of BMP-2 homodimer treatment was investigated on the differentiation of HEPM cells to osteoblasts reflected by changes in morphology, and proliferation after a short, 3 days BMP-2 treatment. Results showed that after three days BMP-2 treatment facilitates cell attachment on a concentration dependent manner however changes in cell morphology and proliferation could not be observed. Continuing the BMP-2 treatment inhibitory effect was measured in cell proliferation, which may refer to cell differentiation.
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- 2016
6. Coating conditions matter to collagen matrix formation regarding von Willebrand factor and platelet binding
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Shlomit Mendelboum Raviv, Naphtali Savion, János Nagy, Boris Shenkman, Katalin Szekeres-Csiki, Jolan Harsfalvi, and Attila Jenei
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Blood Platelets ,Plasma protein binding ,Horseradish peroxidase ,Collagen Type I ,chemistry.chemical_compound ,Coated Materials, Biocompatible ,Von Willebrand factor ,von Willebrand Factor ,Humans ,Platelet ,Elméleti orvostudományok ,Binding site ,Cells, Cultured ,Binding Sites ,biology ,Orvostudományok ,Hematology ,Buffer solution ,Platelet Activation ,Ligand (biochemistry) ,Dissociation constant ,Collagen Type III ,chemistry ,Immunology ,biology.protein ,Biophysics ,Protein Binding - Abstract
Introduction Von Willebrand factor (VWF) and platelet binding needs a uniform collagen matrix therefore we aimed to find an optimal condition for the preparation of human type-I and type-III collagen matrices. Method The effects of pH, salt and ligand concentration and binding time were tested when collagen matrices were prepared by adsorption. Surface-bound collagen and collagen-bound VWF measured by specific antibodies. Platelet adhesion was tested under flow conditions at a shear rate of 1800s −1 for 2min. Matrices and platelets were visualized by atomic force and scanning electron microscope. Results The extent of human collagens type-I and III binding to the surface was 10 and 4 times greater and binding was maximal under 8–16hours, when coated from physiological buffer solution versus acid solution. Collagen fibrils were more developed and platelet adhesion was higher, with more organized and denser aggregates. VWF binding was parallel to the surface bound collagen in both collagen types. Conclusion Collagen coating of surfaces for VWF binding and platelet adhesion studies is very variable from acid solution. Our experiments provide evidences that neutralizing the acid and adding NaCl in physiological concentration, thereby facilitating formation of collagen fibril molecules in solution, results in efficient coating of human type-I and type III collagens, which then bind normal VWF equally well.
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- 2012
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7. Non-Random Distribution of Interleukin Receptors on the Cell Surface
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József Kormos, László Mátyus, Sándor Varga, Sándor Damjanovich, Adrienn J. Veres, Andrea Bodnár, Attila Jenei, and Gergely Szentesi
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Materials science ,Nanostructure ,T-Lymphocytes ,Cell Membrane ,Interleukin-2 Receptor alpha Subunit ,Analytical chemistry ,Colocalization ,Immunogold labelling ,Atomic and Molecular Physics, and Optics ,law.invention ,Membrane ,Interleukin-15 Receptor alpha Subunit ,Microscopy, Electron, Transmission ,law ,Labelling ,Fluorescence Resonance Energy Transfer ,Biophysics ,Humans ,Molecule ,Physical and Theoretical Chemistry ,Absorption (chemistry) ,Electron microscope ,Monte Carlo Method ,Algorithms ,Software - Abstract
Spatial organization of cell surface proteins plays a key role in the process of transmembrane signalling. Receptor clustering and changes in their cell surface distribution are often determining factors in the final outcome of ligand-receptor interactions. There are several techniques for assessing the distribution of protein molecules. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining distance relationships of cell surface molecules. However, it does not provide information on the distribution of molecular clusters. Different kinds of microscopies fill this gap. The evaluation of the images provided by the listed techniques is often questionable. Herein we show the applicability of Ripley's K(t) function as a tool for analyzing the cell surface receptor patterns (Y. Nakamura, et al., Nature 1994, 369, 330-333). We have implemented an effective image processing algorithm for fast localization of gold labels on biological samples. We investigated spatial organization of Interleukin-2R alpha and -15R alpha (IL-2R alpha and IL-15R alpha) on a human CD4+leukaemia T-cell line, Kit225 FT7.10 by using transmission electron microscopy (TEM). TEM analysis showed co-clustering of the two types of alpha-chains even on the few-hundred-nanometer scale. The analysis of our data may contribute to our understanding the action of the IL-2/IL-15 receptor system in T-cell function.
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- 2009
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8. DNA flow cytometry of human spermatozoa: Consistent stoichiometric staining of sperm DNA using a novel decondensation protocol
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Gyöngyi Békési, Zsuzsa Rákosy, Gábor Horváth, Ákos Fábián, Margit Balázs, László Mátyus, Tamás Kovács, and Attila Jenei
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Male ,endocrine system ,Histology ,In situ hybridization ,Biology ,Sensitivity and Specificity ,Pathology and Forensic Medicine ,Flow cytometry ,chemistry.chemical_compound ,medicine ,Humans ,Elméleti orvostudományok ,Propidium iodide ,In Situ Hybridization, Fluorescence ,reproductive and urinary physiology ,Sperm Count ,medicine.diagnostic_test ,urogenital system ,DNA ,Orvostudományok ,Cell Biology ,Flow Cytometry ,Diploidy ,Spermatozoa ,Sperm ,Molecular biology ,Salicylates ,Staining ,Chaotropic agent ,chemistry ,Iodobenzoates ,Ploidy ,Propidium - Abstract
Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate.
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- 2008
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9. Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells
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Thomas A. Waldmann, Sándor Damjanovich, Attila Jenei, Niek F. van Hulst, Erik M. H. P. van Dijk, Maria F. Garcia-Parajo, György Vámosi, Andrea Bodnár, Bärbel I. de Bakker, Optical Sciences, and Faculty of Science and Technology
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Interleukin 2 ,Leukemia, T-Cell ,T-Lymphocytes ,T cell ,Biology ,Lymphoma, T-Cell ,Protein structure ,IR-75533 ,Cell Line, Tumor ,medicine ,Humans ,Elméleti orvostudományok ,Receptor ,Interleukin-15 ,Immunity, Cellular ,Microscopy ,Receptors, Interleukin-15 ,Cell Membrane ,Receptor Aggregation ,Interleukin-2 Receptor alpha Subunit ,Orvostudományok ,METIS-247763 ,Cell Biology ,medicine.disease ,Protein Structure, Tertiary ,Cell biology ,Leukemia ,medicine.anatomical_structure ,Cell culture ,Interleukin 15 ,Interleukin-2 ,Function (biology) ,Signal Transduction ,medicine.drug - Abstract
Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual alpha-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2Ralpha and IL15Ralpha at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2Ralpha and IL15Ralpha at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2Ralpha molecules reside outside the clustered domains, whereas approximately 30% of IL15Ralpha molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2Ralpha and IL15Ralpha domains remained constant, suggesting a 'building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two alpha-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.
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- 2008
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10. BMP-2 hatása a humán embrionális szájpadlásból származó mesenchymalis preosteoblast sejtek morfológiájára és proliferációjára
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Edit Hrubi, László Imre, Zsolt Bacsó, Sándor Bíró, Attila Jenei, and Csaba Hegedűs
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General Medicine - Abstract
Fogászati implantáció során a hiányzó fogat vagy fogakat „műgyökerek”, beültetésével pótoljuk. Az integráció folyamatánakfelgyorsítása érdekében az egyik legújabb törekvés bioaktív anyagokkal, növekedési faktorokkal segíteni azimplantátum körül a csontképződést. Ilyen molekula az amerikai gyógyszer- és élelmiszerügyi hatóság (FDA) által iselfogadott növekedési faktor a Bone Morfogenic Protein 2 (BMP-2) is. A BMP-2 ilyen jellegű alkalmazását in vitro tesztelésrealkalmas sejtvonalakon vizsgálják. Egyike ezeknek az osteoblast progenitor jellegű humán embrionális szájpadlásmesenchyma (HEPM) sejtvonal. Kísérleteink során vizsgáltuk a BMP-2 homodimer fehérjék hatását HEPM sejtek morfológiájáravalamint proliferációjára, rövid idejű, háromnapos kezelés után.Eredményeink azt mutatták, hogy háromnapos kezelés hatására a BMP-2 koncentrációjának függvényében a kezeltsejtek hamarabb tapadtak le, de végleges morfológiájukban, valamint proliferációjukban nem volt különbség.A kezelés idejét növelve azonban a sejtek osztódási képessége lecsökkent, ami utalhat a sejtek differenciációjára.
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- 2015
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11. Steady-state fluorescence quenching applications for studying protein structure and dynamics
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László Mátyus, János Szöllosi, and Attila Jenei
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Protein Conformation ,Biophysics ,Cesium ,Fluorescence in the life sciences ,Photochemistry ,Fluorescence ,Quantitative Biology::Subcellular Processes ,Matrix (chemical analysis) ,Protein structure ,Chlorides ,Fluorescence Resonance Energy Transfer ,Radiology, Nuclear Medicine and imaging ,Elméleti orvostudományok ,Acrylamide ,Quantitative Biology::Biomolecules ,Radiation ,Quenching (fluorescence) ,Radiological and Ultrasound Technology ,Chemistry ,Dynamics (mechanics) ,Temperature ,Tryptophan ,Proteins ,Orvostudományok ,Förster resonance energy transfer ,Energy Transfer ,Chemical physics ,Fluorescence cross-correlation spectroscopy ,Algorithms ,Macromolecule - Abstract
Fluorescence quenching methods are useful to obtain information about the conformational and/or dynamic changes of proteins in complex macromolecular systems. In this review steady-state methods are described and the data interpretation is thoroughly discussed. As a special case of fluorescence quenching mechanism, fluorescence resonance energy transfer (FRET) phenomenon is also presented. Application of a FRET based method to characterize the temperature dependence of the flexibility of protein matrix is clearly demonstrated.
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- 2006
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12. Does mosaicism of the plasma membrane at molecular and higher hierarchical levels in human lymphocytes carry information on the immediate history of cells?
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Sándor Damjanovich, Rezsö Gáspár, Attila Jenei, László Mátyus, László Bene, János Matkó, János Szöllosi, and László Damjanovich
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Immunology ,Transferrin receptor ,Biology ,Cell Fusion ,Cell membrane ,Membrane Microdomains ,Cell surface receptor ,Histocompatibility Antigens ,medicine ,Humans ,Immunology and Allergy ,Lymphocytes ,Elméleti orvostudományok ,Receptors, Immunologic ,Receptor ,Lipid raft ,Receptor Aggregation ,Endoplasmic reticulum ,Cell Membrane ,Models, Immunological ,Receptors, Interleukin ,Orvostudományok ,Cell biology ,Kinetics ,medicine.anatomical_structure ,Signal transduction ,Signal Transduction - Abstract
A theoretical analysis of experimental data is presented in this mini-review on non-random homo- and hetero-associations of cell surface receptors, which can be recruited in the plasma membrane or at the surface of the rough endoplasmic reticulum during the protein synthesis. In the latter case, the likely genetic origin of these supramolecular formations is analyzed, contrasting this concept to the mobility of the cell surface proteins. A model is offered which, on the one hand, allows the mobility in a restricted way even among microdomain-confined receptor proteins through 'swapping partners'. On the other hand, the lack of mixing molecular components of protein clusters will be analyzed, when homo-and hetero-associations are studied through cell fusion experiments. The most frequently studied cell surface patterns have included lipid raft organized HLA class I and II, ICAM-1, tetraspan molecules, IL2 and IL15 and other receptors, as well. On the contrary coated pit-associated transferrin receptors would not mix with the above lipid raft associated receptor patterns, although transferrin receptor would readily oligomerize into homo-associates. The functional consequences of these superstructures are also analyzed. On the 30th anniversary of the Singer-Nicolson fluid mosaic membrane model one has to pay tribute to the authors, because of their deep insight emphasizing also the mosaicism of the membranes in general and that of the plasma membrane, in particular.
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- 2002
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13. [Comperative study of implant surface characteristics]
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Bernadett, Katona, Lajos, Daróczi, Attila, Jenei, József, Bakó, and Csaba, Hegedus
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Dental Implants ,Titanium ,X-Ray Absorption Spectroscopy ,Osseointegration ,Surface Properties ,Microscopy, Electron, Scanning ,Humans - Abstract
The osseointegration between the implant and its' bone environment is very important. The implants shall meet the following requirements: biocompatibility, rigidity, resistance against corrosion and technical producibility. In our present study surface morphology and material characteristics of different implants (Denti Bone Level, Denti Zirconium C, Bionika CorticaL, Straumann SLA, Straumann SLA Active, Dentsply Ankylos and Biotech Kontact implant) were investigated with scanning electron microscopy and energy-dispersive X-ray spectroscopy. The possible surface alterations caused by the manufacturing technology were also investigated. During grit-blasting the implants' surface is blasted with hard ceramic particles (titanium oxide, alumina, calcium phosphate). Properties of blasting material are critical because the osseointegration of dental implants should not be hampered. The physical and chemical features of blasting particles could importantly affect the produced surfaces of implants. Titanium surfaces with micro pits are created after immersion in mixtures of strong acids. On surfaces after dual acid-etching procedures the crosslinking between fibrin and osteogenetic cells could be enhanced therefore bone formation could be directly facilitated on the surface of the implant. Nowadays there are a number of surface modification techniques available. These can be used as a single method or in combination with each other. The effect of the two most commonly used surface modifications (acid-etching and grit-blasting) on different implants are demonstrated in our investigation.
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- 2014
14. CD1d favors MHC neighborhood, GM1 ganglioside proximity and low detergent sensitive membrane regions on the surface of B lymphocytes
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György Vereb, János Szöllősi, Mark A. Exley, Attila Jenei, and Dilip Shrestha
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Octoxynol ,Detergents ,Antigen presentation ,Biophysics ,CD1 ,Receptors, Cell Surface ,chemical and pharmacologic phenomena ,G(M1) Ganglioside ,Major histocompatibility complex ,Biochemistry ,Major Histocompatibility Complex ,Antigen ,parasitic diseases ,MHC class I ,Humans ,Elméleti orvostudományok ,Antigen-presenting cell ,Molecular Biology ,Cells, Cultured ,Antigen Presentation ,B-Lymphocytes ,biology ,Antigen processing ,Cell Membrane ,beta-Cyclodextrins ,Cell Differentiation ,hemic and immune systems ,Orvostudományok ,Flow Cytometry ,Cell biology ,Killer Cells, Natural ,carbohydrates (lipids) ,Cholesterol ,CD1D ,Immunology ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Antigens, CD1d - Abstract
Background Cluster of differentiation 1 (CD1) represents a family of proteins which is involved in lipid-based antigen presentation. Primarily, antigen presenting cells, like B cells, express CD1 proteins. Here, we examined the cell-surface distribution of CD1d, a subtype of CD1 receptors, on B lymphocytes. Methods Fluorescence labeling methods, including fluorescence resonance energy transfer (FRET), were employed to investigate plasma membrane features of CD1d receptors. Results High FRET efficiency was observed between CD1d and MHC I heavy chain (MHC I-HC), β2-microglobulin (β2m) and MHC II proteins in the plasma membrane. In addition, overexpression of CD1d reduced the expression of MHC II and increased the expression of MHC I-HC and β2m proteins on the cell-surface. Surprisingly, β2m dependent CD1d isoform constituted only ~ 15% of the total membrane CD1d proteins. Treatment of B cells with methyl-β-cyclodextrin (MβCD) / simvastatin caused protein rearrangement; however, FRET demonstrated only minimal effect of these chemicals on the association between CD1d and GM1 ganglioside on cell-surface. Likewise, a modest effect was only observed in a co-culture assay between MβCD/simvastatin treated C1R–CD1d cells and invariant natural killer T cells on measuring secreted cytokines (IFNγ and IL4). Furthermore, CD1d rich regions were highly sensitive to low concentration of Triton X-100. Physical proximity between CD1d, MHC and GM1 molecules was also detected in the plasma membrane. Conclusions An intricate relationship between CD1d, MHC, and lipid species was found on the membrane of human B cells. General significance Organization of CD1d on the plasma membrane might be critical for its biological functions.
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- 2014
15. An atomic force microscopy study on the effect of bleaching agents on enamel surface
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Csaba Hegedus, Attila Jenei, E Flóra-Nagy, Tamás Bistey, and Gusztáv Keszthelyi
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Time Factors ,Materials science ,Acrylic Resins ,Dentistry ,Carbamide Peroxide ,Microscopy, Atomic Force ,chemistry.chemical_compound ,stomatognathic system ,Incisor ,Microscopy ,Tooth Bleaching ,Opalescence ,medicine ,Humans ,Urea ,Composite material ,Dental Enamel ,Hydrogen peroxide ,General Dentistry ,Acrylic resin ,Drug Carriers ,Enamel paint ,Atomic force microscopy ,business.industry ,Hydrogen Peroxide ,Oxidants ,Peroxides ,Molecular Weight ,Drug Combinations ,stomatognathic diseases ,medicine.anatomical_structure ,chemistry ,visual_art ,visual_art.visual_art_medium ,Polyvinyls ,business ,Oxidation-Reduction ,After treatment - Abstract
Objectives : The aim of the present study was to evaluate the effect of three peroxide-containing bleaching agents, Opalescence, Nite White and a 30% hydrogen peroxide solution, on enamel surfaces using Atomic Force Microscopy (AFM). Methods : Fifteen non-carious human incisors (ten maxillary and five mandibular, extracted for periodontal reasons) were used. The teeth were divided randomly into three groups of five, according to the bleaching agents. The labial surface of each tooth was imaged by AFM before and after treatment. Each bleaching agent was applied for a total of 28 h (in individual 4 h treatments). The specimens were examined only after 28 h of treatment. Results : On comparing the AFM images of untreated and treated enamel, surface alterations were observed after 28 h of treatment with Opalescence, Nite White and 30% hydrogen peroxide solution. Several grooves present in the enamel surface of untreated teeth became deeper after the bleaching procedure. The depths of the grooves increased in each case. The increase in the depth of grooves was more pronounced in the case of the 30% H 2 O 2 solution. Conclusion : Home-use bleaching agents are capable of causing enamel surface alterations. It is hypothesized that the peroxide-containing bleaching agents affect the organic phase of enamel. Peroxides can affect not only the surface but also the inner structure of enamel. As a result of its low molecular weight, hydrogen peroxide can penetrate into the enamel. Thus, inner oxidative effects are more likely to occur in the subsurface enamel where more organic material is present and oxidation is capable of altering the outer enamel and the surface.
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- 1999
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16. Luminescence quenching by long range electron transfer: A probe of protein clustering and conformation at the cell surface
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Attila Jenei, Michael Edidin, János Matkó, and Taiyin Wei
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Nitroxide mediated radical polymerization ,Protein Conformation ,Surface Immunoglobulin ,Biophysics ,Electrons ,Conjugated system ,Pathology and Forensic Medicine ,Immunoglobulin Fab Fragments ,Electron transfer ,Endocrinology ,Receptors, Transferrin ,Tumor Cells, Cultured ,Humans ,Elméleti orvostudományok ,Cell Line, Transformed ,Fluorescent Dyes ,B-Lymphocytes ,Quenching (fluorescence) ,Chemistry ,Histocompatibility Antigens Class I ,Antibodies, Monoclonal ,Membrane Proteins ,Orvostudományok ,Cell Biology ,Hematology ,Fibroblasts ,Flow Cytometry ,Fluorescence ,Spectrometry, Fluorescence ,Energy Transfer ,Membrane protein ,Luminescent Measurements ,Spin Labels ,Luminescence - Abstract
Quenching of luminescence from fluorescent and phosphorescent probes by nitroxide spin labels with a long range electron transfer (LRET) mechanism (44,45) has been tested as a tool to monitor association/clustering and conformational changes of cell surface proteins. The membrane proteins were labeled with monoclonal antibodies or Fab fragments conjugated with luminescent probes or water-soluble nitroxide spin labels. The method was tested as a probe of 3 different aspects of protein-protein association involving class I MHC molecules: (1) interaction between the heavy and light chains of the MHC molecules, (2) clustering, self-association of MHC molecules, (3) proximity of MHC molecules to transferrin receptors of fibroblasts or surface immunoglobulin molecules of B lymphoblasts. The extent of quenching upon increasing the fractional density of the quencher was sensitive for protein association in accordance with earlier immunoprecipitation and flow cytometric Forster-type energy transfer (FCET) data obtained on the same cells. These data suggest that the LRET quenching can be used as intra- or intermolecular ruler in a 0.5–2.5 nm distance range. This approach is simpler (measurements only on donor side) and faster than many other experimental techniques in screening physical association or conformational changes of membrane proteins by means of spectrofluorimetry, flow cytometry, or microscope based imaging. © 1995 Wiley-Liss, Inc.
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- 1995
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17. Comparative study of the three different fluorophore antibody conjugation strategies
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Adrienn Bagosi, Attila Jenei, János Szöllősi, and Dilip Shrestha
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Fluorophore ,Immunoconjugates ,Antibody Affinity ,Carbohydrates ,Conjugated system ,Biochemistry ,Analytical Chemistry ,Cell Line ,chemistry.chemical_compound ,Animals ,Humans ,Sulfhydryl Compounds ,Amines ,Fluorescent Dyes ,biology ,Sodium periodate ,Carbohydrate ,chemistry ,Yield (chemistry) ,biology.protein ,Amine gas treating ,Electrophoresis, Polyacrylamide Gel ,Antibody ,Oxidation-Reduction ,Conjugate - Abstract
The progression in bioconjugational chemistry has significantly contributed to the evolution and success of protein biology. Mainly, antibody chemistry has been a subject of intensive study owing to the expansion of research areas warranted by using various derivatives of conjugated antibodies. Three reactive moieties (amine, sulfhydryl and carbohydrate) in the antibodies are chiefly favored for the conjugational purpose. This feature is known for decades, nevertheless, amine based conjugation is still the most preferred strategy despite the appreciation the other two methods receive in conserving the antigen binding affinity (ABA). No single report has been published, according to our knowledge, where these three conjugation strategies were applied to the same fluorophore antibody systems. In this study, we evaluated conjugation yield, time demand and cost efficiency of these conjugation procedures. Our results showed that amine based conjugations was by far the best technique due to its simplicity, rapidity, ease of operation, higher conjugate yield, cheaper cost and potential for larger fluorophore/protein labeling ratio without having much effect in ABA. Furthermore, sulfhydryl labeling clearly excelled in terms of reduced non-specific binding and mild effect in ABA but was usually complicated by an asymmetric antibody reduction due to mercaptoethylamine while carbohydrate oxidation based strategy performed the worst during our experiment.
- Published
- 2012
18. Angiotensin II increases the permeability and PV-1 expression of endothelial cells
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János Péter Nagy, Adrienn Németh, Csaba Bödör, Attila Jenei, Attila Sebe, Borbála Végh, László Rosivall, and Shahrokh MirzaHosseini
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medicine.medical_specialty ,Endothelium ,Physiology ,p38 mitogen-activated protein kinases ,Biology ,Caveolae ,Receptor, Angiotensin, Type 1 ,Capillary Permeability ,Mediator ,Internal medicine ,Renin–angiotensin system ,medicine ,Human Umbilical Vein Endothelial Cells ,Humans ,Receptor ,Cells, Cultured ,Effector ,Angiotensin II ,Endothelial Cells ,Gene Expression Regulation, Developmental ,Membrane Proteins ,Cell Biology ,Protein Transport ,Endocrinology ,medicine.anatomical_structure ,Endothelium, Vascular ,Carrier Proteins - Abstract
Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10−7M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.
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- 2011
19. Bare lymphocyte syndrome: an opportunity to discover our immune system
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Dilip Shrestha, János Szöllosi, and Attila Jenei
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Transcriptional Activation ,education ,Immunology ,Antigen presentation ,Human leukocyte antigen ,Major histocompatibility complex ,Regenerative Medicine ,Immune system ,Antigen ,HLA Antigens ,Transplantation Immunology ,hemic and lymphatic diseases ,CIITA ,medicine ,Immunology and Allergy ,Animals ,Humans ,Elméleti orvostudományok ,Antigen Presentation ,biology ,Bare lymphocyte syndrome ,Orvostudományok ,Transporter associated with antigen processing ,medicine.disease ,Immune System ,biology.protein ,Severe Combined Immunodeficiency - Abstract
Bare lymphocyte syndrome (BLS) is a rare immunodeficiency disorder manifested by the partial or complete disappearance of major histocompatibility complex (MHC) proteins from the surface of the cells. Based on this specific feature, it is categorized into three different types depending on which type of MHC protein is affected. These proteins are mainly involved in generating the effective immune responses by differentiating 'self' from 'non-self' antigens through a process referred to as antigen presentation. Investigations on BLS have immensely contributed to our understanding of the transcriptional regulation of these molecules and have led to the discovery of several important proteins of the antigen presentation pathway. Reviews on this subject consistently project type II BLS, MHC II deficiency as BLS syndrome, although literatures' document cases of other types of BLS too. Therefore, in this article, we have assembled information on the BLS syndrome to produce a systematic narration while emphasizing the importance of BLS system in studying various aspects of immune biology.
- Published
- 2011
20. NMR spectroscopic elucidation of the structure and stereochemistry of tricyclic 3-styrylpyrazolines
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Gábor, Tóth, András, Simon, Attila, Jenei, József, Jeko, and Albert, Lévai
- Abstract
Diastereomeric mixtures of tricyclic 3-styrylpyrazolines have been prepared by the reaction of 3-cynnamylidenechroman-4-ones and their 1-thio analogs with hydrazine in hot acetic acid or propionic acid solutions. The diastereomeric mixtures were separated by column chromatography to obtain the pure diastereomers. The elucidation of their structure and stereochemistry and complete (1)H and (13)C assignments have been performed by a combination of various one- and two-dimensional NMR experiments.
- Published
- 2008
21. Effects of vascular endothelial growth factor (VEGF) and angiotensin II (Ang II) on endothelial nano‐channels
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Shahrokh Mirzahosseini, Zsófia Balogh, András Masszi, János Nagy, Csaba Bödör, László Rosivall, and Attila Jenei
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Angiotensin receptor ,medicine.medical_specialty ,biology ,Chemistry ,VEGF receptors ,Biochemistry ,Angiotensin II ,Vascular endothelial growth factor ,chemistry.chemical_compound ,Vascular endothelial growth factor A ,Endocrinology ,Vascular endothelial growth factor C ,Internal medicine ,Genetics ,medicine ,biology.protein ,Molecular Biology ,Biotechnology - Published
- 2008
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22. [In vitro study of the effects of 10-30 % hydrogen peroxide solutions on the surface of human tooth enamel]
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Tamás, Bistey, Attila, Jenei, Zsuzsa, Szondy, P István, Nagy, and Csaba, Hegedus
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Solutions ,Dental Enamel Solubility ,Dose-Response Relationship, Drug ,Surface Properties ,Anti-Infective Agents, Local ,Dental Enamel Permeability ,Humans ,Hydrogen Peroxide ,Sulfhydryl Compounds ,In Vitro Techniques ,Dental Enamel ,Microscopy, Atomic Force ,Tooth - Abstract
The aim of the present study was to investigate the effects of hydrogen peroxide solutions (10-30%) on the enamel surface and the inner structure of human teeth. Prepared enamel samples were exposed to various concentrations of hydrogen peroxide solutions for one hour. Changes in the enamel were analyzed by two different methods. The inner structure was studied by the Ellman's reagent to detect possible changes in the amount of free thiol groups in the remaining organic phase of matured enamel. Surface alterations were imaged by atomic force microscope. Ellman's reaction revealed the presence of three groups of free thiols: one with easy, the second one with medium and the third one with difficult accessibility by hydrogen peroxide. Atomic force microscopy revealed severe alterations on the enamel surface after treatment with both low and high concentration of hydrogen peroxide. Our observations indicated that hydrogen peroxide induced alterations in the surface structure and in the inner phases of mature enamel in both low and high concentrations.
- Published
- 2006
23. Non-Random Patterns of Membrane Proteins and Their Roles in Transmembrane Signaling
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György Vámosi, Sándor Damjanovich, Andrea Bodnár, Katalin Tóth, Attila Jenei, and László Mátyus
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biology ,Chemistry ,Membrane transport protein ,Membrane lipids ,Lipid microdomain ,Cell membrane ,medicine.anatomical_structure ,Membrane ,Membrane protein ,medicine ,biology.protein ,Biophysics ,Integral membrane protein ,Lipid raft - Abstract
The plasma membrane is the first line of encounter with any “own” or “foreign” molecule or hostile “visitor”. Our knowledge of the structure and function of the plasma membrane of mammalian cells was greatly advanced by the famous experiment of Frye and Edidin in 1970, which showed that distinct molecular elements of the surface of mouse and human lymphocytes labeled by green and red fluorescent dyes, respectively, were mixed upon fusing the two cell types. This finding was instrumental in the construction of the Singer–Nicolson fluid mosaic membrane model in 1972, which suggested the free mobility of proteins and other macromolecules in the plasma membrane (Singer and Nicolson 1972). Experimental observations over past decades have led to the “membrane microdomain” concept describing compartmentalization/organization of membrane components into non-random, well-defined patterns. According to recent knowledge, the lateral order of membrane lipids and proteins is a general phenomenon that may have fundamental importance in the function of individual molecules as well as in that of the whole plasma membrane (Edidin 1993, 2001; Vereb et al. 2003). The primary target of external stimuli is the plasma membrane, and nonrandom co-distribution of membrane proteins plays an important role in signal transduction across the cell membrane. The generalized occurrence of such cell-surface patterns of membrane proteins was suggested at the beginning of the 1980s (Damjanovich et al. 1981). Signal transduction processes are often accompanied by dynamic rearrangement of the two-dimensional patterns of the macromolecular constituents at the cell surface (Damjanovich et al. 1992). The structured, and at the same time dynamic, nature of the plasma membrane, i.e., the organization of its components into structures existing at different time and size scales, allows accumulation of the relevant molecules (and exclusion of others) needed for the appropriate response (Vereb et al. 1995, 2003; Edidin 2001). Protein clusters generated by the physical association/molecular proximity of the molecules (nanometer or small-scale clusters) define the basic organization level of membrane proteins (Damjanovich et al. 1998). Different types of such protein clusters can be present in the plasma membrane (Damjanovich et al. 1997b): (1) in many cases, a given “functional unit” is formed by several components/subunits (e. g., the TCR/CD3 complex; Klausner et al. 1990); (2) rearrangeNon-Random Patterns of Membrane Proteins and Their Roles in Transmembrane Signaling
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- 2005
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24. Computer program for analyzing donor photobleaching FRET image series
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Andrea Bodnár, György Vereb, János Matkó, Gergely Szentesi, László Mátyus, Attila Jenei, Sándor Damjanovich, Ákos Fábián, Rezső Gáspár, and Gábor Horváth
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Image Series ,Histology ,Photobleaching ,Pixel ,business.industry ,Computer science ,Digital imaging ,Cell Biology ,computer.file_format ,Pathology and Forensic Medicine ,Förster resonance energy transfer ,Software ,Raw image format ,Histogram ,Cell Line, Tumor ,Histocompatibility Antigens ,Fluorescence Resonance Energy Transfer ,Humans ,business ,Biological system ,beta 2-Microglobulin ,computer ,Algorithms - Abstract
Background The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. Methods New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (β2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. Results The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. Conclusion In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. © 2005 International Society for Analytical Cytology
- Published
- 2005
25. IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells
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Thomas A. Waldmann, Jörg Langowski, János Szöllosi, Carolyn K. Goldman, György Vereb, Katalin Tóth, László Mátyus, Sándor Damjanovich, Attila Jenei, György Vámosi, and Andrea Bodnár
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T cell ,Protein subunit ,T-Lymphocytes ,Biology ,Lymphoma, T-Cell ,Membrane Microdomains ,Heterotrimeric G protein ,Cell Line, Tumor ,MHC class I ,medicine ,Fluorescence Resonance Energy Transfer ,Humans ,Elméleti orvostudományok ,Receptor ,Lipid raft ,HLA-D Antigens ,Multidisciplinary ,Receptors, Interleukin-15 ,Histocompatibility Antigens Class I ,Models, Immunological ,Antibodies, Monoclonal ,Receptors, Interleukin-2 ,Orvostudományok ,Biological Sciences ,Flow Cytometry ,Cell biology ,Protein Subunits ,Förster resonance energy transfer ,medicine.anatomical_structure ,Spectrometry, Fluorescence ,biology.protein ,Signal transduction ,Signal Transduction - Abstract
The private alpha-chains of IL-2 and IL-15 receptors (IL-2R and IL-15R) share the signaling beta- and gamma(c)-subunits, resulting in both common and contrasting roles of IL-2 and IL-15 in T cell function. Knowledge of the cytokine-dependent subunit assembly is indispensable for understanding the paradox of distinct signaling capacities. By using fluorescence resonance energy transfer and confocal microscopy, we have shown that IL-2R alpha, IL-15R alpha, IL-2/15R beta and gamma(c)-subunits, as well as MHC class I and II glycoproteins formed supramolecular receptor clusters in lipid rafts of the T lymphoma line Kit 225 FT7.10. Fluorescence crosscorrelation microscopy demonstrated the comobility of IL-15R alpha with IL-2R alpha and MHC class I. A model was generated for subunit switching between IL-2R alpha and IL-15R alpha upon the binding of the appropriate cytokine resulting in the formation of high-affinity heterotrimeric receptors. This model suggests a direct role for the alpha-subunits, to which no definite function has been assigned so far, in tuning cellular responses to IL-2 or IL-15. In addition, both alpha-chains were at least partially homodimerized/oligomerized, which could be the basis of distinct signaling pathways by the two cytokines.
- Published
- 2004
26. Computer program for determining fluorescence resonance energy transfer efficiency from flow cytometric data on a cell-by-cell basis
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György Vámosi, Attila Jenei, Imre Bori, Sándor Damjanovich, László Mátyus, Gábor Horváth, János Szöllosi, Rezsö Gáspár, and Gergely Szentesi
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Computer science ,Cell ,Population ,Health Informatics ,Gating ,Bioinformatics ,Flow cytometry ,Cell membrane ,Histogram ,medicine ,Fluorescence Resonance Energy Transfer ,Humans ,Computer Simulation ,Elméleti orvostudományok ,education ,Flow Cytometry Standard ,education.field_of_study ,medicine.diagnostic_test ,Basis (linear algebra) ,Computer program ,Orvostudományok ,Flow Cytometry ,Computer Science Applications ,Autofluorescence ,Förster resonance energy transfer ,medicine.anatomical_structure ,Algorithm ,Algorithms ,Software - Abstract
The determination of fluorescence resonance energy transfer (FRET) with flow cytometry (FCET) is one of the most efficient tools to study the proximity relationships of cell membrane components in cell populations on a cell-by-cell basis. Because of the high amount of data and the relatively tedious calculations, this procedure should be assisted by powerful data processing software. The currently available programs are not able to fulfill this requirement. We developed a Windows-based program to calculate fluorescence resonance energy transfer efficiency values from list mode flow cytometry standard (FCS) files. This program displays the measured data in standard plots by generating one- and two-parameter histograms on linear or logarithmic scales. A graphical gating tool allows the user to select the desired cell population according to any combination of the parameter values. The program performs several statistical calculations, including mean, S.D., percent of the gated data. We have implemented two types of data sheet for FRET calculations to aid and guide the user during the analysis: one with population-mean-based autofluorescence correction and the other with spectrum-based cell-by-cell autofluorescence correction. In this paper, we describe the gating algorithms, the file opening procedure and the rules of gating. The structure of the program and a short description of the graphical user-interface (GUI) are also presented in this article.
- Published
- 2003
27. Scanning Near-Field Optical Imaging and Spectroscopy in Cell Biology
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Thomas M. Jovin, Attila Jenei, G. Durack, Achim K. Kirsch, Vinod Subramaniam, and J. P. Robinson
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Optical imaging ,Scanning ion-conductance microscopy ,Scanning confocal electron microscopy ,Biophysics ,Nanotechnology ,Near and far field ,Spectroscopy - Published
- 2003
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28. Class IHLA oligomerization at the surface of B cells is controlled by exogenous beta2-microglobulin: implications in activation of cytotoxic T lymphocytes
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Michael Edidin, János Matkó, Thomas M. Jovin, Sándor Damjanovich, Attila Jenei, Andrea Bodnár, and Zsolt Bacsó
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B-Lymphocytes ,Chemistry ,Beta-2 microglobulin ,T cell ,Immunology ,T-cell receptor ,Histocompatibility Antigens Class I ,General Medicine ,Human leukocyte antigen ,Orvostudományok ,Molecular biology ,CTL ,medicine.anatomical_structure ,Adjuvants, Immunologic ,medicine ,Immunology and Allergy ,Cytotoxic T cell ,Humans ,Elméleti orvostudományok ,Cytotoxicity ,beta 2-Microglobulin ,CD8 ,T-Lymphocytes, Cytotoxic - Abstract
Submicroscopic molecular clusters (oligomers) of class I HLA have been detected by physical techniques [e.g. fluorescence resonance energy transfer (FRET) and single particle tracking of molecular diffusion] at the surface of various activated and transformed human cells, including B lymphocytes. Here, the sensitivity of this homotypic association to exogenous beta(2)-microglobulin (beta(2)m) and the role of free heavy chains (FHC) in class I HLA oligomerization were investigated on a B lymphoblastoid cell line, JY. Scanning near-field optical microscopy and FRET data both demonstrated that FHC and class I HLA heterodimers are co-clustered at the cell surface. Culturing the cells with excess beta(2)m resulted in a reduced co-clustering and decreased molecular homotypic association, as assessed by FRET. The decreased HLA clustering on JY target cells (antigen-presenting cells) was accompanied with their reduced susceptibility to specific lysis by allospecific CD8(+) cytotoxic T lymphocytes (CTL). JY B cells with reduced HLA clustering also provoked significantly weaker T cell activation signals, such as lower expression of CD69 activation marker and lower magnitude of TCR down-regulation, than did the untreated B cells. These results together suggest that the actual level of beta(2)m available at the cell surface can control CTL activation and the subsequent cytotoxic effector function through regulation of the homotypic HLA-I association. This might be especially important in some inflammatory and autoimmune diseases where elevated serum beta(2)m levels are reported.
- Published
- 2003
29. Signal transduction in T lymphocytes and aging
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Sándor Damjanovich, László Mátyus, Attila Jenei, Rezsö Gáspár, and László Bene
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Cell type ,Cell signaling ,Aging ,T-Lymphocytes ,Cell Biology ,Orvostudományok ,Biology ,Lymphocyte Activation ,Biochemistry ,Peripheral blood ,Subclass ,Cell biology ,Endocrinology ,Immune system ,Immune System ,Genetics ,Humans ,Elméleti orvostudományok ,Signal transduction ,Biological regulation ,Molecular Biology ,Neuroscience ,Aged ,Signal Transduction - Abstract
Subclasses of cells in different compartments of the immune system possesses all those attributes, that make them suitable though somewhat limited models for the investigation of cellular processes during aging. Blood samples provide relative easily high amount of cells belonging to the same subclass, all of them having complex cascade processes in their signal transduction mechanisms, therefore being excellent targets for such investigations. One such subclass comprises peripheral blood lymphocytes. The signal-transduction cascade across the plasma membrane of lymphocytes displays many of the general features enabling us to draw conclusions for other cellular signaling problems that may arise during aging in other cell types not directly related to the immune system. The advantage of this approach lies in the fact that sometimes it is extremely difficult to study signal transduction processes in certain cell types under physiological conditions. The simultaneous occurrence of physical, chemical and molecular biological regulation of the immune processes at cellular and network levels make them very good examples for focusing our interest also on similar processes in other systems and cells. The fast developing new measuring techniques and the rapidly accumulating experimental data make it relatively easy to provide interesting new aspects, and ideas in this field. Finally, the immune system itself has its great importance and after all, it has an obvious declination with aging, the immune-senescence.
- Published
- 2003
30. Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes
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Rezsö Gáspár, Thomas A. Waldmann, Sándor Varga, László Mátyus, Andrea Bodnár, Gergely Szentesi, György Vámosi, Sándor Damjanovich, Attila Jenei, Gyorgy Panyi, and Miklós Bagdány
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Potassium Channels ,CD3 Complex ,T-Lymphocytes ,Transfection ,law.invention ,Cell membrane ,Epitopes ,Jurkat Cells ,Mice ,law ,medicine ,Fluorescence Resonance Energy Transfer ,Animals ,Humans ,Elméleti orvostudományok ,Membrane potential ,Multidisciplinary ,Kv1.3 Potassium Channel ,Microscopy, Confocal ,Models, Statistical ,Chemistry ,Cell Membrane ,Colocalization ,Immunogold labelling ,Orvostudományok ,Biological Sciences ,Immunohistochemistry ,Potassium channel ,Cell biology ,Electrophysiology ,Microscopy, Electron ,Förster resonance energy transfer ,medicine.anatomical_structure ,Membrane ,Potassium Channels, Voltage-Gated ,Electron microscope - Abstract
Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C= 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E= 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.
- Published
- 2003
31. Organization of the glycoprotein (GP) IIb/IIIa heterodimer on resting human platelets studied by flow cytometric energy transfer
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Marı́a V. Alvarez, Sándor Damjanovich, László Muszbek, Jolan Harsfalvi, László Mátyus, László Bene, Attila Jenei, and J. González-Rodríguez
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Blood Platelets ,Protein subunit ,Biophysics ,Platelet Glycoprotein GPIIb-IIIa Complex ,Fibrinogen ,Mice ,Protein structure ,medicine ,Animals ,Humans ,Radiology, Nuclear Medicine and imaging ,Platelet ,Elméleti orvostudományok ,Binding site ,chemistry.chemical_classification ,Radiation ,Radiological and Ultrasound Technology ,Antibodies, Monoclonal ,Orvostudományok ,Flow Cytometry ,Molecular biology ,Förster resonance energy transfer ,chemistry ,Energy Transfer ,Glycoprotein ,Glycoprotein IIb/IIIa ,Dimerization ,medicine.drug - Abstract
Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.
- Published
- 2001
32. Cholesterol-dependent clustering of IL-2Rα and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts
- Author
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E. Magyar, Rezső Gáspár, György Vámosi, S. M. Ibrahim, János Matkó, Thomas A. Waldmann, Sándor Varga, János Szöllősi, György Vereb, Sándor Damjanovich, and Attila Jenei
- Subjects
Membrane Fluidity ,Membrane lipids ,T-Lymphocytes ,Transferrin receptor ,Biology ,CD48 Antigen ,Lymphoma, T-Cell ,Filipin ,chemistry.chemical_compound ,Membrane Lipids ,Antigens, CD ,HLA Antigens ,Membrane fluidity ,Tumor Cells, Cultured ,Humans ,Microscopy, Immunoelectron ,Lipid raft ,Multidisciplinary ,Microscopy, Confocal ,Colocalization ,Receptors, Interleukin-2 ,Immunogold labelling ,Biological Sciences ,Immunohistochemistry ,Cell biology ,Neoplasm Proteins ,Cholesterol ,chemistry ,lipids (amino acids, peptides, and proteins) ,Receptor clustering - Abstract
Immunogold staining and electron microscopy show that IL-2 receptor alpha-subunits exhibit nonrandom surface distribution on human T lymphoma cells. Analysis of interparticle distances reveals that this clustering on the scale of a few hundred nanometers is independent of the presence of IL-2 and of the expression of the IL-2R beta-subunit. Clustering of IL-2Ralpha is confirmed by confocal microscopy, yielding the same average cluster size, approximately 600-800 nm, as electron microscopy. HLA class I and II and CD48 molecules also form clusters of the same size. Disruption of cholesterol-rich lipid rafts with filipin or depletion of membrane cholesterol with methyl-beta-cyclodextrin results in the blurring of cluster boundaries and an apparent dispersion of clusters for all four proteins. Interestingly, the transferrin receptor, which is thought to be located outside lipid rafts, exhibits clusters that are only 300 nm in size and are less affected by modifying the membrane cholesterol content. Furthermore, transferrin receptor clusters hardly colocalize with IL-2Ralpha, HLA, and CD48 molecules (crosscorrelation coefficient is 0.05), whereas IL-2Ralpha colocalizes with both HLA and CD48 (crosscorrelation coefficient is between 0.37 and 0.46). This coclustering is confirmed by electron microscopy. The submicron clusters of IL-2Ralpha chains and their coclustering with HLA and CD48, presumably associated with lipid rafts, could underlie the efficiency of signaling in lymphoid cells.
- Published
- 2000
33. Complexity of signal transduction mediated by ErbB2: clues to the potential of receptor-targeted cancer therapy
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Péter Nagy, Thomas M. Jovin, Sándor Damjanovich, Attila Jenei, and J. Szoelloesi
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Cancer Research ,Receptor, ErbB-2 ,Antineoplastic Agents ,Breast Neoplasms ,Biology ,Antibodies, Monoclonal, Humanized ,Pathology and Forensic Medicine ,Breast cancer ,Growth factor receptor ,Neoplasms ,medicine ,Animals ,Humans ,ERBB3 ,Epidermal growth factor receptor ,Kinase activity ,skin and connective tissue diseases ,neoplasms ,ERBB4 ,Antibodies, Monoclonal ,General Medicine ,Genetic Therapy ,Genes, erbB-2 ,Trastuzumab ,medicine.disease ,Oncology ,Immunology ,Cancer research ,biology.protein ,Female ,Signal transduction ,Tyrosine kinase ,Signal Transduction - Abstract
The erbB2 oncogene belongs to the type I trans-membrane tyrosine kinase family of receptors. Its medical importance stems from its widespread over-expression in breast cancer. This review will focus on the signal transduction through this protein, and explains how the overexpression of erbB2 may result in poor prognosis of breast cancer, and finally it will summerize our current understanding about the therapeutic potential of receptor-targeted therapy in breast cancer. ErbB2 does not have any known ligand which is able to bind to it with high affinity. However the kinase activity of erbB2 can be activated without any ligand, if it is overexpressed, and by heteroassociation with other members of the erbB family (erbB1 or epidermal growth factor receptor, erbB3 and erbB4). This interaction substantially increases the efficiency and diversity of signal transduction through these receptor complexes. In addition, erbB2 forms large scale receptor clusters containing hundreds of proteins. These receptor islands may take part in recruiting cytosolic factors which relay the signal towards the nucleus or the cytoplasm. Overexpression of erbB2 was linked to higher transforming activity, increased metastatic potential, angiogenesis and drug resistence of breast tumor in laboratory experiments. As a corollary of these properties, erbB2 amplification is generally thought to be associated with a poor prognosis in breast cancer patients. These early findings lead to the development of antibodies that down-regulate erbB2. Such a therapeutic approach has already been found effective in experimental tumor models and in clinical trials as well. Further understanding of the importance of erbB2 and growth factor receptors in the transformation of normal cells to malignant ones may once give us a chance to cure erbB2 over-expressing breast cancer.
- Published
- 1999
34. Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy
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Thomas M. Jovin, Sándor Damjanovich, Péter Nagy, Achim K. Kirsch, János Szöllosi, and Attila Jenei
- Subjects
medicine.drug_class ,Receptor, ErbB-2 ,CHO Cells ,Biology ,Microscopy, Atomic Force ,Tyrosine-kinase inhibitor ,Receptor tyrosine kinase ,chemistry.chemical_compound ,Epidermal growth factor ,Cricetinae ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,ERBB3 ,Elméleti orvostudományok ,Enzyme Inhibitors ,skin and connective tissue diseases ,Receptor ,neoplasms ,Microscopy, Confocal ,Receptor Protein-Tyrosine Kinases ,Tyrosine phosphorylation ,Cell Biology ,Orvostudományok ,Molecular biology ,Cell biology ,Enzyme Activation ,ErbB Receptors ,chemistry ,biology.protein ,Quinazolines ,Neuregulin ,Signal transduction - Abstract
ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family, is often overexpressed in breast cancer and other malignancies. ErbB2 homodimerizes but also presents as a common auxiliary subunit of the EGF and heregulin receptors (erbB1 or EGFR; and erbB3-4, respectively), with which it heteroassociates. ErbB2 is generally regarded as an orphan (ligand-less) receptor with a very potent kinase domain activated either via its associated partners or constitutively as a consequence of discrete mutations. It follows that the extent and regulation of its cell surface interactions are of central importance. We have studied the large-scale association pattern of erbB2 in quiescent and activated cells labeled with fluorescent anti-erbB2 monoclonal antibodies using scanning near-field optical microscopy (SNOM). ErbB2 was found to be concentrated in irregular membrane patches with a mean diameter of approx. 0.5 microm in nonactivated SKBR3 and MDA453 human breast tumor cells. The average number of erbB2 proteins in a single cluster on nonactivated SKBR3 cells was about 10(3). Activation of SKBR3 cells with EGF, heregulin as well as a partially agonistic anti-erbB2 monoclonal antibody led to an increase in the mean cluster diameter to 0.6-0.9 microm, irrespective of the ligand. The EGF-induced increase in the erbB2 cluster size was inhibited by the EGFR-specific tyrosine kinase inhibitor PD153035. The average size of erbB2 clusters on the erbB2-transfected line of CHO cells (CB2) was similar to that of activated SKBR3 cells, a finding correlated with the increased base-line tyrosine phosphorylation of erbB2 in cells expressing only erbB2. We conclude that an increase in cluster size may constitute a general phenomenon in the activation of erbB2.
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- 1999
35. Picosecond multiphoton scanning near-field optical microscopy
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Donna J. Arndt-Jovin, Thomas M. Jovin, Achim K. Kirsch, Attila Jenei, and Vinod Subramaniam
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Materials science ,Ultraviolet Rays ,Biophysics ,Antibodies ,Chromosomes ,law.invention ,Optics ,Two-photon excitation microscopy ,Optical microscope ,law ,Tumor Cells, Cultured ,Animals ,Humans ,Elméleti orvostudományok ,Fluorescent Dyes ,Photons ,business.industry ,Lasers ,Orvostudományok ,Laser ,Fluorescence ,Photobleaching ,Mitochondria ,Drosophila melanogaster ,Microscopy, Fluorescence ,Picosecond ,Near-field scanning optical microscope ,business ,Excitation ,Research Article - Abstract
We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5–40mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.
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- 1999
36. Fluorescence resonance energy transfer detected by scanning near-field optical microscopy
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Achim K. Kirsch, Thomas M. Jovin, Vinod Subramaniam, Attila Jenei, and Executive board Vrije Universiteit
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[Cell-surface proteins ,Histology ,Analytical chemistry ,Fizikai tudományok ,Fluorescence ,Pathology and Forensic Medicine ,law.invention ,Mice ,Természettudományok ,Optical microscope ,law ,Lectins ,Animals ,SDG 7 - Affordable and Clean Energy ,Fluorescent Dyes ,Membrane Glycoproteins ,Microscopy, Confocal ,business.industry ,Chemistry ,[Cell-surface proteins, Fluorescence resonance ene ,3T3 Cells ,Photobleaching ,Acceptor ,Spectrometry, Fluorescence ,Förster resonance energy transfer ,Excited state ,Optoelectronics ,Near-field scanning optical microscope ,Fluorescence resonance ene ,business ,Fluorescence anisotropy - Abstract
Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the Förster mechanism over a range of 1-10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near-field optical microscope (SNOM) operated in the shared-aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel-by-pixel energy transfer efficiency measurements using SNOM.
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- 1999
- Full Text
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37. Synthesis of spiro-1-pyrazolines by the reaction of exocyclic α,β,γ,δ-unsaturated ketones with diazomethane
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Albert Lévai, Albert Lévai, András Simon, Attila Jenei, Gyula Kálmán, József Jekő, Gábor Tóth, Albert Lévai, Albert Lévai, András Simon, Attila Jenei, Gyula Kálmán, József Jekő, and Gábor Tóth
- Abstract
ARKIVOC: vol. 2009, no. 12, (issn) 1551-7012, (dlps) 5550190.0010.c14, This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 License. Please contact mpub-help@umich.edu to use this work in a way not covered by the license.
- Published
- 2009
38. Flow Cytometric Membrane Potential Measurements
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Carlo Pieri, Rezsö GáspárJr., László Bene, Sándor Damjanovich, and Attila Jenei
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Membrane potential ,Materials science ,Flow (mathematics) ,Measure (physics) ,Biological system - Abstract
Before deciding to use a certain technique to measure membrane potentials, a critical comparison of the available techniques is required.Therefore, we will first briefly describe three different ways to measure membrane potential.
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- 1998
- Full Text
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39. An Introduction to the Working Principles of the Flow Cytometer
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Rezsö GáspárJr., Carlo Pieri, László Bene, Attila Jenei, and Sándor Damjanovich
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education.field_of_study ,Engineering ,Data acquisition ,Flow (mathematics) ,business.industry ,Population ,Electrical engineering ,Sound analysis ,Electronic engineering ,business ,education - Abstract
Flow cytometry is a very fast optical (spectroscopic) analysis of cells on a cell-by-cell basis. The measurement, data acquisition and analysis were made possible by an innovative application of optical spectroscopy and high tech electronics, used originally in nuclear physics. A unique characteristic of the method is the analysis of individual cells at such a high speed that biological variations and their distribution over a conveniently large cell population are accessible to statistically sound analysis within a time scale of minutes among practically physiological conditions of the cells (Shapiro 1985; Ormerod 1994).
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- 1998
- Full Text
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40. HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells
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László Mátyus, Zsolt Bacsó, Andrea Bodnár, Rezső Gáspár, Sándor Damjanovich, Carlo Pieri, László Bene, Sándor Varga, Attila Jenei, and Tibor Farkas
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MHC class II ,Multidisciplinary ,biology ,CD74 ,Cell Membrane ,Histocompatibility Antigens Class I ,Histocompatibility Antigens Class II ,Human leukocyte antigen ,Immunogold labelling ,Biological Sciences ,Major histocompatibility complex ,Cell biology ,Microscopy, Electron ,Förster resonance energy transfer ,Antigen ,MHC class I ,biology.protein ,Tumor Cells, Cultured ,Humans ,Lymphocytes - Abstract
Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.
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- 1997
41. Modification of membrane cholesterol level affects expression and clustering of class I HLA molecules at the surface of JY human lymphoblasts
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László Bene, Andrea Bodnár, Attila Jenei, Sándor Damjanovich, and János Matkó
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Cholesterol ,Effector ,Lymphoblast ,Cell Membrane ,Histocompatibility Antigens Class I ,Immunology ,Antigen presentation ,Orvostudományok ,Lymphocyte Activation ,Epitopes ,chemistry.chemical_compound ,Förster resonance energy transfer ,chemistry ,Biochemistry ,Homoassociation ,Membrane fluidity ,Biophysics ,Humans ,Immunology and Allergy ,Lymphocytes ,Elméleti orvostudományok ,Fluorescence anisotropy ,Cell Line, Transformed - Abstract
Recently we have found that class I HLA molecules, key elements of the antigen presentation system for CD8 - effector cells, show a clustered lateral distribution (homoassociation) at the surface of activated human T- and B-lymphocytes as well as virus-transformed T- and B-lymphoblasts, in contrast to a dispere distribution on resting human PBLs (Matko et al. (1994) J. Immunol. 152, 3353; Bene et al. (1994) Eur. J. Immunol. 24, 2115). Expression of β2m-free HLA heavy chains and exogenous β2m have been shown as potential regulation factors of HLA-I clustering, which in turn may affect cytotoxic activity of CD8 + effector cells. Here we report a study on the effect of plasma membrane-modification (by exogenous cholesterol and phosphatidylcholine) on the expression of free HLA heavy chains and β2m-bound HLA-I molecules on JY human B-lymphoblasts. The modulating effect of these two treatments on the lipid fluidity of cells was demonstrated by fluorescence anisotropy of DPH lipid probe. The lateral clustering (association) of HLA-I molecules was detected by flow cytometric fluorescence resonance energy transfer (FCET) and digital imaging microscopic photobleaching energy transfer (pbFRET) methods, using flourescein-isothiocyanate (FITC) (donor)- and tetramethyl-rhodamine-isothiocyanate (TRITC) (acceptor)-labeled W 6 32 or KE2 antibodies directed against intact HLA-I molecules. Cholesterol enrichment of the plasma membrane increased membrane fluidity and reduced the expression of heavy- and light-chain determinants of HLA-I molecules and free heavy chains (FHCs). This was accompanied with a higher degree of HLA-I clustering as shown by the enhanced intermolecular energy transfer efficiency. In contrast, cholesterol depletion resulted in membrane fluidization and increased expression of HLA-I epitopes. Our results suggest that both cholesterol level and lipid structure/fluidity of the plasma membrane in lymphoblastoid cells may also potentially regulate lateral organization and consequently the presentation efficiency of HLA-I molecules.
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- 1996
42. Structural hierarchy in the clustering of HLA class I molecules in the plasma membrane of human lymphoblastoid cells
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A. Schaper, Thomas M. Jovin, Sándor Damjanovich, János Matkó, J. P. P. Starink, G. Q. Fox, György Vereb, Donna J. Arndt-Jovin, and Attila Jenei
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Adult ,Multidisciplinary ,biology ,Antigen presentation ,Cell Membrane ,Histocompatibility Antigens Class I ,Human leukocyte antigen ,Immunogold labelling ,Orvostudományok ,Gold Colloid ,Major histocompatibility complex ,Photobleaching ,Immunohistochemistry ,Cell biology ,Microscopy, Electron ,Förster resonance energy transfer ,Antigen ,MHC class I ,biology.protein ,Tumor Cells, Cultured ,Humans ,Elméleti orvostudományok ,Lymphocytes ,Research Article - Abstract
Major histocompatibility complex (MHC) class I antigens in the plasma membranes of human T (HUT-102B2) and B (JY) lymphoma cells were probed by immunochemical reagents using fluorescence, transmission electron, and scanning force microscopies. Fluorescent labels were attached to monoclonal antibodies W6/32 or KE-2 directed against the heavy chain of HLA class I (A, B, C) and L368 or HB28 against the beta 2-microglobulin light chain. The topological distribution in the nanometer range was studied by photobleaching fluorescence resonance energy transfer (pbFRET) on single cells. A nonrandom codistribution pattern of MHC class I molecules was observed over distances of 2-10 nm. A second, nonrandom, and larger-scale topological organization of the MHC class I antigens was detected by indirect immunogold labeling and imaging by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Although some differences in antigen distribution between the B- and T-cell lines were detected by pbFRET, both cell lines exhibited similar clustering patterns by TEM and SFM. Such defined molecular distributions on the surfaces of cells of the immune system may reflect an underlying specialization of membrane lipid domains and fulfill important functional roles in cell-cell contacts and signal transduction.
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- 1995
43. Analysis of cell surface molecular distributions and cellular signaling by flow cytometry
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László Bene, János Szöllosi, Péter Nagy, László Mátyus, János Matkó, Andrea Bodnár, Sándor Damjanovich, and Attila Jenei
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Cell signaling ,Sociology and Political Science ,medicine.diagnostic_test ,Membrane permeability ,Chemistry ,Clinical Biochemistry ,Biochemistry ,Molecular biology ,Flow cytometry ,Clinical Psychology ,Förster resonance energy transfer ,Cell surface receptor ,medicine ,Biophysics ,Signal transduction ,Law ,Spectroscopy ,Social Sciences (miscellaneous) ,Intracellular ,Ion channel - Abstract
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 (Ca(2+), 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.
- Published
- 1993
44. Mapping of cell surface protein-patterns by combined fluorescence anisotropy and energy transfer measurements
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János Matkó, László Mátyus, Sándor Damjanovich, Marcel Ameloot, and Attila Jenei
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Radiation ,Radiological and Ultrasound Technology ,Chemistry ,Energy transfer ,Cell Membrane ,Biophysics ,Analytical chemistry ,Membrane Proteins ,Fluorescence Polarization ,Orvostudományok ,DEPT ,Models, Theoretical ,Nuclear magnetic resonance ,Spectrometry, Fluorescence ,Energy Transfer ,Animals ,Radiology, Nuclear Medicine and imaging ,Elméleti orvostudományok ,Surface protein ,Fluorescence anisotropy ,Mathematics - Abstract
LIMBURGS UNIV CTR,PHYSIOL RES GRP,B-3590 DIEPENBEEK,BELGIUM.MATKO, J, DEBRECEN UNIV MED,SCH MED,DEPT BIOPHYS,POB 3,H-4012 DEBRECEN,HUNGARY.
- Published
- 1993
45. Cell fusion experiments reveal distinctly different association characteristics of cell-surface receptors
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János Szöllosi, Péter Nagy, Sándor Damjanovich, Attila Jenei, Rezsö Gáspár, János Matkó, Sándor Varga, László Mátyus, Gyorgy Panyi, and Thomas M. Jovin
- Subjects
Receptors, Cell Surface ,Transferrin receptor ,Gold Colloid ,Biology ,Cell Line ,Cell Fusion ,Membrane Microdomains ,Cell surface receptor ,Humans ,Elméleti orvostudományok ,Lipid raft ,Fluorescent Dyes ,G alpha subunit ,Microscopy ,Cell Membrane ,Histocompatibility Antigens Class I ,Receptor Aggregation ,Histocompatibility Antigens Class II ,Receptors, Interleukin-2 ,Cell Biology ,Orvostudományok ,Photobleaching ,Cell biology ,Membrane ,Förster resonance energy transfer ,Energy Transfer ,Microscopy, Fluorescence ,Membrane protein - Abstract
The existence of small- and large-scale membrane protein clusters, containing dimers, oligomers and hundreds of proteins, respectively, has become widely accepted. However, it is largely unknown whether the internal structure of these formations is dynamic or static. Cell fusion was used to perturb the distribution of existing membrane protein clusters, and to investigate their mobility and associations. Scanning near-field optical microscopy, confocal and electron microscopy were applied to detect the exchange of proteins between large-scale protein clusters, whereas photobleaching fluorescence energy transfer was used to image the redistribution of existing small-scale membrane protein clusters. Large-scale clusters of major histocompatibility complex (MHC)-I exchanged proteins with each other and with MHC-II clusters. Similarly to MHC-I, large-scale MHC-II clusters were also dynamic. Exchange of components between small-scale protein clusters was not universal: intermixing did not take place in the case of MHC-II homoclusters; however, it was observed for homoclusters of MHC-I and for heteroclusters of MHC-I and MHC-II. These processes required a fluid state of the plasma membrane, and did not depend on endocytosis-mediated recycling of proteins. The redistribution of large-scale MHC-I clusters precedes the intermixing of small-scale clusters of MHC-I indicating a hierarchy in protein association. Investigation of a set of other proteins (α subunit of the interleukin 2 receptor, CD48 and transferrin receptor) suggested that a large-scale protein cluster usually exchanges components with the same type of clusters. These results offer new insight into processes requiring time-dependent changes in membrane protein interactions.
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