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Attila Jenei

Publications and source records attributed to Attila Jenei.

8 recordsLinked to original sources

Steady-state fluorescence quenching applications for studying protein structure and dynamics.

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.

Acrylamide↗

Computer program for analyzing donor photobleaching FRET image series.

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 (beta2-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. .

Algorithms↗

[In vitro study of the effects of 10-30 % hydrogen peroxide solutions on the surface of human tooth enamel].

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.

Anti-Infective Agents, Local↗

IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells.

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.

Antibodies, Monoclonal↗

Computer program for determining fluorescence resonance energy transfer efficiency from flow cytometric data on a cell-by-cell basis.

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.

Algorithms↗

Signal transduction in T lymphocytes and aging.

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.

Aged↗

Class I HLA oligomerization at the surface of B cells is controlled by exogenous beta(2)-microglobulin: implications in activation of cytotoxic T lymphocytes.

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.

Adjuvants, Immunologic↗

Does mosaicism of the plasma membrane at molecular and higher hierarchical levels in human lymphocytes carry information on the immediate history of cells?

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.

Cell Fusion↗