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Biomedical subjects

Sándor Damjanovich

Publications and source records attributed to Sándor Damjanovich.

At least 19 recordsLinked to original sources
PubMed2006

ICAM-1 inhibits the homocluster formation of MHC-I in colon carcinoma cells.

ICAM-1 and MHC-I proteins play fundamental roles in antigen presentation, activation of T lymphocytes, and immune responses against tumor cells. Both of them participate in the formation of lipid raft-associated membrane protein clusters. We found significant colocalization between ICAM-1 and MHC-I at the level of large-scale associations. We combined RNA interference and fluorescence resonance energy transfer studies to show that ICAM-1 promotes the partial disassembly of MHC-I homoclusters on LS-174T colon carcinoma cells. Interferon-gamma (IFN-gamma) treatment induced an increase in the expression of MHC-I and ICAM-1 resulting in decreased MHC-I homoassociation. Small interfering RNAs directed against ICAM-1 restored the homoassociation of MHC-I without influencing the expression level of MHC-I by eliminating ICAM-1 molecules interspersed in MHC-I clusters. We conclude that the composition of membrane protein clusters is dynamically altered in response to both physiological and experimentally elicited changes in antigen expression levels.

Cell Line, Tumor↗
PubMed2005

The role of supramolecular protein complexes and membrane potential in transmembrane signaling processes of lymphocytes.

The formation of protein patterns in lymphocyte plasma membranes is analyzed in the light of past and, also, very recent experiments. The analysis surveys the lateral organization of major histocompatibility complex glycoproteins, intercellular adhesion molecule-1, interleukin-2 and -15 receptors, Kv1.3 K+ ion channels and the T-cell receptor as well as their behavior under different conditions. These molecules form small- and large-scale clusters in the membrane of human lymphocytes. Many of the association motifs occur in other investigated cell types. The conclusions point toward a possible role for ion channel activities, membrane potential changes and alterations of the lateral organization of proteins in transmembrane signaling and cytotoxic interactions. In our outlook new factors that potentially affect membrane protein cluster formation and interactions are discussed. A role for MHC glycoproteins in concentrating membrane proteins and organizing protein patterns is suggested, and the possibility that the membrane potential may modulate protein conformation and, thereby, affect protein-protein interactions is pointed out. A well-defined role for the presence of ion channels in the immune synapse is offered, which could explain the significance of ion channel accumulation in the immune synapse together with the T-cell receptor.

Animals↗
PubMed2005

Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements.

Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Ralpha, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.

Antibodies, Monoclonal↗
PubMed2005

Inhibition of human telomerase by oligonucleotide chimeras, composed of an antisense moiety and a chemically modified homo-oligonucleotide.

Most tumor cells attain their immortality by reactivating telomerase. We report here the telomerase inhibitory potential of chimeric oligonucleotides composed of a 13mer antisense sequence targeting the telomerase RNA template region and a (s4dU)n moiety at its 3' or 5'-end. The increase of the thiolated chain length enhances the telomerase inhibitory potential, but decreases specificity, indicated by HIV reverse transcriptase inhibition. Chimeras with 5' (s4dU)(n)s were more potent inhibitors than the antisense alone or the 3' modified ones. Cy5-labeled (s4dU)4AS and (s4dU)8AS proved the internalization of the oligonucleotides, raising the possibility to be tested as cellular anti-telomerase agents.

Base Sequence↗
PubMed2005

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

Calibration↗
PubMed2005

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↗
PubMed2005

Detection of channel proximity by nanoparticle-assisted delaying of toxin binding; a combined patch-clamp and flow cytometric energy transfer study.

Gold nanoparticles of 30 nm diameter bound to cell-surface receptor major histocompatibility complex glycoproteins (MHCI and MHCII), interleukin-2 receptor alpha subunit (IL-2Ralpha), very late antigen-4 (VLA-4) integrin, transferrin receptor, and the receptor-type protein tyrosin phosphatase CD45 are shown by the patch-clamp technique to selectively modulate binding characteristics of Pi(2) toxin, an efficient blocker of K(v)1.3 channels. After correlating the electrophysiological data with those on the underlying receptor clusters obtained by simultaneously conducted flow cytometric energy transfer measurements, the modulation was proved to be sensitive to the density and size of the receptor clusters, and to the locations of the receptors as well. Based on the observation that engagement of MHCII by a monoclonal antibody down-regulates channel current and based on the close nanometer-scale proximity of the MHCI and MHCII glycoproteins, an analogous experiment was carried out when gold nanoparticles bound to MHCI delayed down-regulation of the K(v)1.3 current initiated by ligation of MHCII. Localization of K(v)1.3 channels in the nanometer-scale vicinity of the MHC-containing lipid rafts is demonstrated for the first time. A method is proposed for detecting receptor-channel or receptor-receptor proximity by observing nanoparticle-induced increase in relaxation times following concentration jumps of ligands binding to channels or to receptors capable of regulating channel currents.

Cell Line↗
PubMed2004

Membrane topography of HLA I, HLA II, and ICAM-1 is affected by IFN-gamma in lipid rafts of uveal melanomas.

The lateral distribution and colocalization of HLA I, HLA-DR, and ICAM-1 proteins was studied for the first time in the plasma membrane of two human uveal melanoma cell lines, OCM-1 and OCM-3. Our fluorescence resonance energy transfer and confocal laser scanning microscopic experiments revealed that these molecules are mostly confined to the same membrane regions, where they form similar protein patterns (homo- and hetero-associates) to those found previously on other cell types of lymphoid as well as colorectal carcinoma origin. Confocal microscopic colocalization experiments with GM(1) gangliosides and the GPI-anchored CD59 molecules showed enrichment of HLA I, HLA-DR, and ICAM-1 molecules in specific membrane domains (lipid rafts) excluding the transferrin receptor. IFN-gamma remarkably increased the expression levels of these molecules and rearranged their association patterns, which can affect the adoptive immune response of effector cells.

Flow Cytometry↗
PubMed2004

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↗
PubMed2004

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↗
PubMed2003

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↗
PubMed2003

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↗
PubMed2002

Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2.

The ErbB family of transmembrane receptor tyrosine kinases plays an important role in the pathogenesis of many cancers. The four members of the family, ErbB1-4, form various homo- and heterodimers during the course of signal transduction. A second hierarchical level of molecular associations involving 10(2)-10(3) molecules, termed large-scale clustering, has also been identified, but the regulatory factors and biological consequences of such structures have not been systematically evaluated. In this report, we describe the states of association of ErbB2 and their relationship to local ErbB3 density and lipid rafts based on quantitative fluorescence microscopy of SKBR-3 breast cancer cells. Clusters of ErbB2 colocalized with lipid rafts identified by the GM1-binding B subunit of cholera toxin. Pixel-by-pixel analysis of fluorescence resonance energy transfer between labeled antibodies indicated that the homoassociation (homodimerization) of ErbB2 was proportional to the local density of ErbB2 and inversely proportional to that of ErbB3 and of the raft-specific lipid GM1. Crosslinking lipid rafts with the B subunit of cholera toxin caused dissociation of the rafts and ErbB2 clusters, an effect that was independent of the cytoskeletal anchoring of ErbB2. Crosslinking also decreased ErbB2-ErbB3 heteroassociation and the EGF- and heregulin-induced tyrosine phosphorylation of Shc. When cells were treated with the anti-ErbB2 monoclonal antibody 4D5 (parent murine version of Trastuzumab used in the immunotherapy of breast cancer), internalization of the antibody was inhibited by crosslinking of lipid rafts, but the antiproliferative activity of 4D5 was retained and even enhanced. We conclude that local densities of ErbB2 and ErbB3, as well as the lipid environment profoundly influence the association properties and biological function of ErbB2.

Adaptor Proteins, Signal Transducing↗
PubMed2002

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↗
PubMed2002

Shear-dependent morphology of von Willebrand factor bound to immobilized collagen.

We have developed an immunogold von Willebrand factor (VWF) detection method that permits almost complete coverage of individual VWF molecules, and by this unequivocal localization and morphologic analysis of collagen-bound VWF by atomic force microscopy (AFM). Perfusion of gel filtration-purified VWF in parallel plate perfusion chambers over glass coverslips coated with calf skin collagen, followed by AFM imaging in air, enabled us to assess possible morphologic differences between VWF bound at low (0.07 N/m(2) = 0.7 dynes/cm(2)) and high (4.55 N/m(2) = 45.5 dynes/cm(2)) shear stresses. No significant differences in VWF morphology were found, the molecules were oriented almost randomly, and there were no clear signs of VWF "uncoiling" either at a high or at a low shear regime. After perfusing 1 microg/mL VWF for 5 minutes, surface coverage at high shear was almost twice the one seen at low shear, and some larger and more irregularly shaped VWF molecules could be seen at high shear. This difference disappeared, however, at 15 minutes of perfusion and was probably caused by diffusion kinetics. Moreover, the presence of 68 x 10(9)/L washed fixed platelets in the perfusate did not have any visible effect on VWF morphology at high versus low shear stress. These findings suggest that shear stress does not influence significantly the overall molecular morphology of VWF during its binding to collagen-coated surface and are consistent with a constitutively expressed affinity of collagen-bound VWF for glycoprotein Ib.

Animals↗
PubMed2002

INF-gamma rearranges membrane topography of MHC-I and ICAM-1 in colon carcinoma cells.

Flow-cytometric fluorescence energy transfer (FCET) measurements between fluorescently labeled cell surface MHC-I and ICAM-1 molecules indicated similar receptor patterns in the plasma membrane of interferon-gamma (INF-gamma)-treated colon carcinoma cells as those observed earlier at the surface of lymphoid cells. INF-gamma activation significantly increased the density of MHC-I and ICAM-1 proteins in the membrane. This increase in receptor density was accompanied by decreased proximity level of the homo-associated MHC-I receptors. Hetero-association of MHC-I and ICAM-1 molecules was increased by INF-gamma treatment. INF-gamma changed neither hetero- nor homo-association of transferrin receptors. By staining the sphingomyelin/cholesterol-enriched lipid microdomains with fluorescently labeled cholera toxin B subunit, we found an increase in the amount of lipid-raft associated G(M1)-gangliosides due to INF-gamma treatment. Confocal microscopic results and FCET measurements show that MHC-I and ICAM-1 are components of G(M1)-ganglioside containing lipid-rafts and also support an increase in the size of these lipid-rafts upon INF-gamma treatment.

Carcinoma↗