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Rezso Gáspár

Publications and source records attributed to Rezso Gáspár.

13 recordsLinked to original sources
PubMed2005

Novel alpha-KTx peptides from the venom of the scorpion Centruroides elegans selectively blockade Kv1.3 over IKCa1 K+ channels of T cells.

From the venom of the Mexican scorpion Centruroides elegans Thorell five peptides were isolated to homogeneity by chromatographic procedures and their full amino acid sequence was determined by automatic Edman degradation. They all belong to the Noxiustoxin subfamily of scorpion toxins and were given the systematic names alpha-KTx 2.8 to 2.12, with trivial names Ce1 to Ce5, respectively. They have 39 amino acid residues, except for Ce3 which has only 38, but all of them have three disulfide bridges, and have molecular weights of 4255, 4267, 4249, 4295 and 4255 atomic mass units, respectively for Ce1 to Ce5. The C-terminal residues of Ce2, Ce4 and Ce5 were found to be amidated. The electrophysiological assay (whole-cell patch-clamp) showed that out of the five peptides, Ce1 (alpha-KTx 2.8), Ce2 (alpha-KTX2.9) and Ce4 (alpha-KTx 2.11) were effective blockers of Kv1.3 channels of human T lymphocytes, whereas these peptides did not inhibit the Ca2+-activated K+ channels (IKCa1) of the same cells. The equilibrium dissociation constants of these peptides for Kv1.3 were 0.70, 0.25 and 0.98nM for Ce1, Ce2 and Ce4, respectively. Furthermore, toxins Ce1, Ce2 and Ce4 practically did not inhibit the related voltage gated Shaker K+ channels, and rKv2.1 channels of the Shab family. The high affinity blockage of Kv1.3 channels by these peptides and their selectivity for Kv1.3 over IKCa1 may have significance in the development of novel tools for suppressing the function of those T cell subsets whose proliferation critically depends on the activity of Kv1.3 channels.

Amino Acid Sequence↗
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

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↗
PubMed2004

Anuroctoxin, a new scorpion toxin of the alpha-KTx 6 subfamily, is highly selective for Kv1.3 over IKCa1 ion channels of human T lymphocytes.

The physiological function of T lymphocytes can be modulated selectively by peptide toxins acting on Kv1.3 K(+) channels. Because Kv1.3-specific peptide toxins are considered to have a significant therapeutic potential in the treatment of autoimmune diseases, the discovery of new toxins is highly motivated. Through chromatographic procedures and electrophysiological assays, using patch-clamp methodology, the isolation of a novel peptide named anuroctoxin was accomplished using the venom of the Mexican scorpion Anuroctonus phaiodactylus. It has 35 amino acid residues with a molecular weight of 4082.8, tightly bound by four disulfide bridges whose complete covalent structure was determined. It has a pyroglutamic acid at the N-terminal region and an amidated C-terminal residue. Sequence comparison and phylogenetic clustering analysis classifies anuroctoxin into subfamily 6 of the alpha-KTx scorpion toxins (systematic name, alpha-KTx 6.12). Patch-clamp experiments show that anuroctoxin is a high-affinity blocker of Kv1.3 channels of human T lymphocytes with a K(d) of 0.73 nM, and it does not block the Ca(2+)-activated IKCa1 K(+) channels. These two channels play different but important roles in T-lymphocyte activation. Furthermore, the toxin practically does not inhibit Shaker IR, mKv1.1, and rKv2.1 channels, whereas the affinity of anuroctoxin for hKv1.2 is almost an order of magnitude smaller than for Kv1.3. The pharmacological profile and the selectivity of this new toxin for Kv1.3 over IKCa1 may provide an important tool for the modulation of the immune system, especially in cases in which selective inhibition of Kv1.3 is required.

Animals↗
PubMed2004

pH-dependent modulation of Kv1.3 inactivation: role of His399.

The Kv1.3 K(+) channel lacks N-type inactivation, but during prolonged depolarized periods it inactivates via the slow (P/C type) mechanism. It bears a titratable histidine residue in position 399 (equivalent of Shaker 449), a site known to influence the rate of slow inactivation. As opposed to several other voltage-gated K(+) channels, slow inactivation of Kv1.3 is slowed when extracellular pH (pH(o)) is lowered under physiological conditions. Our findings are as follows. First, when His399 was mutated to a lysine, arginine, leucine, valine or tyrosine, extracellular acidification (pH 5.5) accelerated inactivation reminiscent of other Kv channels. Second, inactivation of the wild-type channel was accelerated by low pH(o) when the ionic strength of the external solution was raised. Inactivation of the H399K mutant was also accelerated by high ionic strength at pH 7.35 but not the inactivation of H399L. Third, after the external application of blocking barium ions, recovery of the wild-type current during washout was slower in low pH(o). Fourth, the dissociation rate of Ba(2+) was pH insensitive for both H399K and H399L. Furthermore, Ba(2+) dissociation rates were equal for H399K and the wild type at pH 5.5 and were equal for H399L and the wild type at pH 7.35. These observations support a model in which the electric field of the protonated histidines creates a potential barrier for potassium ions just outside the external mouth of the pore that hinders their exit from the binding site controlling inactivation. In Kv1.3, this effect overrides the generally observed speeding of slow inactivation when pH(o) is reduced.

Animals↗
PubMed2004

Ion channels and lymphocyte activation.

The ion channels expressed by T lymphocytes play key roles in the control of the membrane potential and calcium signaling, thereby affecting signal transduction pathways that lead to the activation of these cells following antigenic stimulation. Disruption of these pathways can attenuate or prevent the response of T-cells to antigenic challenge resulting in immune suppression. Studies using ion channel blockers of high affinity and specificity have shown that this interference can be achieved at the level of ion channels. Suppression of immune functions by channel blockers has been demonstrated in vitro and in vivo. New information about the molecular structure of ion channels facilitates the design of more potent and more specific inhibitors. Thus, T-cell ion channels are likely to serve as targets for immunomodulatory drugs in the near future. Here, the biophysical properties, tissue distribution, regulation of expression, molecular pharmacology and role in T-cell activation of the voltage-gated Kv1.3 and the Ca(2+)-activated IKCa1 potassium channels and those of the Ca(+) release-activated Ca(2+) (CRAC) channel are reviewed.

Animals↗
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

Active and passive behaviour in the regulation of stiffness of the lateral wall in outer hair cells of the guinea-pig.

The stiffness of the outer hair cell (OHC) lateral wall, measured by the micropipette aspiration technique, is non-linear, decreasing from the ciliary pole (stiffness parameter Sp 1.83+/-0.13 nN/microm n=10) towards the cell base (Sp 1.14+/-0.16 nN/microm, n=10) irrespective of the cochleoapical or cochleobasal origin of the cells. The length of the aspirated lateral wall segment was related exponentially to the duration of the applied negative pressure (6 cm H2O) in the synaptic region of the OHCs whereas an active, sigmoid component was observed between 30 and 60 s in the supranuclear regions. A significant increase of the midlateral wall stiffness (to 1.91+/-0.23 nN/microm; n=10) was observed in calcium-free medium and the sigmoid component of the response of the lateral wall was abolished. Salicylate (5 mM) had no significant effect on the active sigmoid behaviour of the lateral wall (n=10). Gadolinium (5 mM), a non-specific cation channel blocker, increased the stiffness of the lateral wall and attenuated the active component (n=10). The motor protein prestin thus does not seem to be involved in the active stiffness regulation seen in this study. A role for the cortical cytoskeleton in the regulation of stiffness seems reasonable according to our model. The mechanism may involve calcium-dependent metabolic modification of cytoskeletal or membrane proteins.

Animals↗
PubMed2003

Cholesterol modifies the gating of Kv1.3 in human T lymphocytes.

The Kv1.3 potassium channel that belongs to the Shaker family of voltage-gated K(+) channels plays a crucial role in the mitogenic response of T cells. Because it spans the cell membrane its function can be influenced by lipid-protein interactions. In order to study the effect of lipid-protein interactions on the functioning of Kv1.3 we manipulated the membrane cholesterol content in T cells mimicking various physiological conditions by means of the oligosaccharide methyl-beta-cyclodextrin (MbetaCD) and its cholesterol-saturated complex (MbetaCD/C). Fluorescence polarization anisotropy and peak current density were used to monitor the efficiency of cholesterol removal (MbetaCD) and loading (MbetaCD/C). Using whole-cell patch-clamp technique we determined the kinetic and steady-state parameters of activation and inactivation of the Kv1.3 currents under different treatment conditions. Upon elevation of cholesterol content by 1 or 1.5 mg/ml MbetaCD/C the rates of both activation and inactivation were slowed. Moreover, the increased cholesterol level in the membrane resulted in a biphasic activation curve. Cholesterol depletion with MbetaCD (0.95 and 1.425 mg/ml) caused no significant changes in the gating characteristics of Kv1.3. The equilibrium between the open and the closed states of the channels was affected by increased cholesterol content, but at the same time steady-state inactivation was unchanged. We argue that manipulation of membrane cholesterol changed both the kinetic properties of Kv1.3 and steady-state parameters of activation by modifying lipid-protein interactions.

Cholesterol↗
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

Comparative pharmacological studies on the A2 adenosine receptor agonist 5'-n-ethyl-carboxamidoadenosine and its F19 isotope labelled derivative.

Adenosine receptors are expressed in various mammalian tissues where they mediate the effects of adenosine on cellular functions through a number of signalling mechanisms. 18F-NECA is the positron-emitting derivative of the A(2)-receptor agonist NECA (5'-n-ethyl-carboxamidoadenosine) and is a radioligand for PET imaging of adenosine receptors. Contractility and relaxation studies were performed on guinea pig atrial myocardium, pulmonary artery, and thoracic aorta to compare the pharmacological effects of NECA and F-NECA (a non-emitting derivative) on tissues. Furthermore, the effect of NECA and F-NECA on the potassium conductance was investigated in DDT1 MF-2 smooth muscle cells with the patch-clamp technique. Both NECA and F-NECA reduced the contractile force in atrial myocardium and evoked phasic contraction in pulmonary artery (A(1) adenosine-receptor-mediated actions) in a dose dependent manner; however, the apparent affinity was lower for F-NECA. No difference was found in relaxation induced by these compounds in 1 microM noradrenaline-precontracted aorta and pulmonary artery (in the presence of DPCPX, an A(1) adenosine receptor antagonist, tissue containing A(2B) adenosine receptors). NECA (5 microM) and F-NECA (5 microM) also decreased the peak current and accelerated activation and inactivation properties of the potassium channels, but F-NECA was less effective. These results suggest that while NECA and F-NECA are equivalent agonists of vascular A(2B) receptors, they mediate different changes of some parameters. When evaluating the data obtained by the use of radiolabelled ligands, one has to take into consideration the possible physiological effects of the ligands besides its binding properties to tissues.

Adenosine A2 Receptor Agonists↗
PubMedDate not supplied

Nanoparticle energy transfer on the cell surface.

Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2-10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal-fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle-fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range essentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore-nanoparticle distance. The intensity of 90 degrees scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90 degrees light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging microscope. Modulations of 90 degrees light scattering intensity and photobleaching time constant indicate the role of the local field in the fluorescence enhancement. A mathematical simulation based on the electrodynamic theory of fluorescence enhancement showed a consistency between the measured enhancement values, the inter-epitope distances and the quantum yields. The feasibility of realizing proximity sensors operating at distance ranges larger than that of the conventional Forster transfer is demonstrated on the surface of living cells.

Antibodies, Monoclonal↗