Biomedical subjects

S Damjanovich

Publications and source records attributed to S Damjanovich.

At least 19 recordsLinked to original sources

PubMed2001

Clustering of class I HLA oligomers with CD8 and TCR: three-dimensional models based on fluorescence resonance energy transfer and crystallographic data.

Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.

Antigen-Presenting Cells↗

PubMed2001

Effects of toxins Pi2 and Pi3 on human T lymphocyte Kv1.3 channels: the role of Glu7 and Lys24.

Pandinus imperator scorpion toxins Pi2 and Pi3 differ only by a single amino acid residue (neutral Pro7 in Pi2 vs. acidic Glu7 in Pi3). The binding kinetics of these toxins to human Kv1.3 showed that the decreased ON rate (k(ON) = 2.18 x 10(8) m(-1)sec(-1) for Pi2 and 1.28 x 10(7) m(-1)sec(-1) for Pi3) was almost entirely responsible for the increased dissociation constant (K(d)) of Pi3 (K(d) = 795 pm) as compared to Pi2 (K(d) = 44 pm). The ionic strength dependence of the association rates was exactly the same for the two toxins indicating that through-space electrostatic interactions can not account for the different ON rates. Results were further analyzed on the basis of the three-dimensional structural models of the toxins. A 3D structure of Pi3 was generated from the NMR spectroscopy coordinates of Pi2 by computer modeling. The Pi3 model resulted in a salt bridge between Glu7 and Lys24 in Pi3. Based on this finding our interpretation of the reduced ON rate of Pi3 is that the intramolecular salt bridge reduces the local positive electrostatic potential around Lys24 resulting in decreased short-range electrostatic interactions during the binding step. To support our finding, we constructed a 3D model of the Ser-10-Asp Charybdotoxin mutant displaying distinctly reduced affinity for Shaker channels. The mutant Charybdotoxin structure also displayed a salt bridge between residues Asp10 and Lys27 equivalent to the one between Glu7 and Lys24 in Pi3.

Amino Acid Sequence↗

PubMed2001

Multiple binding sites for melatonin on Kv1.3.

Melatonin is a small amino acid derivative hormone of the pineal gland. Melatonin quickly and reversibly blocked Kv1.3 channels, the predominant voltage-gated potassium channel in human T-lymphocytes, acting from the extracellular side. The block did not show state or voltage dependence and was associated with an increased inactivation rate of the current. A half-blocking concentration of 1.5 mM was obtained from the reduction of the peak current. We explored several models to describe the stoichiometry of melatonin-Kv1.3 interaction considering one or four independent binding sites per channel. The model in which the occupancy of one of four binding sites by melatonin is sufficient to block the channels gives the best fit to the dose-response relationship, although all four binding sites can be occupied by the drug. The dissociation constant for the individual binding sites is 8.11 mM. Parallel application of charybdotoxin and melatonin showed that both compounds can simultaneously bind to the channels, thereby localizing the melatonin binding site out of the pore region. However, binding of tetraethylammonium to its receptor decreases the melatonin affinity, and vice versa. Thus, the occupancy of the two separate receptor sites allosterically modulates each other.

Antibodies, Monoclonal↗

PubMed2001

A slow outward current and a hypoosmolality induced anion conductance in embryonic chicken osteoclasts.

In this paper we report on a hypoosmolality induced current, I(osmo), in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward current, I(slo). I(slo) was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and activated at membrane potentials more positive than -44 +/- 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba2+ (1 mM), blockers of K+ conductances in chicken osteoclasts, did not influence I(slo). However, I(slo) was inhibited by 50 microM extracellular verapamil, which allowed us to study I(osmo) in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization activated I(osmo). It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 +/- 0.9 min (n = 9). The current activated within a few ms upon depolarization and did not inactivate during at least 5 sec. I(osmo) reversed around the calculated Nernst potential for Cl- (E(Cl) = +7.3 mV and V(rev) = +5.4 +/- 3.6 mV, n = 9). The underlying conductance, G(osmo) exhibited moderate outward rectification around 0 mV in symmetrical Cl- solutions. Ion substitution experiments showed that G(osmo) is an anion conductance with P(Cl) approximately = P(F) > P(gluc) >> P(Na). I(osmo) was blocked by 0.5 mM SITS but 50 microM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba2+, 50 microM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl- currents have been implicated in charge neutralization during osteoclastic acid secretion for bone resorption. The present results imply that osmolality may be a factor controlling this charge neutralization.

Animals↗

PubMed2000

Blockage of human T lymphocyte Kv1.3 channels by Pi1, a novel class of scorpion toxin.

Using the patch-clamp technique we determined that Pandinus imperator toxin Pi1, a recently described peptide toxin having four disulfide bridges instead of the usual three in scorpion toxins, blocked Kv1.3 channels of human T lymphocytes from the extracellular side with a 1:1 stoichiometry. Kv1.3 block was instantaneous and removable with toxin-free extracellular solution. The toxin did not influence activation or inactivation of the channels. We found that Pi1 blocked Kv1.3 with less affinity (K(d) = 11.4 nM) than the structurally related three disulfide bridge containing toxins Pi2 (50 pM) and Pi3 (0.5 nM). The fourth disulfide bridge in Pi1 had no influence on the channel binding ability of the toxin; the less effective block was due to differences in amino acid side chain properties at positions 11 and 35.

Amino Acid Sequence↗

PubMed2000

Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements.

BACKGROUND: Perrin equation suggests an alternative way for the accurate energy transfer determination on a cell-by-cell basis by measuring polarized donor intensities in a conventional flow cytometer. METHODS: The relationship between energy transfer and fluorescence anisotropy of the donor was investigated by flow cytometric generation of Perrin-lifetime plots of fluorescent antibody-labeled MHC class I and class II molecules on the surface of living cells. The energy transfer reduced the fluorescence lifetime of the donor. RESULTS: Perrin plots have proven to be sensitive to the segmental mobility of the labeling dye and that of antibodies of different isotypes, and homo-transfer due to the multiple labeling of antibodies. A method demonstrating the feasibility of energy transfer determination by measuring anisotropy enhancement of the donor is presented. Flow cytometric histograms of the donor anisotropy and of the deduced energy transfer efficiency are shown, indicating clustering of MHC class I and class II molecules on the surface of human T lymphoblasts. In the Appendix, a method for the simultaneous determination of both energy transfer efficiency and donor fluorescence anisotropy, without need for G-factor measurement, is also presented. CONCLUSIONS: We demonstrate that energy transfer efficiency, i.e., proximity, between suitably selected donor and acceptor, and the rotational relaxation of the donor, i.e., donor mobility, can be simultaneously measured in a flow cytometer.

Adult↗

PubMed2000

Cholesterol-dependent clustering of IL-2Ralpha and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts.

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.

Antigens, CD↗

PubMed2000

Membrane hyperpolarization removes inactivation of Ca2+ channels, leading to Ca2+ influx and subsequent initiation of sperm motility in the common carp.

Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca(2+)-free hypoosmotic solutions, and significant increase in the intracellular Ca(2+) level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10(-7) to 10(-5) M Ca(2+). Ca(2+) channel blockers such as verapamil and omega-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca(2+) influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca(2+) channels, leading to Ca(2+) influx at the initiation of carp sperm motility.

Animals↗

PubMed2000

Ethanol and halothane differently modulate HLA class I and class II oligomerization. A new look at the mode of action of anesthetic agents through fluorescence spectroscopy.

The field of research considering the working mechanism of anesthetic agents is a complex one and the site or sites of action of general anesthetics are yet to be elucidated. Through the years, on the molecular level, the discussion has shifted from the lipid theories to the more specific interaction with the proteins responsible for the signal transduction. While this approach led to several models, they offer, at best, partial explanations for the observed phenomena. Anesthetic agents interact with many systems, of which the neuronal is best studied, leaving interaction with the immune defense system relatively unexplored. In this study we focus on the interaction of ethanol and halothane with the co-localization on the membrane of HLA I and II molecules. We show that ethanol tends to randomize the distribution of HLA I and II molecules, while halothane increases the clustering of HLA I proteins. The notion that anesthetics modulate cell function by disrupting clustering and thereby promoting a random distribution is a novel approach that may explain the general involvement of many systems during exposition to anesthetic drugs. In this study we show the disturbance of co-localization of molecules that may form a functional network. The relevance of this finding depends on the importance of these networks for extracellular and intracellular processes.

Anesthetics, Inhalation↗

PubMed2000

The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells.

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo. The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.

Alemtuzumab↗

PubMed1999

Complexity of signal transduction mediated by ErbB2: clues to the potential of receptor-targeted cancer therapy.

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.

Animals↗

PubMed1999

Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy.

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.

Animals↗

PubMed1998

Hydrocarbon uptake by Streptomyces.

Oligocarbophilic Streptomyces strains capable of hydrocarbon uptake and utilization were isolated from the polluted desert of Kuwait and used in this study. Transmission electron-microscopy of hyphae revealed that they become enriched with large less electron dense areas in the cytoplasm, when biomass samples were incubated with alkanes. The Streptomyces isolate could utilize n-hexadecane as sole carbon and energy source and their fatty acid content showed an increase in the fatty acids with chain length equivalent to those of the alkane substrates. Fluorescence measurements of diphenylhexatriene dissolved in the representative alkane, n-hexadecane, showed that the kinetics of hydrocarbon uptake are quite different in hydrocarbon-utilizer compared with non-utilizer Streptomyces strain. Microviscosity of the cellular membrane of the utilizer strain was also different from that of the non-utilizer control strain Streptomyces griseus after incubation in the presence of n-hexadecane. Very likely the hydrocarbon utilizer transported these compounds more efficiently across their membranes and accumulated them as inclusions in the cytoplasm.

Alkanes↗

PubMed1998

Melatonin increases the intensity of respiratory burst and prevents L-selectin shedding in human neutrophils in vitro.

Effects of melatonin priming of neutrophils and subsequent increase of phorbol 12-miristate 13-acetate stimulated respiratory burst were investigated on the modulation of L-selectin shedding and MAC-1 upregulation. Respiratory burst related H2O2 production and adhesion molecule expression were quantified by flow cytometry. Phorbol 12-miristate 13-acetate dose dependence of intracellular oxidation and adhesion molecule expression showed no relationship between respiratory burst intensity and MAC-1 expression or L-selectin shedding. Treatment of cells with 12.5 nM phorbol 12-miristate 13-acetate resulted in less than 20% of the respiratory burst response, however it induced 91.7% of total MAC-1 expression and 62.8% of L-selectin shedding. Melatonin priming experiments showed also no connection between the extent of respiratory burst and MAC-1 expression, however melatonin priming almost completely prevented L-selectin down-regulation elicited by phorbol 12-miristate 13-acetate, without affecting MAC-1 expression. It is suggested that melatonin may inhibit metalloproteases responsible for L-selectin cleavage.

Dose-Response Relationship, Drug↗

PubMed1998

Supramolecular receptor structures in the plasma membrane of lymphocytes revealed by flow cytometric energy transfer, scanning force- and transmission electron-microscopic analyses.

Receptors in the plasma membrane of blood cells in general and in that of lymphocytes in particular are supposed to move around in a random walk fashion relatively freely driven by thermal diffusion, as described by the Singer-Nicolson fluid mosaic membrane model. In this article we summarized data and techniques that indicated nonrandom codistribution patterns of receptor superstructures under conditions, where the generation of such molecular colocalizations by the methods themselves were excluded. Application of fluorescence energy transfer in a flow cytometer helped to analyze such codistribution patterns in cell populations. After normalizing energy transfer values for possible differences between labeling ratios of the targeting monoclonal antibodies and using the mean values of energy transfer distribution curves, two-dimensional receptor maps were generated from data obtained in a pair-wise fashion between receptors. Major histocompatibility complex (MHC) class I and II, intercellular adhesion molecule-1 (ICAM-1), TcR-CD3-CD4, tetraspan molecules (CD81, CD82, CD53), and the subunits of the multisubunit IL-2 receptor displayed nonrandom codistribution patterns sometimes with, but very frequently without induction by their ligand. Immunogold-bead "sandwich" labeling analyzed by atomic force microscopy has shown that such receptor "islands" existed also in "receptor-island-groups". This indicated the existence of nonrandom receptor distribution of MHC class I and II molecules also at an elevated hierarchical level. An analysis is given herein concerning a standardized approach. The apparent incompatibility of these supramolecular patterns with the Singer-Nicolson type "free-protein and lipid-mobility paradigm" was resolved by recommending an additional emphasis on the mosaicism of the membrane besides receptor mobility.

Antibodies, Monoclonal↗

PubMed1998

Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research.

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.

Diagnostic Tests, Routine↗

PubMed1998

EGF-induced redistribution of erbB2 on breast tumor cells: flow and image cytometric energy transfer measurements.

erbB2, a member of the epidermal growth factor (EGF) receptor-type tyrosine kinase receptor family, is overexpressed in breast carcinomas with poor prognosis. We examined the cell surface association of this receptor with itself and with other cell surface proteins by the Förster-type fluorescence resonance energy transfer using whole antibodies and Fab fragments. We found that erbB2 molecules homoassociate in unstimulated SK-BR-3, BT474 and BT474-M (a metastatic version of the parent BT474 line) breast tumor cells, and that the interaction was enhanced by EGF treatment in suspensions of SK-BR-3 and BT474-M cells. BT474 cells (with low EGF receptor expression) and attached SK-BR-3 cells do not respond to EGF. Image microscopic energy transfer measurements found considerable pixel-by-pixel heterogeneity in the homoassociation state of erbB2. In accordance with the EGF-induced redistribution of erbB2, EGF receptor was found to be in close proximity to erbB2 in FRET measurements. By labeling different epitopes on erbB2 and the lipid bilayer, we were able to prepare an epitope map of erbB2 molecule. Our data suggest the existence of dynamic cell surface patterns of erbB2 and point to functions fulfilled by these molecular complexes.

Breast Neoplasms↗

PubMed1998

Pandinus imperator scorpion venom blocks voltage-gated K+ channels in human lymphocytes.

Using the patch-clamp technique, we determined that Pandinus imperator scorpion venom blocked whole-cell n-type K+ currents in human peripheral blood lymphocytes in a dose-dependent manner with Kd = 0.02 microgram/ml. K+ channel block was instantaneous and removable by washing with venom-free extracellular solution. The venom-induced block was independent of membrane potential. The venom did not influence activation and inactivation kinetics of the K+ channels, however, accelerated recovery from inactivation. Purified peptides Pi1, Pi2, and Pi3 from the P. imperator venom powerfully blocked Kv1.3 channels in human lymphocytes with Kd values of 9.7 nM, 50 pM, and 0.5 nM, respectively. Flow cytometric membrane potential measurements with the oxonol dye showed that Pi2, the most effective peptide toxin of the P. imperator venom, depolarizes human lymphocytes in accordance with its K+ channel blocking effect.

Barbiturates↗