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Biomedical subjects

S Damjanovich

Publications and source records attributed to S Damjanovich.

At least 109 records · Page 6Linked to original sources
PubMed1984

Förster-type energy transfer as a probe for changes in local fluctuations of the protein matrix.

Much evidence, on both theoretical and experimental sides, indicates the importance of local fluctuations (in energy levels, conformational substates, etc.) of the macromolecular matrix in the biological activity of proteins. We describe here a novel application of the Förster-type energy-transfer process capable of monitoring changes both in local fluctuations and in conformational states of macromolecules. A new energy-transfer parameter, f, is defined as an average transfer efficiency, [E], normalized by the actual average quantum efficiency of the donor fluorescence, [phi D]. A simple oscillator model (for a one donor-one acceptor system) is presented to show the sensitivity of this parameter to changes in amplitudes of local fluctuations. The different modes of averaging (static, dynamic, and intermediate cases) occurring for a given value of the average transfer rate, [kt], and the experimental requirements as well as limitations of the method are also discussed. The experimental tests were performed on the ribonuclease T1-pyridoxamine 5'-phosphate conjugate (a one donor-one acceptor system) by studying the change of the f parameter with temperature, an environmental parameter expectedly perturbing local fluctuations of proteins. The parameter f increased with increasing temperature as expected on the basis of the oscillator model, suggesting that it really reflects changes of fluctuation amplitudes (significant changes in the orientation factor, k2, as well as in the spectral properties of the fluorophores can be excluded by anisotropy measurements and spectral investigations). Possibilities of the general applicability of the method are also discussed.

Energy Transfer↗
PubMed1984

Fluorescence energy transfer measurements on cell surfaces: a critical comparison of steady-state fluorimetric and flow cytometric methods.

The energy transfer efficiency E was measured between fluorescein-conjugated concanavalin A (Con A) and rhodamine-conjugated Con A bound to homogeneous tissue culture cells, the HK22 murine lymphoma cell line. Results from a flow cytometric energy transfer method (FCET) and two different steady-state fluorimeter methods were compared. The data were found to be in close agreement after careful correction of the steady-state fluorimetric measurements for contributions from dissociating ligand. The biological variability of the individual cells with respect to E was calculated using an error propagation analysis and were found to be less than the variability in the absolute amount of ligand binding per cell. FCET has a number of advantages over the fluorimetric measurements using suspensions of cells: (1) relatively labile receptor-ligand complexes can be measured; (2) the analysis can be restricted to undamaged cells by gating the data collection on the light-scattering signals; (3) heterogeneous populations of cells with respect to donor and acceptor topology can be distinguished by the correlation of E with other cellular parameters derived from additional signals or combinations thereof; and (4) the dynamics of donor-acceptor redistribution on subpopulations can be measured.

Animals↗
PubMed1984

Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis.

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).

Animals↗
PubMed1984

Flow cytometric analysis of viability of bull sperm cells.

Fluorescein diacetate and propidium iodide double fluorescence labeling of animal sperm cells in combination with 55 degrees C treatment for 15 min was used to monitor the viability and heat tolerance of sperm cells by flow cytometry. The applicability of the elaborated test to detect fertility differences has been shown on sperm samples obtained from bulls of known fertility values. According to preliminary examinations the method seems to be suitable to test the fertility of other male animals, too. The simplicity of the relatively quick procedure may provide a new screening method to qualify male animals before using their semen for large scale artificial insemination.

Animals↗
PubMed1983

Protein fluctuation and enzyme activity.

The possible participation of protein fluctuation in enzyme activity is considered in this paper. Fluctuation is defined as vibrations, involving collective motion of a large number of atoms in protein molecules. Kinetic and thermodynamic aspects of protein fluctuation and enzyme-ligand interactions are discussed, paying special attention to the influence of electric fields and environmental microviscosity. A brief analysis of available experimental and theoretical data strongly suggests an interrelationship between protein fluctuation and enzyme function.

Enzymes↗
PubMed1983

Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.

The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different determinants on the H-2Kk antigen itself were determined by using fluorescence resonance energy-transfer measurements with a dual-laser flow sorter. From the lack of energy transfer between bound monoclonal anti-H-2Kk antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2Kk antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2Kk antigen shows that the two populations are interspersed. Donor/acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2Kk antigen exhibited a degree of energy transfer indicative of a mean separation of 8.6 nm between the sites. Time-resolved phosphorescence anisotropy measurements with anti-H-2Kk antibodies labeled with eosin or erythrosin yielded rotational mobility information for the antigen-antibody complexes on the cell membrane. The rotational correlation time of 10-20 mus and the finite residual anisotropy are compatible with an uniaxial mode of rotation of monomeric antigen around its transmembrane portion and, thus, provide additional evidence for an unclustered distribution. Capping by rabbit anti-mouse IgG immobilized the antigen-antibody complex. Fluorescence recovery after photobleaching was used to calculate an apparent lateral diffusion coefficient of 5 +/- 3 X 10(-10) cm2 . s-1 for the H-2Kk antigen labeled with fluoresceinated IgG or its corresponding Fab fragment.

Animals↗
PubMed1981

Flow cytometric analysis of the uptake of Hoechst 33342 dye by human lymphocytes.

Human peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature-dependent with minimal uptake rate below 16 degrees C. The activation energy of dye transport was found to be 135 kJ/mol above 20 degrees C and about 20 kJ/mol below 16 degrees C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.

Azides↗
PubMed1981

Aging of the erythrocyte. IX. Fluorescence studies on changes in membrane properties.

Fluorescence studies of membranes prepared from density-separated red blood cells demonstrated an increase in lipid viscosity (judging from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 1-aminonaphthalene-8-sulfonate and changes in the physical state of proteins (judging from tryptophan fluorescence intensity and polarization). These alterations may be partly due to lipid peroxidation. In vivo accumulation of products of this process in the membranes was confirmed. Changes were also found in the distribution pattern of cell populations of different mean age in a cell sorter, probably due to alterations in rheological properties of the erythrocytes.

Animals↗
PubMed1981

Characterization of living normal and leukemic mouse lymphocytes by fluorescein diacetate.

The esterases activity of normal and acute leukemic mouse lymphocytes and that of their homogenates was investigated using fluorescein diacetate (FDA) as a fluorogenic substrate. The activity proved to be the same for the two cell populations as well as for the homogenates prepared from them. In cell suspensions, having different osmolalities, the rate of FDA hydrolysis decreased significantly with the increasing osmolality only in the case of intact leukemic lymphocytes. changes in the membrane and cytoplasmic viscosity caused by increased or decreased environmental osmolality of cell suspensions occurred in the same direction and to the same extent for both normal and leukemic cells. Fluorescein, the fluorescent product of the hydrolysis, accumulates in leukemic lymphocytes, whereas it easily effluxes form normal lymphocytes. A flow microfluorimetry analysis of the cell population revealed that the fluorescein content of large leukemic lymphocytes was three times higher than that of small, normal ones. The observed differences specific for leukemic lymphocytes might be useful in detecting leukemic transformation in an early stage of acute lymphoid leukemia.

Animals↗
PubMed1980

Correlation between activity and dynamics of the protein matrix of phosphorylase b.

Quenching of the tryptophan fluorescence of phosphorylase b was studied by using iodide and acrylamide. Steady-state measurements indicated that all indole side chains were accessible to the nonionic quencher, although only 3 out of the total of 12 residues could be quenched by I-. From Stern--Volmer plots and the fluorescence lifetime data, it was concluded that the quenching was mainly of dynamic character. The value of the collisional quenching rate constant was found to be an order of magnitude less than that obtained in the case of fully exposed tryptophans. The relatively high activation energy, 30.9 kJ/mol, of the diffusion-controlled process and the value of the activation entropy suggest that the diffusion takes place in a fluctuating, structured medium. In spite of the application of sensitive fluorescent techniques, no gross conformational changes were found in the presence of acrylamide. However, the catalytic rate of the glycogen synthesis was decreased with the residual activity of the enzyme, proportional to the concentration of the probe. Binding of activator (AMP) and substrates (glucose 1-phosphate and glycogen) was found to be unaffected by acrylamide in concentrations applied (0--0.8 M). In a similar manner, activation enthalpy did not change in the presence of the quencher either. The complete reversibility of both activity inhibition and fluorescence quenching ruled out the irreversible denaturation of the enzyme or the covalent modification of any of the functional groups. We concluded that a model, suggesting the cross-correlation of activity and fluctuation, was consistent with the experimental findings.

Acrylamides↗
PubMed1980

Fluorescence energy transfer studies on normal and leukemic mouse lymphocytes.

Control and acute leukemic cells of AKR:Lac C3H mg:Lac/F1 hybride mice were labeled either with ethidium bromide or ethidium bromide plus fluorescamine. Theoretical and experimental evidence shows that the ethidium bromide binding ability of cells and the average efficiency of Förster-type fluorescence energy transfer between fluorescamine and ethidium bromide inside the cells depend on both the cell and dye concentrations. It is shown further that fluorescamine treatment enhances the uptake of ethidium bromide by both normal and leukemic cells. A sevenfold difference was found in ethidium bromide binding ability between normal and leukemic cells at optimal dye concentration.

Animals↗
PubMed1980

Changes in the intramembrane viscosity of sheep erythrocytes in the presence of membrane bound proteins.

Some dynamic properties of protein- and antibody-treated cell membranes were investigated. The membrane viscosity of sheep erythrocytes increased upon binding various proteins as determined from the fluorescence emission anisotropy of 1,6-diphenyl 1,3,5-hexatriene dissolved in the membranes. However, specific, IgM type immunoglobulins produced against the erythrocytes decreased the intramembrane viscosity of cells. The applicability of the above dye to follow the binding of specific and non-specific proteins to erythrocytes is also discussed.

Animals↗
PubMed1979

A fluorescence energy transfer system for studying the functional properties of chromatin.

3YI-B-1 rat embryo fibroblasts were used for double fluorescent labeling. The labeling was carried out with fluorescamine and ethidium bromide as an energy donor-acceptor pair. The fluorimetric measurements proved the existence of an energy transfer process. The parameters of this transfer depended on both cell and dye concentrations. It is this character that makes this labeling technique promising as a means of distinction between cells with different structures.

Animals↗