Electrokinetic properties of healthy and ß-thalassemia erythrocyte membranes under in vitro exposure to static magnetic field.

Introduction: The current understanding of the biological impacts of a static magnetic field (SMF) is restricted to the direct interactions of the magnetic field with biological membranes. The electrokinetic (zeta) potential is an electrochemical property of erythrocyte surfaces which was negatively charged in physiological media after SMF exposure (0.1‒2.0 T). Methods: The novel data about electrokinetic parameters of the erythrocytes is determined by microelectrophoresis after SMF-exposure in norm and heterozygous ß-thalassemia. The methods of light scattering, lipid peroxidation, fluorescence microscopy are used. Results: The electrokinetic potential of erythrocytes in norm is increased after SMF intensities due to enhanced negatively exposed charges on the outer surface of the membrane accompanied by an increase in light scattering where changes in cell morphology are observed. Conversely, a decrease in the zeta potential of ß-thalassemia erythrocytes upon SMF-treatment was determined because of the reduction in the surface electrical charge of the membranes, where a significant decrease in light scattering at 1.5 T and 2.0 T was recorded. Exposure to SMF (0.5-2.0 T) was associated with an increase in the malondialdehyde content in erythrocytes. Biophysical studies regarding the influence of SMF on the electrostatic free energy of cells shows an increase in negative values in healthy erythrocytes, which corresponds to the implementation of a spontaneous process. This is also the process in ß-thalassemia cells after SMF exposure with lower negative values of free electrostatic energy than erythrocytes in norm. Discussion: The effect of static magnetic field (SMF 0.1-2.0 T) on the electrokinetic and morphological characteristics of erythrocytes in norm and ß-thalassemia is determined and correlated with the increase/reduction in surface charge and shrinkage/swelling of the cells, respectively. Lipid peroxidation of healthy and ß-thalassemia erythrocytes caused an enhancement of lipid peroxidation because of the higher concentrations of TBARS products in cellular suspension. SMF (0.1‒2.0 T) altered the spontaneous chemical processes with negative values of electrostatic free energy of erythrocytes in norm and ß-thalassemia accompanied by a lower FITC-Concanavalin A binding affinity to membrane receptors (SMF 2.0 T). The electrokinetic properties of human erythrocytes in norm and ß-thalassemia upon SMF treatment and their interrelationship with the structural-functional state of the membrane were reported. The presented work would have future fundamental applications in biomedicine.

Erythrocyte Membrane Biophysical Changes Mediated by Pooled Immunoglobulin G and Hematin: Electrokinetic and Lipid Peroxidation Studies.

Pooled Immunoglobulin G (IgG), hematin and the membrane-disruptive amphipathic peptide melittin have received attention as powerful biomacromolecules for biomedical and pharmacology applications. Their action on surface properties, oxidation status and epifluorescence properties measured in vitro provide useful information about the functional activity of upper biomacromolecules in erythrocytes in vivo. The hemolysis of erythrocyte membranes, as well as changes in hematocrit and the morphology of erythrocytes, was investigated here via fluorescence microscopy using FITC-concanavalin A binding to cells. The effect of melittin on the membrane capacitance and resistance of model lipid bilayers was probed via electrochemical impedance spectroscopy. Lipid bilayer capacitance was higher in the presence of 0.10 g/L melittin compared to that in the control, which is likely related to bilayer thinning and alterations of the dielectric permittivity of melittin-treated membranes. The biomolecule interactions with red blood cells were probed in physiological media in which the surface of erythrocyte membranes was negatively charged. Surface parameters of erythrocytes are reported upon IgG/hematin and IgG/melittin treatment. Pooled IgG in the presence of melittin, preincubated IgG/hematin preparations promoted a significant decrease in the electrokinetic potential of erythrocytes (Rh-positive). A malondialdehyde (MDA) assay revealed a high rate of lipid peroxidation in erythrocytes treated with IgG/hematin or IgG/melittin preparations. This finding might be a result of pooled IgG interactions with the hematin molecule and the subsequent conformational changes in the protein molecule altering the electrokinetic properties of the erythrocyte membrane related to the Rh group type of erythrocytes. The pooled IgG and hematin are reported to have important consequences for the biophysical understanding of the immunopathological mechanisms of inflammatory, autoimmune and antibody-mediated pathological processes.

Surface Properties of Synaptosomes in the Presence of L-Glutamic and Kainic Acids: In Vitro Alteration of the ATPase and Acetylcholinesterase Activities.

Morphologically and functionally identical to brain synapses, the nerve ending particles synaptosomes are biochemically derived membrane structures responsible for the transmission of neural information. Their surface and mechanical properties, measured in vitro, provide useful information about the functional activity of synapses in the brain in vivo. Glutamate and kainic acid are of particular interest because of their role in brain pathology (including causing seizure, migraine, ischemic stroke, aneurysmal subarachnoid hemorrhage, intracerebral hematoma, traumatic brain injury and stroke). The effects of the excitatory neurotransmitter L-glutamic acid and its agonist kainic acid are tested on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic membranes prepared from the cerebral cortex of rat brain tissue. The surface parameters of synaptosome preparations from the cerebral cortex in the presence of L-glutamic and kainic acids are studied by microelectrophoresis for the first time. The studied neurotransmitters promote a significant increase in the electrophoretic mobility and surface electrical charge of synaptosomes at 1-4 h after isolation. The measured decrease in the bending modulus of model bimolecular membranes composed of monounsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine provides evidence for softer membranes in the presence of L-glutamate. Kainic acid does not affect membrane mechanical stability even at ten-fold higher concentrations. Both the L-glutamic and kainic acids reduce acetylcholinesterase activity and deviation from the normal functions of neurotransmission in synapses is presumed. The presented results regarding the modulation of the enzyme activity of synaptic membranes and surface properties of synaptosomes are expected by biochemical and biophysical studies to contribute to the elucidation of the molecular mechanisms of neurotransmitters/agonists' action on membranes.

Viper toxins affect membrane characteristics of human erythrocytes.

Elucidating electrokinetic stability by which surface charges regulate toxins interaction with erythrocytes is crucial for understanding the cell functionality. Electrokinetic properties of human erythrocytes upon treatment of Vipoxin, phospholipase A2 (PLA2) and Vipoxin acidic component (VAC), isolated from Vipera ammodytes meridionalis venom were studied using particle microelectrophoresis. PLA2 and Vipoxin treatments alter the osmotic fragility of erythrocyte membranes. The increased stability of cells upon viper toxins is presented by the increased zeta potential of erythrocytes before sedimentation of cells during electric field applied preventing the aggregation of cells. Lipid peroxidation of low dose toxin-treated erythrocytes shows reduced LP products compared to untreated cells. The apparent proton efflux and conductivity assays are performed and the effectiveness PLA2 > Vipoxin>VAC is discussed. The reported results open perspectives to a further investigation of the electrokinetic properties of the membrane after viper toxins treatment to shed light on the molecular mechanisms driving the mechanisms of inflammation and neurodegenerative diseases.

Polylysine effect on thylakoid membranes.

Thylakoid membranes of photosynthetic plant chloroplasts are involved in a wide range of energy producing pathways. Their stacking can be employed in order to provide increased surface area for biocatalytic purposes. Here we probe the aggregate formation of higher plants' thylakoids using low-molecular poly-l-lysine as an electrostatic polymer linker in low ionic strength media. Microelectrophoresis, actinic light scattering, millisecond-delayed fluorescence and free radical production of thylakoid membranes are measured and analyzed in the presence of the cationic polypeptide to track its influence on the surface electrical properties, the electron-transport processes and the proton gradient accumulation across membranes. Enhanced proton gradient in polylysine-treated thylakoids is obtained upon illumination due to alterations of the proton intake across the membrane resulting from the non-specific electrostatic interactions of the cationic polypeptide with thylakoids. We report lower rates of lipid peroxidation in polylysine-treated thylakoids measured both in the dark and under illumination in salt-free medium. The gained insight on the effect of polycations on photosynthetic membranes may be used in future developments of thylakoid-based approaches for energy transfer applications.

Desiccation-induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy, electrokinetic and optical measurements.

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation-induced alterations in surface topography of thylakoids isolated from well-hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well-hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well-hydrated H. rhodopensis had a grainy surface, significantly different from the well-structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross-sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.

Surface electric charge of thylakoid membranes from genetically modified tobacco plants under freezing stress.

In the present study we characterize for the first time electrokinetic and light scattering properties of thylakoids from freezing-tolerant tobacco plants, transformed to accumulate osmoprotectants (proline: AtP5Cs, VacP5Cs; fructan: SacB; glycine betaine: codA). Tobacco plants of wild type (WT) and transformed variants were cultivated at 2°C (cold acclimated) and -2°C (freezing stressed). "Lower salt" thylakoids (I=0.0006) of WT and SacB plants exhibited a decrease in electrophoretic mobility (EPM) after (2°C) treatment. AtP5Cs thylakoids (22°C) show a substantial increase in negative electrical charge (σ) upon illumination. We observed that "low salt"SacB thylakoids at 22°C and 2°C increased the σ on their membrane surfaces during the process of acclimation. WT (22°C) and AtP5Cs thylakoids (2°C) in "low salt" media (I=0.0156) showed a substantial increase in surface electrical charge upon illumination. Cold acclimation on WT and freezing stress on transformed plants resulted in a decrease in aggregation of thylakoids at both ionic strengths. There was a large enhancement in the relaxation capacity of reverse photosynthetic reactions in codA and SacB tobacco after freezing stress. Maximal intensity of the delayed light emission following low temperature stimuli was decreased, revealing a path for tobacco transformants to improve their cold stress tolerance. Here, we suggest the EPM value as an indicator for stability of thylakoids undergone genetic transformation.

Antibodies use heme as a cofactor to extend their pathogen elimination activity and to acquire new effector functions.

Various pathological processes are accompanied by release of high amounts of free heme into the circulation. We demonstrated by kinetic, thermodynamic, and spectroscopic analyses that antibodies have an intrinsic ability to bind heme. This binding resulted in a decrease in the conformational freedom of the antibody paratopes and in a change in the nature of the noncovalent forces responsible for the antigen binding. The antibodies use the molecular imprint of the heme molecule to interact with an enlarged panel of structurally unrelated epitopes. Upon heme binding, monoclonal as well as pooled immunoglobulin G gained an ability to interact with previously unrecognized bacterial antigens and intact bacteria. IgG-heme complexes had an enhanced ability to trigger complement-mediated bacterial killing. It was also shown that heme, bound to immunoglobulins, acted as a cofactor in redox reactions. The potentiation of the antibacterial activity of IgG after contact with heme may represent a novel and inducible innate-type defense mechanism against invading pathogens.

Ferrous ions and reactive oxygen species increase antigen-binding and anti-inflammatory activities of immunoglobulin G.

Polyspecific antibodies represent a first line of defense against infection and regulate inflammation, properties hypothesized to rely on their ability to interact with multiple antigens. We demonstrated that IgG exposure to pro-oxidative ferrous ions or to reactive oxygen species enhances paratope flexibility and hydrophobicity, leading to expansion of the spectrum of recognized antigens, regulation of cell proliferation, and protection in experimental sepsis. We propose that ferrous ions, released from transferrin and ferritin at sites of inflammation, synergize with reactive oxygen species to modify the immunoglobulins present in the surrounding microenvironment, thus quenching pro-inflammatory signals, while facilitating neutralization of pathogens.

Melittin-induced changes in thylakoid membranes: particle electrophoresis and light scattering study.

Thylakoids were used as a model system to evaluate the effect of bee venom peptide melittin (Mt) on membrane surface charge. At neutral pH, thylakoid membrane surfaces carry excess negative electrical charge. Mt strongly altered the electrophoretic mobility (EPM) of 'low-salt' thylakoids and did not significantly change the EPM of 'high-salt' thylakoids. Mt increased the primary ionic-exchange processes across the 'low-salt' thylakoid membranes, while it did not affect those of 'high-salt' thylakoids. Mt decreased the proton gradient generation on the membranes at both ionic strengths, but it affected more strongly the 'high-salt' than that of 'low-salt' thylakoids. The primary photochemical activity of photosystem II, estimated by the ratio Fv/Fm, was not influenced by the low Mt concentrations. It decreased only when chloroplasts had been incubated with higher Mt concentrations and this effect was better expressed in 'low-salt' than in 'high-salt' thylakoid membranes.

Electrokinetic and light scattering properties of pea and Chlamydomonas reinhardtii thylakoid membranes: effect of phytohemagglutinin.

The effects of phytohemagglutinin (PHA) and illumination on the surface charge densities and 90 degrees light scattering properties of pea and Chlamydomonas reinhardtii thylakoids were investigated. The electrophoretic mobility (EPM) of pea thylakoids decreased after treatment by various concentrations of PHA at ionic strengths of I = 0.01 and I = 0.02, while that of C. reinhardtii thylakoids remained stable except for a drop after treatment by PHA at a concentration of 6 ng/mL in a medium with an ionic strength of I = 0.01. Illumination did not influence the EPM of untreated thylakoids. However, if the EPM of thylakoids had been retarded by pretreatment with PHA, light exposure stimulated a recovery of the reduced negative surface charge density up to at least the initial values. In addition to reducing EPM, PHA also induced a decrease of the basal light scattering property of pea thylakoids, which is an indicator of thylakoid aggregation. The physiological role of the membrane surface charges of thylakoid particles in lectin regulated processes of thylakoid stacking and activity is discussed.