Spectral and conformational characteristics of phycocyanin associated with changes of medium pH.

C-phycocyanin (C-PC) is the main component of water-soluble light-harvesting complexes (phycobilisomes, PBS) of cyanobacteria. PBS are involved in the absorption of quantum energy and the transfer of electronic excitation energy to the photosystems. A specific environment of C-PC chromophoric groups is provided by the protein matrix structure including protein-protein contacts between different subunits. Registration of C-PC spectral characteristics and the fluorescence anisotropy decay have revealed a significant pH influence on the chromophore microenvironment: at pH 5.0, a chromophore is more significantly interacts with the solvent, whereas at pH 9.0 the chromophore microenvironment becomes more viscous. Conformations of chromophores and the C-PC protein matrix have been studied by Raman and infrared spectroscopy. A decrease in the medium pH results in changes in the secondary structure either the C-PC apoproteins and chromophores, the last one adopts a more folded conformation.

Na+ i/K+ i imbalance contributes to gene expression in endothelial cells exposed to elevated NaCl.

High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+ i/K+ i-dependent mechanism(s).

Changes in the Frequency of Rhythmic Excitation of Retzius Cells during Thermal Stimulation of Leech Skin.

Thermal stimulation of various parts of the skin in Hirudo medicinalis increases the frequency of spontaneous rhythmic excitation of Retzius neurons in leech ganglia. It was shown that the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion increases only in response to thermal stimulation and returns to initial values upon cooling. This effect was also detected in neurons that are not directly connected by nerve fibers with the particular skin area. Changes in the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion were observed during thermal stimulation of not only leech body, but also of the head and caudal suckers. These changes in spontaneous rhythmic excitation of Retzius cells in the segmental ganglion during thermal stimulation were observed in Hirudo medicinalis, but not in Macrobdella decora.

Heat damage of cytoskeleton in erythrocytes increases membrane roughness and cell rigidity.

The intensity of erythrocyte membrane fluctuations was studied by laser interference microscopy (LIM), which provide information about mechanical properties of the erythrocyte membrane. Atomic force microscopy (AFM) was used to study erythrocyte surface relief; it is related to the cytoskeleton structure of erythrocyte membrane. Intact human erythrocytes and erythrocytes with a destroyed cytoskeleton were used. According to the obtained results, cytoskeleton damage induced by heating up to 50 °Ð¡ results in a reduced intensity of cell membrane fluctuations compared to non-treated cells (20.6 ± 10.2 vs. 30.5 ± 5.5 nm, correspondingly), while the roughness of the membrane increases (4.5 ± 1.5 vs. 3.4 ± 0.5 nm, correspondingly).

Visualization and Cytotoxicity of Fluorescence-Labeled Dimeric Magnetite-Gold Nanoparticles Conjugated with Prostate-Specific Membrane Antigen in Mouse Macrophages.

We demonstrated the possibility of penetration of magnetite-gold nanoparticles conjugated with prostate-specific membrane antigen into mouse macrophages. It was found that after 3-h incubation with nanoparticles in a concentration of 15 mg/liter at 37oC, they were seen in only 13% macrophages. In about 90% cells, the nanoparticles were detected within the cytoplasm. Under these conditions, membrane damage was revealed in 25% cells. These results should be taken into account in further development and application of nanomaterials for diagnostic and therapeutic purposes in oncology.

Role of Purinergic Receptors of Erythrocytes in the Regulation of Conformation and Oxygen- and NO-Transporting Capacity of Hemoglobin.

Raman spectroscopy was used to investigate changes in hemoglobin conformation and its ability to transfer O2 and NO induced by activation of purinergic receptors of erythrocytes with extracellular ATP. It was found that addition of ATP in a final concentration of 0.1 mM and higher to erythrocyte suspension was followed by changes in hematoporphyrin conformation, while addition of ATP in a concentration of 1 mM and higher increased the proportion of oxyhemoglobin and NO-associated hemoglobin complexes. In case of purinergic receptors activation in blood erythrocytes, significant changes in hemoglobin conformation were observed only at a final ATP concentration of 5 mM, probably due to buffer properties of the blood.

Changes in the state of hemoglobin in patients with coronary heart disease and patients with circulatory failure.

Morphology of erythrocytes and conformation of hemoglobin-derived hematoporphyrin were studied in patients with coronary heart disease (CHD) and patients with circulatory failure using laser interference microscopy and Raman spectroscopy. Correlation was revealed (r=0.81) between hemoglobin oxygen saturation and oxyhemoglobin fraction in erythrocytes evaluated by Raman spectroscopy. Patients with CHD and patients with circulatory failure showed reduced oxygen-releasing capacity of hemoglobin and hemoglobin content and increased oxygen-binding capacity of hemoglobin, and hemoglobin affinity for oxygen. Significant differences from the control were observed only in patients with circulatory failure. It was found that hemoglobin content, hematocrit, and the shape of erythrocytes during CHD and circulatory failure did not differ from the control, whereas the area of erythrocytes was increased.

Thermal inactivation of volume-sensitive K⁺,Cl⁻ cotransport and plasma membrane relief changes in human erythrocytes.

Previously, we reported that in mammalian erythrocytes irreversible annealing of spectrin heterodimers at 49-50 °C abolished cell volume-dependent regulation of ion carriers, thus suggesting an implication of a two-dimensional (2D) membrane carcass in volume sensing and/or signal transduction. To further examine this hypothesis, we employed atomic force microscopy. This method revealed folded membrane relief of fixed human erythrocytes with an average wave height of 3-5 nm covered by globular structures with a diameter of 40-50 nm and an average height of 1-2 nm. Erythrocyte swelling caused by reduction of medium osmolality decreased the height of membrane surface waves by 40 % and increased K(+),Cl(-) cotransport by approximately sixfold. Both volume-sensitive changes of membrane relief and activity of K(+),Cl(-) cotransporter were abolished by a 10-min preincubation at 50 °C. Our results strongly suggest that volume-dependent alterations of the human erythrocyte membrane relief are caused by reorganization of the 2D spectrin-actin network contributing to regulation of the activity of volume-sensitive ion transporters.

Oxygen-binding characteristics of erythrocyte in children with type I diabetes mellitus of different duration.

Oxygen-binding properties of erythrocyte hemoglobin were studied in children with type 1 diabetes mellitus by Raman spectroscopy. The content of hemoglobin-oxygen complexes increased significantly only in children with lasting disease (more than 1 year); oxygen-binding capacity of hemoglobin is significantly changed, while its capacity to release oxygen remained unchanged. These changes were paralleled by alteration of hemoglobin affinity for oxygen. The area and content of hemoglobin were studied by laser interference microscopy. Hemoglobin content increased significantly in erythrocytes of patients with a more than 1-year history of type 1 diabetes mellitus. In these children, a significant inverse correlation between oxyhemoglobin fraction, oxygen binding capacity, and cholesterol content was found, this clinical parameter positively correlated with affinity for oxygen measured by Raman spectroscopy.

Laser interference microscopy of amphibian erythrocytes: impact of cell volume and refractive index.

This study examined the action of anisosmotic media on the volume of nucleated erythrocytes isolated from Rana temporaria. Elevation of medium osmolarity from 100 to 345 mOsm resulted in attenuation of mean cell volume by more than 3-fold, estimated by hematocrit measurement. By contrast to this 'classic' erythrocyte volume evaluation technique, we did not observe any significant cell volume modulation by examining the 3D reconstruction of erythrocyte interference images obtained by laser interference microscopy. Comparative analysis of mean cell volume, phase height and cell area appraised by laser interference microscopy showed that the lack of visible alterations of phase image geometry was caused by sharp elevation of the average refractive index of the cytoplasm in shrunken cells. Thus, our results show for the first time that laser interference microscopy in combination with a direct method for cell volume measurement may be employed for estimation of the refractory index of intracellular milieu and for assessment of changes of physical chemical properties of the cytoplasm evoked by diverse stimuli including osmotic stress.

Evaluation of erythrocyte shape and status by laser interference microscopy.

The area, thickness, and volume of erythrocytes of different types (discocytes, stomatocytes, and echinocytes) from normal subjects and coronary patients were studied by laser interference microscopy. Increase of pH value leading to the stomatocyte-discocyte-echinocyte transformations resulted in a slight decrease of cell volume. In coronary patients, erythrocyte had larger area and volume and exhibited increased aggregation capacity compared to erythrocytes from controls. The results recommend laser interference microscopy as an adequate method for erythrocyte evaluation in laboratory diagnostic measurements.

Non-invasive study of nerve fibres using laser interference microscopy.

This paper presents the results of a laser interference microscopy study of the morphology and dynamical properties of myelinated nerve fibres. We describe the principles of operation of the phase-modulated laser interference microscope and show how this novel technique allows us to obtain information non-invasively about the internal structure of different regions of a nerve fibre. We also analyse the temporal variations in the internal optical properties in order to detect the rhythmic activity in the nerve fibre at different time scales and to shed light on the underlying biological processes. We observe pronounced frequencies in the dynamics of the optical properties and suggest that the oscillatory modes have similar origin in different regions, but different strengths and mutual modulation properties.

Unraveling cell processes: interference imaging interwoven with data analysis.

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1-0.6 Hz) result from plasma membrane processes and that higher frequency variations (20-26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties.

Dipole potentials indicate restructuring of the membrane interface induced by gadolinium and beryllium ions.

The dipole component of the membrane boundary potential, phi(d), is an integral parameter that may report on the conformational state of the lipid headgroups and their hydration. In this work, we describe an experimental approach to measurements of the dipole potential changes, Deltaphi(d), and apply it in studies of Be(2+) and Gd(3+) interactions with membranes composed of phosphatidylserine (PS), phosphatidylcholine (PC), and their mixtures. Deltaphi(d) is determined as the difference between the changes of the total boundary potential, phi(b), measured by the IFC method in planar lipid membranes and the surface potential, phi(s), determined from the electrophoretic mobility of liposomes. The Gouy-Chapman-Stern formalism, combined with the condition of mass balance, well describes the ion equilibria for these high-affinity cations. For the adsorption of Be(2+) and Gd(3+) to PC membranes, and of Mg(2+) to PS membranes, the values of Deltaphi(b) and Deltaphi(s) are the same, indicative of no change of phi(d). Binding of Gd(3+) to PS-containing membranes induces changes of phi(d) of opposite signs depending on the density of ionized PS headgroups in the bilayer. At low density, the induced Deltaphi(d) is negative (-30 mV), consistent with the effect of dehydration of the surface. At maximal density (pure PS, neutral pH), adsorption of Be(2+) or Gd(3+) induces an increase of phi(d) of 35 or 140 mV, respectively. The onset of the strong positive dipole effect on PS membranes with Gd(3+) is observed near the zero charge point and correlates with a six-fold increase of membrane tension. The observed phenomena may reflect concerted reorientation of dipole moments of PS headgroups as a result of ion adsorption and lipid condensation. Their possible implications to in-vivo effects of these high-affinity ions are discussed.