New nanocomposites for SERS studies of living cells and mitochondria.

A great enhancement in Raman scattering (SERS) from heme-containing submembrane biomolecules inside intact erythrocytes and functional mitochondria is demonstrated for the first time using silver-silica beads prepared using a new method involving aerosol pyrolysis with aqueous diamminesilver(i) hydroxide as a unique source of plasmonic nanoparticles for SiO2 microspheres. The recorded SERS spectra reveal a set of characteristic peaks at 750, 1127, 1170, 1371, 1565, 1585 and 1638 cm-1, resulting from the normal group vibrations of the pyrrole rings, methine bridges and side radicals in the heme molecules. The SERS spectra of functional mitochondria are sensitive to the activity of the mitochondrial electron transport chain, thus making the method a novel label-free approach to monitor the redox state and conformation of cytochromes in their natural cell environment. The developed nanocomposites are highly suitable for the analysis of biological objects due to their robust synthesis and superior spatial and temporal signal reproducibility, which was preserved for a period of at least one year.

[Studies of the blood antioxidant system and oxygen-transporting properties of human erythrocytes during 105-day isolation].

Effects of strict 105-d isolation on blood antioxidant status, erythrocyte membrane processes and oxygen-binding properties of hemoglobin were studied in 6 male volunteers (25 to 40 y.o.) in ground-based simulation of a mission to Mars (experiment Mars-105). The parameters were measured using venous blood samples collected during BDC, on days 35, 70 and 105 of the experiment and on days 7 and 14-15 after its completion. Methods of biochemistry (determination of enzyme activity and thin-layer chromatography) and biophysical (laser interference microscopy, Raman spectroscopy) showed changes in relative content of lipid and phospholipid fractions suggesting growth of membrane microviscosity and increase in TBA-AP (active products of lipids peroxidation interacting with thiobarbituric acid). A significant increase in glucose-6-phosphate dehydrogenase and superoxide dismutase activities against reduction of catalase activity points to both reparative processes in erythrocytes and disbalance between the number of evolving active forms of oxygen and antioxidant protection mechanisms in cells. Hemoglobin sensitivity of oxygen and blood level of oxyhemoglobin were found to increase, too. It is presumed that adaptation of organism to stresses experienced during and after the experiment may destroy balance of the antioxidant protection systems which is conducive to oxidation of membrane phospholipids, alteration of their content, increase of membrane microviscosity and eventual failure of the gas-exchange function of erythrocytes.

[The use of nanoparticles for the study of conformations submembrane hemoglobin].

The advantages and features of the integrated application of methods of atomic force microscopy, laser interference microscopy and Raman microscopy in the study of erythrocytes was discussed. For the successful application of Raman microscopy in surface enhanced Raman spectroscopy mode the silver colloids was using. The dependence of the enhancement of Raman signals from silver colloids size was demonstrated. The using of developed methods for clinic diagnostic was discussed.

Dynamical patterns of calcium signaling in a functional model of neuron-astrocyte networks.

We propose a functional mathematical model for neuron-astrocyte networks. The model incorporates elements of the tripartite synapse and the spatial branching structure of coupled astrocytes. We consider glutamate-induced calcium signaling as a specific mode of excitability and transmission in astrocytic-neuronal networks. We reproduce local and global dynamical patterns observed experimentally.

[Role of erythrocyte cytoplasmic structures in changes in the affinity of haemoglobin for oxygen].

Changes in the refractive index of the cytoplasm and the affinity of haemoporphyrin of erythrocyte haemoglobin to oxygen (pH, 2,3-diphosphoglycerate) have been investigated using laser interference microscopy and Raman spectroscopy. It has been established that a decrease in pH and an increase in the content of 2,3-diphosphoglycerate are accompanied by changes in both the form of the cell and the refractive index of the cytoplasm and the affinity of haemoporphyrin of hemoglobin to oxygen. It has been shown that as pH is reduced, the capacity of haemoporphyrin for binding oxygen decreases and as the concentration of 2,3-diphosphoglycerate is increased, the ability of haemoporphyrin for oxygen reabsorption increases.

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.

Giant glial cell: new insight through mechanism-based modeling.

The paper describes a detailed mechanism-based model of a tripartite synapse consisting of P- and R-neurons together with a giant glial cell in the ganglia of the medical leech (Hirudo medicinalis), which is a useful object for experimental studies in situ. We describe the two main pathways of the glial cell activation: (1) via IP(3) production and Ca(2 +) release from the endoplasmic reticulum and (2) via increase of the extracellular potassium concentration, glia depolarization, and opening of voltage-dependent Ca(2 +) channels. We suggest that the second pathway is the more significant for establishing the positive feedback in glutamate release that is critical for the self-sustained activity of the postsynaptic neuron. This mechanism differs from the mechanisms of the astrocyte-neuron signaling previously reported.

Effect of proteins from the spirochete Borrelia burgdorferi sensu lato on myelinated nerve excitability.

We studied the effect of spirochete Borrelia burgdorferi sensu lato cell membrane proteins on excitability of myelinated nerve fiber. It was found that cell surface proteins of spirochetes B. burgdorferi s. s. bind to Ranvier nodes of the axon and to Schwann cells. Binding of B. burgdorferi s. s. and B. garinii to the nerve fiber modulates the amplitude and conduction velocity of the action potential, while B. afzelii had no effect on these parameters. The decrease in the spike amplitude and conduction velocity during sorption of B. burgdorferi s. s. or cell wall proteins was accompanied by desorption of membrane-bound calcium.

[Role of viscosity and permeability of erythrocyte plasmatic membrane in controlling the oxygen transport effectiveness by human hemoglobin on completion of space flight].

Plasmatic membrane viscosity and permeability and hemoporphyrine conformation in human hemoglobin were studied on completion of long-duration space flight (LSF). Reversible increases in viscosity and selective permeability (Na+/H+ -turnover) of erythrocyte plasmatic membrane were observed immediately after and in the period of recovery from LSF. Viscosity of lipids in both external and internal locations of plasmatic membrane in human erythrocytes was changed after LSF. The reversible rise of the Na+/H+ -turnover is likely to alter intracellular pH and oxygen binding with hemoglobin. The former is confirmed by the concurrent reversible decline in the deoxyhemoglobin ability to bind oxygen and the oxyhemoglobin ability to retain oxygen. In LSF and during readaptation to the normal gravity blood levels of hemoglobin and free iron are known to be reduced and may be answerable for the hypoxic state of human organism.

Ion transport, membrane fluidity and haemoglobin conformation in erythrocyte from patients with cardiovascular diseases: Role of augmented plasma cholesterol.

Tissue hypoxia, which plays a key role in the development of renal and vascular complications of cardiovascular diseases (CVD), might be considered a consequence of vascular remodeling and/or attenuated oxygen (O(2)) delivery by erythrocytes. Using Raman spectroscopy (RS), we observed that erythrocytes from patients with CVD exhibit changes in the conformation of haemoglobin (Hb) haemoporphyrin (HP), reflecting its lower O(2) transport capacity. Hypertriglyceridemia and hypercholesterolemia are well-known hallmarks of CVD. This study examined the role of plasma lipids in the regulation of erythrocyte membrane viscosity, oxy-Hb content as well as Na(+)/H(+) exchange and Ca(2+)-ATPase, whose activities are altered in patients with CVD. HP conformation was assessed by RS of blood samples. Membrane fluidity was estimated at depths of 0.6-0.8 and 2.2nm by electron-paramagnetic resonance spectroscopy of erythrocytes loaded with spin-labeled 5-doxylstearic acid and 16-doxylstearic acid, respectively. Ion-selective electrodes were employed for the study of H(+) and Ca(2+) fluxes. Both oxy-Hb content and erythrocyte membrane fluidity were decreased in essential hypertension and coronary artery disease patients and negatively correlated with plasma cholesterol but not triglyceride content. This observation allows us to assume that decreased oxy-Hb content in patients with CVD is caused by high plasma cholesterol via attenuation of erythrocyte membrane fluidity and its permeability to O(2). Plasma cholesterol level correlated positively and negatively with erythrocyte Na(+)/H(+) exchange and Ca(2+)-ATPase, respectively. However, in contrast to membrane fluidity, the impact of these ion transporters in oxy-Hb regulation under baseline conditions seems to be negligible. We propose that decreased oxy-Hb content contributes to the reduced O(2) tissue supply seen in patients with CVD.

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.

[The role of plasmatic erythrocytic membrane in formation of hypoxia in patients with chronic cardiac failure].

AIM: To study oxygen supply to erythrocytes in patients with chronic cardiac failure as a factor provoking hypoxia. MATERIAL AND METHODS: The trial enrolled 12 men with chronic cardiac failure (CCF) of functional NYHA class II aged 37 to 74 years (group A). Group B--healthy donors (n = 10) aged 20-40 years. Conformation changes of hemoporphirine were detected with degradation spectroscopy, viscosity of plasmatic membrane--with resonance spectroscopy. Degradation spectroscopy estimated content of Hb-ligant complexes as 0.34 +/- 0.013; complexes I Hb-NO 0.73 +/- 0.11, complexes II Hb-NO 0.28 +/- 0.08 (group A). RESULTS: Electronic-paramagnetic spectroscopy has estimated: S--0.716 +/- 0.005 (group A), 0.667 +/- 0.011 (group B). In group B- 0.5 +/- 0.08; 0.41 +/- 0.22; 0.55 +/- 0.12, respectively. CONCLUSION: Patients with CHF have high microviscosity of plasmatic erythrocytic membrane near location of polar lipid groups; subnormal content of oxyhemoglobin and elevation of the number of complexes of nitric hemoglobin-oxide; altered binding of nitric oxide with hemoglobin.

Interference microscopy under double-wavelet analysis: a new approach to studying cell dynamics.

This Letter combines a novel experimental approach to the study of intracellular processes with a newly developed technique for multimode time-series analysis. Experiments are performed on isolated pond snail (Lymnaea stagnalis) neurons. Local variations in the cellular refractive index as detected by laser interference microscopy are related to the processes in the cell. A wavelet analysis shows the presence of several identifiable modes in the membrane and intracellular dynamics, and a double-wavelet analysis reveals nonlinear interactions between the regulatory processes in the form of mutual frequency and amplitude modulations.