卷 12, 编号 2 (2018)

In order to investigate quantitatively the role of lipopolysaccharides (LPS) from outer bacterial membrane at the initial state of bacterium adhesion to a host cell membrane, a model system for single cell force spectroscopy was developed and used. The system comprised of an LPS-coated microsphere placed into optical trap and a J774 macrophage being approached the microsphere to initiate their binding and then moved back to rupture the bond. An “object shadow” phenomenon was discovered, manifested as large-scale variations of the signal of photodetector registering the trapped microsphere displacement, such variations emerging long before the actual interaction between the macrophage and microsphere. The theory and the measurements technique were developed for registration of the force required for detachment of bounded microsphere from the object investigated by means of optical tweezers under the “object shadow” conditions. Characteristic spectra of binding force between J774 macrophage and microspheres functionalized with various LPS, as well as LPS plus complementary antibodies preparations were obtained at the rate of detachment force application of 3–6 pN/s. Force spectrum characteristic of Yersinia pseudotuberculosis LPS possessing O-antigen had a maximum at ~14 pN with half-width of ~23 pN. The treatment of O-antigen with complementary antibodies resulted in transformation of this spectrum into a spectrum with maximum at ~10 pN and half-width of ~14 pN, being almost identical to the spectrum of Y. pestis LPS devoid of O-antigen, with a maximum at ~9 pN and half-width of ~13 pN. A possible mechanism of force spectra formation has been proposed under assumptions of nonspecific binding of O-antigen and probable receptor-type binding of LPS core region to the macrophage surface. The elastic modulus of macrophage envelope, as estimated using analysis of displacement of the contacting microsphere as an indenter, was ≈0.17 pN/nm.

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.

A decrease of the plasma membrane H⁺-ATPase activity in plant cells is associated with the formation of response to adverse factors and reception of signals. A theoretical analysis of the influence of the H⁺-ATPase activity on the flow of CO₂ into a plant cell has been conducted. With this purpose the model of transport processes and electrogenesis in the plant cell developed previously was used, which takes into account transport systems, including H⁺-ATPase, in the plasma membrane and tonoplast. The CO₂ fraction (\({P_{c{o_2}}}\) ) in the total amount of inorganic carbon (C_i) in the external medium was used as an indicator of the CO₂ amount entering the plant cell; this parameter depends on the extracellular pH, which, in particular, is influenced by the H⁺-ATPase activity. Excitable and non-excitable cells were simulated. It was shown that a decrease of the H+-ATPase activity causes a \({P_{c{o_2}}}\) reduction in both variants of the model, and this reduction has an extremum: after passing through minimal values, \({P_{c{o_2}}}\) reaches a stationary level. The dynamics of \({P_{c{o_2}}}\) decrease may be related to the Ca²⁺ influx into the cytoplasm of the plant cell. The reduction of \({P_{c{o_2}}}\) depended on the extent of the H⁺-ATPase inactivation and on its initial activity. As a whole, it was shown that the inactivation of the H⁺-ATPase can affect the CO₂ uptake in a plant cell and thereby regulate photosynthetic processes.

The effectiveness of live cell mRNA detection for neurobiological studies was evaluated. We modified the commercial protocol for the use of RNA detection probes (SmartFlareTM, Merck) for primary hippocampal cultures. It was shown that RNA probes could be used both as an independent evaluation system and in combination with Ca²⁺ imaging. The complex of these methods provided the unique possibility of performing simultaneous studies of the functional calcium homeostasis of neuronal-glial networks and differential analysis of the plasticity of cells with different levels of mRNA expression.

The binding of peptide VEGEGEEGEEY to the surface of ovarian cancer cells was studied by confocal microscopy. It has been demonstrated that the peptide competes with hyaluronan for the binding to RHAMM (receptor for hyaluronan-mediated motility). It was found that peptide VEGEGEEGEEY specifically bound to RHAMM on ovarian cancer cell surface, and this interaction was blocked by anti-RHAMM antibodies. The peptide did not bind to RHAMM-knockout fibroblasts RHAMM^(–/–) but interacted with transfected fibroblasts RHAMM^(+/+), which further confirms the binding of the peptide to RHAMM. Evaluation of the peptide binding selectivity showed that the peptide reacts with cancer cells and does not bind to the surface of fibroblasts and normal cells. Thus, the specificity of binding of peptide FITC-VEGEGEEGEEY to RHAMM suggests a possibility of its application as a molecular probe for imaging and diagnosis of ovarian cancer at early stages.

Using immunocytochemistry and confocal laser microscopy, the distribution of nucleolin (protein C23) in neurons of the human substantia nigra was investigated. Fragments of the human midbrain (n = 8) with the verified lack of neurodegeneration were used in the study. The material was fixed in zinc ethanol formaldehyde, a special fixative that provides high preservation of antigenic determinants. New morphological data on the distribution of nucleolin in neurons of the human substantia nigra were obtained. Nucleolin was found in the nucleolus and nucleoplasm of dopaminergic neurons but not in the cytoplasm or cell surface. In the nucleolus and nucleoplasm, protein C23 is distributed irregularly in the form of some accumulations (clumps) with fuzzy borders. Moreover, nucleolin distribution varied in structurally similar neurons containing cytoplasmic neuromelanin. The Marinesco bodies typically not exhibiting nucleolin expression were identified in the nucleus, in addition to nucleoli, by confocal laser microscopy with simultaneous use of transmitted light detector. The findings can contribute to elucidation of the role of nucleolin in the specific functions of normal dopaminergic neurons in the human substantia nigra and assessment of probable involvement of C23 in the development of nucleolar stress and neurodegeneration.