The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presence of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating. The apparent activation constant of Ca2+ accumulation is 53.9 +/- 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation me- dium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.

/pdf/ca2-accumulation-study-in-isolated-smooth-muscle-1wpijsnmkc.pdf

[Ca2+ accumulation study in isolated smooth muscle mitochondria using fluo-4 AM].

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

/pdf/evaluation-of-functioning-of-mitochondrial-electron-2fol9jmivl.pdf

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

In this paper, a Petri net-based model of the fluorescence of the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). NADH and FAD nucleotides from the electron transport chain and the swelling of isolated mitochondria is presented. The model describes selected aspects of these processes under the influence of the chosen electron transport chain chemical modificators. The model expressed in the language of the Petri net theory has an intuitive graphical interpretation and can be analyzed using rigorous mathematical methods. For the simulation experiment, we chose the Cell Illustrator v.3 software (Human Genome Center, University of Tokyo, Japan). An analysis of the fluorescent response of NADH/FAD in the isolated mitochondria to specific electron transport chain inhibitors (rotenone, antimycin A, and sodium cyanide (NaCN)) and protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) revealed a correlation between the changes in the NADH/FAD fluorescence (their redox state) and the functions of particular complexes in the inner mitochondrial membrane. The inhibition of the electron transport chain resulted in the organelle’s swelling; we obtained mathematical equations through modeling to formalize the process of mitochondria swelling and NADH/FAD fluorescence changes in a medium with sodium azide. In particular, these equations adequately and simultaneously described the time characteristics of the reduction of the fluorescence of nucleotides and the swelling of mitochondria. Our model enabled us to predict the changes in the organelle NADH/FAD fluorescence and their hydrodynamic diameters in time. Furthermore, it helped us to optimize the experimental procedures, allowing us to analyze the process dynamics and to compare the modeling results with actual observations under changing compositions of the incubation media and the presence of activators/inhibitors.

/pdf/methodology-of-petri-networks-for-simultaneous-evaluation-of-4cwlct2i17.pdf

Methodology of Petri networks for simultaneous evaluation of the impact of different modifiers on the fluorescence of nucleotides from electron transport chain in isolated mitochondria and on the process of swelling

Using photon correlation spectroscopy, which allows investigating changes in the hydrodynamic dia­meter of the particles in suspension, it was shown that ultrahigh concentrations of Ca2+ (over 10 mM) induce swelling of isolated mitochondria. An increase in hydrodynamic diameter was caused by an increase of non-specific mitochondrial membrane permeability to Ca ions, matrix Ca2+ overload, activation of ATP- and Ca2+-sensitive K+-channels, as well as activation of cyclosporin-sensitive permeability transition pore. To formalize the experimental data and to assess conformity of experimental results with theoretical predictions we developed a simulation model using the hybrid functional Petri net method.

/pdf/the-use-of-the-petri-net-method-in-the-simulation-modeling-2sohyd6ono.pdf

The use of the Petri net method in the simulation modeling of mitochondrial swelling.

Immune aggression to transplanted allogeneic bone marrow, i.e. the graft-versus-host disease (GVHD), could be decreased by the suppression of effector and/or activation of T- regulatory cells (Treg). This task could be solved by co-transplantaiton of allogeneic bone marrow and mesenchymal stromal cells (MSCs). This study demonstrated the elevated immune modulating activity of MSCs by their culturing in vitro on Al₂0₃ oxide nanocoatings. Introduction of the cells to the animals with GVHD resulted in an increased content of Treg in the spleen of bone marrow recipients, reduced severity of the pathology, and higher survival of animals. Thefindings could be the basis for developing the new approaches to optimize the GVHD treatment methods involving the oxide nanocoating cultured MSCs.

/pdf/experimental-therapy-of-graft-versus-host-disease-by-1z8omavq5c.pdf

Experimental therapy of graft-versus-host disease by mesenchymal stromal cells grown on oxide nanocoatings.

The influence of modulators of calcium exchange in mitochondria--oligomycin, Mg2+ and ruthenium red (RuR)--on the myometrium mitochondria size and granularity was studied in the work. The study of the mitochondria size was carried out using the photon correlation spectroscopy. It was shown that the average hydrodynamic diameter was 655 +/- 14 nm (n = 5; control). The addition of oligomycin (1 microg/ml)--the inhibitor of ATP-synthase F0-component, increases the mitochondria average hydrodynamic diameter to 913 +/- 75 nm (n = 5), that is by 39% more than the control. In the presence of RuR (10 microM) (Ca2(+)-uniporter inhibitor) and Mg2+ (7 mM) the mitochondria average hydrodynamic diameter increases to 788 +/- 28 and 788 +/- 38 nm (n = 5) respectively, that is by 17% more than the control. Using flow cytometry it was shown, that oligomycin (1 microg/ml) causes the increase of side scattering of the mitochondria. Addition of RuR (10 microM) and Mg2+ (7 mM) does not lead to significant changes in side scattering of the mitochondria. So it was shown that oligomycin significantly increases mitochondria granularity, but Mg2+ and RuR have no influence on this parameter

Kandaurova Nv

Papers

Modulators of transmembrane calcium exchange in myometrium mitochondria change their hydrodynamic diameter