PubMed Health⌕ Search

Biomedical subjects

A N Tikhonov

Publications and source records attributed to A N Tikhonov.

At least 19 recordsLinked to original sources
PubMed2005

Biological and polymeric self-assembled hybrid systems: structure and properties of thylakoid/polyelectrolyte complexes.

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.

Acrylic Resins↗
PubMed2004

A dynamic phase microscopic study of optical characteristics of individual chloroplasts.

Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), h(x,y,t) approximately integraln(x,y,z,t)dz, an integral refractive index projection of the medium, n(x,y,z,t), in optically transparent biological specimens at high spatial and temporal resolutions. In this study, DPM was used for the analysis of fluctuations in the optical characteristics of individual bean chloroplasts in various metabolic states. A "phase image" of an individual chloroplast, which represents a three-dimensional plot of the "phase height", was obtained for the first time, and the frequency spectra of the fluctuations of h(x,y,t) were investigated. The fluctuation patterns, i.e., the intensity and the frequency spectra of phase height fluctuations in bean chloroplasts (Class B) were found to depend on their metabolic state. Under conditions of noncyclic (or pseudocyclic) electron transport, the fluctuations displayed characteristic frequencies in the range of 0.25-0.6 Hz and were space-time-correlated in the chloroplast domains with the cross sizes of approximately 2 microm. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 microM). A stronger (in comparison with 20 microM valinomycin) effect of 20 microM nigericin suggests that the light-induced generation of the transmembrane pH difference (DeltapH) makes the main contribution to the increment of space-correlated fluctuations of h(x,y,t). Studies of chloroplasts incubated in media of various osmolarity (50-500 mM sucrose) have shown that structural changes in thylakoids are among other factors responsible for phase height fluctuations.

Chloroplasts↗
PubMed2004

Effects of diffusion and topological factors on the efficiency of energy coupling in chloroplasts with heterogeneous partitioning of protein complexes in thylakoids of grana and stroma. A mathematical model.

In this work, we studied theoretically the effects of diffusion restrictions and topological factors that could influence the efficiency of energy coupling in the heterogeneous lamellar system of higher plant chloroplasts. Our computations are based on a mathematical model for electron and proton transport in chloroplasts coupled to ATP synthesis in chloroplasts that takes into account the nonuniform distribution of electron transport and ATP synthase complexes in the thylakoids of grana and stroma. Numerical experiments allowed the lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids to be simulated under different metabolic conditions (in the state of photosynthetic control and under conditions of photophosphorylation). This model also provided an opportunity to simulate the effects of steric constraints (the extent of appression of thylakoids in grana) on the rates of non-cyclic electron transport and ATP synthesis. This model demonstrated that there might be two mechanisms of regulation of electron and proton transport in chloroplasts: 1) slowing down of non-cyclic electron transport due to a decrease in the intra-thylakoid pH, and 2) retardation of plastoquinone reduction due to slow diffusion of protons inside the narrow gap between the thylakoids of grana. Numerical experiments for model systems that differ with respect to the arrangement of thylakoids in grana allowed the effects of osmolarity on the photophosphorylation rate in chloroplasts to be explained.

Adenosine Triphosphate↗
PubMed2000

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains.

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.

Biophysical Phenomena↗
PubMed1998

Superoxide radical production by sponges Sycon sp.

Using the catechol Tiron as an O2-. scavenger, we showed that sea sponges (Sycon sp.) produce superoxide radicals in sea water at a high rate without any stimuli added. The rate of O2-. outflow from sponges to their water surroundings reaches a value of 0.5 nmol/min per sponge at pH 6.5. The generation of O2-. was inhibited by Cu,Zn-superoxide dismutase, and restored by the addition of KCN. We also confirmed the abiotic production of O2-. in sea water, detected earlier with a different method by Petasne and Zika [Nature 325 (1987) 516-518].

Animals↗
PubMed1998

Oxygen permeability of thylakoid membranes: electron paramagnetic resonance spin labeling study.

Oxygen transport in thylakoid membranes of spinach chloroplasts (Spinacia oleracea) has been studied by observing the collisions of molecular oxygen with spin labels, using line broadening electron paramagnetic resonance (EPR) spectroscopy. Stearic acid spin labels were used to probe the local oxygen diffusion-concentration product. The free radical moiety was located at various distances from the membrane surface, and collision rates were estimated from linewidths of the EPR spectra measured in the presence and absence of molecular oxygen. The profile of the local oxygen diffusion-concentration product across the membrane determined at 20 degrees C demonstrates that this product, at all membrane locations, is higher than the value measured in water. From the profile of the oxygen diffusion-concentration product, the membrane oxygen permeability coefficient has been estimated using the procedure developed earlier (W.K. Subczynski, J.S. Hyde, A. Kusumi, Proc. Natl. Acad. Sci. USA 86 (1989) 4474-4478). At 20 degrees C, the oxygen permeability coefficient for the lipid portion of the thylakoid membrane was found to be 39.5 cm s-1. This value is 20% higher than the oxygen permeability coefficient of a water layer of the same thickness as the thylakoid membrane. The high permeability coefficient implies that the oxygen concentration difference across the thylakoid membrane generated under the illumination of the leaf by saturating actinic light is negligible, smaller than 1 microM.

Cell Membrane Permeability↗
PubMed1994

Oxygen production and consumption by chloroplasts in situ and in vitro as studied with microscopic spin label probes.

A new spin-label oximetry approach able to measure the oxygen partial pressure in complex photosynthetic systems has been developed using bovine serum albumin (BSA)-coated light paraffin oil particles containing cholestane spin label (CSL). Paraffin oil particles protect the spin label against the action of chemically active metabolites. The amplitude of the electron paramagnetic resonance (EPR) signal from CSL measured at a saturating microwave power is sensitive to the concentration of oxygen. We demonstrate here the ability of this method to monitor the kinetics of light-induced oxygen production in situ, i.e., in the interior of a bean leaf. The oxygen release, observed during leaf illumination with continuous light, exhibits an overshoot that correlates with the well-known nonmonotonous behaviour of the Photosystem I reaction center, P700. Short-term illumination of isolated bean chloroplasts, suspended in the presence of the electron mediator methylviologen, induces a reversible uptake of oxygen. However, after prolonged illumination, chloroplasts lose their ability to regenerate oxygen in the dark. The exhaustion of oxygen (and oxygen active forms) is accompanied by the loss of CSL paramagnetism and the capacity to photooxidize P700. Comparison of the kinetics of P700 redox transients with oximetric data demonstrates that oxygen concentration is the essential factor controlling electron transport in leaves and isolated chloroplasts.

Chlorophyll↗
PubMed1994

The structural differences between the embryos of viable and nonviable wheat seeds as studied with the EPR spectroscopy of lipid-soluble spin labels.

Dried and hydrated embryos of wheat seeds (viable and nonviable, harvested in 1992 and 1976, respectively) were studied by the EPR method with the use of the spin-labeling technique. Spin label Tempone was used for testing the plasmalemma integrity. It has been demonstrated that the loss of seed viability correlates with the loss of external membrane integrity. Spin-labeled derivatives of stearic acids, 5-doxylstearate I(12.3) and 16-doxylstearate I(1.14), were used to monitor the changes in structural characteristics of embryo cell membranes. The EPR spectra of these spin labels represent the superpositions of at least two signals from the molecules located in domains characterized by different fluidity. The comparison of the EPR spectra from I(12.3) in embryo cells and model systems (total fraction of lipids and purified seed oil) indicates that the majority of spin label molecules is located in the lipid surroundings, while the minor portion of I(12.3) is localized in so-called lipid bodies which contain seed oil. The embryo cells of viable and nonviable seeds differ in the sizes of these 'solid' and 'fluid' intracellular domains. The environment of spin label molecules located in cell membranes of nonviable seeds is more rigid, as compared with that in the membranes of the viable cells. The study of dehydration-rehydration effects has demonstrated that the loss of water causes the restriction of spin label mobility in embryo cells from both kinds of seeds.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Relationships between the effects of redox potential, alpha-thenoyltrifluoroacetone and malonate on O(2) and H2O2 generation by submitochondrial particles in the presence of succinate and antimycin.

The rate of the antimycin-induced H2O2 and O(2) generation in beef heart submitochondrial particles is maximal at the [succinate]/[fumarate] ratio of approximately 1:5 and decays at both higher and lower redox potentials. Succinate dehydrogenase inhibitors, such as TTFA or malonate, stimulate active oxygen production in the presence of excess succinate but are inhibitory at Eh values more positive than the optimal. The modulation of O(2) and H2O2 generation by these inhibitors can be explained by their effects on the steady-state redox potential(s) of the component(s) of mitochondrial site 2.

Animals↗
PubMed1984

Physical state of the mitochondrial inner membrane as a factor controlling the proteolysis of mitochondrial translation products in yeast.

The degradation of mitochondrial translation products has been studied in Saccharomyces cerevisiae yeast. A high rate of degradation is observed in the early exponential phase of aerobic growth. Maturation of the yeast and glucose repression suppress the degradation. Anaerobic growth is also marked by a low breakdown rate of mitochondrial translation products. These variations did not correlate with the cytochrome c hydrolase activity of sonic submitochondrial particles (this activity was shown to reflect the general state of the proteolytic system of the inner mitochondrial membrane that is responsible for the breakdown of mitochondrially made polypeptides; see Novikova, L.A., et al. (1981) FEBS Lett. 135, 245-248). Experiments with lipid-soluble paramagnetic probes revealed significant variations in the physical state of the mitochondrial inner membrane, as judged from the comparison of the temperature-dependence plots of structural parameters obtained from the EPR spectra of the probes. The breakdown of mitochondrial translation products was, in general, the more rapid the lower was the temperature of the structural transition in the mitochondrial inner membrane and the higher was the relative content of unsaturated fatty acyl chains in the membrane phospholipids.

Electron Spin Resonance Spectroscopy↗
PubMed1984

The effect of phospholipids on the activity of mitochondrial pyrophosphatases. Lipidized soluble mitochondrial pyrophosphatase: the coupling factor for oxidation and phosphorylation.

The effect of temperature on the hydrolase activity of mitochondrial pyrophosphatases, i.e. soluble (PPase I) and membrane (PPase II), has been studied. In contrast to the soluble species, the membrane form has inflexions in the Arrhenius curves. However, after lipidization of PPase I by various phospholipids, the curves also acquire inflexion points, which prove similar or identical with the phase separation points of the lipids used. The closeness of the inflexion points of PPase II, containing phosphatidylcholine, and PPase I lipidized by mitochondria phosphatidylcholine, to the phase separation points of this lipid indicates that the inflexions on the PPase II curves should be ascribed to this phospholipid. It has been shown that the hydrolysis of PPi by SMP is affected by the cooperative rearrangements of the entire lipid component of the membrane rather than by the change of the phase state of PPase II phosphatidylcholine. Reconstitution experiments on the PPi synthesis system have shown that after lipidization PPase I is able to incorporate into SMP and become a coupling factor for respiration and PPi synthesis, like PPase II.

Animals↗
PubMed1983

Correlation between the rate of proteolysis of mitochondrial translation products and fluidity of the mitochondrial inner membrane in Saccharomyces cerevisiae yeast. Alteration of the rate of proteolysis under glucose repression.

Our previous results [Kalnov, Novikova, Zubatov & Luzikov (1979) FEBS Lett. 101, 355-358; Biochem. J. 182, 195-202] suggested that in yeast the mitochondrial translation products localized in the mitochondrial inner membrane are rapidly broken down by a proteolytic system inherent in the membrane. In the present work, it is demonstrated that, on glucose repression in undividing cells of Saccharomyces cerevisiae, there is no proteolysis of the mitochondrial translation products. This effect is not likely to be associated with lower activity of the proteolytic system of the mitochondrial inner membrane. Nor is the cessation of proteolysis due to qualitative changes in the composition of mitochondrial translation products. What repression does cause is a considerable alteration in the physical state (i.e. structure of the lipid bilayer) of the mitochondrial inner membrane; this was established by experiments involving lipid-soluble spin probes. The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondrial phospholipids.

Cycloheximide↗
PubMed1983

Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain.

Antimycin, 2-nonyl-4-hydroxyquinoline N-oxide and funiculosin induce O.2(-) generation by submitochondrial particles oxidizing succinate, whereas KCN, mucidin, myxothiazol or 2,3-dimercaptopropanol inhibit O.2(-) generation. Thenoyltrifluoroacetone does not induce superoxide production by itself but slightly stimulates the reaction initiated by antimycin. The results indicate that auto-oxidation of unstable ubisemiquinone formed in centre o of the Q-cycle generates most of the O.2(-) radicals in the cytochrome bc1-site of the mitochondrial respiratory chain.

Alkenes↗
PubMed1982

[Inhibition of H2O2 and O2-. generation in the respiratory chain, treated with 2,3-dimercaptopropanol].

Inhibition of electron transport in bovine heart submitochondrial particles resulting from treatment with British antilewisite is accompanied by suppression of superoxide radical and H2O2 generation at the b-c1 site of the respiratory chain. In conjunction with the previously obtained data, the results suggest that the effect of British antilewisite is directed towards the step of ubihydroqinone oxidation to ubisemiquinone by the Rieske FeS-protein.

Animals↗
PubMed1982

[Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts].

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.

Chlorophyll↗
PubMed1982

[Interaction of ubisemiquinone with succinate dehydrogenase and the cytochrome chain of mitochondria].

The free radical EPR signals of ubisemiquinone in mitochondria and submitochondrial particles (SMP) were investigated. One of the signals observed under the conditions of the respiratory chain highly oxidized and characterized by an unusually short time of the spin-lattice relaxation has previously been termed as SQ-2. The intensity of SQ-2 in SMP strongly depends on pH, the maximal concentration of QH. is reached at about 8.5. The signal is absent in the succinate dehydrogenase-depleted SMP and is highly sensitive to specific inhibitors of succinate: CoQ-oxidoreductase, such as alpha-thenoyltrifluoroacetone and carboxin. In SMP SQ-2 disappears in the presence of low concentrations of ferricyanide, while in mitochondria this non-penetrating oxidant provokes the appearance of SQ-2. The data obtained suggest that SQ-2 belongs to a stable ubisemiquinone which forms a complex with a FeS center of succinate dehydrogenase, is localized at the M-side of the membrane, and is kinetically isolated from the cytochrome chain. Oxidation of the terminal segment of the respiratory chain of mitochondria and SMP reduced by succinate in the presence of antimycin, is in some cases accompanied by an appearance of a strong free radical EPR signal which is stable at 77K but disappears rapidly in the frozen samples at -30- -40 degrees C. It is suggested that the signal is generated by an antimycin-insensitive oxidation of QH2 to QH. via the branch of the respiratory chain comprised of the Rieske FeS-protein and cytochrome c1. The mechanisms of how the two-electron oxidation-reduction of CoQ is coupled with the one-electron transfer through the cytochromes and FeS centers in the respiratory chain are discussed.

Animals↗
PubMed1980

Platelet adhesion to fluid and solid phospholipid membranes.

We have studied platelet adhesion to phospholipid model membranes in vitro. Our results showed that films made of egg lecithin, dioleoyllecithin or phosphatidylethanolamine from two different sources (egg yolk and E. coli) are unadhesive for platelets. Platelets adhered to films made of distearoyllecithin, dipalmitoyllecithin and N--stearoylsphingomyelin. According to electron spin resonance measurements, the former lipids were present during incubation with platelet-rich plasma above the phase transition temperature, whereas the latter were present below this temperature. Cross-linking of phosphatidylethanolamine films with glutaraldehyde or egg lecithin, as well as dioleoyllecithin with OsO4, abolishes the phase transition of the lipids in these films, transforming them to the solid state. After such treatment the films become adhesive for platelets. Thus fluid liquid crystalline phospholipid membranes are unadhesive for platelets; solid crystalline (gel) films are adhesive for these cells. We suggest that the fluidity of the plasma membrane has an essential role in making the endothelium unadhesive for platelets in vivo.

Animals↗