[Transmembrane transport of K+ and Cl- during pollen grain activation in vivo and in vitro].

We studied the possibility of K+ and Cl- efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl- efflux; potassium channel inhibitor (tetraethylammonium chloride) partially reduced K+ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba2+ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K+ and Cl- release. An important role in these processes is played by NPPB-, TEA- and Ba(2+)-sensitive plasmalemma ion channels.

[Sporopollenin accumulation in Nicotiana tabacum L. microspore wall during its development].

Accumulation of sporopollenin components in microspore wall, its polymerization dynamics and possible participation of reactive oxygen species (ROS) in this process has been studied. For this purpose fluorescent and electron microscopy (TEM) was used. It has been determined that phenylpropanoid components of sporopollenin that form the exine accumulate in the microspore cell wall at the middle and late tetrad stages. At the late tetrad stage, they fully cover the microspore surface and accumulate abundantly in aperture areas. In accordance with this, numerous thick sporopollenin lamellae, electron-dense and acetolysis-resistant, emerge in aperture areas. Exine in the areas between apertures includes both acetolysis-resistant sporopollenin and washout components. These particular parts of the wall are intensively stained with fluorescent dye MitoSOX, which detects the presence of ROS. The staining disappeared after the treatment of microspore with superoxide dismutase, demonstrating the presence of superoxide in the exine. Superoxide easily converts to hydrogen peroxide, which can cause oxidative polymerization of sporopollenin components, leading to the formation of chemically stable biopolymer. The data obtained favor the hypothesis of ROS involvement in the formation of sporopollenin.

[Antioxidant properties of the pollen exine polymer matrix].

The antioxidant properties of exine polymer matrix which forms the outer layer of pollen grain wall were studied. The main component of this matrix is sporopollenin - a unique biopolymer resistant to mechanical and chemical damage. The samples of isolated exine, purified from soluble compounds, were studied with EPR using stable nitroxyl radical TEMPO and DMPO spin trap. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduces TEMPO radical and eliminates hydroxyl radical. Also, the fluorometric analysis demonstrated that the exine eliminated H2O2, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4M NaOH, 170 degrees C) of ethers bonds a large amount of hydroxycinnamic acids has been released, and exines have lost their antioxidant capacity almost completely. The obtained results point to the ability of extracellular polymer matrix of the exine to eliminate free radicals and H2O2 during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.

[Another discussion of membrane voltage alterations in growing pollen tube].

Here we give a critical analysis of the opinion of Andreev (2011) on membrane potential distribution along the pollen tube plasmalemma. He assumes that a lateral gradient of dipole potential exists, but suggests a lateral gradient of transmembrane potential impossible. We demonstrate by concrete examples that the argumentation of the initiator of discussion is based on inaccurate citation of our experimental data (Breygina et al., 2009) and incomplete analysis of previously published articles. Speaking about transmembrane potential, he doesn't consider numerous facts demonstrating the uneven distribution of transmembrane ion fluxes and ion-transport proteins in the pollen tube plasmalemma, as well as data obtained by modeling of transmembrane potential distribution in objects of different shape. In addition, the assumption on the uneven distribution of dipole potential doesn't have an experimental basis neither in studies of the pollen tube, nor in the practice of using fluorescent voltage-sensitive dyes DiBAC4(3) and Di-4-ANEPPS. We are expecting the author to obtain experimental data in support of his position.

[Effects of anion channel blockers NPPB and DIDS on tobacco pollen tube growth and its mitochondria state].

Influence of anion channel blockers NPPB and DIDS on pollen tube growth and its mitochondria functioning was studied by means of fluorescence microscopy and flow cytometry. NPPB (40 microM) blocked pollen tube growth completely, but did not change its diameter. DIDS (20-80 microM) caused pollen tube swelling and bursting, suggesting that DIDS-sensitive channels take part in the regulation of pollen tube osmotic balance. The osmotic effect of low DIDS concentration (20 (Mkappa)M) was not accompanied by changes in the tube growth rate. The mapping of membrane potential on the pollen tube plasmalemma using Di-4-ANEPPS revealed the involvement of NPPB-sensitive but not DIDS-sensitive anion channels in the maintenance of the longitudinal membrane potential gradient along the tube surface. The study of isolated pollen mitochondria showed that DIDS increased their capacity to take up potential-dependent dye DiOC5(3), i. e. caused hyperpolarization of mitochondrial membranes. At the same time DIDS influenced on intramitochondrial ROS content and excretion of ROS from mitochondria. Thus, NPPB and DIDS differently influenced on transmembrane potential distribution along pollen tube plasmalemma, on its osmotic balance, and on mitochondria functioning. This set of data suggests that pollen tube growth is dependent on activity of anion channels that differ in localization and functions.

[Membrane potential changes during pollen germination and tube growth].

We applied quantitative fluorescent microscopy to study membrane potential alterations during pollen germination and in growing pollen tube. Two voltage-sensitive dyes were applied: DiBAC4(3) was used to detect average membrane potential values in pollen grains and isolated protoplasts; Di-4-ANEPPS gave an option of membrane potential mapping on pollen protoplast and pollen tube surfaces. We have found out that tobacco pollen grain activation is accompanied by hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (-108 mV) with respect to the pollen grains (-23 mV) from which they were isolated. We found polar distribution of the membrane potential along the protoplast surface, and longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H(+)-ATPase inhibitor, sodium orthovanadate (1 mM) or its activator fusicoccin (1 microM), the longitudinal voltage gradient altered but did not disappear. Anion channel blocker, NPPB (40 microM), fully discarded the gradient in pollen tubes. Obtained results give evidence of the plasma membrane hyperpolarization during pollen germination and uneven potential distribution on pollen grain and tube surfaces. Inhibitory analysis showed involvement of the plasma membrane H(+)-ATPase and anion channels in membrane potential regulation.

[Formation of reactive oxygen species during pollen grain germination].

The formation of reactive oxygen species in pollen at the early germination stage, which precedes the formation of the pollen tube, was studied. During this period, pollen grain is being hydrated, abruptly increasing its volume, and it passes from the resting state to active metabolism. Fluorescent methods have made it possible to reveal reactive oxygen species in the cytoplasm and inner layer of the pollen wall, intine. The cytoplasmic reactive oxygen species were mostly found in mitochondria, while extracellular ones were localized in aperture zones of intine, as well as in the solution surrounding pollen grains in vitro. The content of extracellular reactive oxygen species decreased after superoxide dismutase (100 units per ml) and diphenylene iodonium (100 microM), which indicates NADPH oxidase as one of possible producent of them. In conditions of suppression of extracellular reactive oxygen species production (100 microM diphenilene iodonium) or their promoted removal (after addition of 10 to 100 microM ascorbic acid), the number of germinating pollen grains increased. This effect disappeared after further increase in the concentration of the listed reagents. The result is evidence of the significance of processes of generation/removal of extracellular reactive oxygen species for pollen germination.

[The role of H+/- ATPase and alternative oxidase in the regulation of intracellular pH at the different stages of development of the tobacco male gametophyte]. / Rol' H+/- ATFazy i al'ternativnoi oksidazy v reguliatsii velichiny vnutrikletochnogo pH na raznykh stadiiakh razvitiia muzhskogo gametofita tabaka.

In order to determine the role of the plasma membrane H(+)-ATPase and alternative oxidase (alternative pathway of respiration) in the regulation of intracellular pH during development of the tobacco male gametophyte, we studied the changes in pH due to the inhibition of these enzymes by orthovadanate and benzhydroxamic acid, respectively. The inhibition of these enzymes decreased the intracellular pH at all three studied stages of the male gametophyte development: middle and late binuclear pollen grains and activated mature pollen grain. The data obtained suggest that H(+)-ATPase and alternative oxidase are involved in the regulation of intracellular pH of the pollen grain during its differentiation and activation that precede germination. At the same time, during the recovery of intracellular pH after its acidification by propionic acid, it was found that other mechanisms, not related to the above mentioned, greatly contribute to the regulation of pH.

[Determination of a position of a functional pore in the tobacco pollen]. / Determinatsiia polozheniia funktsional'noi pory v pyl'tsevom zerne tabaka.

Pollen hydration and germination on the "wet" stigma of Nicotiana tabacum L. were studied by SEM and TEM to reveal the role of the stigma in selecting the germinative pore, and in establishing the axis of polarity in the pollen grain. Pollinated stigmas were fixed with glutaraldehyde or osmium tetroxide vapour, or processed with rapid freeze fixation and freeze substitution. Fixation was performed in 5, 15 or 30 min and 3.5 h after pollination. The tube easily emerged from either pore, this process not depending on the pollen grain orientation relative to the stigma. The orientation of pollen tubes remained random till their length becomes longer than the pollen grain diameter. The TEM analysis of ultrastructural changes in poral regions during pollen hydration and germination showed that the germinative pore was positioned just near the generative cell and vegetative nucleus. Within the first 5 min after pollination a new layer of the electron-lucent wall adjacent to the plasma membrane was formed in the region of a future germinative pore. Following 15 min, marked changes were revealed in the cytoplasm region, close to the germinative pore. Minute dictyosome vesicles were accumulated near the plasma membrane. Small mitochondria and short ER cisternae were distal to a zone of secretory vesicles. The data suggest that the axis of polarity in the germinating pollen grain is predetermined by a spatial organization of the vegetative cell.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of acute and prolonged irradiation.

Somatic mutant frequencies at glycophorin A (GPA) and T-cell receptor (TCR) loci were assessed. The dependence of the GPA mutant frequency on doses of acute and prolonged irradiation was shown. In the case of acute irradiation the GPA mutant frequency displayed a three-fold greater dose-related increase as compared to prolonged irradiation. A dose-dependent increase in the TCR variant frequency was found only in a group of subjects with recent exposures. In Chernobyl clean-up workers the TCR mutant frequency was significantly higher than in control non-irradiated individuals.

[Inorganic ion dynamics in the microspore and pollen grain of tobacco during the development of the male gametophyte]. / Dinamika neorganicheskikh ionov v mikrospore i pyl'tsevom zerne tabaka v protsesse razvitiia muzhskogo gametofita.

We studied the dynamics of mobile potassium, chloride, and nitrate ions during development of the micro-spore and differentiation of the pollen grain in Nicotiana tabacum L. by measuring their concentration in aqueous extracts from cells destroyed by freezing-thawing using ion-selective electrodes. Stage-specific changes in the ion content and intracellular concentration in the male gametophyte were found. A relationship of the dynamics of ions to growth processes and changes in metabolic activity during gametophytogenesis has been discussed. The changes in the potassium and chloride ion concentrations have been interpreted as regulatory changes controlling protein synthesis in the pollen grain vegetative cell.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of prolonged irradiation.

PURPOSE: To determine the variant frequencies (VF) at glycophorin A (GPA) and T-cell receptor (TCR) loci in persons exposed to prolonged ionizing radiation at different doses and to assess the significance of the GPA and TCR assays for biodosimetry of prolonged irradiation. MATERIALS AND METHODS: The VF values were determined by means of flow cytometry in 120 persons exposed between 1968-1996. Most exposures were in Chernobyl clean-up workers in 1986-1987. RESULTS: A significant correlation was shown between the NO GPA variant cell frequency and dose (r = 0.61, p < 0.0001). The slope of the linear regression was 6.3 x 10(-6) NO mutant cells/Gy. Dose-dependent increase in the TCR VF was found in the group with recent exposures (slope 2.1 x 10(-4) variant cells/Gy, r = 0.75, p = 0.0002). In the Chernobyl clean up workers who received doses less than 0.25 Gy the TCR VF unlike the GPA VF was significantly higher than in the control non-irradiated individuals (p < 0.01 and p > 0.05 respectively). CONCLUSIONS: The GPA assay has limited potential to be used as a biodosimeter of prolonged irradiation, at least in dose interval up to 2.0 Gy. The TCR assay is likely to have greater potential in estimation of recent radiation exposure than the GPA assay.

[Determination of mutation frequency at loci of glycophorin A and T-cell receptor: study of Chernobyl AES accident cleanup workers]. / Opredelenie chastoty mutatsii po lokusam glikoforina A i T-kletochnogo retseptora: obsledovanie likvidatorov avarii na CHAES.

The frequencies of somatic mutations at loci of glycophorin A (GPA) and T-cell receptor (TCR) were determined in control unexposed donors and Chernobyl clean up workers, who received low doses of irradiation up to 0.25 Gy. High variability of mutant rates for two investigated genes was shown in the clean up workers. No significant difference in the GPA (NO) mutant frequencies was observed between the clean up workers and control donors (p > 0.05), though there is a tendency for increasing the GPA mutation rate in the clean up workers. Meanwhile, the TCR mutation rate was significantly increased the clean up workers (p < 0.01), perhaps because of acceleration of spontaneous mutagenesis and possible genome instability. Persons with elevated levels of mutations at two loci can present a group at high risk in respect to oncological diseases.

Phase equilibria of absorbed liquids and the structure of porous media.

The phase equilibria of water within porous silica has been studied by proton and deuteron magnetic resonance. Proton signal amplitude as a function of filling factor was measured. These protons arise from the proton-deuterium equilibrium that is established between the liquid and the absorbed layer on the pore wall. The results for temperatures below 0 degree C show a maximum as a function of filling factor, theta. This suggests that the pores fill from either a surface layer or from the crevices and interstices into the center. Another experiment used cryoporometry to study the size of crystals formed within the pores as a function of theta and leads to the same conclusion.

[A comprehensive radioisotope assessment of the effectiveness of Ferrum Lek in patients with iron-deficiency anemia]. / Kompleksnaia radioizotopnaia otsenka éffektivnosti preparata Ferrum Lek u bol'nykh zhelezodefitsitnoi anemiei.

Before and after treatment iron metabolism was evaluated in 37 iron deficient women given intravenous Ferrum Lek. In addition to standard blood counts and serum iron tests, iron metabolism was investigated with Fe-59 whole body radiometry in a low-background camera. The treatment effect was assessed in 1.3 and 6 months. The red blood cells and iron content in the serum returned to normal values. The effect persisted for 6 months. The initial 3 months iron accumulated in the depot, being further distributed in the blood. Total body iron 6 months after the treatment was at the baseline levels. It is thought adequate to use Ferrum Lek in the management of women with iron deficiency anemia.

[Heterogeneity of a peripheral blood lymphocyte population stimulated by phytohemagglutinin]. / Geterogennost' populiatsii limfotsitov perifericheskoi krovi, stimulirovannykh fitogemaggliutininom

Lymphocytes of human peripheral blood treated with PHA in short-living cultures can be divided into two subpopulations of cells according to their capacity to bind acridine orange. Cells of the first subpopulation are similar to unincubated ones, whereas the second subpopulation contains cells with higher dye-binding properties.

[Binding of acridine orange by chromatin in human lymphocytes with different capabilities for adhesion]. / Sviazyvanie akridinovogo oranzhevogo khromatinom limfotsitov cheloveka s razlichnoi sposobnost'iu k adgezii

A population of morphologically homogenous peripheral blood lymphocytes separated on a column with glass beads was studied microfluorimetrically. It was found that high adhesive lymphocytes bound acridine orange in a greater extent than did low adhesive cells.

[Effect of acid extraction of basic proteins on the binding of acridine orange by cell chromatin]. / Vliianie kislotnoi ékstraktsii osnovnykh belkov na sviazyvanie khromatinom kletok akridinovnogo oranzhevogo

The binding of acridine orange to chromatin of fixed human lymphocytes is increased after stepwise removal of histones by hydrochloric acid. No changes in the dye-binding features of the cells were found after f1 histone extraction (0.01 N HC1). Additional extraction of f2 and f3 by 0.05 N HC1 (partial extraction) and by 0.25 N HC1 (more complete extraction) resulted in increased binding of acridine orange (1.9 and 2.5 of that of the control, respectively).9222