[ROS induction and structural modification in human lymphocyte membrane under the influence of carbon nanotubes].

To assess the potential risks of using the artificial nanostructures the structural state of the human lymphocyte membrane and lipid peroxidation under the influence of multi-walled carbon nanotubes with metal impurities was studied. The ability of carbon nanotubes to induce the formation of reactive oxygen species in cells was examined. A dose-dependent increase in reactive oxygen species formation in lymphocytes, which was not registered in cells pre-incubated with N-acetylcystein, after exposure to carbon nanotubes was shown. The addition of iron chelator deferoxamine to carbon nanotubes has also resulted in a decrease of reactive oxygen species. The mechanism of the activation of lipid peroxidation under the influence of carbon nanotubes and a structural modification of human lymphocyte membranes were discussed.

[The formation of stable aggrregates of human platelets induced by lectin from Solanum tuberosum].

It has been demonstrated for the first time that GlcNAc-specific lectin from Solanum tuberosum induces the formation of haptenic sugar-resistant intercellular contacts (HSR-contacts) in platelet aggregation and does not induce stable neutrophil and lymphocyte aggregation. The formation of HSR-contacts in platelets was significantly impaired by the inhibitors of cAMP phosphodiesterase (papaverine) and arachidonic acid methabolism (indomethacin, aristolochic acid, and MK-886) as well as by the sulfhydryl reagent N-ethylmaleimide. The results obtained indicate that STA can be used to study the mechanisms of stable platelet aggregation, to screen drugs with potential antithrombotic activity, and to develop new cell engineering techniques.

Disaggregation of platelet aggregates formed by the action of thrombin in the presence of hydrogen peroxide.

The aggregation and change in the intracellular Ca2+ concentration induced by thrombin (0.005-0.22 U/ml) in the presence of H2O2 (0.05-0.6 mM) was investigated. Under the chosen experimental conditions (incubation time of platelets with H2O2 not more than 15 sec), H2O2 neither accelerated nor inhibited the thrombin-induced platelet aggregation. However, platelet aggregates formed by the action of thrombin in the presence of H2O2 were unstable and disaggregated. Disaggregation was abolished by catalase added after thrombin. The disaggregation effect was dose-dependent; the process of disaggregation was confirmed by electron microscopy. Hydrogen peroxide did not influence thrombin-induced increase in the intracellular Ca2+ concentration, but dose-dependently accelerated Ca2+ extrusion from the platelet cytoplasm.

H2O2-induced platelet aggregation and increase in intracellular Ca2+ concentration are blocked by inhibitors of intracellular signaling.

Inhibitors of signaling enzymes such as guanosine-5;-O-2-thiodiphosphate, aristolochic acid, aspirin, indomethacin, and trifluoperazine block H2O2-induced platelet aggregation and H2O2-induced increase in the intracellular concentration of Ca2+. These findings suggest that the effect of H2O2 on platelets is associated with activation of signal pathways responsible for increase in the concentration of intracellular Ca2+. On H2O2-induced platelet aggregation, the concentration of cAMP in the cytoplasm decreases and that of cGMP increases.

Platelet aggregation and change in intracellular Ca(2+) induced by low frequency ultrasound in vitro.

Treatment of platelet-rich plasma and washed platelets with low frequency ultrasound (US, 22 kHz) at intensity range of 1.0-8.8 W/cm(2) resulted in intensity- and time-dependent platelet aggregation. The effect was absent in calcium-free medium and was initiated by adding supernatant from sonicated suspension (16 W/cm(2), 2 min) to non-treated platelets. A marked decrease in the rate of US-induced aggregation was observed in the presence of specific inhibitors of platelet activation dipyridamole, pentoxifillin, aspirin and verapamil. Concentration of intracellular calcium in washed platelets evaluated with fluorescent probe quin-2 acetoxymethyl ester (quin-2) increased upon sonication in both the calcium containing and calcium free media. It is suggested that US increase of [Ca(2+)](i) is involved in platelet aggregation induced by low frequency US.

N-Ethylmaleimide induces disaggregation of platelets preaggregated by ADP and thrombin and decreases cytoplasmic calcium level.

The effect of N-ethylmaleimide (NEM, 1-200 microM) on ADP- and thrombin-induced platelet aggregation and thrombin-induced increase in intracellular Ca2+ concentration was studied. Addition of NEM to platelets preaggregated with ADP or thrombin induces platelet disaggregation. The anti-aggregant activity of NEM was different for ADP- and thrombin-induced aggregations. At 200 microM concentration, NEM completely disaggregated ADP-induced aggregates and only partially disaggregated thrombin-aggregated platelets. NEM did not influence the thrombin-induced increase in cytoplasmic Ca2+ and had no effect on the basal level of Ca2+ in the cytosol of non-activated platelets. However, NEM decreased the level of thrombin-mobilized Ca2+ in the cytosol of activated platelets. Thus, NEM can induce disaggregation of ADP- and thrombin-preaggregated platelets by activating a system which removes Ca2+ from the platelet cytosol.

Formation of lactose-resistant aggregates of human platelets induced by the mistletoe lectin and differential signaling responses to cell contact formation by the lectin or thrombin.

Human platelets afford a suitable and physiologically relevant model to study receptor-dependent cell aggregation and ensuing biosignaling reactions. Since cell surface glycoconjugates can serve as ligands in recognitive protein--carbohydrate interactions, it is of interest to investigate the reactivity of such epitopes for a plant lectin and the elicited intracellular responses. Therefore, the galactose-specific lectin (Viscum album agglutinin, VAA) was employed as a tool for this purpose. It was found that VAA induced platelet aggregation at a concentration of 2.5 microgram/ml using 2.5. 108 cells/ml, composed of the formation of both lactose-sensitive (Lac+) and lactose-resistant (Lac-) intercellular contacts. Lac- aggregates were formed only by metabolically active platelets of about 70% of the samples from the group of studied volunteers. The requirement of metabolic activity for formation of these contacts which no longer depend on lectin--ligand recognition was underscored by the lack of their appearance in the presence of metabolic inhibitors such as nordihydroguaiaretic acid, trifluoperazine, N-ethylmaleimide and menadione. With respect to biosignaling, the effective aggregation of platelets did not affect the basal level of Ca2+ in cells and reduced the rate of the menadione-dependent generation of H2O2. In parallel series platelet aggregation induced by bovine thrombin (0.03 U/ml) triggered an increase in the cytoplasmic Ca2+ level and an enhancement of the H2O2 generation. Overall, these results imply metabolically controlled post-binding reactions which strengthen the lectin-induced cell association and demonstrate differential responses with respect to the Ca2+ level and H2O2-generation between lectin- or thrombin-mediated aggregation of human platelets.

Effects of metabolic inhibitors and lectins on the menadione-dependent generation of H2O2 by rat thymocytes.

The capacity of oxidative metabolism and its regulation is an important factor in disease control. Using scopoletin as a fluorescent substrate of peroxidase the extent of menadione-dependent production of H2O2 by rat thymocytes was determined. The reaction was inhibited by 2,4-dinitrophenol, papaverine, nordihydroguaiaretic acid and iodacetamide. The membrane-penetrating SH reagent N-ethylmaleimide primed the reaction, probably due to an inhibition of glutathione peroxidase. To delineate an influence of cell-surface protein-carbohydrate interactions by exogenous lectins, the impact of cell binding was analyzed for several plant lectins, namely concanavalin A, phytohemagglutinin, the lectins from Triticum vulgaris and from Sambucus nigra. Except for the alpha-NeuNAc(2-6)gal/galNAc-binding agglutinin, the other three plant proteins with specificities to different parts of N-linked oligosaccharides primed the reaction. This activity of lectins did not coincide with their ability to aggregate cells. The given results indicate that biosignaling pathways triggered by lectins are involved in the regulation of the intracellular reduction of menadione.