A. N. Osipov

Carnivorous pitcher plant uses free radicals in the digestion of prey.

Abstract A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex.

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

Study of antioxidant and membrane activity of rosmarinic acid using different model systems

Rosmarinic acid is found in many species of different families of higher plants and its chemical structure is phenol propanoid with various biological activity. In this paper, we conducted a comparative study of antioxidant (radical-scavenging) properties of rosmarinic acid in systems of 2,2tāzo-bis(2-methylpropionamidin)dihydrochloride-luminol and hemoglobin-hydrogen peroxide-luminol, determined its protective potential in preventing peroxidation of linoleic acid, and evaluated the effect on the permeability of planar bilayer lipid membranes. Linoleic acid peroxidation was assessed by iron-thiocyanate method. In these studies, trolox was used as a reference antioxidant, and ascorbic acid, and dihydroquercetin were taken as standards. Rosmarinic acid is significantly superior to trolox, ascorbic acid and dihydroquercetin in the tests for antioxidant activity in the systems studied, as well as in inhibition of linoleic acid peroxidation. According to their activity the investigated substances can be arranged in the following order: rosmarinic acid > dihydroquercetin trolox > ascorbic acid. Rosmarinic acid does not cause significant changes in the permeability of planar bilayer membranes in a dose range of 0.5 to 10 μg/mL. Antioxidant activity of rosmarinic acid is due to the neutralization of reactive oxygen species and/or luminol radicals generated in model systems. The observed features of the antioxidant and membrane activity of rosmarinic acid, which may underlie the previously mentioned pharmacological effects are discussed.

Cytochrome c produces pores in cardiolipin-containing planar bilayer lipid membranes in the presence of hydrogen peroxide

Interaction of cytochrome c with cardiolipin in the presence of hydrogen peroxide induces peroxidase activity in cytochrome c and the ability to oxidize membrane lipids. These cytochrome c properties play a substantial role in the cytochrome c-mediated apoptotic reactions. In the present study the electric properties (specific capacitance and integral conductance) of the cardiolipin-containing asolectin planar bilayer lipid membranes (pBLM) in the presence of cytochrome c and hydrogen peroxide were studied. Cytochrome c interaction with cardiolipin-containing pBLM in the presence of hydrogen peroxide resulted in the dramatic increase of the conductance, pore production, their growth up to 3.5 nm diameter and subsequent membrane destruction. In the absence of hydrogen peroxide cytochrome c demonstrated almost no effect on the membrane capacitance and conductance. The data obtained prove the pivotal role of cytochrome c and membrane lipids in the permeabilization of pBLM. Correlation of apoptotic reactions and cytochrome c-mediated membrane permeability is discussed.

Effects of laser and LED radiation on mitochondrial respiration in experimental endotoxic shock.

Effects of low-level laser therapy (442 and 532xa0nm) and LED radiation (650xa0nm) on mitochondrial respiration in experimental endotoxic shock have been studied. A model of experimental endotoxic shock in rats was obtained by intraperitoneal injection of lipopolysaccharide B. It was found that low-level laser therapy and LED radiation dramatically affected the rate of mitochondrial respiration in third and fourth states both in LPS-treated animals and in control experiments. The maximal increase of the mitochondrial respiration rate (of about 40xa0%) in LPS-treated animals was observed when blue laser was applied at the dose of 6xa0J/cm2. Measurements of inner mitochondrial membrane surface potential with fluorescence probe JC-1 in LPS-treated rats showed in approximately 10xa0% decrease of potential in LPS-treated animals compared to control.

Are the mitochondrial respiratory complexes blocked by NO the targets for the laser and LED therapy

Effects of laser (442 and 532xa0nm) and light-emitting diode (LED) (650xa0nm) radiation on mitochondrial respiration and mitochondrial electron transport rate (complexes II–III and IV) in the presence of nitric oxide (NO) were investigated. It was found that nitric oxide (300xa0nM–10xa0μM) suppresses mitochondrial respiration. Laser irradiation of mitochondria (442xa0nm, 3xa0Jxa0cm−2) partly restored mitochondrial respiration (approximately by 70xa0%). Irradiation with green laser (532xa0nm) or red LED (650xa0nm) in the same dose had no reliable effect. Evaluation of mitochondrial electron transport rate in complexes II–III and IV and effects of nitric oxide demonstrated almost similar sensitivity of complex II–III and IV to NO, with approximately 50xa0% inhibition at NO concentration of 3xa0μM. Subsequent laser or LED irradiation (3xa0Jxa0cm−2) showed partial recovery of electron transport only in complex IV and only under irradiation with blue light (442xa0nm). Our results support the hypothesis of the crucial role of cytochrome c oxidase (complex IV) in photoreactivation of mitochondrial respiration suppressed by NO.

Can Summary Nitrite+Nitrate Content Serve as an Indicator of NO Synthesis Intensity in Body Tissues?

Studies with the use of a highly specific enzymatic sensor demonstrated that, contrary to the common opinion, normally nitrate is in fact not present in the most important physiological fluids. NO metabolites in the amniotic fluid and semen are mainly presented by NO donor compounds. Therefore, the intensity of NO synthesis can be evaluated by the total content of all its metabolites, but not by the widely used summary nitrite+nitrate content.

Nitrite and nitroso compounds can serve as specific catalase inhibitors

Objective: We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness. Methods: Catalase activity was evaluated by the permanganatometric and calorimetric assays. Results: This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide. Discussion: We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO+ group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.

Effects of low-level laser therapy on mitochondrial respiration and nitrosyl complex content

Among the photochemical reactions responsible for therapeutic effects of low-power laser radiation, the photolysis of nitrosyl iron complexes of iron-containing proteins is of primary importance. The purpose of the present study was to compare the effects of blue laser radiation on the respiration rate and photolysis of nitrosyl complexes of iron-sulfur clusters (NO-FeS) in mitochondria, subjected to NO as well as the possibility of NO transfer from NO-FeS to hemoglobin. It was shown that mitochondrial respiration in State 3 (V3) and State 4 (V4), according to Chance, dramatically decreased in the presence of 3xa0mM NO, but laser radiation (λu2009=u2009442xa0nm, 30xa0J/cm2) restored the respiration rates virtually to the initial level. At the same time, electron paramagnetic resonance (EPR) spectra showed that laser irradiation decomposed nitrosyl complexes produced by the addition of NO to mitochondria. EPR signal of nitrosyl complexes of FeS-clusters, formed in the presence of 3xa0mM NO, was maximal in hypoxic mitochondria, and disappeared in a dose-dependent manner, almost completely at the irradiation dose 120xa0J/cm2. EPR measurements showed that the addition of lysed erythrocytes to mitochondria decreased the amount of nitrosyl complexes in iron-sulfur clusters and produced the accumulation of NO-hemoglobin. On the other hand, the addition of lysed erythrocytes to mitochondria, preincubated with nitric oxide, restored mitochondrial respiration rates V3 and V4 to initial levels. We may conclude that there are two possible ways to destroy FeS nitrosyl complexes in mitochondria and recover mitochondrial respiration inhibited by NO: laser irradiation and ample supply of the compounds with high affinity to nitric oxide, including hemoglobin.

Investigation of the antioxidant and membranotropic activity of the quinozaline alkaloid triptantrin in different model systems

A comparative study of antioxidant (radical-scavenging) properties of triptantrin, which is a quinazoline alkaloid with anti-inflammatory activity that occurs in many species of higher plants and microorganisms, including the human microbiome, in systems containing 2,2’-azobis (2-methylpropionamidin) dihydrochloride and luminol, or hemoglobin, hydrogen peroxide, and luminol was performed and the impact of the alkaloid on the permeability of planar bilayer lipid membranes was assessed. Trolox was used as a reference antioxidant, and ascorbic acid and dihydroquercetin were used as standards. The antioxidant activity of triptantrin in both systems was much lower than that of the reference and standard antioxidants. The substances formed the following series according to antioxidant activity: dihydroquercetin > trolox > ascorbic acid > triptantrin, with the antioxidant potential of the latter being 1000 times lower than that of trolox and 3000 times lower than that of the bioflavonoid dihydroquercetin. Triptantrin did not cause significant changes in permeability of planar bilayer membranes when applied at concentrations of 0.5–10 μg/mL. The data show that triptantrin lacks considerable radical-scavenging and membranotropic activities; therefore one can assume that the high antiinflammatory activity that has been reported for triptantrin is neither related to an ability to neutralize reactive oxygen species nor to effects on cell membrane permeability. The putative mechanisms that underlie the biological effects of triptantrin are discussed.