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.     

Electrical capacitance of lipid bilayer membranes of hydrogenated egg lecithin at the temperature phase transition

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.     

A study of the antioxidant and membranotropic activities of equinochrome a using different model systems

Echinochrome A (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone) isolated from the body of sand dollar Scaphechinus mirabilis is an active substance of cardioprotective medication Histochrome and exerts a wide spectrum of anti-inflammatory activities. In the present paper, we conducted a comparative study of the antioxidant (radical-scavengering) properties of echinochrome A in 2,2′-azobis(2-methylpropionamidine) dihydrochloride?luminol and hemoglobin?hydrogen peroxide?luminol systems and assessed its impact on permeability of planar bilayer lipid membranes. Trolox was used as a reference antioxidant and ascorbic acid and dihydroquercetin are taken as standards. Echinochrome A shows moderate antioxidant activity, possessing higher antioxidant capacity than Trolox and ascorbic acid, but exhibiting lower antioxidant potential compared with dihydroquercetin in tests for antioxidant activity in both investigated systems. The test substances can be arranged in the following order according to the effectiveness of the antioxidant effect: dihydroquercetin > echinochrome A > Trolox > ascorbic acid. Echinochrome A does not lead to significant changes in the permeability of planar bilayer membranes in a dose range of 1.5 to 30 μМ. Our data indicate that echinochrome A has a rather high level of radical-scavengering activity without a primary membranotropic effect. It is thought that the high levels of the cardioprotective and anti-inflammatory activities of echinochrome A may be due not only to the ability of this substance to neutralize reactive oxygen species, but also to its capacity to generate physiological concentrations of hydrogen peroxide molecules in biological systems as signaling messengers of various metabolic processes and biochemical pathways. The suspected mechanisms of the biological activity of echinochrome A are discussed.     

Electric capacitance of bilayer lipid membranes from hydrogenated egg lecithin during phase transition from liquid crystalline state to gel

The electrical capacity of planar bilayer lipid membranes (BLM) from natural hydrogenated egg lecithin (HEL) in n-decane at a temperature of phase transition was measured. The temperature of phase transition was determined calorimetrically to be 51 degrees C. The data obtained revealed a phase separation of HEL in BLM into two fractions, one freezing at 42-44 degrees C and one that is converted to a liquid-crystal state at 51-59 degrees C. It was assumed that the first fraction is rich in dipalmitoyl lecithin, and the second fraction is rich in distearoyl lecithin. Freezing and the transition to the liquid-crystal state were accompanied by an increase and decrease in membrane thickness, respectively, in part due to a displacement of the solvent from the torus to the planar part of the bilayer. The displacement of the solvent is explained by changes in the disjoining pressure in BLM, which arises across the lipid bilayer due to van der Waals forces of attraction between water layers on both sides of the BLM.     

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein

Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose binding protein (MBP) and a His(8)-tag. Sixty-eight percent of targeted proteins were expressed in high yield (>30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose binding protein as a fusion tag.     

Effect of polymyxin B on ion permeability of bacterial membranes

Effect of polymyxin B on the movement of K+ and H+ in polymyxin-sensitive cells of E. coli under different metabolic states has been studied. It was shown that polymyxin B induced the efflux of K+, decreased the efflux of H+ and inhibited the consumption of oxygen in bacterial cells. The effect of antibiotic on ion movement was independent of respiratory conditions. It was suggested that polymyxin B increased ion permeability and destroyed lipid-protein interactions of the respiratory chain simultaneously.     

Fragment-based discovery of a potent NAMPT inhibitor

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.     

Soft perforation of cardiolipin-containing planar lipid bilayer membrane by cytochrome c and H2O2

The release of cytochrome c (cyt c) from mitochondria is responsible for initiation of cell apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanism remains unclear. Cyt c binds to and penetrates lipid bilayer membranes of specific phospholipid cardiolipin (CL) contained in mitochondria. We present here the experimental results of monitoring planar BLM (pBLM) from mixtures of azolectin and of CL (4/1 by moles) by triangle voltage pulses of 100 mV in amplitude and frequency of 2 Hz. The BLM were modified by a successive addition of cyt c and of H₂O₂ in water solution. It is shown that the addition of cyt c alone leads to a stepwise increase in the ionic conductance of the pBLM, indicating the appearance of transmembrane pores. Pore lifetimes then reached several seconds at an average pore diameter of ~2 nm. Current–voltage characteristics were then linear and passed through the origin which is characteristic for broad, nonselective ion pores. Subsequent addition of H₂O₂ caused a dramatic increase in transmembrane current at retention of average pore size constant. Observed increase in membrane current is due to growth of a number of pores in an open state. We suggest that hydrogen peroxide in the presence of cyt c promotes a peroxidation of membrane phospholipids to form lysolipids, the embedding of which stabilizes the edge of the pore and the surface of lipid bilayer.     

Effect of the polycationic antibiotic polymyxin B on the respiratory activity of rat liver mitochondria

the mechanism of the effect of the peptide antibiotic polymixin B on respiration of rat liver mitochondria was studied. It was shown that polymixin B at concentrations of (1,6--2,0) . 10(-3) M inhibits mitochondrial respiration in state 3 and 3u irrespective of the oxidation substrate used and activates oxygen consumption in state 4 at lower concentrations. It is assumed that the antibiotic affects mitochondrial respiration by changing the ionic permeability of the membranes.