Cytochrome c autocatalyzed carbonylation in the presence of hydrogen peroxide and cardiolipins.

Cytochrome c (cyt c) is a small hemoprotein involved in electron shuttling in the mitochondrial respiratory chain and is now also recognized as an important mediator of apoptotic cell death. Its role in inducing programmed cell death is closely associated with the formation of a complex with the mitochondrion-specific phospholipid cardiolipin (CL), leading to a gain of peroxidase activity. However, the molecular mechanisms behind this gain and eventual cyt c autoinactivation via its release from mitochondrial membranes remain largely unknown. Here, we examined the kinetics of the H2O2-mediated peroxidase activity of cyt c both in the presence and absence of tetraoleoyl cardiolipin (TOCL)- and tetralinoleoyl cardiolipin (TLCL)-containing liposomes to evaluate the role of cyt c-CL complex formation in the induction and stimulation of cyt c peroxidase activity. Moreover, we examined peroxide-mediated cyt c heme degradation to gain insights into the mechanisms by which cyt c self-limits its peroxidase activity. Bottom-up proteomics revealed >50 oxidative modifications on cyt c upon peroxide reduction. Of note, one of these by-products was the Tyr-based "cofactor" trihydroxyphenylalanine quinone (TPQ) capable of inducing deamination of Lys ϵ-amino groups and formation of the carbonylated product aminoadipic semialdehyde. In view of these results, we propose that autoinduced carbonylation, and thus removal of a positive charge in Lys, abrogates binding of cyt c to negatively charged CL. The proposed mechanism may be responsible for release of cyt c from mitochondrial membranes and ensuing inactivation of its peroxidase activity.

2-Hexadecenal inhibits growth of C6 glioma cells.

2-Hexadecenal (2HD) formation in the organism occurs via irreversible enzymatic degradation of sphingosine-1-phosphate or nonenzymatic γ-, UV-, or HOCl-induced destruction of a number of sphingolipids including S1P. The current research focuses on the study of 2HD effects on C6 glioma cells growth. The results obtained show that 2HD causes a dose-dependent decrease in proliferative and mitotic indices. The change in the mitotic index is due to the redistribution of cells in the different phases of mitosis. These processes are accompanied by cytoskeleton rearrangement and changes in cell morphology, which are expressed in F-actin redistribution, change in the number and type of filopodia and fibrils, leading to cell shape changes, decrease in intercellular contacts and monolayer rarefaction. Cells treatment with 2HD leads to apoptosis induction and signalling pathways modification, including activation of JNK, p38, and ERK1/2 MAPK but not PI3K. The effects observed are not related to the cytotoxicity of 2HD. Significance of the study: 2HD-an unsaturated aldehyde, which level can rise under conditions of oxidative stress as a result of nonenzymatic sphingolipids' destruction. The mechanisms of 2HD action on various cell types have not been sufficiently studied. Therefore, the study on functional role of this aldehyde in different cell types that may be its target is relevant. This study demonstrated that 2HD inhibits growth of C6 glioma cells due to modification of intracellular processes of signal transduction, cytoskeleton rearrangement, change in the mitotic regimen and apoptosis induction.

2-Hexadecenal Regulates ROS Production and Induces Apoptosis in Polymorphonuclear Leucocytes.

2-Hexadecenal (2-HD)-a biologically active long-chain fatty aldehyde formed in organism enzymatically or nonenzymatically in the reaction of free-radical destruction of sphingolipids under the action of hypochlorous acid, producing by myeloperoxidase. This research aimed to study 2-HD effects on polymorphonuclear leukocytes' (PMNLs) functions. It has been shown that at submicromolar concentrations, 2-HD causes an elevation in ROS production by PMNLs. It has been found that such effect is associated with signal transduction pathways modification and expressed in elevation of NADPH oxidase, MPO, and JNK-MAPK contributions to this process. At higher concentrations, 2-HD induces apoptosis, which correlates with a significant increase in free Ca2+ in the cytoplasm, a decrease in ROS production, and a decline in mitochondrial potential. Both of these processes are accompanied by cytoskeleton reorganization.

Radical-regulating and antiviral properties of ascorbic acid and its derivatives.

The ability of ascorbic acid and a number of its derivatives to suppress replication of Herpes simplex virus type I was investigated in human rhabdomyosarcoma cell line. In parallel, interaction of the test compounds with carbon- and oxygen-centered radicals formed on radiolysis of hydroxyl-containing organic compounds was studied using the steady state radiolysis method. It has been shown that 2-O-glycoside of ascorbic acid, displaying marked antiviral properties against Herpes simplex virus type I, is also capable of inhibiting fragmentation and recombination reactions of α-hydroxyl-containing carbon-centered radicals while not affecting processes involving oxygen-centered radicals.

The influence of H/D kinetic isotope effect on radiation-induced transformations of hydroxyl-containing compounds in aqueous solutions.

Vicinal diols and its derivatives can be exploited as model compounds for the investigation of radiation-induced free-radical transformations of hydroxyl-containing biomolecules such as carbohydrates, phospholipids, ribonucleotides, amino acids, and peptides. In this paper, for the first time, the prospects of isotope reinforcement approach in inhibiting free-radical transformations of hydroxyl-containing compounds in aqueous solutions are investigated on the example of radiolysis of 1,2-propanediol and 1,2-propanediol-2-d1 aqueous solutions. At an absorbed dose rate of 0.110 ± 0.003 Gy·s-1 a profound kinetic isotope effect (KIE) is observed for the non-branched chain formation of acetone, which is a final dehydration product of predominant carbon-centred radicals CH3·C(OH)CH2OH. In 0.1 and 1 M deaerated solutions at pH 7.00 ± 0.01, the values of KIE are 8.9 ± 1.7 and 15.3 ± 3.1, respectively. A rationale for the fact that a strong KIE takes place only in the case of chain processes, which may occur during free-radical transformations of vicinal diols, is also provided herein based on the results of 2-propanol and 2-propanol-2-d1 indirect radiolysis. Lastly, the lack of KIE is shown in the case of 2-butanone formation from 2,3-butanediol or 2,3-butanediol-2,3-d2. This indicates that the type (primary, secondary) of the ß-carbonyl radicals formed as a result of CH3·C(OH)CH(OH)R (R = H, CH3) dehydration determines the manifestation of the effect.

Effects of quinones and nitroazoles on free-radical fragmentation of glycerol-1-phosphate and 1,2-dimyristoyl-glycero-3-phosphatidyl-glycerol.

Effects of quinones and azoles on the formation of steady-state radiolysis products in aqueous solutions of glycerol-1-phosphate and aqueous dispersions of 1,2-dimyristoyl-glycero-3-phosphatidyl-glycerol has been investigated. The data obtained by LC-MS-ESI and spectrophotometric measurements shows that the compounds having quinoid structures, including the antitumor agent doxorubicin, and azoles having nitro groups effectively inhibit free-radical fragmentation of glycerol-1-phosphate and 1,2-dimyristoyl-glycero-3-phosphatidyl-glycerol, decreasing the radiation-chemical yields of either inorganic phosphate or phosphatidic acid respectively. The observed effects of blocking free-radical processes are believed to be related to the ability of the tested compounds to oxidize α-hydroxyl-containing carbon-centered radicals of starting substrates, which give rise to fragmentation reaction. The possibility of using the discovered properties of quinones, doxorubicin and nitroazoles to provide practical solutions in oncological radiotherapy and pathophysiology is discussed.

Formation of phosphatidic acid in stressed mitochondria.

Mitochondria are an important intracellular source of ROS as well as a sensitive target for oxidative damage under certain pathological conditions such as iron or copper overload. Mitochondrial membranes are rich in the tetraacyl phospholipid cardiolipin. Its integrity is important for efficient oxidative phosphorylation. Mouse liver mitochondria were subjected to oxidative stress by the Cu(2+)(Fe(2+))/H(2)O(2)/ascorbate system. Phosphatidic acid was detected in oxidized mitochondria, but not in unperturbed mitochondria. The Cu(2+)/H(2)O(2)/and (or not) ascorbate system caused the formation of phosphatidic acid and phosphatidylhydroxyacetone in cardiolipin liposomes. These products proceed via an HO*-radical induced fragmentation taking place in the polar moiety of cardiolipin. Mass spectrometry analysis of phosphatidic acid newly formed in mitochondria revealed that it has been derived from fragmentation of cardiolipin. Thus, free-radical fragmentation of cardiolipin in its polar part with the formation of phosphatidic acid is a likely mechanism that damages mitochondria under conditions of oxidative stress.

Synthesis, characterization, antifungal and anti-HIV activities of metal(II) complexes of 4,6-di-tert-butyl-3-[(2-hydroxyethyl)thio]benzene-1,2-diol.

Co(II) and Ni(II) complexes with 4,6-di-tert-butyl-3-[(2-hydroxyethyl)thio]benzene-1,2-diol (L) have been synthesized and characterized by means of elemental analysis, TG/DTA, FT-IR, ESR, UV-vis, XRD, magnetic susceptibility, cyclic voltammetry and conductance measurements. According to the data obtained the organic compound acts as a bidentate O,S-coordinated ligand and yields Co(II) and Ni(II) complexes of the stoichiometry ML(2) which is characterized by square planar geometry. Antifungal and anti-HIV activities of the ligand and its metal(II) complexes were found to decrease in the sequence CuL(2)>CoL(2) ~ NiL(2)>HL, along with their reducing ability (determined electrochemically).

Effects of various vitamins and coenzymes Q on reactions involving alpha-hydroxyl-containing radicals.

Effects of vitamins B, C, E, K and P, as well as coenzymes Q, on formation of final products of radiation-induced free-radical transformations of ethanol, ethylene glycol, alpha-methylglycoside and glucose in aqueous solutions were studied. Based on the obtained results, it can be concluded that there are substances among vitamins and coenzymes that effectively interact with alpha-hydroxyl-containing radicals. In the presence of these substances, recombination reactions of alpha-hydroxyalkyl radicals and fragmentation of alpha-hydroxy-beta-substituted organic radicals are suppressed. It has been established that the observed effects are due to the ability of the vitamins and coenzymes under study to either oxidize alpha-hydroxyl-containing radicals yielding the respective carbonyl compounds or reduce them into the initial molecules.

Homolytic cleavage of the O-glycoside bond in carbohydrates: a steady-state radiolysis study.

The formation of products resulting from the O-glycoside bond cleavage following radiolysis of aqueous solutions of methyl-alpha-D-glucopyranoside (I), 3-O-methyl-alpha-D-glucopyranose (II), maltose, lactose, gentiobiose and cellobiose were studied. Radiation-induced destruction yields were also determined for dextran, laminarin and trimethylcelulose upon irradiation of their aqueous solutions. Oxygen, quinones and compounds capable of forming quinoid structures were found to inhibit radiation-induced homolytic destruction processes taking place in glycosides, di- and polysaccharides. The data obtained in this study enabled the authors to demonstrate an important role played by the fragmentation reaction of C-2 radicals which were generated from the starting substances in the formation of final radiolysis products.

A novel pathway of photoinduced decomposition of sphingolipids.

A possibility of a novel photodestruction process has been shown for sphingolipids and their low-molecular analogues, including the stage of Norrish Type-I decomposition of excited molecules of the starting compounds followed by fragmentation of nitrogen-centered radicals via rupture of the C-C and O-H bonds. Realization of such transformation during photolysis of ceramide, sphingomyelin and galactocerebroside results in formation of 2-hexadecenal, which possess a wide spectrum of biological activity.

Effects of curcumin and related compounds on processes involving α-hydroxyethyl radicals.

Effects of curcumin and related compounds on product formation in radiolysis of aerated and deaerated ethanol were studied. Ab initio calculations of enthalpy values relating to O-H bond dissociation and H-atom addition to > C = O bonds of the compounds under study have been performed. The obtained data allowed the conclusion that the presence of a 7-carbon chain containing conjugated > C = C < and > C = O bonds in the structures of curcumin and its analogues makes these compounds capable of inhibiting the reactions involving α-hydroxyl-containing carbon-centered radicals. This finding broadens the existing views concerning radical-regulating properties of curcuminoids, and it should be taken into account when practical use of these compounds is envisaged.

A new mechanism for photo- and radiation-induced decomposition of sphingolipids.

Data have been obtained showing regularities in product formation following radiolysis of serinol, lysosphingomyelin and photolysis of N-(2-hydroxypropyl)hexanamide, sphingomyelin, which point to the possibility of photo- and radiation-induced destruction of the named substrates via a C-C bond rupture. The key stage of this process is the formation and decomposition of N-centered radicals generated from the starting compounds.

Synthesis and biological evaluation of copper (II) complexes of sterically hindered o-aminophenol derivatives as antimicrobial agents.

Cu(II) complexes with 4,6-di(tert-butyl)-2-aminophenol (I) and 2-anilino-4,6-di(tert-butyl)phenol (II) have been synthesized and characterized by means of elemental analysis, TG/DTA, FT-IR, UV-vis, ESR, and conductance measurements. The compounds I and II can coordinate in their singly deprotonated forms and behave as bidentate O,N-coordinated ligands; their CuL(2) complexes are characterized by CuN(2)O(2) coordination modes and square planar geometry. In vitro antimicrobial screening against Gram-positive and Gram-negative bacteria, yeasts, and moulds indicated that the compound I and its Cu(II) complex were more active than Questiomycin B, the compound II, and its Cu(II) complex.

Synthesis and properties of 11-(3,5-di-tert-butyl-2-hydroxyphenylcarbamoyl)undecanoic acid, a new amphiphilic antioxidant.

Based on the membrane addressing concept, designing and synthesis of 11-(3,5-di-tert-butyl-2-hydroxyphenylcarbamoyl)undecanoic acid have been carried out. Antioxidant properties of the prepared compound were investigated in comparison with its non-amphiphilic analogues.

Novel (3,5-di-tert-butyl-2-hydroxy-phenylcarbamoyl)-alkanoic acids as potent antioxidants.

A series of novel phenolic antioxidants of amphiphilic structure has been synthesized. Investigations into the influence of aliphatic spacer length and nature of a hydrophilic anchor on the antioxidant activity allowed elucidating certain structure requirements for the membrane-addressed antioxidant designing.