The cardiolipin antigen: chemistry and composition

Cardiolipin, the primary lipid hapten in the antigen suspension used for the detection of antitreponemal antibodies in the sera of syphilitic patients, was successfully coupled to glucose oxidase, peroxidase, and some other enzymes using different crosslinking agents. These complexes were used to replace the pure uncomplexed cardiolipin for the preparation of the antigen suspension. When these suspensions were allowed to react with serum that contained anticardiolipin antibodies the activity of the enzyme was inhibited. In the absence of antibody, no enzyme inhibition was observed.     

Prevention of lipid peroxidation by NAD(P)H in rat liver submitochondrial particles

In rat liver submitochondrial particles both NADH and NADPH inhibit lipid peroxidation induced by cumene hydroperoxide. Concomitantly with the inhibition of lipid peroxidation, NADH and NADPH strongly stimulate the peroxidase activity of rat liver submitochondrial particles. Rotenone slightly prevents both the protective effect on malondialdehyde formation and peroxidase activity. The peroxidase activity of rat liver submitochondrial particles was attributed to the NAD(P)H-mediated reduction of mitochondrial cytochrome P-450 which can act upon hydroperoxides, by decomposing them to alcohols.     

Horseradish peroxidase inhibition and antioxidant activity of ebselen and related organoselenium compounds

Horseradish peroxidase (HRP) inhibition and glutathione peroxidase (GPx) activities of ebselen and some related derivatives are described. These studies show that ebselen and ebselen ditelluride (EbTe(2)) with significant antioxidant activity, inhibit the HRP-catalyzed oxidation reactions. In addition, inhibition of lipid peroxidation and singlet oxygen quenching studies were carried out. Although the inhibition of HRP by ebselen is comparable with that of EbTe(2), the inhibitory effect on gamma-radiation induced lipid peroxidation and the GPx activity of ebselen is found to be much higher than that of EbTe(2).     

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.     

Modulation of creatine kinase activity by ruthenium complexes

Creatine kinase is a crucial enzyme for brain, heart and skeletal muscle energy homeostasis, and a decrease of its activity has been associated with cell death. Many biological properties have been attributed to ruthenium complexes. In this context, this work was performed in order to evaluate creatine kinase activity from rat brain, heart and skeletal muscle (quadriceps) after administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)] (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV). Our results showed that complex I caused inhibition of creatine kinase activity in hippocampus, striatum, cerebral cortex, heart and skeletal muscle. Besides, complex II did not affect the enzyme activity. complexes III and IV increased creatine kinase activity in hippocampus, striatum, cerebral cortex and heart, but not in skeletal muscle. Besides, none of the complexes in vitro altered creatine kinase activity, suggesting that enzymatic activity is indirectly affected by complexes I, III and IV. It is believed that diminution of creatine kinase in brain of rats caused by complex I may be related to results from other study reporting memory impairment caused by the same complex. Further research is necessary in order to elucidate the effects of ruthenium complexes in other important metabolic enzymes.     

Pharmacological Activity of Ruthenium Complexes trans-[RuCl2(L)4] (L = Nicotinic or i-Nicotinic acid) on Anxiety and Memory in Rats

Many biological properties have been attributed to ruthenium complex I (trans-[RuCl₂(nic)₄]) and ruthenium complex II (trans-[RuCl₂ (i-nic) ₄]) including nitric oxide synthase inhibition. In this study, we evaluated pharmacological effects of these complexes on anxiety and memory formation. Memory was evaluated with inhibitory avoidance and habituation to an open-field and anxiety was tested with elevated plus-maze. Adult male Wistar rats (250 to 350 g) received intraperitoneal injections of vehicle, ruthenium complex I (45.2, 90.4, or 180.7 μmol/kg), or ruthenium complex II (0.08, 4.5, or 13.6 μmol/kg) 30 min prior open-field training or elevated plus-maze test and 30 min or 0 h after training. No effects were observed in the anxiety parameters and habituation to an open-field. The ruthenium complexes impaired memory retention compared with vehicle group in the inhibitory avoidance, as when administrated 30 min prior as immediately after training. The memory impairment induced by ruthenium complexes may be due to their nitric oxide synthase inhibition capacity.     

The role of selenium in the regulation of lipid peroxidation in biological membranes and glutathione peroxidase activity

The antioxidative effect of selenium cannot be exclusively due to the functioning of the selenium-dependent glutathione peroxidase mechanism of utilization of various hydroperoxides. This hypothesis is based on the following experimental evidence. Selenium ions are readily incorporated into animal organs and tissues immediately after injection (2 hours) as well as into cell organelles and cytosol where they inhibit lipid peroxidation. The activity of glutathione peroxidase (EC 1.1.1.19) in rat liver and guinea pig cytosol is thereby unchanged but increases drastically after 12 hours reaching a maximum an the 3rd-4th day. The effectiveness of lipid peroxidation inhibition does not increase under these conditions. Although the glutathione peroxidase activity is absent in the nuclei and microsomes, exogenous selenium inhibits lipid peroxidation in these organelles. The activity of the rat liver cytosolic enzyme markedly exceeds that of its guinea pig counterpart. However, lipid peroxidation in guinea pig liver occurs less intensively than that in rat liver cytosol.     

DNA binding, photocleavage, and topoisomerase inhibition of functionalized ruthenium(II)-polypyridine complexes

The interaction of ruthenium(II)-polypyridyl complexes with DNA has attracted considerable interests during the past two decades. This paper presents some recent progresses in our laboratory on the interaction of Ru(II)-polypyridyl complexes with DNA. The first part describes the effect of modulating the intercalative ligand on the DNA-binding behaviors of the complexes, such as DNA-binding affinity, DNA-binding enantioselectivity, DNA molecular 'light switch' effect, and DNA sequence selectivity. The second part focuses on the DNA photocleavage by the complexes and its mechanism. In the final part, we discuss the topoisomerase inhibition and its mechanism, as well as the antitumor activity of the Ru(II)-polypyridyl complexes.     

Neutron activation increases activity of ruthenium-based complexes and induces cell death in glioma cells independent of p53 tumor suppressor gene

Novel metal complexes have received great attention in the last decades due to their potential anticancer activity. Notably, ruthenium-based complexes have emerged as good alternative to the currently used platinum-based drugs for cancer therapy, providing less toxicity and side effects to patients. Glioblastoma is an aggressive and invasive type of brain tumor and despite of advances is the field of neurooncology there is no effective treatment until now. Therefore, we sought to investigate the potential antiproliferative activity of phosphine-ruthenium-based complexes on human glioblastoma cell lines. Due to its octahedral structure as opposed to the square-planar geometry of platinum(II) compounds, ruthenium(II) complexes exhibit different structure–function relationship probably acting through a different mechanism from that of cisplatin beyond their ability to bind DNA. To better improve the pharmacological activity of metal complexes we hypothesized that neutron activation of ruthenium in the complexes would allow to decrease the effective concentration of the compound needed to kill tumor cells. Herein we report on the effect of unmodified and neutron activated phosphine ruthenium II complexes on glioblastoma cell lines carrying wild-type and mutated p53 tumor suppressor gene. Induction of apoptosis/authophagy as well as generation of reactive oxygen species were determined. The phosphine ruthenium II complexes tested were highly active against glioblastoma cell lines inducing cell death both through apoptosis and autophagy in a p53 independent fashion. Neutron activation of ruthenium compounds rendered them more active than their original counterparts suggesting a new strategy to improve the antitumor activity of these compounds.     

Inhibitory effect of platinum and ruthenium bipyridyl complexes on porcine pancreatic phospholipase A2

Pancreatic phospholipase A(2) (PLA(2)) plays an important role in cellular homeostasis as well as in the process of carcinogenesis. Effects of metallo-drugs used as chemotherapeutics on the activity of this enzyme are unknown. In this work, the interaction between porcine pancreatic PLA(2) and two selected transition metal complexes--tetrachloro(bipyridine) platinum(IV) ([PtCl(4)(bipy)]) and dichloro (bipyridine) ruthenium(III)chloride ([RuCl(2)(bipy)(2)]Cl)--was studied. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence spectroscopy have been used to analyse the enzyme activity in the absence and presence of metal complexes and to verify potential binding of these drugs to the enzyme. The tested metal complexes decreased the activity of phospholipase A(2) in an uncompetitive inhibition mode. A binding of the ruthenium complex near the active site of the enzyme could be evidenced and possible modes of interaction are discussed.     

Change in the reaction of the antioxidant system of wheat sprouts after UV-irradiation of seeds

The response of the antioxidant system of sprouts of wheat Triticum aestivum L. to preliminary irradiation of seeds with UV light was studied. The dependence of lipid peroxidation and the extent of antioxidant activity on the duration of irradiation was studied. It was shown that low doses of UV radiation (5-15 min) stimulate the antioxidant protection of green wheat sprouts grown for eight days. Increasing the irradiation time to 30-60 min leads to the inhibition of lipid peroxidation by the antioxidant system. A more prolonged irradiation of seeds with UV light (for 1-6 h) led to an increase in the level of lipid peroxidation in sprouts. However, 1-2-day-old sprouts from seeds irradiated for 5-6 h, adapted themselves to the influence due to the compensatory mechanisms. By the 8th day of germination of preliminarily irradiated seeds, the content of antioxidants and malone dialdehyde returned to the norm. The dynamics of activity of peroxidase in seeds irradiated with low doses of UV light for 30 min was studied. It was found that on the third day of seed germination, a decrease in peroxidase activity followed by its slight increase occurred. The maximum activity of the enzyme in the endosperm was observed on day 5-6, and in roots and green sprouts, on day 3-5 of germination. It was concluded that antioxidants and peroxidase are involved in the compensatory mechanisms of inhibition of free radicals formed upon UV irradiation of seeds.     

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.     

Antioxidant effect of Ebselen (PZ 51): peroxidase mimetic activity on phospholipid and cholesterol hydroperoxides vs free radical scavenger activity.

The selenocompound Ebselen (PZ 51) is a potent inhibitor of lipid peroxidation. This antioxidant effect has been previously attributed both to a peroxidase mimetic activity and to a free radical scavenging capability. In the present paper the latter is ruled out by competition kinetic analysis based on the inhibition of carotenoid bleaching by hydroperoxyl radicals. Furthermore, evidence is reported indicating that Ebselen exhibits a peroxidase activity extended to cholesterol and cholesterol ester hydroperoxides, besides phospholipid hydroperoxides. According to this, we propose that the unique mechanism of the antioxidant capacity of Ebselen is the reduction of lipid hydroperoxides present in liposomes or lipoproteins, eventually leading to the prevention of hydroperoxide-dependent peroxidation.     

4-Hydroxynonenal inhibits glutathione peroxidase: protection by glutathione.

4-Hydroxy-2,3-trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3-trans-nonenal or 2,3-trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or beta-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or beta-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3-trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3-trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.     

Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage

Ultraviolet B (UVB medium wave, 280–315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/μmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.     

Characteristics of the antioxidant system of alveolar surfactant in immediate-type allergies

The antioxidant system (glutathione peroxidase, glutathione reductase, superoxide dismutase, total antioxidant activity) of the lung surfactant has been studied for and intensity of peroxidation in that surfactant after administration of sensitizing and resolving doses of the allergen to animals. An increase in the amount of lipid peroxidation products as well as in activity of superoxide dismutase followed by a fall of gamma-glutamyl transpeptidase activity was observed in the lung surfactant 3 and 12 days after introduction of a sensitizing dose of the allergen. Intensification of 5-lipoxygenase activity and accumulation of malonic dialdehyde in the lung surfactant under the anaphylactic shock were accompanied by inhibition of activity of the glutathione-dependent antioxidant system glutathione reductase and glutathione peroxidase) as well as by a fall of antioxidative activity of the surfactant. The data obtained have evidenced for a imbalance between the induction and metabolism systems of lipid hydroperoxides in the respiratory organs under immediate allergies.     

Novel enzymatic properties of DNA-Pt complexes

DNA-Pt complexes have shown novel enzymatic activity as a peroxidase similar to that of horseradish peroxidase in the colorimetric reaction with its substrate. The enzymatic activity of these complexes increased with increasing reaction time and pH in reaction solutions of DNA and K2[PtCl4]. This enhanced enzymatic activity was attributed to the increase in Pt conjugated to DNA in the complex. The enzymatic activity per unit mole of the DNA-Pt complex was significantly higher for complexes prepared with high molecular weight DNA because the enzymatic activity of the complex per repeat unit of DNA was almost constant for these complexes prepared under the same reaction conditions. All the DNA-Pt complexes in this study prepared with different DNA sequences (i.e., [A]20, [G]20, [C]20, [T]20, and [AG]10) exhibited peroxidase enzymatic activity. These complexes showed good thermal stability as compared to native horseradish peroxidase.     

Further studies on toxic and radiosensitizing properties of ruthenium complexes of 4-nitroimidazoles

Transition metal complexes containing nitroimidazole ligands have been shown previously to act as radiosensitizers of hypoxic cells in vitro. As part of our study on metal-radiosensitizer complexes, we were encouraged by a ruthenium (Ru) sensitizer, RuCl2(DMSO)2(4(5)-nitroimidazole)2, 1, which showed better radiosensitizing properties and lower toxicity than the free ligand. In this study, we have extended our investigation to include the various other substituted 4-nitroimidazoles as ligands. The new Ru complexes, analogues of 1, were synthesized, identified and characterized and their toxicity and radiosensitizing abilities examined in vitro using Chinese hamster ovary (CHO) cells. Like 1, each of these ruthenium complexes has lower CHO hypoxic toxicity than the free ligands alone at equimolar concentration. These newer complexes gave sensitizing enhancement ratio (SER) values of 1.1 to 1.3 at 1.0 X 10(-4) mol dm-3 compared with 1.6 for 1. Unlike complex 1, the new complexes do not bind to plasmid DNA (assessed by inhibition of restriction endonuclease activity), possibly because the chloride (Cl-) ligand does not dissociate. In addition, the redox potential of the coordinated imidazole ligands is relatively unchanged compared to that of the free ligand. These factors may explain the more favourable properties of 1 compared with those of the new 4-nitroimidazole complexes of Ru.     

Outer membrane protein OmpF involved in the transportation of polypyridyl ruthenium complexes into Escherichia coli

The discovery that OmpF was related to the transportation of ruthenium complexes through cell membrane was achieved with proteomics technologies. An integral ruthenium complex exists inside the cell as identified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. An inhibition assay with Escherichia coli was used to demonstrate the relationship between the transportation of the polypyridyl ruthenium complexes and the presence of OmpF (outer membrane protein F). For instance, the amount of [Ru(tpy)(bpy)Cl]⁺ (tpy: teripyridine; bpy: bipyridine) that entered the cells was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) of cell extracts and was measured to be approximately 0.55 μM. In the presence of 10% sucrose solution which is known to reduce the OmpF concentration, the ruthenium complex concentration was reduced to approximately 0.28 μM, which is a 50% reduction. These data suggest that OmpF plays a key role in the transportation of positively charged polypyridyl chlororuthenium complexes into E. coli.     

Disulfide between Cys392 and Cys438 of human serum albumin is redox-active, which is responsible for the thioredoxin-supported lipid peroxidase activity

Human serum albumin (HSA) is an abundant protein found in blood plasma and extracellular fluids. Previously, we found that HSA has a distinct thioredoxin (Trx)-dependent lipid peroxidase activity in the presence of palmitoyl-CoA. In this paper, we identified the redox-active disulfide, which can be specifically reduced by Trx, responsible for the Trx-dependent lipid peroxidase activity. The IIB-III fragment of HSA (Pro299-Leu585) sustained the Trx-dependent lipid peroxidase activity. Chemical modification of the Trx-reduced IIB-III with a thiol-specific modification agent resulted in a complete loss of the peroxidase activity. The analysis of tryptic-peptides derived from the inactivated HSA and IIB-III revealed that Cys392 and Cys438, which exist as an intramolecular disulfide bond in HSA, were preferentially modified in both HSA and IIB-III. Taken together, these results suggested that HSA has a capability to reduce lipid hydroperoxide with the use of Trx as an in vivo electron donor, and that the redox-active disulfide between Cys392 and Cys438 acts as a primary site of the catalysis for the Trx-linked lipid peroxidase activity.