Julius Liobikas

Uncoupling and antioxidant effects of ursolic acid in isolated rat heart mitochondria.

Ursolic acid (1), a pentacyclic triterpene acid, is one of the major components of certain traditional medicinal plants and possesses a wide range of biological effects, such as anti-inflammatory, antioxidative, and cytotoxic activities. Furthermore, 1, when present at 1.6-5 ng/mL concentrations in commercial herbal preparations used for patients with cardiac disorders, may also exert pro-cardiac activities. There are several indirect suggestions that the cardioprotective mechanism of ursolic acid could involve the mitochondria; however the mechanism of action is still not known. Therefore, the effects of 0.4-200 ng/mL ursolic acid (1) on the functions of isolated rat heart mitochondria oxidizing either pyruvate and malate, succinate, or palmitoyl-l-carnitine plus malate were investigated. It was found that 1 induced a statistically significant uncoupling of oxidative phosphorylation. A statistically significant decrease in H₂O₂ production in the mitochondria was observed after incubation with 5 ng/mL 1. This effect was comparable to the effectiveness of the classical uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Since mild mitochondrial uncoupling has been proposed as one of the mechanisms of cardioprotection, the present results indicate that ursolic acid (1) has potential use as a cardioprotective compound.

Diazoxide and Pinacidil Uncouple Pyruvate-Malate-Induced Mitochondrial Respiration

We investigated the effects of KATP channel openers diazoxide and pinacidil on the respiration rate and membrane potential (ΔΨ) of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8–1348.3 μM) increased the V2 (-ADP) respiration rate accordingly by 13–208% and 30–273% and decreased the ΔΨ by 2–17% and 6–55%. These effects were also similar in the respiration medium without K+. Moreover, carboxyatractyloside completely abolished diazoxide- and pinacidil-induced uncoupling, indicating a role for the mitochondrial adenine nucleotide translocase in this process.

Anthocyanins block ischemia-induced apoptosis in the perfused heart and support mitochondrial respiration potentially by reducing cytosolic cytochrome c.

Anthocynanins, found in fruits and vegetables, have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, antioxidants are generally strong reductants, and some reductants have been found to block apoptosis by reducing cytosolic cytochrome c, which prevents caspase activation. We tested the ability of various anthocyanins: to reduce cytochrome c, to support cytochrome c-induced mitochondrial respiration and to inhibit apoptosis induced by heart ischemia. Anthocyanins such as delphinidin-3-glucoside (Dp3G) and cyanidin-3-glucoside (Cy3G) were able to reduce cytochrome c directly and rapidly, whereas pelargonidin-3-glucoside (Pg3G), malvinidin-3-glucoside (Mv3G) and peonidin-3-glucoside (Pn3G) had relatively low cytochrome c reducing activities. Dp3G and Cy3G but not Pg3G supported mitochondrial state 4 respiration in the presence of exogenous cytochrome c. Pre-perfusion of hearts with 20 μM Cy3G but not Pg3G prevented ischemia-induced caspase activation. This suggests that the ability of anthocyanins to block caspase activation may be due to their ability to reduce cytosolic cytochrome c. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Anthocyanins as substrates for mitochondrial complex I – protective effect against heart ischemic injury

Anthocyanins, a subclass of flavonoids, are known to protect against myocardial ischemia; however, little is known about their direct, acute effects on mitochondria injured by the ischemic insult. In this study, the effects of delphinidin 3‐O‐glucoside (Dp3G), cyanidin 3‐O‐glucoside (Cy3G) and pelargonidin 3‐O‐glucoside (Pg3G) on the activity of complex I of the mitochondrial respiratory chain were studied in mitochondria isolated from normal rat hearts and rat hearts subjected to ischemia for 45 min. Cy3G and Dp3G increased the activity of complex I, measured in the presence or absence of coenzyme Q1 (CoQ1), in ischemia‐damaged mitochondria, whereas in nonischemic mitochondria the effect was observed only in the absence of CoQ1. Dp3G and Cy3G but not Pg3G increased state 3 respiration and ATP synthesis with NADH‐dependent substrates in mitochondria after ischemia. The results suggest that certain anthocyanins can act as electron acceptors at complex I, and bypass ischemia‐induced inhibition, resulting in increased ATP production after ischemia. This study provides new information on a possible role of certain anthocyanins in the regulation of energy metabolism in mammalian cells.

Direct effects of K(ATP) channel openers pinacidil and diazoxide on oxidative phosphorylation of mitochondria in situ.

KATP channel openers protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, however, the protection mechanisms have not been fully clarified yet. Since the skinned fibers technique gives an opportunity to investigate an entire population of mitochondria in their native milieu, in this study we have investigated the effects of KATP channel openers pinacidil and diazoxide on the respiration rate of rat heart mitochondria in situ, oxidizing physiological substrates pyruvate and malate (6+6 mM). Respiration rates were recorded by the means of Clark-type oxygen electrode in the physiological salt solution (37°C). Our results showed that both pinacidil and diazoxide (60-1250 µM) in a concentration-dependent manner increased pyruvate-malate supported State 2 respiration rate of skinned cardiac fibers (59.1 ± 5.1 nmol O/min/mg fiber dry weight, RCI 2.6 ± 0.2, n=4) by 15-120%. Moreover, diazoxide did not affect, whereas pinacidil (60-1250 µM) decreased the State 3 respiration rate of skinned cardiac fibers (116.6 ± 13.6 nmol O/min/mg fiber dry weight, RCI 2.3 ± 0.2, n=4) by 4-27%. Thus, common effect for both KATP channel openers is uncoupling of pyruvate and malate oxidizing mitochondria in skinned cardiac fibers, whereas pinacidil under same conditions also inhibits mitochondrial respiratory chain. Since mitochondria in situ resemble to the great extent mitochondria in vivo, our results suggest that uncoupling and/or respiratory chain inhibition could play a role in the cardioprotection by KATP channel openers.

Anthocyanins in cardioprotection: A path through mitochondria

Constantly growing experimental data from in vitro, in vivo and epidemiological studies show the great potential of anthocyanin-containing fruit and berry extracts or pure individual anthocyanins as cardioprotective food components or pharmacological compounds. In general it is regarded that the cardioprotective activity of anthocyanins is related to their antioxidant properties. However there are recent reports that certain anthocyanins may protect the heart against ischemia/reperfusion-induced injury by activating signal transduction pathways and sustaining mitochondrial functions instead of acting solely as antioxidants. In this review, we summarize the proposed mechanisms of direct or indirect actions of anthocyanins within cardiac cells with the special emphasis on recently discovered their pharmacological effects on mitochondria in cardioprotection: reduction of cytosolic cytochrome c preventing apoptosis and sustainment of electron transfer between NADH dehydrogenase and cytochrome c supporting oxidative phosphorylation in ischemia-damaged mitochondria.

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP

The present study on saponin‐treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl‐CoA+carnitine, palmitoyl‐L‐carnitine and octanoyl‐L‐carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K m of oxidative phosphorylation for ADP. Octanoyl‐D‐carnitine, as well as palmitate, palmitoyl‐CoA, and palmitoyl‐L‐carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.

5-Hydroxy-1,4-naphthalenedione exerts anticancer effects on glioma cells through interaction with the mitochondrial electron transport chain

Survival of patients with glioblastoma remains within the range of several months despite advances in therapeutic options. We have already shown that 5-hydroxy-1,4-naphthalenedione (juglone) exerts antiproliferative, anti-invasive, and cytotoxic effects on glioma C6 cells. Here, we further revealed that juglone is relatively selective to glioma cells as compared to the normal glial culture, and investigated its mechanisms of action. The incubation of glioma C6 cells with juglone generated high levels of reactive oxygen species (ROS). The produced ROS accounted for the anticancer effect of juglone as antioxidant N-acetylcysteine reduced both cytotoxic and antiproliferative activities of juglone. Furthermore, high resolution respirometry revealed that juglone decreased oxygen consumption mainly by affecting pyruvate/malate- and glutamate/malate-induced mitochondrial respiration. The inhibition of respiratory complex I by amytal decreased juglone-triggered generation of ROS and diminished its anticancer activity. Thus, our results indicate that juglone generates ROS through interaction with respiratory complex I in glioma C6 cells, and, in turn, induces the anticancer effects.

Computational analysis of thaumatin-II allergenicity and prediction of antigenic elements of thaumatin-like family proteins@@@Taumatino II alergeniškumo įvertinimas ir antigeniškumo elementų nustatymas bioinformatikos metodais

Thaumatin-II is an intense sweet tasting protein isolated from fruits of Thaumatococcus daniel lii. Thaumatin is a member of the pathogenesis-related protein family referred to as thaumatinlike proteins (TLPs). Expression of thaumatin-II in plants was associated with an enhanced resistance against pathogens. In addition, thaumatin was used to provide the sweet taste quality to plant products. Thus, TLP proteins have a potential application for improving plant stress resistance and taste qualities using the recombinant DNA technology. However, several members of the TLP family were characterized as food allergens. The allergenic properties of thaumatinII have not been characterized. In this study, a putative allergenicity of thaumatin-II was established using computational analysis of sequence similarity with known human allergens. Antigenicity analysis and multiple sequence alignment of related TLP sequences identified six putative allergenic epitopes. The residues Thr12, Leu74, Gln133 and Thr161 of thaumatin-II or equivalent residues of other TLPs were proposed as a target for mutagenesis aimed to develop protein isoforms with reduced allergenicity.

Long-term regulation of gene expression in muscle cells by systemically delivered siRNA

Abstract Many muscular dystrophies, including lethal Duchenne muscular dystrophy, are incurable and require the sustained application of drugs that have only minor treatment effects and serious negative side effects. The mechanism of siRNA‐mediated transcriptional gene regulation (TGR) appears to have a long‐lasting effect and may be a viable solution to treat muscle disorders because single or at least rarely repeated therapies would be used. For the best results, siRNA should be delivered to all disease affected muscles, and systemic delivery of siRNA through blood vessels is probably the only applicable choice to achieve this goal. Unfortunately, there are many challenges to overcome such as siRNA degradation in blood, renal clearance, blood–muscle barrier, cell entry and endosomal escape. By exploiting and considering the unique features of muscles and the mechanism of TGR, we will discuss the possible ways to induce TGR in muscles by using non‐viral systemic siRNA delivery methods. Graphical abstract Figure. No Caption available.