Under several pathological conditions, reactive oxygen species-induced damages play important roles in pathogenesis (1-3). High levels of reactive oxygen species are generated from a variety of sources such as the xanthine oxidase system (1), the leakage of electrons from the mitochondrial respiratory chain (2, 4), the cyclooxygenase pathway of arachidonic acid metabolism (3,5), and the respiratory burst of phagocyte cells (6, 7), and they can cause DNA damage-generating singlestranded DNA breaks (8). Poly(ADP-ribose)polymerase (PARP-1, 2 EC 2.4.2.30) is a multifunctional nuclear enzyme (9) that is activated by DNA strand breaks and catalyzes the covalent coupling of branched chains of ADP-ribose units to various nuclear proteins such as histone proteins and PARP-1 itself. PARP-1 is involved in chromatin remodeling, DNA repair, replication, transcription, and the maintenance of genomic stability by, in part, poly(ADP-ribosyl)ation (9). With moderate amounts of DNA damage, PARP-1 is thought to participate in the DNA repair process (10, 11). However, oxidative stress, which induces a large amount of DNA damage, can cause excessive activation of PARP-1, leading to depletion of its substrate NAD ϩ ; and in an effort to resynthesize NAD ϩ , ATP is also depleted, resulting in cell death as a consequence of energy loss (12-15). PARP inhibitors show pronounced protection against myocardial ischemia (16), neuronal ischemia (17, 18), acute lung inflammation (19), acute septic shock (20), zymogen-induced multiple organ failure (21), and diabetic pancreatic damage (22-24), providing evidence for the role of excessive PARP-1 activation in cell death. It is believed that by preventing excessive NAD ϩ and ATP utilization, PARP inhibitors protect cells against oxidative damage, but some recent data suggest a more complex mechanism for the cytoprotection (25, 26). There is evidence that PARP activation can contribute to exaggeration of mitochondrial damage (27) and mitochondrial reactive oxygen species production (28), indicating that PARP activation can modulate processes outside of the nucleus. Recent works reported the existence of mitochondrial PARP that can be blocked with PARP-1 inhibitors (29); therefore, it would be important to clarify whether the mitochondrial protection by PARP inhibitors is a direct consequence of the inhibition of mitochondrial ADP-ribosylation or the inhibition of nuclear PARP modulation by yet unidentified processes that are responsible for the mitochondrial protection. Our previous works demonstrated that PARP inhibitors induced the phosphorylation and activation of Akt in the liver, lung, and spleen of lipopolysaccharide-treated mice, raising the possibility that the protective effect of PARP inhibition can be mediated through the PI3-kinase/Akt pathway (30). These observations indicate that the protective effect of PARP inhibitors should be far more In the present study, we analyzed the effect of PARP inhibition by pharmacologic agents, by the transdominant expression of the PARP N-terminal DNA...

Multiple pregnancy is a risk for prematurity and preterm birth. The goal of assisted reproduction is to achieve a single pregnancy, by transferring a single embryo. This requires improved methods to identify the competent embryo. Here, we describe such a test, based on flow cytometric determination of the nucleic acid (PI+) containing extracellular vesicle (EV) count in day 5 embryo culture media. 88 women undergoing IVF were included in the study. More than 1 embryos were transferred to most patients. In 58 women, the transfer resulted in clinical pregnancy, whereas in 30 women in implantation failure. In 112 culture media of embryos from the “clinical pregnancy” group, the number of PI+ EVs was significantly lower than in those of 49 embryos, from the “implantation failure” group. In 14 women, transfer of a single embryo resulted in a singleton pregnancy, or, transfer of two embryos in twin pregnancy. The culture media of 19 out of the 20 “confirmed competent” embryos contained a lower level of PI+ EVs than the cut off level, suggesting that the competent embryo can indeed be identified by low PI+ EV counts. We developed a noninvasive, simple, inexpensive, quick test, which identifies the embryos that are most likely to implant.

Earlier evidence suggests, that the embryo signals to the maternal immune system. Extracellular vesicles (EVs) are produced by all types of cells, and because they transport different kinds of molecules from one cell to the other, they can be considered as means of intercellular communication. The aim of this work was to test, whether the embryo is able to produce sufficient amounts of EVs to alter the function of peripheral lymphocytes. Embryo-derived EVs were identified by their Annexin V biding capacity, and sensitivity to Triton X dependent lysis, using flow cytometry. Transmission electron microscopy was used to detect EVs at the implantation site. Progesterone-induced blocking factor (PIBF) expression in embryo-derived EVs was demonstrated with immuno-electron microscopy. The % of IL-10 + murine lymphocytes was determined by flow cytometry. EVs were present in embryo culture media, but not in empty media. Mouse embryo-derived EVs adhere to the surface of both CD4+ and CD8+ murine peripheral T lymphocytes, partly, via phosphatidylserine binding. The number of IL-10+ murine peripheral CD8+ cells increases in the presence of embryo-derived EVS, and this effect is counteracted by pre-treatment of EVs with an anti-PIBF antibody, suggesting that the embryo communicates with the maternal immune system via EVs.

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase-Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase-Akt cytoprotective pathway.

Amiodarone is a widely used and potent antiarrhythmic agent that is metabolized to desethylamiodarone. Both amiodarone and its metabolite possess antiarrhythmic effect, and both compounds can contribute to toxic side effects. Here, we compare the effect of amiodarone and desethylamiodarone on mitochondrial energy metabolism, membrane potential, and permeability transition and on mitochondria-related apoptotic events. Amiodarone but not desethylamiodarone protects the mitochondrial energy metabolism of the perfused heart during ischemia in perfused hearts. At low concentrations, amiodarone stimulated state 4 respiration due to an uncoupling effect, inhibited the Ca 2ϩ -induced mitochondrial swelling, whereas it dissipated the mitochondrial membrane potential (⌬⌿), and prevented the ischemia-reperfusion-induced release of apoptosis-inducing factor (AIF). At higher concentrations, amiodarone inhibited the mitochondrial respiration and simulated a cyclosporin A (CsA)-independent mitochondrial swelling. In contrast to these, desethylamiodarone did not stimulate state 4 respiration, did not inhibit the Ca 2ϩ -induced mitochondrial permeability transition, did not induce the collapse of ⌬⌿ in low concentrations, and did not prevent the nuclear translocation of AIF in perfused rat hearts, but it induced a CsA-independent mitochondrial swelling at higher concentration, like amiodarone. That is, desethylamiodarone lacks the protective effect of amiodarone seen at low concentrations, such as the inhibition of calcium-induced mitochondrial permeability transition and inhibition of the nuclear translocation of the proapoptotic AIF. On the other hand, both amiodarone and desethylamiodarone at higher concentration induced a CsA-independent mitochondrial swelling, resulting in apoptotic death that explains their extracardiac toxic effect.Amiodarone (2-butyl-3-benzofuranyl 4-[2-(diethylamino)-ethoxy]-3,5-diiodophenyl-ketone hydrochloride) is one of the most effective antiarrhythmic drugs and is frequently used in the clinical practice for treating ventricular and supraventricular arrhythmias. It is a class III antiarrhythmic agent, prolonging action potential duration whose effect may involve blocking of ␤-adrenergic receptors, sodium channels, and L-type calcium channels (Singh and Vaughan Williams, 1970;Nokin et al., 1983;Nattel et al., 1987;Varro et al., 1996). It may also have a role in preventing mortality after myocardial infarction (Julian et al., 1997). Despite its effective antiarrhythmical properties, the use of amiodarone is often limited by its toxic side effects, including thyroid dysfunction, liver, and pancreas fibrosis (Amico et al., 1984;Martin and Howard, 1985). However, the most severe adverse effect of the drug is pulmonary fibrosis, occurring in up to 13% of the patients receiving the amiodarone in doses higher than 400 mg day Ϫ1 (Martin and Rosenow, 1988). The etiology of the amiodarone-induced pulmonary toxicity is unknown.Desethylamiodarone, the major metabolite of amiodarone, also has antiarrhyth...