Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors.

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1.

Yen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or the increase in the first incision rate by assembly of a dimer on a HJ, despite minor differences. However, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5'-3' exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfils the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

Holliday Junction Resolution.

Holliday junctions are four-way DNA structures that may arise during meiotic recombination, double-strand break repair, or postreplicative repair by the reciprocal exchange of single strands between two DNA molecules. Given their ability to effectively bridge two sister chromatids or homologous chromosomes, cells have implemented various pathways to ensure their timely removal. One of them is the nucleolytic processing of the Holliday junctions by specialized structure-selective endonucleases termed resolvases, which sever the connection between the linked molecules. These Holliday junction resolvases are essential tools of the DNA damage repair machinery to ensure accurate chromosomal segregation, whose activities can be modulated by posttranslational modifications like phosphorylation. Here, we describe a protocol to purify S. cerevisiae Yen1 resolvase in two different phosphorylation states (high and low) and to set up a biochemical assay to compare their ability to process a synthetic, oligonucleotide-based Holliday junction structures.

Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins.

The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

An unusual cause of neonatal cyanosis .

We report a case of a female neonate whose pulse oximetry screening for congenital heart disease at 40 h of life was positive. The pregnancy was uneventful with no relevant family history. The neonate presented with bluish discolouration of the skin lasting until day 15. Cardiovascular examination and chest radiography were normal. Septic screening was negative. Oxygen therapy was started with poor response; investigations revealed a methaemoglobinaemia of 7.4%. The methaemoglobin level reached a peak of 15% on day 10, falling thereafter. The infant was discharged by day 20 with a normal physical examination and a methaemoglobinaemia of 11.4%. By 2 months of age this had fallen to 2.4%. Further investigation revealed a haemoglobin M variant: a heterozygous mutation of the γ globin gene known as Hb F-M Viseu. The mutation occurs in the γ chain, therefore the methaemoglobinaemia is transitory, resolving with the transition from fetal to adult haemoglobin.

MitoTimer: a novel tool for monitoring mitochondrial turnover.

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, in which fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial-targeting sequence (coined "MitoTimer") and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and production was induced with doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Dox addition for as little as 1 h was sufficient to induce MitoTimer expression within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent transition from green to red over 48 h and was amenable to analysis by fluorescence microscopy and flow cytometry of whole cells or isolated mitochondria. A second Dox administration 48 h after the initial induction resulted in a second round of expression of green MitoTimer. The extent of new protein incorporation during a second pulse was increased by administration of a mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal new insights into mitochondrial dynamics within cells. Coupled with organelle flow cytometry, it offers new opportunities to investigate mitochondrial subpopulations by biochemical or proteomic methods.

Mitochondrial therapeutics for cardioprotection.

Mitochondria represent approximately one-third of the mass of the heart and play a critical role in maintaining cellular function-however, they are also a potent source of free radicals and pro-apoptotic factors. As such, maintaining mitochondrial homeostasis is essential to cell survival. As the dominant source of ATP, continuous quality control is mandatory to ensure their ongoing optimal function. Mitochondrial quality control is accomplished by the dynamic interplay of fusion, fission, autophagy, and mitochondrial biogenesis. This review examines these processes in the heart and considers their role in the context of ischemia-reperfusion injury. Interventions that modulate mitochondrial turnover, including pharmacologic agents, exercise, and caloric restriction are discussed as a means to improve mitochondrial quality control, ameliorate cardiovascular dysfunction, and enhance longevity.

Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury.

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury.

Efeito da remoção de fenilalanina sobre o perfil peptídico de hidrolisados proteicos da farinha de trigo / Phenylalanine removal effect on the peptide profile of hydrolyzed protein from wheat flour

Para avaliar o efeito da remoção de fenilalanina no perfil peptídico dos hidrolisados proteicos de farinha de trigo, foram preparados nove hidrolisados empregando-se a associação sucessiva de pancreatina e de extrato enzimático bruto obtido da casca de abacaxi (EB). Foram testados o efeito da ordem de adição das enzimas, da temperatura de reação, da relação enzima: substrato (E:S) e do tratamento físico da amostra. A análise do perfil peptídico dos hidrolisados foi realizada em duas etapas: antes e após a remoção da fenilalanina. A cromatografia líquida de alta eficiência de exclusão molecular foi utilizada para efetuar o fracionamento e a quantificação dos peptídeos e aminoácidos livres pela técnica da Área Corrigida da Fração. O processo de remoção de fenilalanina melhorou o perfil peptídico de três hidrolisados, mas não afetou no de cinco hidrolisados. O efeito benéfico desse processo está associado ao aumento no teor de di- e tripeptídeos ou à redução na quantidade de peptídeos grandes. O melhor perfil peptídico foi obtido após a remoção de fenilalanina, utilizando-se pancreatina E:S de 4:100 a 50°C, durante 210 min, seguidade EB E:S de 10:100 a 70°C durante 90 min.

The effect of phenylalanine removal on the peptide profile of the protein hydrolysates from wheat flour wasinvestigated. Nine hydrolysates were prepared, using a successive association of a pancreatin and a crudeenzymatic extract obtained from pineapple peel (CE), and the effect of the order of enzymes addition, thereaction temperature, the enzyme: substrate (E:S) ratio, and the physical treatment of sample were examined.The analysis of peptide profile of hydrolysates was performed in two stages, that is before and after removingphenylalanine. The size-exclusion high performance liquid chromatography was used for performing the fractionation, followed by Correct Fraction Area technique for quantifying peptides and free amino acids. The process of phenylalanine removal improved the peptide profile of three hydrolyzed samples, but it did not affect the five hydrolysates. The beneficial effect of this process is correlated with the increase of di- and tripeptides contents, or in reducing the amount of large peptides. The best peptide profile was obtained after phenylalanine removal by using pancreatin at E:S ratio of 4:100 at 50ºC for 210min, followed by CE at E:S ratio of 10:100 at 70ºC for 90 min.

Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells.

Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) than cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.

Autophagy in health and disease. 5. Mitophagy as a way of life.

Our understanding of autophagy has expanded greatly in recent years, largely due to the identification of the many genes involved in the process and to the development of better methods to monitor the process, such as green fluorescent protein-LC3 to visualize autophagosomes in vivo. A number of groups have demonstrated a tight connection between autophagy and mitochondrial turnover. Mitochondrial quality control is the process whereby mitochondria undergo successive rounds of fusion and fission with a dynamic exchange of components to segregate functional and damaged elements. Removal of the mitochondrion that contains damaged components is accomplished via autophagy (mitophagy). Mitophagy also serves to eliminate the subset of mitochondria producing the most reactive oxygen species, and episodic removal of mitochondria will reduce the oxidative burden, thus linking the mitochondrial free radical theory of aging with longevity achieved through caloric restriction. Mitophagy must be balanced by biogenesis to meet tissue energy needs, but the system is tunable and highly dynamic. This process is of greatest importance in long-lived cells such as cardiomyocytes, neurons, and memory T cells. Autophagy is known to decrease with age, and the failure to maintain mitochondrial quality control through mitophagy may explain why the heart, brain, and components of the immune system are most vulnerable to dysfunction as organisms age.

Validação intralaboratorial de metodologia quantitativa para determinação de L-fenilalanina em farinha de trigo por espectrofotometria derivada segunda / Single-laboratory validation of a quantitative methodology for determination of L-phenylalanine in wheat flour by second derivative spectrophotometry

O objetivo deste trabalho foi validar um método quantitativo para determinação de L-fenilalanina (Fen) em farinha de trigo por espectrofotometria derivada segunda. A amostra de farinha de trigo, na quantidade de 0,525g, foi submetida à hidrólise ácida com HCl a 5,7 mol/L, a 110 °C, por 24 h. O material hidrolisado foi reconstituído para 50 mL com tampão fosfato de sódio a 0,1 mol/L, pH 7,0. As soluções preparadas a partir dessa amostra foram submetidas às leituras de absorvância, entre 230 nm e 280 nm, em espectrofotômetro UV/VIS. Os espectros de derivada segunda foram traçados e os valores das áreas dos picos negativos foram utilizados para estimar os teores de Fen. A linearidade do método foi demonstrada na faixa de 0,010 mg/mL a 0,035 mg/mL (correspondente a teores de 251 mg/100g a 877 mg/100g de Fen em farinha de trigo). Efeitos de matriz foram observados. A determinação de Fen não sofreu interferência de compostos como L-tirosinae L-triptofano. As porcentagens de recuperação variaram de 81% a 118% e os desvios padrão relativos de repetitividade e reprodutibilidade parcial foram respectivamente 11% e 15%, para amostras contendo 354 mg/100g, demonstrando adequada recuperação e precisão do método. Os limites de detecção e quantificação foram, respectivamente, 63 mg/100ge 175 mg/100g. Os parâmetros de desempenho estudados indicaram adequação do método para o monitoramento e controle de teores de Fen em farinha de trigo.

Ação de fatores enzimáticos na obtenção de hidrolisados protéicos de farinha de trigo com baixo teor de fenilalanina / Action of enzymatic factors in obtaining protein hydrolysates of wheat flour, low-phenylalanine

Visando ao preparo de farinha de trigo com baixo teor de fenilalanina (Phe), extraiu-se, enzimaticamente as proteínas, empregando-se uma protease alcalina de Bacillus licheniformis. Em seguida, os extratos protéicos foram hidrolisados pela ação de enzimas comerciais (pancreatina e bromelina) e de extratos enzimáticos obtidos da casca de abacaxi (bruto e purificado), avaliando-se alguns parâmetros enzimáticos, tais como tipo de enzima, tipo de ação enzimática, tipo de associação enzimática e ordem de ação enzimática. O carvão ativado (CA) foi empregado como meio adsorvente e a eficiência da remoção de Phe foi avaliada por espectrofotometria derivada segunda, determinando-se o teor de Phe na farinha de trigo e em seus hidrolisados, após tratamento com CA. O melhor resultado foi encontrado ao se empregar a associação sucessiva do extrato bruto seguida da pancreatina, tendo atingido 66,28 por cento de remoção e o teor final de Phe de 522,44 mg/100 g de hidrolisado.

With the aim of producing wheat flour with low phenylalanine (Phe) content to be introduced in phenylketonuric's diet, the proteins were enzymaticaly extracted, using an alkaline protease from Bacillus licheniformis. Then, the protein extracts were hydrolyzed by the action of commercial enzymes (pancreatin and bromelain) and of enzymatic extracts obtained from pineapple peel (crude and purified). Some enzymatic parameters were evaluated, such as type of enzyme, type of enzyme action, type of enzymatic association and order of enzyme action. The activated carbon (AC) was used as adsorbent and the efficiency of Phe removal was evaluated by second derivative spectrophotometry measuring the Phe content in wheat flour and in their hydrolysates after AC treatment. The best result was found for the successive association of crude extract followed by pancreatin obtaining 66.28 percent of removal and a final Phe content of 522.44 mg/100 g of hydrolysate.

LPS-induced autophagy is mediated by oxidative signaling in cardiomyocytes and is associated with cytoprotection.

Bacterial endotoxin lipopolysaccharide (LPS) is responsible for the multiorgan dysfunction that characterizes septic shock and is causal in the myocardial depression that is a common feature of endotoxemia in patients. In this setting the myocardial dysfunction appears to be due, in part, to the production of proinflammatory cytokines. A line of evidence also indicates that LPS stimulates autophagy in cardiomyocytes. However, the signal transduction pathway leading to autophagy and its role in the heart are incompletely characterized. In this work, we wished to determine the effect of LPS on autophagy and the physiological significance of the autophagic response. Autophagy was monitored morphologically and biochemically in HL-1 cardiomyocytes, neonatal rat cardiomyocytes, and transgenic mouse hearts after the administration of bacterial LPS or TNF-alpha. We observed that autophagy was increased after exposure to LPS or TNF-alpha, which is induced by LPS. The inhibition of TNF-alpha production by AG126 significantly reduced the accumulation of autophagosomes both in cell culture and in vivo. The inhibition of p38 MAPK or nitric oxide synthase by pharmacological inhibitors also reduced autophagy. Nitric oxide or H(2)O(2) induced autophagy in cardiomyocytes, whereas N-acetyl-cysteine, a potent antioxidant, suppressed autophagy. LPS resulted in increased reactive oxygen species (ROS) production and decreased total glutathione. To test the hypothesis that autophagy might serve as a damage control mechanism to limit further ROS production, we induced autophagy with rapamycin before LPS exposure. The activation of autophagy by rapamycin suppressed LPS-mediated ROS production and protected cells against LPS toxicity. These findings support the notion that autophagy is a cytoprotective response to LPS-induced cardiomyocyte injury; additional studies are needed to determine the therapeutic implications.

Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia-reperfusion.

Ischemia followed by reperfusion is known to negatively affect mitochondrial function by inducing a deleterious condition termed mitochondrial permeability transition. Mitochondrial permeability transition is triggered by oxidative stress, which occurs in mitochondria during ischemia-reperfusion as a result of lower antioxidant defenses and increased oxidant production. Permeability transition causes mitochondrial dysfunction and can ultimately lead to cell death. A drug able to minimize mitochondrial damage induced by ischemia-reperfusion may prove to be clinically effective. We aimed to analyze the effects of nicorandil, an ATP-sensitive potassium channel agonist and vasodilator, on mitochondrial function of rat hearts and cardiac HL-1 cells submitted to ischemia-reperfusion. Nicorandil decreased mitochondrial swelling and calcium uptake. It also decreased reactive oxygen species formation and thiobarbituric acid reactive substances levels, a lipid peroxidation biomarker. We thus confirm previous reports that nicorandil inhibits mitochondrial permeability transition and demonstrate that nicorandil inhibits this process by preventing oxidative damage and mitochondrial calcium overload induced by ischemia-reperfusion, resulting in improved cardiomyocyte viability. These results may explain the good clinical results obtained when using nicorandil in the treatment of ischemic heart disease.

Ischemic preconditioning enhances fatty acid-dependent mitochondrial uncoupling.

This study tests the hypothesis that ischemic preconditioning (IP) changes fatty acid (FA)-dependent uncoupling between mitochondrial respiration and oxidative phosphorylation. We found that IP does not alter mitochondrial membrane integrity or FA levels, but enhances membrane potential decreases when FA are present, in an ATP-sensitive manner. FA hydroperoxides had equal effects in control and preconditioned mitochondria, and GTP did not abrogate the IP effect, suggesting uncoupling proteins were not involved. Conversely, thiol reductants and atractyloside, which inhibits the adenine nucleotide translocator, eliminated the differences in responses to FA. Together, our results suggest that IP leads to thiol oxidation and activation of the adenine nucleotide translocator, resulting in enhanced FA transport and mild mitochondrial uncoupling.

Nicorandil preserves the function of the mitochondrial phosphorylative and oxidative system in an animal model of global ischemia-reperfusion.

Ischemia followed by reperfusion (IR) negatively affects mitochondrial function. At the level of the oxidative-phosphorylative system, IR inhibits the respiratory complexes and ATP synthase, and increases the passive leak of protons through the inner mitochondrial membrane, uncoupling respiration from phosphorylation, decreasing mitochondrial potential and, consequently, ATP production. Drugs that minimize the mitochondrial damage induced by IR may prove to be clinically effective. In the present work, we analyzed the impact of nicorandil, a mitochondrial ATP-sensitive potassium channel agonist, on mitochondrial dysfunction at the level of the oxidative-phosphorylative system of rat hearts subjected to IR. The decrease in the respiratory control ratio (RCR) induced by IR leads to the conclusion that IR has a negative impact on the activity of the mitochondrial respiratory system, uncoupling oxidation from phosphorylation. This effect is reversed by nicorandil, which increases not only RCR, but also the ADP/O ratio. Regarding respiratory rate, state 3 rate was approximately the same for all the experimental groups, while state 4 rate was lower for the group where IR was induced in the presence of nicorandil. This result is in accordance with the data obtained for the RCR and ADP/O. State 4 rate is most affected by uncoupling, given that it is controlled by proton leak. Mitochondria subjected to IR in the presence of nicorandil have a lower state 4 rate, i.e. they are less uncoupled. From these results we conclude that nicorandil preserves the function of mitochondria subjected to IR in terms of both respiration and phosphorylative capacity.

Ischemic preconditioning requires increases in reactive oxygen release independent of mitochondrial K+ channel activity.

Mitochondrial ATP-sensitive K+ channels (mitoKATP) mediate ischemic preconditioning, a cardioprotective procedure. MitoKATP activity has been proposed to either enhance or prevent the release of reactive oxygen species. This study tested the redox effects of mitoKATP in order to clarify the role of these channels during preconditioning. We found no evidence that mitoKATP channels increase mitochondrial reactive oxygen species release directly. In addition, neither ischemic preconditioning nor the mitoKATP agonist diazoxide increased antioxidant defenses. Furthermore, increases in reactive oxygen species observed during ischemic preconditioning were not inhibited by mitoKATP antagonists, suggesting that they occur upstream of channel activity. Antioxidants were tested to verify if diazoxide-promoted ischemic protection was dependent on reactive oxygen species. N-Acetylcysteine proved to be an inadequate antioxidant for these tests since it directly interfered with the ability of diazoxide to activate mitoKATP. Catalase reversed the beneficial effect of preconditioning, but not of diazoxide, indicating that reactive oxygen species mediating preconditioning occur upstream of mitoKATP. Taken together, these results demonstrate that ischemic preconditioning increases reactive oxygen release independently of mitoKATP and suggest that the activity of this channel prevents oxidative reperfusion damage by decreasing reactive oxygen species production.