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Find video protocols related to scientific articles indexed in Pubmed.
Iodoaromatization reactions of enyne-dioxinones: syntheses of 4H-1,3-benzodioxin-4-ones, masked pentasubstituted arenes.
J. Org. Chem.
PUBLISHED: 08-28-2014
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Sequential reaction of a keto-dioxinone with dimethylformamide dimethyl acetal and a range of magnesium acetylides gave the corresponding enyne-dioxinones as mixtures of E and Z isomers (E > Z). Subsequent reaction with iodine monochloride resulted in cycloaromatization, presumably via an iodovinyl cation, giving a range of 4H-1,3-benzodioxin-4-ones.
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Decreasing mitochondrial fission alleviates hepatic steatosis in a murine model of nonalcoholic fatty liver disease.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 07-31-2014
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Mitochondria produce the majority of cellular ATP through oxidative phosphorylation, and their capacity to do so is influenced by many factors. Mitochondrial morphology is recently suggested as an important contributor in controlling mitochondrial bioenergetics. Mitochondria divide and fuse continuously, which is affected by environmental factors, including metabolic alterations. Underscoring its bioenergetic influence, altered mitochondrial morphology is reported in tissues of patients and in animal models of metabolic dysfunction. In this study, we found that mitochondrial fission plays a vital role in the progression of nonalcoholic fatty liver disease (NAFLD). The development of hepatic steatosis, oxidative/nitrative stress, and hepatic tissue damage, induced by a high-fat diet, were alleviated in genetically manipulated mice suppressing mitochondrial fission. The alleviation of steatosis was recapitulated in primary hepatocytes with the inhibition of mitochondrial fission. Mechanistically, our study indicates that fission inhibition enhances proton leak under conditions of free fatty acid incubation, implicating bioenergetic change through manipulating mitochondrial fission. Taken together, our results suggest a mechanistic role for mitochondrial fission in the etiology of NAFLD. The efficacy of decreasing mitochondrial fission in the suppression of NAFLD suggests that mitochondrial fission represents a novel target for therapeutic treatment of NAFLD.
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DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.
Free Radic. Biol. Med.
PUBLISHED: 04-19-2014
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Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.
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SIRT3 deficiency exacerbates ischemia-reperfusion injury: implication for aged hearts.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 04-18-2014
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Ischemia-reperfusion (IR) injury is significantly worse in aged hearts, but the underlying mechanisms are poorly understood. Age-related damage to mitochondria may be a critical feature, which manifests in an exacerbation of IR injury. Silent information regulator of transcription 3 (SIRT3), the major mitochondrial NAD(+)-dependent lysine deacetylase, regulates a variety of functions, and its inhibition may disrupt mitochondrial function to impact recovery from IR injury. In this study, the role of SIRT3 in mediating the response to cardiac IR injury was examined using an in vitro model of SIRT3 knockdown (SIRT3(kd)) in H9c2 cardiac-derived cells and in Langendorff preparations from adult (7 mo old) wild-type (WT) and SIRT3(+/-) hearts and aged (18 mo old) WT hearts. SIRT3(kd) cells were more vulnerable to simulated IR injury and exhibited a 46% decrease in mitochondrial complex I (Cx I) activity with low O2 consumption rates compared with controls. In the Langendorff model, SIRT3(+/-) adult hearts showed less functional recovery and greater infarct vs. WT, which recapitulates the in vitro results. In WT aged hearts, recovery from IR injury was similar to SIRT3(+/-) adult hearts. Mitochondrial protein acetylation was increased in both SIRT3(+/-) adult and WT aged hearts (relative to WT adult), suggesting similar activities of SIRT3. Also, enzymatic activities of two SIRT3 targets, Cx I and MnSOD, were similarly and significantly inhibited in SIRT3(+/-) adult and WT aged cardiac mitochondria. In conclusion, decreased SIRT3 may increase the susceptibility of cardiac-derived cells and adult hearts to IR injury and may contribute to a greater level of IR injury in the aged heart.
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Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.
Nature
PUBLISHED: 03-24-2014
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Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.
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Acute cellular rejection: impact of donor-specific antibodies and C4d.
Transplantation
PUBLISHED: 01-17-2014
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Mixed rejection in kidney transplantation consists of histologic and/or serological evidence of both cellular and humoral components. As it is not confined to a distinct category in the Banff classification, how to best manage these patients is not clear. The aim of this study was to determine the incidence and outcome of morphological T-cell-mediated rejection (TCMR) with a humoral component, defined as the presence of either DSA or C4d, compared with the outcome of pure TCMR.
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Internet publicity of data problems in the bioscience literature correlates with enhanced corrective action.
PeerJ
PUBLISHED: 01-01-2014
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Several online forums exist to facilitate open and/or anonymous discussion of the peer-reviewed scientific literature. Data integrity is a common discussion topic, and it is widely assumed that publicity surrounding such matters will accelerate correction of the scientific record. This study aimed to test this assumption by examining a collection of 497 papers for which data integrity had been questioned either in public or in private. As such, the papers were divided into two sub-sets: a public set of 274 papers discussed online, and the remainder a private set of 223 papers not publicized. The sources of alleged data problems, as well as criteria for defining problem data, and communication of problems to journals and appropriate institutions, were similar between the sets. The number of laboratory groups represented in each set was also similar (75 in public, 62 in private), as was the number of problem papers per laboratory group (3.65 in public, 3.54 in private). Over a study period of 18 months, public papers were retracted 6.5-fold more, and corrected 7.7-fold more, than those in the private set. Parsing the results by laboratory group, 28 laboratory groups in the public set had papers which received corrective action, versus 6 laboratory groups in the private set. For those laboratory groups in the public set with corrected/retracted papers, the fraction of their papers acted on was 62% of those initially flagged, whereas in the private set this fraction was 27%. Such clustering of actions suggests a pattern in which correction/retraction of one paper from a group correlates with more corrections/retractions from the same group, with this pattern being stronger in the public set. It is therefore concluded that online discussion enhances levels of corrective action in the scientific literature. Nevertheless, anecdotal discussion reveals substantial room for improvement in handling of such matters.
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Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism.
J. Biol. Chem.
PUBLISHED: 10-19-2013
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We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.
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Mitochondrial dysfunction and permeability transition in osteosarcoma cells showing the Warburg effect.
J. Biol. Chem.
PUBLISHED: 10-07-2013
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Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.
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Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I.
Nat. Med.
PUBLISHED: 04-15-2013
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Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.
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Kir6.2 is not the mitochondrial KATP channel but is required for cardioprotection by ischemic preconditioning.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 04-12-2013
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ATP-sensitive K(+) (KATP) channels that contain K(+) inward rectifier subunits of the 6.2 isotype (Kir6.2) are important regulators of the cardiac response to ischemia-reperfusion (I/R) injury. Opening of these channels is implicated in the cardioprotective mechanism of ischemic preconditioning (IPC), but debate surrounds the contribution of surface KATP (sKATP) versus mitochondrial KATP (mKATP) channels. While responses to I/R injury and IPC have been examined in Kir6.2(-/-) mice before, breeding methods and other technical obstacles may have confounded interpretations. The aim of this study was to elucidate the role of Kir6.2 in cardioprotection and mKATP activity, using conventionally bred Kir6.2(-/-) mice with wild-type littermates as controls. We found that perfused hearts from Kir6.2(-/-) mice exhibited a normal baseline response to I/R injury, were not protected by IPC, and showed a blunted response to the IPC mimetic drug diazoxide. These data suggest that the loss of IPC in Kir6.2(-/-) hearts is not due to an underlying difference in I/R sensitivity. Furthermore, mKATP channel activity was identical in cardiac mitochondria isolated from wild-type versus Kir6.2(-/-) mice, suggesting no role for Kir6.2 in the mKATP. Collectively, these data indicate that Kir6.2 is required for the full response to IPC or diazoxide but is not involved in mKATP formation.
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Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts.
Transplantation
PUBLISHED: 03-08-2013
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BACKGROUND.: Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. METHODS.: We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. RESULTS.: C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). CONCLUSIONS.: The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.
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Role of p90(RSK) in regulating the Crabtree effect: implications for cancer.
Biochem. Soc. Trans.
PUBLISHED: 01-30-2013
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High glucose inhibits mitochondrial respiration, known as the Crabtree effect, in cancer cells and possibly other cell types. The upstream pathways regulating this phenomenon are poorly understood. In diabetes, where glucose levels are elevated, the p90(RSK) (p90 ribosomal S6 kinase) has received much attention as a potential upstream mediator of the effects of high glucose. Evidence is also emerging that p90(RSK) may play a role in cancer cell signalling, although the role of p90(RSK) in regulating cancer cell metabolism is unclear. In the present paper, we provide an overview of the Crabtree effect and its relationship to mitochondrial metabolism. Furthermore, preliminary data are presented suggesting a role for p90(RSK) and its upstream components, the ERK (extracellular-signal-regulated kinase) family of MAPKs (mitogen-activated protein kinases), in the Crabtree effect.
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BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells.
Cell Stem Cell
PUBLISHED: 01-17-2013
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Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed "ROS-low"). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation.
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Physiological consequences of complex II inhibition for aging, disease, and the mKATP channel.
Biochim. Biophys. Acta
PUBLISHED: 01-02-2013
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In recent years, it has become apparent that there exist several roles for respiratory complex II beyond metabolism. These include: (i) succinate signaling, (ii) reactive oxygen species (ROS) generation, (iii) ischemic preconditioning, (iv) various disease states and aging, and (v) a role in the function of the mitochondrial ATP-sensitive K(+) (mKATP) channel. This review will address the involvement of complex II in each of these areas, with a focus on how complex II regulates or may be involved in the assembly of the mKATP. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.
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A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel.
PeerJ
PUBLISHED: 01-01-2013
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Opening of BK-type Ca(2+) activated K(+) channels protects the heart against ischemia-reperfusion (IR) injury. However, the location of BK channels responsible for cardioprotection is debated. Herein we confirmed that openers of the SLO1 BK channel, NS1619 and NS11021, were protective in a mouse perfused heart model of IR injury. As anticipated, deletion of the Slo1 gene blocked this protection. However, in an isolated cardiomyocyte model of IR injury, protection by NS1619 and NS11021 was insensitive to Slo1 deletion. These data suggest that protection in intact hearts occurs by a non-cardiomyocyte autonomous, SLO1-dependent, mechanism. In this regard, an in-situ assay of intrinsic cardiac neuronal function (tachycardic response to nicotine) revealed that NS1619 preserved cardiac neurons following IR injury. Furthermore, blockade of synaptic transmission by hexamethonium suppressed cardioprotection by NS1619 in intact hearts. These results suggest that opening SLO1 protects the heart during IR injury, via a mechanism that involves intrinsic cardiac neurons. Cardiac neuronal ion channels may be useful therapeutic targets for eliciting cardioprotection.
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Nitroalkenes confer acute cardioprotection via adenine nucleotide translocase 1.
J. Biol. Chem.
PUBLISHED: 12-09-2011
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Electrophilic nitrated lipids (nitroalkenes) are emerging as an important class of protective cardiovascular signaling molecules. Although species such as nitro-linoleate (LNO(2)) and nitro-oleate can confer acute protection against cardiac ischemic injury, their mechanism of action is unclear. Mild uncoupling of mitochondria is known to be cardioprotective, and adenine nucleotide translocase 1 (ANT1) is a key mediator of mitochondrial uncoupling. ANT1 also contains redox-sensitive cysteines that may be targets for modification by nitroalkenes. Therefore, in this study we tested the hypothesis that nitroalkenes directly modify ANT1 and that nitroalkene-mediated cardioprotection requires ANT1. Using biotin-tagged LNO(2) infused into intact perfused hearts, we obtained mass spectrometric (MALDI-TOF-TOF) evidence for direct modification (nitroalkylation) of ANT1 on cysteine 57. Furthermore, in a cell model of ischemia-reperfusion injury, siRNA knockdown of ANT1 inhibited the cardioprotective effect of LNO(2). Although the molecular mechanism linking ANT1-Cys(57) nitroalkylation and uncoupling is not yet known, these data suggest that ANT1-mediated uncoupling may be a mechanism for nitroalkene-induced cardioprotection.
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SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.
PLoS ONE
PUBLISHED: 09-30-2011
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Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance "big" K(+) channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K(+) transport and APC. The canonical Ca(2+)-activated BK (or "maxi-K") channel SLO1 was dispensable for both mitochondrial K(+) transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K(+) channel SLO2 was required, and that SLO2-dependent mitochondrial K(+) transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K(+).
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Ischemic preconditioning: the role of mitochondria and aging.
Exp. Gerontol.
PUBLISHED: 08-25-2011
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Aging represents a triple threat for myocardial infarction (MI). Not only does the incidence of MI increase with age, but the heart becomes more susceptible to MI induced damage and protective interventions such as ischemic preconditioning (IPC) become less effective. Therefore, any rational therapeutic strategy must be built around the ability to combat the detrimental effects of ischemia in aged individuals. To accomplish this, we need to develop a better understanding of how ischemic damage, protection, and aging are linked. In this regard, mitochondria have emerged as a common theme. First, mitochondria contribute to cell damage during ischemia-reperfusion (IR) and are central to cell death. Second, the protective signaling pathways activated by IPC converge on mitochondria, and the opening of mitochondrial ion channels alone is sufficient to elicit protection. Finally, mitochondria clearly influence the aging process, and specific defects in mitochondrial activity are associated with age-related functional decline. This review will summarize the effects of aging on myocardial IR injury and discuss relevant and emerging strategies to protect against MI with an emphasis on mitochondrial function.
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SIRT1-mediated acute cardioprotection.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 08-19-2011
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Overexpression studies have revealed a role for silent information regulator of transcription 1 (SIRT1) lysine deacetylase in cardioprotection against ischemia-reperfusion injury via long-term transcriptional effects. However, short-term SIRT1-mediated lysine deacetylation, within the context of acute cardioprotection, is poorly understood. In this study, the role of SIRT1 in the acute cardioprotective paradigm of first window ischemic preconditioning (IPC) was studied using SIRT1-deficient (SIRT1(+/-)) and SIRT1-overexpressing (SIRT1(+++)) mice. In wild-type hearts, cytosolic lysine deacetylation was observed during IPC, and overacetylation was observed upon pharmacological SIRT1 inhibition. Consistent with a role for SIRT1 in IPC, SIRT1(+/-) hearts could not be preconditioned and exhibited increased cytosolic lysine acetylation. Furthermore, SIRT1(+++) hearts were endogenously protected against ischemia-reperfusion injury and exhibited decreased cytosolic acetylation. Both of these effects in SIRT1(+++) mice were reversed by pharmacological SIRT1 inhibition on an acute timescale. Several downstream targets of SIRT1 were examined, with data suggesting possible roles for endothelial nitric oxide synthase phosphorylation, NF-?B, and stimulation of autophagy. In conclusion, these data suggest that SIRT1, acting on nontranscriptional targets, is required for cardioprotection by acute IPC and that SIRT1-dependent lysine deacetylation occurs during IPC and may play a role in cardioprotective signaling.
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Kidney transplantation with minimized maintenance: alemtuzumab induction with tacrolimus monotherapy--an open label, randomized trial.
Transplantation
PUBLISHED: 08-13-2011
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Immunosuppressive regimens for kidney transplantation which reduce the long-term burden of immunosuppression are attractive, but little data are available to judge the safety and efficacy of the different strategies used. We tested the hypothesis that the simple, cheap, regimen of alemtuzumab induction combined with tacrolimus monotherapy maintenance provided equivalent outcomes to the more commonly used combination of interleukin-2 receptor monoclonal antibody induction with tacrolimus and mycophenolate mofetil combination maintenance, both regimens using steroid withdrawal after 7 days.
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Antibody-mediated rejection after alemtuzumab induction: incidence, risk factors, and predictors of poor outcome.
Transplantation
PUBLISHED: 06-04-2011
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Antibody-mediated rejection (AMR) is associated with allograft loss. Identification of factors associated with poor outcome has not been extensively studied.
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Mice lacking TR4 nuclear receptor develop mitochondrial myopathy with deficiency in complex I.
Mol. Endocrinol.
PUBLISHED: 05-26-2011
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The estimated incidence of mitochondrial diseases in humans is approximately 1:5000 to 1:10,000, whereas the molecular mechanisms for more than 50% of human mitochondrial disease cases still remain unclear. Here we report that mice lacking testicular nuclear receptor 4 (TR4(-/-)) suffered mitochondrial myopathy, and histological examination of TR4(-/-) soleus muscle revealed abnormal mitochondrial accumulation. In addition, increased serum lactate levels, decreased mitochondrial ATP production, and decreased electron transport chain complex I activity were found in TR4(-/-) mice. Restoration of TR4 into TR4(-/-) myoblasts rescued mitochondrial ATP generation capacity and complex I activity. Further real-time PCR quantification and promoter studies found TR4 could modulate complex I activity via transcriptionally regulating the complex I assembly factor NDUFAF1, and restoration of NDUFAF1 level in TR4(-/-) myoblasts increased mitochondrial ATP generation capacity and complex I activity. Together, these results suggest that TR4 plays vital roles in mitochondrial function, which may help us to better understand the pathogenesis of mitochondrial myopathy, and targeting TR4 via its ligands/activators may allow us to develop better therapeutic approaches.
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p90 ribosomal S6 kinase regulates activity of the renin-angiotensin system: a pathogenic mechanism for ischemia-reperfusion injury.
J. Mol. Cell. Cardiol.
PUBLISHED: 05-03-2011
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Increasing evidence suggests that local renin-angiotensin system (RAS) plays an important role in cardiac diseases. Elevated p90 ribosomal S6 kinase (RSK) activity has been observed in diabetic animal, as well as in human failing hearts. We hypothesize that RSK mediates cardiac dysfunction by up regulating local RAS signaling. In the present study, we show that the prorenin mRNA level was significantly increased (~5.6-fold) in transgenic mouse hearts with cardiac specific expression of RSK (RSK-Tg). The RSK-Tg mice were more vulnerable to ischemia/reperfusion (I/R) injury than non-transgenic littermate controls (NLC). To further understand the direct contribution of cardiac renin to I/R injury, we used a Langendorff system to evaluate the effect of renin inhibition by aliskiren in RSK-Tg mouse hearts. In the vehicle-perfused group, I/R significantly decreased left ventricular developed pressure (LVDP) in RSK-Tg hearts compared to NLC (7% versus 60% of the baseline). However, aliskiren perfusion significantly increased LVDP in RSK-Tg (7% to 61%, p<0.01) but not in NLC hearts (60% to 62%, n.s.). The protective effect of aliskiren in RSK-Tg hearts was further demonstrated with positive (contraction) dp/dt (6.5% to 63%, p<0.01) and rate pressure product (RPP) (5% to 51%, p<0.01). Moreover, aliskiren significantly decreased I/R induced infarction in RSK-Tg (60% to 32%, p<0.01), compared to NLC hearts (37% to 32%, n.s.). These results suggest that RSK plays a crucial role in regulating local cardiac renin, which contributes to I/R induced cardiac injury and dysfunction. Thus, renin inhibition may provide an alternative therapeutic strategy under conditions of increased RAS.
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A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways.
Plant J.
PUBLISHED: 02-16-2011
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The use of small molecules has great power to dissect biological processes. This study presents the identification and characterisation of an inhibitor of peroxisome matrix protein import. A mini-screen was carried out to identify molecules that cause alteration in peroxisome morphology, or mislocalization of a peroxisome targeted fluorescent reporter protein. A benzimidazole lead compound (LDS-003655) was identified that resulted in reduced GFP fluorescence in peroxisomes and cytosolic GFP accumulation. The effect of the compound was specific to peroxisomes as Golgi bodies, endoplasmic reticulum and the actin cytoskeleton were unaffected even at 25 ?M, whereas peroxisome import via the PTS1 pathway was compromised at 100 nM. When seedlings were grown on 25 ?M LDS-003655 they displayed morphology typical of seedlings grown in the presence of auxin, and expression of the auxin reporter DR5::GFP was induced. Analysis of a focussed library of LDS-003655 derivatives in comparison with known auxins led to the conclusion that the auxin-like activity of LDS-003655 is attributable to its in situ hydrolysis giving rise to 2,5-dichlorobenzoic acid, whereas the import inhibiting activity of LDS-003655 requires the whole molecule. None of the auxins tested had any effect on peroxisome protein import. Matrix import by the PTS2 import pathway was relatively insensitive to LDS-003655 and its active analogues, with effects only seen after prolonged incubation on high concentrations. Steady-state protein levels of PEX5, the PTS1 import pathway receptor, were reduced in the presence of 100 nM LDS-003655, suggesting a possible mechanism for the import inhibition.
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The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival: a reductase-based peptide counters ?-adrenergic receptor ligand-mediated cardiac dysfunction.
FASEB J.
PUBLISHED: 09-27-2010
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HO-2 oxidizes heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca(2)-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of ?-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ? 96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligands stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC(281) and KYCCSRK(296), respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.
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Lysine deacetylation in ischaemic preconditioning: the role of SIRT1.
Cardiovasc. Res.
PUBLISHED: 09-07-2010
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Acute ischaemic preconditioning (IPC) induces protection against cardiac ischaemia-reperfusion (IR) via post-translational modification of key proteins. Lysine (Lys) acetylation is an important regulator of protein function, but this type of modification has not been studied in the context of IPC. We investigated Lys acetylation in IPC and its upstream regulation by SIRT1.
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The mitochondrial complex II and ATP-sensitive potassium channel interaction: quantitation of the channel in heart mitochondria.
Acta Biochim. Pol.
PUBLISHED: 08-31-2010
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The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in cardioprotection, although the channel remains molecularly undefined. Several studies have demonstrated that mitochondrial complex II inhibitors activate the mK(ATP), suggesting a potential role for complex II in channel composition or regulation. However, these inhibitors activate mK(ATP) at concentrations which do not affect bulk complex II activity. Using the potent complex II inhibitor Atpenin A5, this relationship was investigated using tight-binding inhibitor theory, to demonstrate that only 0.4 % of total complex II molecules are necessary to activate the mK(ATP). These results estimate the mK(ATP) content at 15 channels per mitochondrion.
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Measurement of extracellular (exofacial) versus intracellular protein thiols.
Meth. Enzymol.
PUBLISHED: 06-20-2010
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In recent years, the importance of compartmentalization in redox signaling has been realized. A number of specific thiol pools exist both inside and outside the cell, and these thiols are regulated via unique mechanisms and serve specific roles in cell signaling. This chapter covers some of the methodologies available for the interrogation of thiol status in various cellular compartments, with a focus on mitochondrial, cytosolic, and exofacial thiols. Finally, the relevance of these thiols to pathological disease states, in particular cancer, will be discussed. The chapters in the remainder of this volume more than adequately cover the diversity of thiol modifications, describing the specific biochemical nature of these reactions, ranging from S-nitrosation through glutathionylation, to oxidation and beyond. Therefore, this topic will not be further addressed here. Similarly, general methodological considerations are considered to have been dealt with in the remainder of this volume, including requirements for subdued lighting, avoidance of reducing agents and transition metals in media, and rapid sample preparation with adequate control over temperature and pH.
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An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays.
Toxicol. Appl. Pharmacol.
PUBLISHED: 06-17-2010
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Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays--a measure of ATP production--under rapid phosphorylation conditions to explore sites of Mn(2+) inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn(2+) inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn(2+) inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F?F? ATP synthase. In mitochondria fueled by either succinate or glutamate+malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn(2+) inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.
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Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.
Biochem. J.
PUBLISHED: 06-11-2010
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The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and alpha-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.
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Redox regulation of the mitochondrial K(ATP) channel in cardioprotection.
Biochim. Biophys. Acta
PUBLISHED: 06-04-2010
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The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.
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SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress.
FASEB J.
PUBLISHED: 04-12-2010
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Sirtuin1 (SIRT1) deacetylase levels are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. We determined the mechanism of SIRT1 redox post-translational modifications leading to its degradation. Human lung epithelial cells exposed to hydrogen peroxide (150-250 microM), aldehyde-acrolein (10-30 microM), and cigarette smoke extract (CSE; 0.1-1.5%) in the presence of intracellular glutathione-modulating agents at 1-24 h, and oxidative post-translational modifications were assayed in cells, as well as in lungs of mice lacking and overexpressing glutaredoxin-1 (Glrx1), and wild-type (WT) mice in response to cigarette smoke (CS). CSE and aldehydes dose and time dependently decreased SIRT1 protein levels, with EC(50) of 1% for CSE and 30 microM for acrolein at 6 h, and >80% inhibition at 24 h with CSE, which was regulated by modulation of intracellular thiol status of the cells. CS decreased the lung levels of SIRT1 in WT mice, which was enhanced by deficiency of Glrx1 and prevented by overexpression of Glrx1. Oxidants, aldehydes, and CS induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. Proteomics studies revealed alkylation of cysteine residue on SIRT1. Our data suggest that oxidants/aldehydes covalently modify SIRT1, decreasing enzymatic activity and marking the protein for proteasomal degradation, which has implications in inflammatory conditions.
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A novel mitochondrial K(ATP) channel assay.
Circ. Res.
PUBLISHED: 02-25-2010
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The mitochondrial ATP sensitive potassium channel (mK(ATP)) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mK(ATP) activity is disputed.
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Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
Nat. Biotechnol.
PUBLISHED: 01-12-2010
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Most cells have the inherent capacity to shift their reliance on glycolysis relative to oxidative metabolism, and studies in model systems have shown that targeting such shifts may be useful in treating or preventing a variety of diseases ranging from cancer to ischemic injury. However, we currently have a limited number of mechanistically distinct classes of drugs that alter the relative activities of these two pathways. We screen for such compounds by scoring the ability of >3,500 small molecules to selectively impair growth and viability of human fibroblasts in media containing either galactose or glucose as the sole sugar source. We identify several clinically used drugs never linked to energy metabolism, including the antiemetic meclizine, which attenuates mitochondrial respiration through a mechanism distinct from that of canonical inhibitors. We further show that meclizine pretreatment confers cardioprotection and neuroprotection against ischemia-reperfusion injury in murine models. Nutrient-sensitized screening may provide a useful framework for understanding gene function and drug action within the context of energy metabolism.
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Mitochondrial biotransformation of omega-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids: a prodrug strategy for targeting cytoprotective antioxidants to mitochondria.
Bioorg. Med. Chem.
PUBLISHED: 01-06-2010
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Mitochondrial reactive oxygen species (ROS) generation and the attendant mitochondrial dysfunction are implicated in a range of disease states. The objective of the present studies was to test the hypothesis that the mitochondrial beta-oxidation pathway could be exploited to deliver and biotransform the prodrugs omega-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids to the corresponding phenolic antioxidants or methimazole. 3- and 5-(Phenoxy)alkanoic acids and methyl-substituted analogs were biotransformed to phenols; rates of biotransformation decreased markedly with methyl-group substitution on the phenoxy moiety. 2,6-Dimethylphenol formation from the analogs 3-([2,6-dimethylphenoxy]methylthio)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid was greater than that observed with omega-(2,6-dimethylphenoxy)alkanoic acids. 3- and 5-(1-Methyl-1H-imidazol-2-ylthio)alkanoic acids were rapidly biotransformed to the antioxidant methimazole and conferred significant cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. Both 3-(2,6-dimethylphenoxy)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid also afforded cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. These results demonstrate that mitochondrial beta-oxidation is a potentially useful delivery system for targeting antioxidants to mitochondria.
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Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.
Cardiovasc. Res.
PUBLISHED: 07-09-2009
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Both mitochondria and nitric oxide (NO*) contribute to cardioprotection by ischaemic preconditioning (IPC). IPC causes mild uncoupling of mitochondria via uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT), and mild uncoupling per se is cardioprotective. Although electrophilic lipids are known to activate mitochondrial uncoupling, the role of such species in IPC-induced uncoupling and cardioprotection is unclear. We hypothesized that endogenous formation of NO*-derived electrophilic lipids (nitroalkenes such as nitro-linoleate, LNO2) during IPC may stimulate mitochondrial uncoupling via post-translational modification of UCPs and ANT, thus affording cardioprotection.
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A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-15-2009
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Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.
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Modulation of cell surface protein free thiols: a potential novel mechanism of action of the sesquiterpene lactone parthenolide.
PLoS ONE
PUBLISHED: 05-29-2009
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There has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols.
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Oxygen sensitivity of mitochondrial reactive oxygen species generation depends on metabolic conditions.
J. Biol. Chem.
PUBLISHED: 04-14-2009
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The mitochondrial generation of reactive oxygen species (ROS) plays a central role in many cell signaling pathways, but debate still surrounds its regulation by factors, such as substrate availability, [O2] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of [O2]. In the current study, we used a wide variety of substrates and inhibitors to probe the O2 sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent Km for O2 of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 microM O2 for the complex I flavin site, complex I electron backflow, complex III QO site, and electron transfer flavoprotein quinone oxidoreductase of beta-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying [O2]. Based on these data, we hypothesize that at physiological [O2], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high [O2]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental [O2]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.
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Cardioprotection by metabolic shut-down and gradual wake-up.
J. Mol. Cell. Cardiol.
PUBLISHED: 02-03-2009
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Mitochondria play a critical role in cardiac function, and are also increasingly recognized as end effectors for various cardioprotective signaling pathways. Mitochondria use oxygen as a substrate, so by default their respiration is inhibited during hypoxia/ischemia. However, at reperfusion a surge of oxygen and metabolic substrates into the cell is thought to lead to rapid reestablishment of respiration, a burst of reactive oxygen species (ROS) generation and mitochondrial Ca(2+) overload. Subsequently these events precipitate opening of the mitochondrial permeability transition (PT) pore, which leads to myocardial cell death and dysfunction. Given that mitochondrial respiration is already inhibited during hypoxia/ischemia, it is somewhat surprising that many respiratory inhibitors can improve recovery from ischemia-reperfusion (IR) injury. In addition ischemic preconditioning (IPC), in which short non-lethal cycles of IR can protect against subsequent prolonged IR injury, is known to lead to endogenous inhibition of several respiratory complexes and glycolysis. This has led to a hypothesis that the wash-out of inhibitors or reversal of endogenous inhibition at reperfusion may afford protection by facilitating a more gradual wake-up of mitochondrial function, thereby avoiding a burst of ROS and Ca(2+) overload. This paper will review the evidence in support of this hypothesis, with a focus on inhibition of each of the mitochondrial respiratory complexes.
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NDUFS4: creation of a mouse model mimicking a Complex I disorder.
Mitochondrion
PUBLISHED: 01-29-2009
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The Complex I NADH dehydrogenase-ubiquinone-FeS 4 (NDUFS4) subunit gene is involved in proper Complex I function such that the loss of NDUFS4 decreases Complex I activity resulting in mitochondrial disease. Therefore, a mouse model harboring a point mutation in the NDUFS4 gene was created. An embryonic lethal phenotype was observed in homozygous (NDUFS4(-/-)) mutant fetuses. Mitochondrial function was impaired in heterozygous animals based on oxygen consumption, and Complex I activity in NDUFS4 mouse mitochondria. Decreased Complex I activity with unaltered Complex II activity, along with an accumulation of lactate, were consistent with Complex I disorders in this mouse model.
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In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine.
J. Mol. Cell. Cardiol.
PUBLISHED: 01-21-2009
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The reversible S-nitrosation and inhibition of mitochondrial complex I is a potential mechanism of cardioprotection, recruited by ischemic preconditioning (IPC), S-nitrosothiols, and nitrite. Previously, to exploit this mechanism, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed, and protected perfused hearts and isolated cardiomyocytes against ischemia-reperfusion (IR) injury. In the present study, the murine left anterior descending coronary artery (LAD) occlusion model of IR injury was employed, to determine the protective efficacy of SNO-MPG in vivo. Intraperitoneal administration of 1 mg/kg SNO-MPG, 30 min prior to occlusion, significantly reduced myocardial infarction and improved EKG parameters, following 30 min occlusion plus 2 or 24 h reperfusion. SNO-MPG protected to the same degree as IPC, and notably was also protective when administered at reperfusion. Cardioprotection was accompanied by increased mitochondrial protein S-nitrosothiol content, and inhibition of complex I, both of which were reversed after 2 h reperfusion. Finally, hearts from mice harboring a heterozygous mutation in the complex I NDUSF4 subunit were refractory to protection by either SNO-MPG or IPC, suggesting that a fully functional complex I, capable of reversible inhibition is critical for cardioprotection. Overall, these results are consistent with a role for mitochondrial S-nitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.
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The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial KATP channels.
Basic Res. Cardiol.
PUBLISHED: 01-18-2009
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The cardioprotective effects of ischemic preconditioning (IPC) can be mimicked or blocked by pharmacologic agents, which modulate the mitochondrial ATP-sensitive potassium (mK(ATP)) channel, thereby implicating this channel in the mechanism of IPC. Cardioprotection can also be achieved via inhibition of mitochondrial respiratory complex II, and significant pharmacologic overlap exists between complex II inhibitors and mK(ATP) channel agonists. However, the relationship between complex II and the mK(ATP) channel remains unclear. Atpenin A5 (AA5) is a potent and specific complex II inhibitor, and herein we report that AA5 (1 nM) also activates the mK(ATP) channel and protects against simulated ischemia-reperfusion (IR) injury in isolated cardiomyocytes. Similar to known mK(ATP) agonists, AA5-mediated protection was sensitive to the mK(ATP) antagonists 5-hydroxydecanoate (5HD) and glyburide. Notably, the optimal mK(ATP) opening and protective concentration of AA5 had no effect on complex II enzymatic activity, suggesting an interaction of AA5 with complex II, but not inhibition of the complex per se, is necessary for protection. A cardioprotective effect of AA5 was also observed in isolated perfused hearts, wherein AA5 increased post-IR contractile function and decreased infarct size, in a 5HD-sensitive manner. In conclusion, the specific complex II inhibitor AA5 is the most potent mK(ATP) activator discovered to date, and provides a novel method of activating mK(ATP) channels and protecting the heart from IR injury.
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Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion.
Free Radic. Biol. Med.
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The carbon dioxide/bicarbonate (CO(2)/HCO(3)(-)) pair is the main biological pH buffer. However, its influence on biological processes, and in particular redox processes, is still poorly explored. Here we study the effect of CO(2)/HCO(3)(-) on ischemic injury in three distinct models (cardiac HL-1 cells, perfused rat heart, and Caenorhabditis elegans). We found that, although various concentrations of CO(2)/HCO(3)(-) do not affect function under basal conditions, ischemia-reperfusion or similar insults in the presence of higher CO(2)/HCO(3)(-) resulted in greater functional loss associated with higher oxidative damage in all models. Because the effect of CO(2)/HCO(3)(-) was observed in all models tested, we believe this buffer is an important determinant of oxidative damage after ischemia-reperfusion.
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Familial granulomatosis with polyangiitis: three cases of this rare disorder in one Indoasian family carrying an identical HLA DPB1 allele.
BMJ Case Rep
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The aetiology of granulomatosis with polyangiitis (GPA) remains unclear. There is likely a complex interplay between environmental triggers and genetic predisposition. Early diagnosis and treatment improve outcome in this condition with an untreated reported mortality of 90%. There are a few cases of familial GPA in the literature, but we report here the interesting and rare scenario of three members of the same Indoasian family with GPA who share an identical human leucocyte antigen (HLA) haplotype. In particular, all three members of the family share the HLADPB1 allele *04 : 01. This is the first reported Indoasian family with GPA. The HLA antigens found on genotyping our family and, in particular, HLADPB1 has been linked with GPA in previous studies. Genome-wide association studies in Anti Neutrophil Cytoplasm Antibody (ANCA) associated vasculitis (AAV) are currently in progress and should improve understanding of the genetic aspects of this disease. There may be important implications for patients with GPA and for their relatives.
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Mitochondria as a drug target in ischemic heart disease and cardiomyopathy.
Circ. Res.
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Ischemic heart disease is a significant cause of morbidity and mortality in Western society. Although interventions, such as thrombolysis and percutaneous coronary intervention, have proven efficacious in ischemia and reperfusion injury, the underlying pathological process of ischemic heart disease, laboratory studies suggest further protection is possible, and an expansive research effort is aimed at bringing new therapeutic options to the clinic. Mitochondrial dysfunction plays a key role in the pathogenesis of ischemia and reperfusion injury and cardiomyopathy. However, despite promising mitochondria-targeted drugs emerging from the laboratory, very few have successfully completed clinical trials. As such, the mitochondrion is a potential untapped target for new ischemic heart disease and cardiomyopathy therapies. Notably, there are a number of overlapping therapies for both these diseases, and as such novel therapeutic options for one condition may find use in the other. This review summarizes efforts to date in targeting mitochondria for ischemic heart disease and cardiomyopathy therapy and outlines emerging drug targets in this field.
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Guidelines for the use and interpretation of assays for monitoring autophagy.
Daniel J Klionsky, Fábio C Abdalla, Hagai Abeliovich, Robert T Abraham, Abraham Acevedo-Arozena, Khosrow Adeli, Lotta Agholme, Maria Agnello, Patrizia Agostinis, Julio A Aguirre-Ghiso, Hyung Jun Ahn, Ouardia Ait-Mohamed, Slimane Ait-Si-Ali, Takahiko Akematsu, Shizuo Akira, Hesham M Al-Younes, Munir A Al-Zeer, Matthew L Albert, Roger L Albin, Javier Alegre-Abarrategui, Maria Francesca Aleo, Mehrdad Alirezaei, Alexandru Almasan, Maylin Almonte-Becerril, Atsuo Amano, Ravi Amaravadi, Shoba Amarnath, Amal O Amer, Nathalie Andrieu-Abadie, Vellareddy Anantharam, David K Ann, Shailendra Anoopkumar-Dukie, Hiroshi Aoki, Nadezda Apostolova, Giuseppe Arancia, John P Aris, Katsuhiko Asanuma, Nana Y O Asare, Hisashi Ashida, Valerie Askanas, David S Askew, Patrick Auberger, Misuzu Baba, Steven K Backues, Eric H Baehrecke, Ben A Bahr, Xue-Yuan Bai, Yannick Bailly, Robert Baiocchi, Giulia Baldini, Walter Balduini, Andrea Ballabio, Bruce A Bamber, Edward T W Bampton, Gábor Bánhegyi, Clinton R Bartholomew, Diane C Bassham, Robert C Bast, Henri Batoko, Boon-Huat Bay, Isabelle Beau, Daniel M Béchet, Thomas J Begley, Christian Behl, Christian Behrends, Soumeya Bekri, Bryan Bellaire, Linda J Bendall, Luca Benetti, Laura Berliocchi, Henri Bernardi, Francesca Bernassola, Sébastien Besteiro, Ingrid Bhatia-Kiššová, Xiaoning Bi, Martine Biard-Piechaczyk, Janice S Blum, Lawrence H Boise, Paolo Bonaldo, David L Boone, Beat C Bornhauser, Karina R Bortoluci, Ioannis Bossis, Fréderic Bost, Jean-Pierre Bourquin, Patricia Boya, Michaël Boyer-Guittaut, Peter V Bozhkov, Nathan R Brady, Claudio Brancolini, Andreas Brech, Jay E Brenman, Ana Brennand, Emery H Bresnick, Patrick Brest, Dave Bridges, Molly L Bristol, Paul S Brookes, Eric J Brown, John H Brumell, Nicola Brunetti-Pierri, Ulf T Brunk, Dennis E Bulman, Scott J Bultman, Geert Bultynck, Lena F Burbulla, Wilfried Bursch, Jonathan P Butchar, Wanda Buzgariu, Sérgio P Bydlowski, Ken Cadwell, Monika Cahova, Dongsheng Cai, Jiyang Cai, Qian Cai, Bruno Calabretta, Javier Calvo-Garrido, Nadine Camougrand, Michelangelo Campanella, Jenny Campos-Salinas, Eleonora Candi, Lizhi Cao, Allan B Caplan, Simon R Carding, Sandra M Cardoso, Jennifer S Carew, Cathleen R Carlin, Virginie Carmignac, Leticia A M Carneiro, Serena Carra, Rosario A Caruso, Giorgio Casari, Caty Casas, Roberta Castino, Eduardo Cebollero, Francesco Cecconi, Jean Celli, Hassan Chaachouay, Han-Jung Chae, Chee-Yin Chai, David C Chan, Edmond Y Chan, Raymond Chuen-Chung Chang, Chi-Ming Che, Ching-Chow Chen, Guang-Chao Chen, Guo-Qiang Chen, Min Chen, Quan Chen, Steve S-L Chen, WenLi Chen, Xi Chen, Xiangmei Chen, Xiequn Chen, Ye-Guang Chen, Yingyu Chen, Yongqiang Chen, Yu-Jen Chen, Zhixiang Chen, Alan Cheng, Christopher H K Cheng, Yan Cheng, Heesun Cheong, Jae-Ho Cheong, Sara Cherry, Russ Chess-Williams, Zelda H Cheung, Eric Chevet, Hui-Ling Chiang, Roberto Chiarelli, Tomoki Chiba, Lih-Shen Chin, Shih-Hwa Chiou, Francis V Chisari, Chi Hin Cho, Dong-Hyung Cho, Augustine M K Choi, DooSeok Choi, Kyeong Sook Choi, Mary E Choi, Salem Chouaib, Divaker Choubey, Vinay Choubey, Charleen T Chu, Tsung-Hsien Chuang, Sheau-Huei Chueh, Taehoon Chun, Yong-Joon Chwae, Mee-Len Chye, Roberto Ciarcia, Maria R Ciriolo, Michael J Clague, Robert S B Clark, Peter G H Clarke, Robert Clarke, Patrice Codogno, Hilary A Coller, María I Colombo, Sergio Comincini, Maria Condello, Fabrizio Condorelli, Mark R Cookson, Graham H Coombs, Isabelle Coppens, Ramón Corbalán, Pascale Cossart, Paola Costelli, Safia Costes, Ana Coto-Montes, Eduardo Couve, Fraser P Coxon, James M Cregg, José L Crespo, Marianne J Cronjé, Ana Maria Cuervo, Joseph J Cullen, Mark J Czaja, Marcello D'Amelio, Arlette Darfeuille-Michaud, Lester M Davids, Faith E Davies, Massimo De Felici, John F de Groot, Cornelis A M de Haan, Luisa De Martino, Angelo De Milito, Vincenzo De Tata, Jayanta Debnath, Alexei Degterev, Benjamin Dehay, Lea M D Delbridge, Francesca Demarchi, Yi Zhen Deng, Jörn Dengjel, Paul Dent, Donna Denton, Vojo Deretic, Shyamal D Desai, Rodney J Devenish, Mario Di Gioacchino, Gilbert Di Paolo, Chiara Di Pietro, Guillermo Díaz-Araya, Inés Díaz-Laviada, Maria T Diaz-Meco, Javier Diaz-Nido, Ivan Dikic, Savithramma P Dinesh-Kumar, Wen-Xing Ding, Clark W Distelhorst, Abhinav Diwan, Mojgan Djavaheri-Mergny, Svetlana Dokudovskaya, Zheng Dong, Frank C Dorsey, Victor Dosenko, James J Dowling, Stephen Doxsey, Marlène Dreux, Mark E Drew, Qiuhong Duan, Michel A Duchosal, Karen Duff, Isabelle Dugail, Madeleine Durbeej, Michael Duszenko, Charles L Edelstein, Aimee L Edinger, Gustavo Egea, Ludwig Eichinger, N Tony Eissa, Suhendan Ekmekcioglu, Wafik S El-Deiry, Zvulun Elazar, Mohamed Elgendy, Lisa M Ellerby, Kai Er Eng, Anna-Mart Engelbrecht, Simone Engelender, Jekaterina Erenpreisa, Ricardo Escalante, Audrey Esclatine, Eeva-Liisa Eskelinen, Lucile Espert, Virginia Espina, Huizhou Fan, Jia Fan, Qi-Wen Fan, Zhen Fan, Shengyun Fang, Yongqi Fang, Manolis Fanto, Alessandro Fanzani, Thomas Farkas, Jean-Claude Farré, Mathias Faure, Marcus Fechheimer, Carl G Feng, Jian Feng, Qili Feng, Youji Feng, László Fésüs, Ralph Feuer, Maria E Figueiredo-Pereira, Gian Maria Fimia, Diane C Fingar, Steven Finkbeiner, Toren Finkel, Kim D Finley, Filomena Fiorito, Edward A Fisher, Paul B Fisher, Marc Flajolet, Maria L Florez-McClure, Salvatore Florio, Edward A Fon, Francesco Fornai, Franco Fortunato, Rati Fotedar, Daniel H Fowler, Howard S Fox, Rodrigo Franco, Lisa B Frankel, Marc Fransen, José M Fuentes, Juan Fueyo, Jun Fujii, Kozo Fujisaki, Eriko Fujita, Mitsunori Fukuda, Ruth H Furukawa, Matthias Gaestel, Philippe Gailly, Malgorzata Gajewska, Brigitte Galliot, Vincent Galy, Subramaniam Ganesh, Barry Ganetzky, Ian G Ganley, Fen-Biao Gao, George F Gao, Jinming Gao, Lorena Garcia, Guillermo Garcia-Manero, Mikel Garcia-Marcos, Marjan Garmyn, Andrei L Gartel, Evelina Gatti, Mathias Gautel, Thomas R Gawriluk, Matthew E Gegg, Jiefei Geng, Marc Germain, Jason E Gestwicki, David A Gewirtz, Saeid Ghavami, Pradipta Ghosh, Anna M Giammarioli, Alexandra N Giatromanolaki, Spencer B Gibson, Robert W Gilkerson, Michael L Ginger, Henry N Ginsberg, Jakub Golab, Michael S Goligorsky, Pierre Golstein, Candelaria Gomez-Manzano, Ebru Goncu, Céline Gongora, Claudio D Gonzalez, Ramon Gonzalez, Cristina González-Estévez, Rosa Ana González-Polo, Elena Gonzalez-Rey, Nikolai V Gorbunov, Sharon Gorski, Sandro Goruppi, Roberta A Gottlieb, Devrim Gozuacik, Giovanna Elvira Granato, Gary D Grant, Kim N Green, Aleš Gregorc, Frédéric Gros, Charles Grose, Thomas W Grunt, Philippe Gual, Jun-Lin Guan, Kun-Liang Guan, Sylvie M Guichard, Anna S Gukovskaya, Ilya Gukovsky, Jan Gunst, Asa B Gustafsson, Andrew J Halayko, Amber N Hale, Sandra K Halonen, Maho Hamasaki, Feng Han, Ting Han, Michael K Hancock, Malene Hansen, Hisashi Harada, Masaru Harada, Stefan E Hardt, J Wade Harper, Adrian L Harris, James Harris, Steven D Harris, Makoto Hashimoto, Jeffrey A Haspel, Shin-Ichiro Hayashi, Lori A Hazelhurst, Congcong He, You-Wen He, Marie-Josee Hebert, Kim A Heidenreich, Miep H Helfrich, Gudmundur V Helgason, Elizabeth P Henske, Brian Herman, Paul K Herman, Claudio Hetz, Sabine Hilfiker, Joseph A Hill, Lynne J Hocking, Paul Hofman, Thomas G Hofmann, Jörg Höhfeld, Tessa L Holyoake, Ming-Huang Hong, David A Hood, Gökhan S Hotamisligil, Ewout J Houwerzijl, Maria Høyer-Hansen, Bingren Hu, Chien-An A Hu, Hong-Ming Hu, Ya Hua, Canhua Huang, Ju Huang, Shengbing Huang, Wei-Pang Huang, Tobias B Huber, Won-Ki Huh, Tai-Ho Hung, Ted R Hupp, Gang Min Hur, James B Hurley, Sabah N A Hussain, Patrick J Hussey, Jung Jin Hwang, Seungmin Hwang, Atsuhiro Ichihara, Shirin Ilkhanizadeh, Ken Inoki, Takeshi Into, Valentina Iovane, Juan L Iovanna, Nancy Y Ip, Yoshitaka Isaka, Hiroyuki Ishida, Ciro Isidoro, Ken-Ichi Isobe, Akiko Iwasaki, Marta Izquierdo, Yotaro Izumi, Panu M Jaakkola, Marja Jäättelä, George R Jackson, William T Jackson, Bassam Janji, Marina Jendrach, Ju-Hong Jeon, Eui-Bae Jeung, Hong Jiang, Hongchi Jiang, Jean X Jiang, Ming Jiang, Qing Jiang, Xuejun Jiang, Alberto Jiménez, Meiyan Jin, Shengkan Jin, Cheol O Joe, Terje Johansen, Daniel E Johnson, Gail V W Johnson, Nicola L Jones, Bertrand Joseph, Suresh K Joseph, Annie M Joubert, Gábor Juhász, Lucienne Juillerat-Jeanneret, Chang Hwa Jung, Yong-Keun Jung, Kai Kaarniranta, Allen Kaasik, Tomohiro Kabuta, Motoni Kadowaki, Katarina Kågedal, Yoshiaki Kamada, Vitaliy O Kaminskyy, Harm H Kampinga, Hiromitsu Kanamori, Chanhee Kang, Khong Bee Kang, Kwang Il Kang, Rui Kang, Yoon-A Kang, Tomotake Kanki, Thirumala-Devi Kanneganti, Haruo Kanno, Anumantha G Kanthasamy, Arthi Kanthasamy, Vassiliki Karantza, Gur P Kaushal, Susmita Kaushik, Yoshinori Kawazoe, Po-Yuan Ke, John H Kehrl, Ameeta Kelekar, Claus Kerkhoff, David H Kessel, Hany Khalil, Jan A K W Kiel, Amy A Kiger, Akio Kihara, Deok Ryong Kim, Do-Hyung Kim, Dong-Hou Kim, Eun-Kyoung Kim, Hyung-Ryong Kim, Jae-Sung Kim, Jeong Hun Kim, Jin Cheon Kim, John K Kim, Peter K Kim, Seong Who Kim, Yong-Sun Kim, Yonghyun Kim, Adi Kimchi, Alec C Kimmelman, Jason S King, Timothy J Kinsella, Vladimir Kirkin, Lorrie A Kirshenbaum, Katsuhiko Kitamoto, Kaio Kitazato, Ludger Klein, Walter T Klimecki, Jochen Klucken, Erwin Knecht, Ben C B Ko, Jan C Koch, Hiroshi Koga, Jae-Young Koh, Young Ho Koh, Masato Koike, Masaaki Komatsu, Eiki Kominami, Hee Jeong Kong, Wei-jia Kong, Viktor I Korolchuk, Yaichiro Kotake, Michael I Koukourakis, Juan B Kouri Flores, Attila L Kovács, Claudine Kraft, Dimitri Krainc, Helmut Krämer, Carole Kretz-Remy, Anna M Krichevsky, Guido Kroemer, Rejko Krüger, Oleg Krut, Nicholas T Ktistakis, Chia-Yi Kuan, Róza Kucharczyk, Ashok Kumar, Raj Kumar, Sharad Kumar, Mondira Kundu, Hsing-Jien Kung, Tino Kurz, Ho Jeong Kwon, Albert R La Spada, Frank Lafont, Trond Lamark, Jacques Landry, Jon D Lane, Pierre Lapaquette, Jocelyn F Laporte, Lajos László, Sergio Lavandero, Josée N Lavoie, Robert Layfield, Pedro A Lazo, Weidong Le, Laurent Le Cam, Daniel J Ledbetter, Alvin J X Lee, Byung-Wan Lee, Gyun Min Lee, Jongdae Lee, Ju-Hyun Lee, Michael Lee, Myung-Shik Lee, Sug Hyung Lee, Christiaan Leeuwenburgh, Patrick Legembre, Renaud Legouis, Michael Lehmann, Huan-Yao Lei, Qun-Ying Lei, David A Leib, José Leiro, John J Lemasters, Antoinette Lemoine, Maciej S Lesniak, Dina Lev, Victor V Levenson, Beth Levine, Efrat Levy, Faqiang Li, Jun-lin Li, Lian Li, Sheng Li, Weijie Li, Xue-Jun Li, Yan-Bo Li, Yi-Ping Li, Chengyu Liang, Qiangrong Liang, Yung-Feng Liao, Pawel P Liberski, Andrew Lieberman, Hyunjung J Lim, Kah-Leong Lim, Kyu Lim, Chiou-Feng Lin, Fu-Cheng Lin, Jian Lin, Jiandie D Lin, Kui Lin, Wan-Wan Lin, Weei-Chin Lin, Yi-Ling Lin, Rafael Linden, Paul Lingor, Jennifer Lippincott-Schwartz, Michael P Lisanti, Paloma B Liton, Bo Liu, Chun-Feng Liu, Kaiyu Liu, Leyuan Liu, Qiong A Liu, Wei Liu, Young-Chau Liu, Yule Liu, Richard A Lockshin, Chun-Nam Lok, Sagar Lonial, Benjamin Loos, Gabriel Lopez-Berestein, Carlos Lopez-Otin, Laura Lossi, Michael T Lotze, Péter Low, Binfeng Lu, Bingwei Lu, Bo Lu, Zhen Lu, Fredéric Luciano, Nicholas W Lukacs, Anders H Lund, Melinda A Lynch-Day, Yong Ma, Fernando Macian, Jeff P MacKeigan, Kay F Macleod, Frank Madeo, Luigi Maiuri, Maria Chiara Maiuri, Davide Malagoli, May Christine V Malicdan, Walter Malorni, Na Man, Eva-Maria Mandelkow, Stéphen Manon, Irena Manov, Kai Mao, Xiang Mao, Zixu Mao, Philippe Marambaud, Daniela Marazziti, Yves L Marcel, Katie Marchbank, Piero Marchetti, Stefan J Marciniak, Mateus Marcondes, Mohsen Mardi, Gabriella Marfè, Guillermo Mariño, Maria Markaki, Mark R Marten, Seamus J Martin, Camille Martinand-Mari, Wim Martinet, Marta Martinez-Vicente, Matilde Masini, Paola Matarrese, Saburo Matsuo, Raffaele Matteoni, Andreas Mayer, Nathalie M Mazure, David J McConkey, Melanie J McConnell, Catherine McDermott, Christine McDonald, Gerald M McInerney, Sharon L McKenna, BethAnn McLaughlin, Pamela J McLean, Christopher R McMaster, G Angus McQuibban, Alfred J Meijer, Miriam H Meisler, Alicia Meléndez, Thomas J Melia, Gerry Melino, Maria A Mena, Javier A Menendez, Rubem F S Menna-Barreto, Manoj B Menon, Fiona M Menzies, Carol A Mercer, Adalberto Merighi, Diane E Merry, Stefania Meschini, Christian G Meyer, Thomas F Meyer, Chao-Yu Miao, Jun-Ying Miao, Paul A M Michels, Carine Michiels, Dalibor Mijaljica, Ana Milojkovic, Saverio Minucci, Clelia Miracco, Cindy K Miranti, Ioannis Mitroulis, Keisuke Miyazawa, Noboru Mizushima, Baharia Mograbi, Simin Mohseni, Xavier Molero, Bertrand Mollereau, Faustino Mollinedo, Takashi Momoi, Iryna Monastyrska, Martha M Monick, Mervyn J Monteiro, Michael N Moore, Rodrigo Mora, Kevin Moreau, Paula I Moreira, Yuji Moriyasu, Jorge Moscat, Serge Mostowy, Jeremy C Mottram, Tomasz Motyl, Charbel E-H Moussa, Sylke Müller, Sylviane Muller, Karl Münger, Christian Münz, Leon O Murphy, Maureen E Murphy, Antonio Musarò, Indira Mysorekar, Eiichiro Nagata, Kazuhiro Nagata, Aimable Nahimana, Usha Nair, Toshiyuki Nakagawa, Kiichi Nakahira, Hiroyasu Nakano, Hitoshi Nakatogawa, Meera Nanjundan, Naweed I Naqvi, Derek P Narendra, Masashi Narita, Miguel Navarro, Steffan T Nawrocki, Taras Y Nazarko, Andriy Nemchenko, Mihai G Netea, Thomas P Neufeld, Paul A Ney, Ioannis P Nezis, Huu Phuc Nguyen, Daotai Nie, Ichizo Nishino, Corey Nislow, Ralph A Nixon, Takeshi Noda, Angelika A Noegel, Anna Nogalska, Satoru Noguchi, Lucia Notterpek, Ivana Novak, Tomoyoshi Nozaki, Nobuyuki Nukina, Thorsten Nürnberger, Beat Nyfeler, Keisuke Obara, Terry D Oberley, Salvatore Oddo, Michinaga Ogawa, Toya Ohashi, Koji Okamoto, Nancy L Oleinick, F Javier Oliver, Laura J Olsen, Stefan Olsson, Onya Opota, Timothy F Osborne, Gary K Ostrander, Kinya Otsu, Jing-hsiung James Ou, Mireille Ouimet, Michael Overholtzer, Bulent Ozpolat, Paolo Paganetti, Ugo Pagnini, Nicolas Pallet, Glen E Palmer, Camilla Palumbo, Tianhong Pan, Theocharis Panaretakis, Udai Bhan Pandey, Zuzana Papackova, Issidora Papassideri, Irmgard Paris, Junsoo Park, Ohkmae K Park, Jan B Parys, Katherine R Parzych, Susann Patschan, Cam Patterson, Sophie Pattingre, John M Pawelek, Jianxin Peng, David H Perlmutter, Ida Perrotta, George Perry, Shazib Pervaiz, Matthias Peter, Godefridus J Peters, Morten Petersen, Goran Petrovski, James M Phang, Mauro Piacentini, Philippe Pierre, Valérie Pierrefite-Carle, Gérard Pierron, Ronit Pinkas-Kramarski, Antonio Piras, Natik Piri, Leonidas C Platanias, Stefanie Pöggeler, Marc Poirot, Angelo Poletti, Christian Poüs, Mercedes Pozuelo-Rubio, Mette Prætorius-Ibba, Anil Prasad, Mark Prescott, Muriel Priault, Nathalie Produit-Zengaffinen, Ann Progulske-Fox, Tassula Proikas-Cezanne, Serge Przedborski, Karin Przyklenk, Rosa Puertollano, Julien Puyal, Shu-Bing Qian, Liang Qin, Zheng-Hong Qin, Susan E Quaggin, Nina Raben, Hannah Rabinowich, Simon W Rabkin, Irfan Rahman, Abdelhaq Rami, Georg Ramm, Glenn Randall, Felix Randow, V Ashutosh Rao, Jeffrey C Rathmell, Brinda Ravikumar, Swapan K Ray, Bruce H Reed, John C Reed, Fulvio Reggiori, Anne Regnier-Vigouroux, Andreas S Reichert, John J Reiners, Russel J Reiter, Jun Ren, Jose L Revuelta, Christopher J Rhodes, Konstantinos Ritis, Elizete Rizzo, Jeffrey Robbins, Michel Roberge, Hernan Roca, Maria C Roccheri, Stéphane Rocchi, H Peter Rodemann, Santiago Rodríguez de Córdoba, Bärbel Rohrer, Igor B Roninson, Kirill Rosen, Magdalena M Rost-Roszkowska, Mustapha Rouis, Kasper M A Rouschop, Francesca Rovetta, Brian P Rubin, David C Rubinsztein, Klaus Ruckdeschel, Edmund B Rucker, Assaf Rudich, Emil Rudolf, Nelson Ruiz-Opazo, Rossella Russo, Tor Erik Rusten, Kevin M Ryan, Stefan W Ryter, David M Sabatini, Junichi Sadoshima, Tapas Saha, Tatsuya Saitoh, Hiroshi Sakagami, Yasuyoshi Sakai, Ghasem Hoseini Salekdeh, Paolo Salomoni, Paul M Salvaterra, Guy Salvesen, Rosa Salvioli, Anthony M J Sanchez, José A Sánchez-Alcázar, Ricardo Sánchez-Prieto, Marco Sandri, Uma Sankar, Poonam Sansanwal, Laura Santambrogio, Shweta Saran, Sovan Sarkar, Minnie Sarwal, Chihiro Sasakawa, Ausra Sasnauskiene, Miklós Sass, Ken Sato, Miyuki Sato, Anthony H V Schapira, Michael Scharl, Hermann M Schätzl, Wiep Scheper, Stefano Schiaffino, Claudio Schneider, Marion E Schneider, Regine Schneider-Stock, Patricia V Schoenlein, Daniel F Schorderet, Christoph Schüller, Gary K Schwartz, Luca Scorrano, Linda Sealy, Per O Seglen, Juan Segura-Aguilar, Iban Seiliez, Oleksandr Seleverstov, Christian Sell, Jong Bok Seo, Duska Separovic, Vijayasaradhi Setaluri, Takao Setoguchi, Carmine Settembre, John J Shacka, Mala Shanmugam, Irving M Shapiro, Eitan Shaulian, Reuben J Shaw, James H Shelhamer, Han-Ming Shen, Wei-Chiang Shen, Zu-Hang Sheng, Yang Shi, Kenichi Shibuya, Yoshihiro Shidoji, Jeng-Jer Shieh, Chwen-Ming Shih, Yohta Shimada, Shigeomi Shimizu, Takahiro Shintani, Orian S Shirihai, Gordon C Shore, Andriy A Sibirny, Stan B Sidhu, Beata Sikorska, Elaine C M Silva-Zacarin, Alison Simmons, Anna Katharina Simon, Hans-Uwe Simon, Cristiano Simone, Anne Simonsen, David A Sinclair, Rajat Singh, Debasish Sinha, Frank A Sinicrope, Agnieszka Sirko, Parco M Siu, Efthimios Sivridis, Vojtech Skop, Vladimir P Skulachev, Ruth S Slack, Soraya S Smaili, Duncan R Smith, María S Soengas, Thierry Soldati, Xueqin Song, Anil K Sood, Tuck Wah Soong, Federica Sotgia, Stephen A Spector, Claudia D Spies, Wolfdieter Springer, Srinivasa M Srinivasula, Leonidas Stefanis, Joan S Steffan, Ruediger Stendel, Harald Stenmark, Anastasis Stephanou, Stephan T Stern, Cinthya Sternberg, Björn Stork, Peter Stralfors, Carlos S Subauste, Xinbing Sui, David Sulzer, Jiaren Sun, Shi-Yong Sun, Zhi-Jun Sun, Joseph J Y Sung, Kuninori Suzuki, Toshihiko Suzuki, Michele S Swanson, Charles Swanton, Sean T Sweeney, Lai-King Sy, Gyorgy Szabadkai, Ira Tabas, Heinrich Taegtmeyer, Marco Tafani, Krisztina Takács-Vellai, Yoshitaka Takano, Kaoru Takegawa, Genzou Takemura, Fumihiko Takeshita, Nicholas J Talbot, Kevin S W Tan, Keiji Tanaka, Kozo Tanaka, Daolin Tang, Dingzhong Tang, Isei Tanida, Bakhos A Tannous, Nektarios Tavernarakis, Graham S Taylor, Gregory A Taylor, J Paul Taylor, Lance S Terada, Alexei Terman, Gianluca Tettamanti, Karin Thevissen, Craig B Thompson, Andrew Thorburn, Michael Thumm, Fengfeng Tian, Yuan Tian, Glauco Tocchini-Valentini, Aviva M Tolkovsky, Yasuhiko Tomino, Lars Tönges, Sharon A Tooze, Cathy Tournier, John Tower, Roberto Towns, Vladimir Trajkovic, Leonardo H Travassos, Ting-Fen Tsai, Mario P Tschan, Takeshi Tsubata, Allan Tsung, Boris Turk, Lorianne S Turner, Suresh C Tyagi, Yasuo Uchiyama, Takashi Ueno, Midori Umekawa, Rika Umemiya-Shirafuji, Vivek K Unni, Maria I Vaccaro, Enza Maria Valente, Greet Van den Berghe, Ida J van der Klei, Wouter van Doorn, Linda F van Dyk, Marjolein van Egmond, Leo A van Grunsven, Peter Vandenabeele, Wim P Vandenberghe, Ilse Vanhorebeek, Eva C Vaquero, Guillermo Velasco, Tibor Vellai, Jose Miguel Vicencio, Richard D Vierstra, Miquel Vila, Cécile Vindis, Giampietro Viola, Maria Teresa Viscomi, Olga V Voitsekhovskaja, Clarissa von Haefen, Marcela Votruba, Keiji Wada, Richard Wade-Martins, Cheryl L Walker, Craig M Walsh, Jochen Walter, Xiang-Bo Wan, Aimin Wang, Chenguang Wang, Dawei Wang, Fan Wang, Fen Wang, Guanghui Wang, Haichao Wang, Hong-Gang Wang, Horng-Dar Wang, Jin Wang, Ke Wang, Mei Wang, Richard C Wang, Xinglong Wang, Xuejun Wang, Ying-Jan Wang, Yipeng Wang, Zhen Wang, Zhigang Charles Wang, Zhinong Wang, Derick G Wansink, Diane M Ward, Hirotaka Watada, Sarah L Waters, Paul Webster, Lixin Wei, Conrad C Weihl, William A Weiss, Scott M Welford, Long-Ping Wen, Caroline A Whitehouse, J Lindsay Whitton, Alexander J Whitworth, Tom Wileman, John W Wiley, Simon Wilkinson, Dieter Willbold, Roger L Williams, Peter R Williamson, Bradly G Wouters, Chenghan Wu, Dao-Cheng Wu, William K K Wu, Andreas Wyttenbach, Ramnik J Xavier, Zhijun Xi, Pu Xia, Gengfu Xiao, Zhiping Xie, Zhonglin Xie, Da-zhi Xu, Jianzhen Xu, Liang Xu, Xiaolei Xu, Ai Yamamoto, Akitsugu Yamamoto, Shunhei Yamashina, Michiaki Yamashita, Xianghua Yan, Mitsuhiro Yanagida, Dun-Sheng Yang, Elizabeth Yang, Jin-Ming Yang, Shi Yu Yang, Wannian Yang, Wei Yuan Yang, Zhifen Yang, Meng-Chao Yao, Tso-Pang Yao, Behzad Yeganeh, Wei-Lien Yen, Jia-Jing Yin, Xiao-Ming Yin, Ook-Joon Yoo, Gyesoon Yoon, Seung-Yong Yoon, Tomohiro Yorimitsu, Yuko Yoshikawa, Tamotsu Yoshimori, Kohki Yoshimoto, Ho Jin You, Richard J Youle, Anas Younes, Li Yu, Long Yu, Seong-Woon Yu, Wai Haung Yu, Zhi-Min Yuan, Zhenyu Yue, Cheol-Heui Yun, Michisuke Yuzaki, Olga Zabirnyk, Elaine Silva-Zacarin, David Zacks, Eldad Zacksenhaus, Nadia Zaffaroni, Zahra Zakeri, Herbert J Zeh, Scott O Zeitlin, Hong Zhang, Hui-Ling Zhang, Jianhua Zhang, Jing-Pu Zhang, Lin Zhang, Long Zhang, Ming-Yong Zhang, Xu Dong Zhang, Mantong Zhao, Yi-Fang Zhao, Ying Zhao, Zhizhuang J Zhao, Xiaoxiang Zheng, Boris Zhivotovsky, Qing Zhong, Cong-Zhao Zhou, Changlian Zhu, Wei-Guo Zhu, Xiao-feng Zhu, Xiongwei Zhu, Yuangang Zhu, Teresa Zoladek, Wei-Xing Zong, Antonio Zorzano, Jürgen Zschocke, Brian Zuckerbraun.
Autophagy
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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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Peritubular capillary basement membrane multilayering on electron microscopy: a useful marker of early chronic antibody-mediated damage.
Transplantation
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Chronic antibody-mediated rejection is an important cause of late graft failure. Developing an early marker of the disease may allow diagnosis and treatment before irreversible graft damage has occurred. The aim of this study was to assess whether, on electron microscopy examination, peritubular capillary (PTC) basement membrane multilayering precedes and predicts the development of transplant glomerulopathy (TG).
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De novo DQ donor-specific antibodies are associated with a significant risk of antibody-mediated rejection and transplant glomerulopathy.
Transplantation
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The importance of human leukocyte antigen (HLA) matching in renal transplantation is well recognized, with HLA-DR compatibility having the greatest influence. De novo DQ donor-specific antibodies (DSAbs) are the predominant HLA class II DSAb after transplantation. The aim of this study was to establish the incidence and outcomes after the development of DQ DSAbs along with the impact of class II HLA mismatch on their development.
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A cell-based phenotypic assay to identify cardioprotective agents.
Circ. Res.
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Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS).
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The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives.
Blood
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Synthetic triterpenoids are multitarget compounds exhibiting promise as preventative and therapeutic agents for cancer. Their proposed mechanism of action is by forming Michael adducts with reactive nucleophilic groups on target proteins. Our previous work demonstrates that the 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives promote B-lymphoid cell apoptosis through a mitochondria-mediated pathway linked to mitochondrial protein aggregation. As one function of the Lon protease is to eliminate abnormal mitochondrial proteins, we hypothesized that CDDO-induced protein aggregation and lymphoma apoptosis occur by inactivating this enzyme. Here, we show that CDDO and its derivatives directly and selectively inhibit Lon. CDDO blocks Lon-mediated proteolysis in biochemical and cellular assays, but does not inhibit the 20S proteasome. Furthermore, a biotinylated-CDDO conjugate modifies mitochondrial Lon. A striking common phenotype of CDDO-treated lymphoma cells and Lon-knockdown cells is the accumulation of electron-dense aggregates within mitochondria. We also show that Lon protein levels are substantially elevated in malignant lymphoma cells, compared with resting or activated B cells. Finally, we demonstrate that Lon knockdown leads to lymphoma cell death. Together, these findings suggest that Lon inhibition plays a contributory role in CDDO-induced lymphoma cell death, and support the concept that mitochondrial Lon is a novel anticancer drug target.
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Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.
FEBS Lett.
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Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.
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Mitochondrial targeted nitro-linoleate, a tool compound for studying cardioprotection.
Br. J. Pharmacol.
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Cardiac ischemia-reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids such as nitro-linoleate (LNO2 ), occurs via a mechanism involving covalent modification of mitochondrial adenine nucleotide translocator 1 (ANT1). Thus it was hypothesized that conjugation of LNO2 to the mitochondriotropic triphenylphosphonium (TPP(+) ) moiety would enhance its protective properties.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.