Carbonyl Cyanide P-trifluoromethoxyphenylhydrazone Research Articles

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

Abstract 12308: Presenilin-2 Regulates Mitophagy via Promoting Intracellular Translocation of Parkin to Mitochondria

Introduction: Presenilin-2 (PS2) is a component of the γ-secretase complex, intracellular protease. Variant in PSEN2 , the causative genes of Alzheimer's disease, are also implicated in the development of dilated cardiomyopathy. Hypoxia increases PSEN2 expression 10-fold over normal, and PSEN2 knockout (PS2KO) cells exhibit significantly lower ATP production. However, the mechanism of the mitochondrial dysfunction has not been fully elucidated. Hypothesis: PS2 regulates mitochondrial selective autophagy (mitophagy) by promoting the translocation of Parkin to mitochondria. Methods: Expression of autophagy and mitophagy markers was examined using cultured rat cardiomyocytes (H9c2) silenced for PSEN2 and PSEN1 , exposed to 10 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler used to induce mitophagy. Results: Mitochondrial respiration of PS2KO-H9c2 was showed reduced (maximal respiration capacity, 68.4 ± 27.0 pmol/min/cells) compared to PS1KO-H9c2 (147.6 ± 27.2 pmol/min/cells) or normal cells (276.6 ± 11.9 pmol/min/cells) (P<0.001). When mitophagy-dye modified with perylene-3,4-dicarboxylic anhydride was used, mitophagy detection rates were significantly increased by the addition of FCCP to normal H9c2 (34% increased, P=0.041) and PS1KO-H9c2 (46% increased, P=0.035), but there was no change with PS2KO-H9c2 (25% decreased, P=0.232). When FCCP was added to PS2KO-H9c2, Parkin was not only enhanced in whole cell lysate of PS2KO-H9c2, but also increased even at mRNA level; however, in the isolated mitochondrial fraction, Parkin was significantly reduced, and PINK1 and phosphorylated ubiquitin (serine 65) were also attenuated. In Immunofluorescent staining confirmed that Parkin was not co-localized with mitochondria in PS2KO-H9c2, implicating PS2 in promoting the transfer of Parkin from the cytoplasm to mitochondria. As mitochondrial depolarization causes PINK1 stabilization needed for Parkin recruitment, we interrogated mitochondrial membrane potential measurement using Rhodamine-123 and examined expression of OPA1 isoforms; no significant results were obtained. Conclusions: PS2 contributes to the intracellular transport of Parkin to regulate mitophagy.

The Busy Division of Scientific Affairs

Leadership Perspectives| May 2022 The Busy Division of Scientific Affairs Andrew D. Rosenberg, MD, FASA Andrew D. Rosenberg, MD, FASA Search for other works by this author on: This Site PubMed Google Scholar ASA Monitor May 2022, Vol. 86, 13–14. https://doi.org/10.1097/01.ASM.0000830800.95339.81 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Search Site Citation Andrew D. Rosenberg; The Busy Division of Scientific Affairs. ASA Monitor 2022; 86:13–14 doi: https://doi.org/10.1097/01.ASM.0000830800.95339.81 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll PublicationsASA Monitor Search Advanced Search Topics: carbonyl cyanide p-trifluoromethoxyphenylhydrazone, editorial board, happiness, knowledge acquisition, pain, patient safety, perioperative care, surgical procedures, operative, point-of-care ultrasonography, geriatric anesthesia As your Vice President for Scientific Affairs, it is my pleasure to provide you with an update of activities in the area of scientific affairs. I want to thank our ASA members and leaders who have worked so hard over the past year by demonstrating their incredible dedication and commitment to the care of patients while promoting science. The Division of Scientific Affairs is composed of five Sections: Clinical Care (Joy L. Hawkins, MD, FASA, Chair), Publications (Daniel J. Cole, MD, FASA, Chair), Annual Meeting (Santhanam Suresh, MD, FASA, Chair), Education and Research (David Martin, MD, PhD, FASA, Chair), and Subspecialties (Erin A. Sullivan, MD, FASA, Chair). Rounding out the Division, and also a component of the Committee on Professional Education Oversight, is representation from the Committee on Scientific Affairs headed by David Martin, MD, PhD, FASA, who is already on the committee. Within each of these sections are hard-working... You do not currently have access to this content.

Protein kinase C signalling involved in prothoracicotropic hormone-stimulated prothoracic glands in the silkworm, Bombyx mori.

In the present study, the participation of protein kinase C (PKC) signalling in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx prothoracic glands (PGs) is demonstrated and characterized. PTTH stimulated phosphorylation of a 37-kDa protein in Bombyx PGs both in vitro and in vivo, as recognized by a PKC substrate antibody. Treatment with either A23187 or thapsigargin also stimulated this 37-kDa protein phosphorylation. PTTH-stimulated phosphorylation of the 37-kDa protein was markedly attenuated in the absence of Ca2+ . The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated phosphorylation of this protein, indicating the involvement of Ca2+ and PLC. A mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) and a chemical activator of adenosine 5'-monophosphate-activated protein kinase (AMPK) (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) did not affect PTTH-stimulated phosphorylation of the 37-kDa protein, implying that ERK and PI3K/AMPK are not the upstream signalling pathways for PKC-dependent protein phosphorylation. The mitochondrial oxidative phosphorylation inhibitors (the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone and diphenylene iodonium) inhibited PTTH-stimulated phosphorylation of the 37-kDa protein, indicating its redox regulation. Treatment with PKC inhibitors (either calphostin C, chelerythrine C or rottlerin) reduced PTTH-stimulated phosphorylation of the 37-kDa protein. PTTH-stimulated ecdysteroidogenesis was also inhibited by treatment with rottlerin, thus further confirming participation of PKC-dependent phosphorylation in PTTH signalling. From these results, we demonstrated that redox-regulated PTTH-stimulated PKC signalling is involved in ecdysteroid secretion in Bombyx PGs.

Passive Influx and Ion Trapping Are More Relevant to the Cellular Accumulation of Highly Permeable Low-Molecular-Weight Acidic Drugs than Is Organic Anion Transporter 2.

Recently published work suggests that highly permeable low-molecular-weight (LMW) acidic drugs are transported by organic anion transporter 2 (OAT2). However, an asymmetric distribution of ionizable drugs in subcellular organelles where pH gradients are significant may occur in the presence of an inhibitor relative to its absence (e.g., lysosomal trapping). In the present study, OAT2-mediated transport of highly permeable LMW anions could not be demonstrated using OAT2 transfected cells, despite robust transport of the OAT2 substrate penciclovir. Moreover, a rifamycin SV (RifSV)-dependent reduction in the accumulation of highly permeable LMW anions previously observed in hepatocytes could be qualitatively reproduced using HepG2 cells and also in Madin-Darby canine kidney (MDCK) cells, which lack expression of OAT2. Neither HepG2 nor MDCK cells demonstrated meaningful penciclovir transport, nor was the cellular accumulation of the highly permeable LMW anions sensitive to competitive inhibition by the neutral OAT2 substrate penciclovir. Both cell lines, however, demonstrated sensitivity to the mitochondrial uncoupler p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) in a manner similar to RifSV. Furthermore, the transepithelial MDCK permeability of the highly permeable LMW anions was measured in the absence and presence of RifSV and FCCP at concentrations that reduced the cellular accumulation of anions. Neither inhibitor, nor the OAT2 inhibitor ketoprofen, reduced the transepithelial flux of the anions as would be anticipated for transported substrate inhibition. The findings presented here are aligned with cellular accumulation of highly permeable LMW anions being significantly determined by ion trapping sensitive to mitochondrial uncoupling, rather than the result of OAT2-mediated transport. SIGNIFICANCE STATEMENT: The manuscript illustrates that passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low-molecular-weight acidic drugs than is the previously proposed mechanism of OAT2-mediated transport. The outcome illustrated here highlights a rare, and perhaps previously not reported, observation of anionic drug trapping in a compartment sensitive to mitochondrial uncoupling (e.g., the mitochondrial matrix) that may be confused for transporter-mediated uptake.

Abstract 14800: Presenilin 1 and Presenilin 2 Contribute to Mitophagy Including Alternative Pathway

Introduction: Presenilin 1 (PS1) and presenilin 2 (PS2) are proteins that compose γ-secretase, which is a protease present in late endosome lysosomes. Recently, amyloid beta accumulation was found in the cardiomyocytes of Alzheimer's disease patients and cause cardiac dysfunction. Mutations in PSEN1 and PSEN2 , the causative genes of familial Alzheimer's disease, are involved in the development of dilated cardiomyopathy (DCM); however, their roles in cardiomyocytes have not been elucidated thoroughly. Hypothesis: We hypothesized that PS1 and PS2 might be playing a role in mitochondria-selective autophagy (mitophagy) of cardiomyocytes. Methods: Whole-exome sequencing of 32 DCM patients was focused on 29 autophagy-related genes and 16 genes encoding autophagy-related proteins. Based on the results, the expression of autophagy and mitophagy markers was examined using cultured rat cardiomyocytes silenced for PSEN1 or PSEN2 and exposed to starvation, 100 μM bafilomycin A1, inhibiting fusion between autophagosomes and lysosomes, or 10 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler used to induce mitophagy. Results: A variant of PSEN2 (c.1262C>T) may be the cause of DCM in a 50-year-old Japanese man. Electron microscopic observation of myocardium revealed a fibrillar substance in the expanded T-tube, suggesting failure to clear aggregates. FCCP treatment caused an increase in microtubule-associated protein 1 light chain 3 (LC3)-II and p62 which was further enhanced in cells with silenced PS1 or PS2. Rab9, which is associated with the alternative pathway of mitophagy, was also increased in cells silenced for PS1 or PS2. Parkin was decreased with FCCP treatment, suggesting its clearance via mitophagy, and this was further enhanced when PS1 was silenced. Voltage-dependent anion channel (VDAC) levels, a reflection of mitochondrial content, were slightly increased when PS1 and PS2 were silenced. LC3-II increased with starvation and further increased with bafilomycin A1 treatment: however, silencing PS2 did not result in further increase, suggesting that flux was impaired. Conclusions: PS2 and probably PS1 are required to support autophagic flux necessary for cardiomyocyte homeostasis.

Bicarbonate suppresses mitochondrial membrane depolarization induced by conventional uncouplers

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.

Ibrutinib in Combination with Venetoclax Decreases Mitochondrial Bioenergetics through the Impaired BTK, AKT and AMPK/SIRT/PGC-1α Signaling Pathway in CLL

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), and venetoclax, an inhibitor of B-cell lymphoma 2 (Bcl-2) protein are approved therapies for CLL. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of BTK, AKT, and AMPK/SIRT/PGC-1α signaling pathway have to be explored yet in CLL. Ibrutinib and venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of p-BTK, p-AKT, Bcl-2, p-AMPK, SIRT1, and PGC-1α were analyzed. We hypothesize that ibrutinib, along with venetoclax, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of BTK, AKT and AMPK/SIRT/PGC-1α signaling pathway and enable lower doses and profound effects for combination therapies rather than single agent therapy. Methods: The high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Austria), a Clarke-type oxygen electrode, is used to measure mitochondrial respiration rates of CLL cells at 370C. Freshly isolated CLL cells (10 mil. cells/ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone (2-12 uM), and antimycin A (2 uM). Oxygen consumption rate is expressed in pmol oxygen/s /mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by the flow cytometer (NovoCyte). Results: CLL cells obtained ex vivo from ibrutinib treated patients had decreased mitochondrial bioenergetics compared to pre-treatment samples. Primary CLL cells treated in vitro with sub-lethal doses of ibrutinib for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by low doses (0.5 - 2 nM) of venetoclax. The combination treatment of ibrutinib with venetoclax for 24 or 48h significantly decreased these bioenergetics parameters compared to each individual drug alone except for the spare respiratory capacity vs venetoclax alone at 24 h. MMP was significantly decreased by these drug treatments and its combinations compared to DMSO. Cell viability was used to confirm a sub-lethal phenotype. ROS levels were significantly increased by venetoclax compared to DMSO, and in combination with ibrutinib vs ibrutinib alone. However, ROS levels by ibrutinib were not significantly changed compared to DMSO or venetoclax. Protein levels of p-BTK, p-AKT, p-AMPK, SIRT1, and PGC-1α were decreased by ibrutinib or venetoclax alone compared to DMSO and further decreased by the combination of these two drugs compared to single agent. Conclusion: CLL cells from ibrutinib treated patients, a clinically approved BTK inhibitor, demonstrated decreased bioenergetics similar to normal B-lymphocytes suggesting ibrutinib treatment normalizes the mitochondrial bioenergetics in CLL. Ibrutinib and venetoclax affected bioenergetics profiles in CLL cells at low doses. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each BTK or Bcl-2 inhibitor agent alone. Protein levels of p-BTK, p-AKT, p-AMPK, SIRT1, and PGC-1α in ibrutinib and venetoclax treated samples were reduced compared to DMSO and each single agent inhibitor. These novel data suggest the involvement of BTK, AKT and AMPK/SIRT/PGC-1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. Disclosures Johnston: Janssen: Research Funding. Banerji:Janssen: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Licensing fee, Research Funding; Abbvie: Consultancy, Honoraria; CIHR: Research Funding; LLSC: Research Funding; Research Manitoba: Research Funding; Astra-Zeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Dana-Farber Cancer Institute: Other: Licencing fee; CCMF: Research Funding; CancerCare Manitoba/University of Manitoba: Employment; CAPhO: Honoraria; BIOGEN: Other: Licensing fee.

Pilot study of mitochondrial bioenergetics in subjects with acute porphyrias

Background and aimsThe acute porphyrias are characterized by defects in heme synthesis, particularly in the liver. In some affected patients, there occurs a critical deficiency in a regulatory heme pool within hepatocytes that leads to up-regulation of 5-aminolevulinic acid [ALA] synthase-1, which is the first and normally rate-controlling enzyme in the pathway. In earlier work, we described defects in mitochondrial functions in cultured skin fibroblasts from patients with acute intermittent porphyria [AIP]. Others described defects in livers of murine models of AIP. Here, we explored mitochondrial energetics in peripheral blood mononuclear cells [PBMCs] and platelets in persons with AIP and hereditary coproporphyria [HCP]. Our hypotheses were that there are deficits in bioenergetic capacity in acute porphyrias and that subjects with more severe acute porphyria have more pronounced reductions in mitochondrial oxygen consumption rates [OCR]. MethodsWe studied 17 subjects with acute hepatic porphyrias, 14 with classical AIP, one with severe AIP due to homozygous deficiency of hydroxymethylbilane synthase [HMBS], 2 with HCP, and 5 non-porphyric controls. We collected peripheral blood, isolated PBMCs, which we assayed either immediately or after frozen storage [80C] for up to 14 days. Using Seahorse XF-24-3, we measured OCR in the presence of glucose + pyruvate under basal condition, and after additions of oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone [FCCP], and antimycin+rotenone. ResultsMost subjects [13/17, 76%] were female. Subjects with moderate/severe symptoms associated with acute porphyria had significantly lower basal and maximal-OCR than those with no/mild symptoms who were the same as controls. We observed significant inverse correlation between urinary porphobilinogen [PBG] excretion and OCR. The subject with homozygous AIP had a much lower-OCR than his asymptomatic parents. Summary/conclusionsResults support the hypothesis that active acute hepatic porphyria is characterized by a deficiency in mitochondrial function that is detectable in PBMCs, suggesting that limitations in electron transport and ATP production exist in such individuals.

Ischemia With Preconditioning in Wistar Rats Maintains Mitochondrial Respiration, Even With Mild Hepatocellular Disturbance

IntroductionIn hepatectomy or liver transplantation, preconditioning is a procedure indicated to protect the organ from ischemia-reperfusion injury (I-R). ObjectiveEvaluate the effect of preconditioning after hepatic I-R in Wistar rats, through mitochondrial respiration, liver histology, and profile. MethodTwenty male Wistar rats, weighing on average 307.1 g, were anesthetized with sodium thiopental (25 mg/kg) intravenously and xylazine hydrochloride (30 mg/kg) intramuscularly. The animals were divided into 2 groups: the preconditioning group (PCG), which contained 10 animals, and the hepatic pedicle was isolated and submitted to clamping with microvascular clamp (10 minutes of ischemia and 10 minutes of reperfusion, followed by 30 minutes of ischemia and 30 minutes of reperfusion); and the simulated operation group (SOG), which contained 10 animals submitted to manipulation of the hepatic pedicle and observation for the same length of time, with blood collected for transaminase dosage measurements, and liver biopsy for evaluation of mitochondrial respiration and histologic liver analysis and after sacrificed under anesthesia. The project was approved by the Ethics Committee on Animal Experimentation CEEA/UNICAMP under protocol number 3905-1. ResultThe PCG mitochondria showed the same respiration level as the SOG, when stimulated with the addition of adenosine diphosphate or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the respiratory control ratio and resting of velocity of respiration the groups behaved in a similar way. The PCG presented high aspartate and alanine transaminases (P < .03) and about 60% of sinusoidal congestion and venous congestion in the histologic analysis when compared with SOG. ConclusionWe found that ischemia with preconditioning in Wistar rats can lead to mild histologic and biochemical dysfunction without leading to impairment of mitochondrial respiration.

Effect of Cyanide on Mitochondrial Membrane Depolarization Induced by Uncouplers.

In this work, it was found that the ability of common uncouplers - carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) - to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor - cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.

Later Evaluation of Ischemia and Reperfusion by the Pringle Maneuver in Wistar Rats, Demonstrating That Hepatic Lesions Can Be Reversible

ObjectiveThere has been much research on hepatic ischemia and reperfusion by means of short or longer interruption of the portal triad. The aim of this work was to evaluate the mitochondrial respiratory activity and liver histology at 2 different times after the Pringle maneuver. MethodsTwenty-eight male Wistar rats, weighing ∼308 g, with histologic and mitochondrial study: immediate ischemic group (IIG; 40 minutes; 9 animals) and late ischemic group (LIG; 28 days; 9 animals). The rats were anesthetized and underwent a U-incision in the abdomen. In a simulated operation, manipulation of the hepatic pedicle was performed (5 animals immediate [ISG] and 5 late [LSG]). The hepatic pedicle was clamped for 20 minutes of ischemia foloowed by 20 minutes of reperfusion. The animals were killed under anesthesia. ResultsMitochondria when stimulated by adenosine diphosphate or carbonylcyanide p-trifluoromethoxyphenylhydrazone had a significant respiratory reduction (P < .001). The respiratory control ratio in the LIG was altered (P < .02) compared with IIG. In the resting state, there was no change in the velocity of respiration between ischemic groups. Histopathologic findings showed 55.5% sinusoidal dilatation in IIG and 66.6% in LIG; 77.7% ballooning in IIG and 55.5% in LIG; and 11.1% focal necrosis in both IIG and LIG. ConclusionsThe oxidative phosphorylation system recovered with improvement in mitochondrial respiration; however, morphologic recovery was associated with the type and intensity of injury.

Effect of 50-Hz Sinusoidal Magnetic Field on the Production of Superoxide Anion and the Expression of Heat-shock Protein 70 in RAW264 Cells

There is a growing concern if the power-line frequency (50/60 Hz) magnetic field (termed in this paper ELF-MF) increases cancer risks. Since one of the major causes of cancer is cellular oxidative stress, whether the ELF-MF increases the oxidative stress is a central problem in the studies on the biological effect of the ELF-MF. Here, we have investigated the effect of 50-Hz sinusoidal magnetic field on the production of O2-, the expression of heat shock protein (HSP) 70 and the mitochondrial membrane potential in cell line macrophage RAW264 cells. Macrophages were exposed to or not exposed to 0.1-mT or 0.5-mT, 50-Hz sinusoidal magnetic field and were subjected to (1) assay for O2- (2) analysis of the expression of HSP70, and (3) measurement of the mitochondrial membrane potential with a fluorescent indicator. The 50-Hz magnetic field enhanced production of O2- and the expression of HSP70, both of which are consistent with previous studies. The exposure to 50-Hz magnetic field decreased mitochondrial membrane potential indicating the diminished activity of mitochondria. The uncoupler of mitochondrial function, carbonyl cyanide p-trifluoromethoxyphenylhydrazone diminished the membrane potential, as expected. On the other hand, it increased the production of O2-. The results collectively suggest that the 50-Hz magnetic field diminished the mitochondrial membrane potential, which led to the increase in the production of O2- and the expression of HSP70 protein.

Sulfated lentinan induced mitochondrial dysfunction leads to programmed cell death of tobacco BY-2 cells

Sulphated lentinan (sLTN) is known to act as a resistance inducer by causing programmed cell death (PCD) in tobacco suspension cells. However, the underlying mechanism of this effect is largely unknown. Using tobacco BY-2 cell model, morphological and biochemical studies revealed that mitochondrial reactive oxygen species (ROS) production and mitochondrial dysfunction contribute to sLNT induced PCD. Cell viability, and HO/PI fluorescence imaging and TUNEL assays confirmed a typical cell death process caused by sLNT. Acetylsalicylic acid (an ROS scavenger), diphenylene iodonium (an inhibitor of NADPH oxidases) and protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (a protonophore and an uncoupler of mitochondrial oxidative phosphorylation) inhibited sLNT-induced H2O2 generation and cell death, suggesting that ROS generation linked, at least partly, to a mitochondrial dysfunction and caspase-like activation. This conclusion was further confirmed by double-stained cells with the mitochondria-specific marker MitoTracker RedCMXRos and the ROS probe H2DCFDA. Moreover, the sLNT-induced PCD of BY-2 cells required cellular metabolism as up-regulation of the AOX family gene transcripts and induction of the SA biosynthesis, the TCA cycle, and miETC related genes were observed. It is concluded that mitochondria play an essential role in the signaling pathway of sLNT-induced ROS generation, which possibly provided new insight into the sLNT-mediated antiviral response, including PCD.

Studies on the influence of different metabolic uncouplers on the biodegradation of toluene in a differential biofilter reactor

One of the biggest challenges in a traditional biofilter is to overcome the low volumetric degradation rate, which often makes the footprint excessive. The volumetric degradation rate or elimination capacity (EC) is directly influenced by the specific biodegradation rate of the microorganisms involved in degrading the pollutants. Application of metabolic uncouplers in biofiltration could improve the biodegradation rate of microorganisms, which in turn could increase the EC. The addition of metabolic uncouplers to the growth system decreases the biomass growth whereas in energy-excess, non-growth systems like biofiltration, it is expected to increase the specific substrate uptake rate since the substrate requirement for maintenance energy should increase, which in turn should increase the EC. Seven potential metabolic uncouplers were screened in batch serum bottles and subsequently tested in a continuous biofilter reactor with soil as the biofilter medium. The metabolic uncouplers tested were benzoic acid, carbonylcyanide p-trifluoromethoxy phenylhydrazone (FCCP), carbonylcyanide m-chloromethoxy phenylhydrazone (CCCP), pentachlorophenol (PCP), malonic acid, 2, 4, 6-trichlorophenol (TCP) and m-chlorophenol (mCP). Only PCP and 2, 4, 6-TCP increased the toluene degradation rate significantly. PCP increased the toluene degradation rate by 35% at 140 µM, whereas 4051 µM TCP increased the rate by 18%. FCCP did not significantly affect the degradation rate and the other metabolic uncouplers decreased the degradation rate.

Structure-activity relationships of furazano[3,4-b]pyrazines as mitochondrial uncouplers.

Chemical mitochondrial uncouplers are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. These uncouplers have potential for the treatment of diseases such as obesity, Parkinson's disease, and aging. We have previously identified a novel mitochondrial protonophore, named BAM15, which stimulates mitochondrial respiration across a broad dosing range compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Herein, we report our investigations on the structure-activity relationship profile of BAM15. Our studies demonstrate the importance of the furazan, pyrazine, and aniline rings as well as pKa in maintaining its effective protonophore activity.

No Protective Function Found in Wistar Rats Submitted to Long Ischemia Time and Reperfusion After Intermittent Clamping of the Total Hepatic Pedicle

BackgroundAlthough the intermittent Pringle maneuver is used for major transplant surgery, traumas, and hepatic protection, long ischemia time and reperfusion may limit some protection in Wistar rats. The aim of the study was to evaluate the protection effects of intermittent clamping in the total hepatic pedicle after a long period of ischemia and reperfusion in Wistar rats. MethodsForty-two male Wistar rats, weighing ±327.7 g, were anesthetized intravenously with sodium thiopental and given a U-shaped incision in the abdomen. The total hepatic pedicle was isolated and subjected to clamping with a microvascular clamp. Groups included were the continuous group (CG, n = 14, 40 minutes of ischemia/40 minutes of reperfusion); the intermittent group (IG, n = 14, 4 cycles a 10 minute ischemia/reperfusion 10 minutes); and the sham group (SG, n = 14, 80 minutes of observation time). Blood collection for transaminase dosage was carried out, and hepatic biopsy specimens were taken for mitochondrial respiration and histological evaluation. ResultsIn groups CG and IG, aspartate aminotransferase and alanine aminotransferase enzymes were elevated in comparison to group SG (P < .008); mitochondrias, when stimulated by use of adenosine diphosphate or carbonylcyanide p-trifluoromethoxyphenylhydrazone, had a significant decrease in mitochondrial respiration (P < .05), and the respiratory control ratio in the ischemic groups was lower (P < .03) when compared with the GS. On histological examination, 100% of the GC had lesions: 33% focal hemorrhagic necrosis, 17% sinuzoidal congestion and/or vacuolization, and 50% venous congestion; in the IG, 100% had lesions: 43% sinusoidal congestion and/or vacuolization and 57% venous congestion. ConclusionsThe intermittent total hepatic pedicle clamping for a long period of time in the Wistar rats had no efficacy in protection of liver injury.

The slow S to M rise of chlorophyll a fluorescence reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii.

The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between photosystem I (PS I) and photosystem II (PS II) to provide balanced photosynthesis. Chlorophyll (Chl) a fluorescence induction (the so-called OJIPSMT transient) is a signature of several photosynthetic reactions. Here, we show that the slow (seconds to minutes) S to M fluorescence rise is reduced or absent in the stt7 mutant (which is locked in state 1) in C. reinhardtii. This suggests that the SM rise in wild type C. reinhardtii may be due to state 2 (low fluorescence state; larger antenna in PS I) to state 1 (high fluorescence state; larger antenna in PS II) transition, and thus, it can be used as an efficient and quick method to monitor state transitions in algae, as has already been shown in cyanobacteria (Papageorgiou et al. 1999, 2007; Kaňa et al. 2012). We also discuss our results on the effects of (1) 3-(3,4-dichlorophenyl)-1,4-dimethyl urea, an inhibitor of electron transport; (2) n-propyl gallate, an inhibitor of alternative oxidase (AOX) in mitochondria and of plastid terminal oxidase in chloroplasts; (3) salicylhydroxamic acid, an inhibitor of AOX in mitochondria; and (4) carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an uncoupler of phosphorylation, which dissipates proton gradient across membranes. Based on the data presented in this paper, we conclude that the slow PSMT fluorescence transient in C. reinhardtii is due to the superimposition of, at least, two phenomena: qE dependent non-photochemical quenching of the excited state of Chl, and state transitions.

Antioxidant effects of JM-20 on rat brain mitochondria and synaptosomes: mitoprotection against Ca²⁺-induced mitochondrial impairment.

Because mitochondrial oxidative stress and impairment are important mediators of neuronal damage in neurodegenerative diseases and in brain ischemia/reperfusion, in the present study, we evaluated the antioxidant and mitoprotective effect of a new promising neuroprotective molecule, JM-20, in mitochondria and synaptosomes isolated from rat brains. JM-20 inhibited succinate-mediated H₂O₂ generation in both mitochondria and synaptosomes incubated in depolarized (high K(+)) medium at extremely low micromolar concentration and with identical IC₅₀ values of 0.91 μM. JM-20 also repressed glucose-induced H₂O₂ generation stimulated by rotenone or by antimycin A in synaptosomes incubated in high sodium-polarized medium at extremely low IC₅₀ values of 0.395 μM and 2.452 μM, respectively. JM-20 was unable to react directly with H₂O₂ or with superoxide anion radicals but displayed a cathodic reduction peak at -0.71V, which is close to that of oxygen (-0.8V), indicating high electron affinity. JM-20 also inhibited uncoupled respiration in mitochondria or synaptosomes and was a more effective inhibitor in the presence of the respiratory substrates glutamate/malate than in the presence of succinate. JM-20 also prevented Ca(2+)-induced mitochondrial permeability transition pore opening, membrane potential dissipation and cytochrome c release, which are key pathogenic events during stroke. This molecule also prevented Ca(2+) influx into synaptosomes and mitochondria; the former effect was a consequence of the latter because JM-20 inhibition followed the patterns of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), which is a classic mitochondrial uncoupler. Because the mitochondrion is considered an important source and target of neuronal cell death signaling after an ischemic insult, the antioxidant and protective effects of JM-20 against the deleterious effects of Ca(2+) observed at the mitochondrial level in this study may endow this molecule with the ability to succeed in mitochondrion-targeted strategies to combat ischemic brain damage.

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.