• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.108-109
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• 2013

Mitochondria play key roles in the production of cell's energy. Their dominant function is the synthesis of adenosine 5'-triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (Pi) through the oxidative phosphorylation. Evaluation of drug-induced mitochondrial toxicity has become increasingly important since mitochondrial dysfunction has recently been implicated in numerous diseases including cancer and diabetes mellitus. Mitochondrial functions have been monitored via oxygen consumption, mitochondrial membrane potential, and more importantly via ATP synthesis since ATP synthesis is the most essential function of mitochondria. Various analytical methods have been employed to investigate ATP synthesis in mitochondria, including high performance liquid chromatography (HPLC), bioluminescence technique, and pH measurement. However, most of these methods are based on destructive analysis or indirect monitoring through the enzymatic reaction. Infrared absorption spectroscopy (IRAS) is one of the useful techniques for real-time, label-free, and direct monitoring of biological reactions [1,2]. However, the strong water absorption requires very short path length in the order of several micrometers. Transmission measurements with thin path length are not suitable for mitochondrial assays because solution handlings necessary for evaluating mitochondrial toxicity, such as rapid mixing of drugs and oxygen supply, are difficult in such a narrow space. On the other hand, IRAS in the multiple internal reflection (MIR) geometry provides an ideal optical configuration to combine solution handling and aqueous-phase measurement. We have recently reportedon a real-time monitoring of drug-induced necrotic and apoptotic cell death using MIR-IRAS [3,4]. Clear discrimination between viable and damaged cells has been demonstrated, showing a promise as a label-free and real-time detection for cell-based assays. In the present study, we have applied our MIR-IRAS system to mitochondria-based assays by monitoring ATP synthesis in isolated mitochondria from rat livers. Mitochondrial ATP synthesis and hydrolysis were in situ monitored with MIR-IRAS, while dissolved oxygen level and solution pH were simultaneously monitored with O2 and pH electrodes, respectively. It is demonstrated that ATP synthesis and hydrolysis can be monitored by the IR spectral changes in phosphate groups in adenine nucleotides and MIR-IRAS is useful for evaluating time-dependent drug effects of mitochondrial toxicants.

• BMB Reports
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• v.42 no.2
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• pp.113-118
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• 2009

Despite the growing body of evidence suggesting a role for MsrA in antioxidant defense, little is currently known regarding the function of MsrB in cellular protection against oxidative stress. In this study, we overexpressed the mammalian MsrB and MsrA genes in Saccharomyces cerevisiae and assessed their subcellular localization and antioxidant functions. We found that the mitochondrial MsrB3 protein (MsrB3B) was localized to the cytosol, but not to the mitochondria, of the yeast cells. The mitochondrial MsrB2 protein was detected in the mitochondria and, to a lesser extent, the cytosol of the yeast cells. In this study, we report the first evidence that MsrB3 overexpression in yeast cells protected them against $H_2O_2$-mediated cell death. Additionally, MsrB2 overexpression also provided yeast cells with resistance to oxidative stress, as did MsrA overexpression. Our results show that mammalian MsrB and MsrA proteins perform crucial functions in protection against oxidative stress in lower eukaryotic yeast cells.

• YAKHAK HOEJI
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• v.43 no.6
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• pp.851-860
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• 1999

The effects of antimycin A(AA), dodium azide ($NaN_3$) and 2,4-dinitrophenol (DNP), which inhibit mitochondrial ATP production, on cellular functions of cultured astrocytes were studied. High concentrations of AA $(50{\;}\mu\textrm{g}/ml),{\;}NaN_3$ (100mM) and DNP (20mM) significantly decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, which was known to be related to mitochondrial function and then cel viability. AA ($50{\;}\mu\textrm{g}/ml$) increased lactate dehydrogenase (LDH) release and decreased [$^3H$] glutamate uptake, suggesting severe damage of cellular function by the concentrations of the compounds. Meanwhile, low concentrations of AA $(\leq{;\}10{\;}\mu\textrm{g}/ml),{\;}NaN_3{;\}(\leq{\;}50mM)$ and DNP ($\leq{\;}5mM$) significantly increased MTT reduction, the effect of which was specific to astrocytes. AA (5 and $10{\;}\mu\textrm{g}/ml$) did not affect LDH release and [$^3H$] glutamate uptake, indicating that these compounds increased MTT reduction at the low concentrations without cellular membrane damage. However, the low concentrations of AA produced significant decrease of MTT reduction in a glucose-free medium. Low concentrations of AA (1 and $5{\;}\mu\textrm{g}/ml$) did not change ATP production of astrocytes in the medium containing 10 mM glucose, but completely inhibited in a glucose-free medium, suggesting marked increase of cytosolic ATP production by the blockade of mitochondrial ATP production with low concentrations of AA. These results suggest that astrocytes have ability to enhance neuronal function or survival under conditions of incomplete ischemia or early by enhancement of glycolysis, and that cellular reduction of MTT occurs not only mitochondrially but also extramitchondrially.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.199-209
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• 2023

Background: Due to the interrupted blood supply in cerebral ischemic stroke (CIS), ischemic and hypoxia results in neuronal depolarization, insufficient NAD+, excessive levels of ROS, mitochondrial damages, and energy metabolism disorders, which triggers the ischemic cascades. Currently, improvement of mitochondrial functions and energy metabolism is as a vital therapeutic target and clinical strategy. Hence, it is greatly crucial to look for neuroprotective natural agents with mitochondria protection actions and explore the mediated targets for treating CIS. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stems and leaves was demonstrated to have neuroprotective effects against cerebral ischemia/reperfusion injury. However, the potential mechanisms have been not completely elaborate. Methods: The model of middle cerebral artery occlusion and reperfusion (MCAO/R) was adopted to verify the neuroprotective effects and potential pharmacology mechanisms of PNGL in vivo. Antioxidant markers were evaluated by kit detection. Mitochondrial function was evaluated by ATP content measurement, ATPase, NAD and NADH kits. And the transmission electron microscopy (TEM) and pathological staining (H&E and Nissl) were used to detect cerebral morphological changes and mitochondrial structural damages. Western blotting, ELISA and immunofluorescence assay were utilized to explore the mitochondrial protection effects and its related mechanisms in vivo. Results: In vivo, treatment with PNGL markedly reduced excessive oxidative stress, inhibited mitochondrial injury, alleviated energy metabolism dysfunction, decreased neuronal loss and apoptosis, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL significantly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions, and regulated its related downstream SIRT1/2/3-MnSOD/PGC-1α pathways. Conclusion: The study finds that the mitochondrial protective effects of PNGL are associated with the NAMPT-SIRT1/2/3-MnSOD/PGC-1α signal pathways. PNGL, as a novel candidate drug, has great application prospects for preventing and treating ischemic stroke.

• BMB Reports
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• v.49 no.2
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• pp.116-121
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• 2016

Although proteomic analyses have revealed the presence of mitochondrial oxidative phosphorylation (OXPHOS) proteins in the plasma membrane, there have been no in-depth evaluations of the presence or function of OXPHOS I-V in the plasma membrane. Here, we demonstrate the in situ localization of OXPHOS I-V complexes to the sarcolemma of skeletal muscle by immunofluorescence and immunohistochemistry. A portion of the OXPHOS I-V complex proteins was not co-stained with MitoTracker but co-localized with caveolin-3 in the sarcolemma of mouse gastrocnemius. Mitochondrial matrix-facing OXPHOS complex subunits were ectopically expressed in the sarcolemma of the non-permeabilized muscle fibers and C2C12 myotubes. The sarcolemmal localization of cytochrome c was also observed from mouse gastrocnemius muscles and C2C12 myotubes, as determined by confocal and total internal resonance fluorescence (TIRF) microscopy. Based on these data, we conclude that a portion of OXPHOS complexes is localized in the sarcolemma of skeletal muscle and may have non-canonical functions.

• Clinical and Experimental Pediatrics
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• v.60 no.12
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• pp.408-412
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• 2017

Combined oxidative phosphorylation deficiency-17 (COXPD-17) is very rare and is caused by homozygous or compound heterozygous mutations in the ELAC2 gene on chromosome 17p12. The ELAC2 gene functions as a mitochondrial tRNA processing gene, and only 4 different pathogenic mutations have been reported in ELAC2-associated mitochondrial dysfunction involving oxidative phosphorylation. Affected patients show various clinical symptoms and prognosis, depending on the genotype. We report a novel mutation in the ELAC2 gene (c.95C>G [p.Pro32Arg], het), in an infant with COXPD-17 who presented with encephalopathy including central apnea and intractable epilepsy, and growth and developmental retardation. During hospitalization, consistently elevated serum lactic acid levels were noted, indicative of mitochondrial dysfunction. The patient suddenly died of shock of unknown cause at 5 months of age. This is the first case report of COXPD-17 in Korea and was diagnosed based on clinical characteristics and genetic analysis.

• BMB Reports
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• v.39 no.5
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• pp.556-559
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• 2006

The Bcl-2 family of proteins regulates mitochondrial functions during cell death by modulating the efflux of death-promoting proteins such as cytochrome c and endonuclease G. Upon the binding of death ligands to their receptors, caspase-8 cleaves Bid, a BH3-only protein, into tBid that causes the mitochondrial damages resulting in the release of cytochrome c and endonuclease G. Also, another BH3-only protein, hNoxa, has been shown to induce the efflux of cytochrome c from the mitochondria. Whether the efflux proteins from the mitochondria in response to tBid or hNoxa are the same or different, however, has not been addressed. We have demonstrated that endonuclease G activities are not detectable among the proteins released from isolated mitochondria by hNoxa but are detectable in that by tBid. These results suggest that the efflux of proteins from the mitochondria are differentially modulated by tBid and hNoxa.

• Archives of Pharmacal Research
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• v.26 no.11
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• pp.951-959
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• 2003

A series of typical (chlorpromazine, haloperidol and thioridazine) and atypical (risperidone, quetiapine, clozapine and olanzapine) antipsychotics were tested for effects on integrated bioenergetic functions of isolated rat liver mitochondria. Polarographic measurement of oxygen consumption in freshly isolated mitochondria showed that electron transfer activity at respiratory complex I is inhibited by chlorpromazine, haloperidol, risperidone, and quetiapine, but not by clozapine, olanzapine, or thioridazine. Chlorpromazine and thioridazine act as modest uncouplers of oxidative phosphorylation. The typical neuroleptics inhibited NADH-coenzyme Q reductase in freeze-thawed mitochondria, which is a direct measure of complex I enzyme activity. The inhibition of NADH-coenzyme Q reductase activity by the atypicals risperidone and quetiapine was 2-4 fold less than that for the typical neuroleptics. Clozapine and olanzapine had only slight effects on NADH-coenzyme Q reductase activity, even at 200 $\mu$ M. The relative potencies of these neuroleptic drugs as inhibitors of mitochondrial bioenergetic function is similar to their relative potencies as risk factors in the reported incidence of extrapyramidal symptoms, including tardive dyskinesia (TD). This suggests that compromised bioenergetic function may be involved in the cellular pathology underlying TD.

• Herbal Formula Science
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• v.30 no.3
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• pp.155-163
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• 2022

Objectives : Lonicera japonica is known for anti-inflammation and antibiotic effect in Korean medicine. This study aimed for investigating the cytoprotective effect of Lonicera japonica extract (LJE) for HepG2 cells against arachidonic acid (AA)+iron-induced oxidative stress. Methods : The effect of LJE on cell viability was assessed by MTT assay. ROS assay was selected to assess antioxidant effect of LJE. To assess LJE's effect on mitochondrial function, flow cytometric analysis was operated. And immunoblot analysis was used to establish the underlying mechanism of LJE. Results : LJE protected HepG2 cells against AA+iron-induced oxidative stress by phosphorylation of liver kinase B1 and blocked the decline of procaspase 3. Also, LJE preserved the mitochondrial membrane permeability induced by AA+iron. Conclusion : LJE protected the hepatocyte from AA+iron-induced oxidative stress by activation of LKB1 by the preservation of mitochondrial functions.

• BMB Reports
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• v.32 no.5
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• pp.506-510
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• 1999

The newly-found thiol peroxidases (TPx) with a conserved cysteine as the primary site of catalysis are capable of catalyzing the thiol-dependent reduction of peroxides. However, the cellular distributions of the isoforms remain poorly understood. As a first step in understanding the physiological functions of the TPx isoforms, we examined the cellular and tissue distribution of the isoenzymes in various bovine tissues. The tissue distributions of TPx isoenzymes indicate that two types of TPx are widely distributed throughout all of the tested tissues. These two forms are the predominant proteins, with levels of the proteins being quite different from each other. The level of predominant TPx proteins, named type II (TPx II) and type V (TPx V), appeared to be very different with respect to tissue type. The cellular distribution and level of TPx isoenzymes also varied with the types of cells. Immunoblot analysis of the mitochondrial and cytosol fractions from various tissues indicates that TPx III is a unique mitochondrial form. Based on the different tissue and cellular distribution of TPx isoenzymes, we discuss the physiological function of TPx isoenzymes, especially the ubiquitous TPx II.