• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.778-783
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• 2008

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be down regulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.

• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.97-97
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• 1997

It was reported that ATP depletion occurs and accelerates cell damage during ischemia and reperfusion. To determine the mechanism of cell damage, the change of energy metabolism in liver was studied during ischemia/reperfusion. The groups were divided into four categories : sham-operated group, ischemia/reperfusion group, and two types of ATP-MgCl$_2$ treatment groups(one was treated during ischemia and the another during reperfusion). Rats were administered intravenously saline or ATP-MgCl$_2$. Rats were anesthetized and blood vessels in the left and median lobes of the liver were occluded. After 60min of ischemia, the clamp at those vessels were removed. After ischemia, one and five hours after reflow, energy metabolites(ATP, ADP, AMP, inosine, adenosine, hypoxanthine, xanthine) in liver were measured with HPLC. To observe mitochondrial function, aterial keton body ratio in blood and mitochondrial glutamate dehydrogenase activity in liver were measured. And lipid peroxidation was measured to evalutate the involvement of free radicals. In this study, ATP and ADP were catabolized to their metabolites(AMP, inosine, adenosine, hypoxanthine, xanthine) during ischemia and they resynthesized ATP and ADP during reperfusion. But total purine base were not restored to level of normal rat. The main source of resynthesizing ATP and ADP was AMP. In both ATP-MgCl$_2$ treated groups, mitochondrial function was protected and lipid peroxidation was significantly reduced. Our findings suggest that ischemia/reperfusion impairs hepatic energy metabolism.

• The Korean Journal of Mycology
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• v.17 no.2
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• pp.99-104
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• 1989

Effects of organic compounds, photosensitizers and influx of metal ions on the light-induced mitochondrial ATP synthase in Lentinus edodes purified by stepped sucrose density gradient centrifugation were studied. In our previous work, the activation wavelength and the illumination time of mitochondrial ATP synthase were 470 nm and 15 sec, respectively. This enzyme was activated 85% by 1 mmole 2,6-dichlorophenol indopheol and inhibited by 1 mmole 2,-4-dinitrophenol, $10\;{\mu}mole$ 2-heptyl-4-hydroxyquinoline-N-oxide and $100\;{\mu}g$ oligomycin per ml of ethanol. Particularly, the enzyme was activated 414% by 10 mmole phenazine methosulfate as photosensitizer at 470 nm light. In the influx effects of $Fe^{3+}$ and $Fe^{2+}$ ion, the activity of the above enzyme increased under the optimal light condition compared with nonillumination state.

• The Korean Journal of Mycology
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• v.19 no.1
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• pp.32-40
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• 1991

Mitochondria in Pleurotus ostreatus were isolated and purified by the stepped sucrose density gradient centrifugation, to investigate the effects of the light on the enzymatic activity of the mitochondrial ATP synthase. This enzyme, which was illuminated by the light ranging from 400 nm to 700 nm, showed that the specific activity was stimulated at 490 nm for 15 sec. Effects of organic compounds on the mitochondrial ATP synthase were also investigated at the optimum conditions; The activities of this enzyme were increased to 168 percent by the addition of 2,6-dichlo­rophenol indophenol(DCPIP), 224 percent by phenazine methosulfate(PMS), but inhibited 91 per­cent by oligomycin, 14 percent by 2-heptyl-4-hydroxyquinoline-N-oxide(HQNO) and 75 percent by 2,4-dinitrophenol (DNP), respectively. Effects of metal ions of the mitochondrial ATP synthase were investigated at the optimum conditions. The activities of the enzyme were inhibited 35 percent by $Ca^{2+}$, 14 percent by $Co^{2+}$ and 73 percent by $Mn^{2+}$. For effects of anions, the activities of this enzyme were inhibited 80 percent by $CN^{-}$, 52 percent by $SO_{4}\;^{2-}$, 28 percent by each of $CO_{3}\;^{2-}$­and $NO_{3}\;^{-}$, respectively.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.4
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• pp.189-194
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• 2011

ATP-sensitive $K^+$ channels ($K_{ATP}$) are major component of preventing ischemia-reperfusion injury. However, there is little information regarding to the expressional difference of $K_{ATP}$ and its function between left and right ventricles. In this study, we measured the lactate dehydrogenase release of rabbit heart slices in vitro and determined the difference of the $K_{ATP}$ expression at the both ventricles by measuring the level of $K_{ATP}$-forming Kir6.2 (OcKir6.2) mRNA using in situ hybridization. The hearts were preconditioned with 15 min hypoxia and reoxygenated for 15 min before a hypoxic period of 60 min, followed by reoxygenation for 180 min. With hypoxic preconditioning (100% $N_2$) with 15 min, left ventricles (LV) showed higher release of LDH comparing with right ventricles (RV). Adding $K_{ATP}$ blocker glibenclamide ($10{\mu}M$) prior to a hypoxic period of 60 min, hypoxic preconditioning effect of RV was more abolished than LV. With in situ hybridization, the optical density of OcKir6.2 was higher in RV. Therefore, we suggest that different $K_{ATP}$ expression between LV and RV is responsible for the different response to hypoxia and hypoxic preconditioning of rabbit hearts.

• Investigative Magnetic Resonance Imaging
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• v.2 no.1
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• pp.89-95
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• 1998

Purpose : To investigate the phosphorus metabolic abnormalities in skeletal muscle of patients with mitochondrial myopathy using in vivo $^{31}P$ magnetic resonance spectroscopy(MRS). Materials and Methods : Patients with mitochondrial myopathy(N=10) and normal control subjects (N=10) participated. All in vivo $^{31}P$ MRS examinations were performed on 1.5T whole-body MRI/MRS system by using an image selected in vivo spectroscopy (ISIS) pulse sequence that provided a $4{\times}4{\times}4{\;}cm^{3}$ volume of interest (VOI) in the right thigh muscle tissue. Peak areas for each phophorus methabolite were measured using a Marquart algorithm. Results : The specific features in patients with mitochondrial myopathy were a significant increase of Pi/PCr ratio (p=0.003) and a significant decrease of ATP/PCr ratio (p=0.004) as compared with normal controls. In particular, the ${\beta}-ATP/PCr$ ratio between controls and patients with mitochondrial myopathy was predominantly altered. Conclusions : In vivo $^{31}P$ MRS may be a useful modality in the clinical evaluation of patients with mitochondrial myopathy based on ATP/PCr and Pi/PCr ratios in skeletal muscle tissue and provides a valuable information in further understanding disorders of muscle metabolism.

• BMB Reports
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• v.50 no.4
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• pp.214-219
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• 2017

Mitochondria play pivotal roles in the ATP production, apoptosis and generation of reactive oxygen species. Although dynamic regulation of mitochondria morphology is a critical step to maintain cellular homeostasis, the regulatory mechanisms are not yet fully elucidated. In this study, we identified miR-200a-3p as a novel regulator of mitochondrial dynamics by targeting mitochondrial fission factor (MFF). We demonstrated that the ectopic expression of miR-200a-3p enhanced mitochondrial elongation, mitochondrial ATP synthesis, mitochondrial membrane potential and oxygen consumption rate. These results indicate that miR-200a-3p positively regulates mitochondrial elongation by downregulating MFF expression.

• Animal Bioscience
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• v.34 no.12
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• pp.1912-1920
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• 2021

Objective: This study investigated the effects of 5-aminolevulinic acid (5-ALA) on the motility parameters, mitochondrial membrane depolarization, and ATP levels in chicken sperm. Methods: The pooled semen from Barred Plymouth Rock males was used. In the first experiment, the semen was diluted 4-times with phosphate-buffered saline (PBS (-)) containing various concentrations (0, 0.01, 0.05, and 0.1 mM) of 5-ALA, and then the sperm motility parameters after incubation were evaluated by computer-assisted sperm analysis (CASA). In the second experiment, the semen was diluted 4-times with PBS (-) containing 0.05 mM 5-ALA, and then sperm mitochondrial membrane depolarization and ATP levels after 1.5 h of incubation were analyzed with the MitoPT^® JC-1 Assay and ATP Assay kits, respectively. In the third experiment, the semen was removed from the seminal plasma and resuspended with the mediums of PBS (-), PBS (-) supplemented with CaCl₂ and MgCl₂ (PBS (+)) + 5-ALA, PBS (+) + caffeine, and PBS (+) + caffeine + 5-ALA. Then, the sperm motility parameters after incubation were evaluated by CASA. In the last experiment, the semen was treated with the mediums of PBS (-), PBS (-) + 5-ALA, 5.7% glucose, 5.7% glucose + 5-ALA after removing the seminal plasma, and then the sperm motility parameters were evaluated by CASA. Results: The addition of 0.05 mM 5-ALA significantly increased the chicken sperm motility, progressive motility, linearity, average path velocity, curvilinear velocity, straight-line velocity, and the wobble. The sperm mitochondrial membrane depolarization was also increased by the 5-ALA treatment. The 5-ALA treatment decreased the sperm ATP levels. Both the caffeine treatment and glucose treatment decreased the sperm motility during incubation period. Conclusion: 5-ALA might increase sperm mitochondrial membrane depolarization to utilize the ATP for enhancing sperm movement.

• BMB Reports
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• v.41 no.2
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• pp.153-157
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• 2008

The objective of the present study was to identify mitochondrial components associated with the damage caused by iron to the rat heart. Decreased cell viability was assessed by increased presence of lactate dehydrogenase (LDH) in serum. To assess the functional integrity of mitochondria, Reactive Oxygen Species (ROS), the Respiratory Control Ratio (RCR), ATP and chelatable iron content were measured in the heart. Chelatable iron increased 15-fold in the mitochondria and ROS increased by 59%. Deterioration of mitochondrial function in the presence of iron was demonstrated by low RCR (46% decrease) and low ATP content (96% decrease). Using two dimensional gel electrophoresis (2DE), we identified alterations in 21 mitochondrial proteins triggered by iron overload. Significantly, expression of the $\alpha$, $\beta$, and d subunits of $F_1F_o$ ATP synthase increased along with the loss of ATP. This suggests that the $F_1F_o$ ATP synthase participates in iron metabolism.