• BMB Reports
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• 제38권6호
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• pp.690-694
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• 2005

Transcription termination of the human mitochondrial genome requires specific binding to termination factor mTERF. In this study, mTERF was produced in E. coli and purified by two-step chromatography. mTERF-binding DNA sequences were isolated from a pool of randomized sequences by the repeated selection of bound sequences by gel-mobility shift assay and polymerase chain reaction. Sequencing and comparison of the 23 isolated clones revealed a 16-bp consensus sequence of 5'-GTG$\b{TGGC}$AGANCCNGG-3' in the light-strand (underlined residues were absolutely conserved), which nicely matched the genomic 13-bp terminator sequence 5'-$\b{TGGC}$AGAGCCCGG-3'. Moreover, mTERF binding assays of heteroduplex and single-stranded DNAs showed mTERF recognized the light strand in preference to the heavy strand. The preferential binding of mTERF with the light-strand may explain its distinct orientation-dependent termination activity.

• BMB Reports
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• 제53권1호
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• pp.3-9
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• 2020

The mitochondrial genome encodes 13 proteins that are components of the oxidative phosphorylation system (OXPHOS), suggesting that precise regulation of these genes is crucial for maintaining OXPHOS functions, including ATP production, calcium buffering, cell signaling, ROS production, and apoptosis. Furthermore, heteroplasmy or mis-regulation of gene expression in mitochondria frequently is associated with human mitochondrial diseases. Thus, various approaches have been developed to investigate the roles of genes encoded by the mitochondrial genome. In this review, we will discuss a wide range of techniques available for investigating the mitochondrial genome, mitochondrial transcription, and mitochondrial translation, which provide a useful guide to understanding mitochondrial gene expression.

• Journal of Radiation Protection and Research
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• 제36권3호
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• pp.119-126
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• 2011

Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated ${\beta}$-galactosidase (SA-${\beta}$-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and $H_2O_2$-treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and $H_2O_2$-treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-${\beta}$-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

• Nutrition Research and Practice
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• 제16권2호
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• pp.147-160
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• 2022

BACKGROUND/OBJECTIVES: Patients with chronic kidney disease (CKD) have a high concentration of uremic toxins in their blood and often experience muscle atrophy. Indoxyl sulfate (IS) is a uremic toxin produced by tryptophan metabolism. Although an elevated IS level may induce muscle dysfunction, the effect of IS on physiological concentration has not been elucidated. Additionally, the effects of ursolic acid (UA) on muscle hypertrophy have been reported in healthy models; however, it is unclear whether UA ameliorates muscle dysfunction associated with chronic diseases, such as CKD. Thus, this study aimed to investigate whether UA can improve the IS-induced impairment of mitochondrial biogenesis. MATERIALS/METHODS: C2C12 cells were incubated with or without IS (0.1 mM) and UA (1 or 2 μM) to elucidate the physiological effect of UA on CKD-related mitochondrial dysfunction and its related mechanisms using real-time reverse transcription-polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay. RESULTS: IS suppressed the expression of differentiation marker genes without decreasing cell viability. IS decreased the mitochondrial DNA copy number and ATP levels by downregulating the genes pertaining to mitochondrial biogenesis (Ppargc1a, Nrf1, Tfam, Sirt1, and Mef2c), fusion (Mfn1 and Mfn2), oxidative phosphorylation (Cycs and Atp5b), and fatty acid oxidation (Pdk4, Acadm, Cpt1b, and Cd36). Furthermore, IS increased the intracellular mRNA and secretory protein levels of interleukin (IL)-6. Finally, UA ameliorated the IS-induced impairment in C2C12 cells. CONCLUSIONS: Our results indicated that UA improves the IS-induced impairment of mitochondrial biogenesis by affecting differentiation, ATP levels, and IL-6 secretion in C2C12 cells. Therefore, UA could be a novel therapeutic agent for CKD-induced muscle dysfunction.

• International Journal of Oral Biology
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• 제43권2호
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• pp.69-76
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• 2018

Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancy. Numerous therapies have been proposed for its cure. Research is continually being conducted to develop new forms of treatment as current therapies are associated with numerous side-effects. Luteolin, a common dietary flavonoid, has been demonstrated to possess strong anti-cancer activity against various human cancer cell lines. Nevertheless, research into luteolin-based anticancer activity against oral cancer remains scarce. Thus, the objective of this study was to assess the effect of luteolin as an anti-cancer agent. After treatment with luteolin, Ca9-22 and CAL-27 oral cancer cells showed condensed nuclei and enhanced apoptotic rate with evidence of mitochondria-mediated apoptosis. Epithelialmesenchymal transition (EMT) is closely related to tumor migration and invasion. Luteolin suppressed cancer cell invasion and migration in the current study. Elevated expression of E-cadherin, an adherens junction protein, was evident in both cell lines after luteolin treatment. Luteolin also significantly inhibited transcription factors (i.e., N-cadherin, Slug, Snail, Twist, and ZEB-1) that regulated expression of tumor suppressors such as E-cadherin based on Western blot analysis and quantitative PCR. Thus, luteolin could induce mitochondrial apoptosis and inhibit cancer cell invasion and migration by suppressing EMT-induced transcription factors.

• The Korean Journal of Physiology and Pharmacology
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• 제26권5호
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• pp.357-365
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• 2022

Simultaneous myofibril and mitochondrial development is crucial for the cardiac differentiation of pluripotent stem cells (PSCs). Specifically, mitochondrial energy metabolism (MEM) development in cardiomyocytes is essential for the beating function. Although previous studies have reported that MEM is correlated with cardiac differentiation, the process and timing of MEM regulation for cardiac differentiation remain poorly understood. Here, we performed transcriptome analysis of cells at specific stages of cardiac differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected MEM genes strongly upregulated at cardiac lineage commitment and in a time-dependent manner during cardiac maturation and identified the protein-protein interaction networks. Notably, MEM proteins were found to interact closely with cardiac maturation-related proteins rather than with cardiac lineage commitment-related proteins. Furthermore, MEM proteins were found to primarily interact with cardiac muscle contractile proteins rather than with cardiac transcription factors. We identified several candidate MEM regulatory genes involved in cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpα, and Cdkn2a in mESC-derived cells, and CCK and NOS3 in hiPSC-derived cells) and cardiac maturation (Ppargc1α, Pgam2, Cox6a2, and Fabp3 in mESC-derived cells, and PGAM2 and SLC25A4 in hiPSC-derived cells). Therefore, our findings show the importance of MEM in cardiac maturation.

• KSBB Journal
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• 제20권5호
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• pp.363-370
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• 2005

중추신경계의 신경간세포가 파킨슨병과 뇌졸중과 같은 퇴행성 뇌 질환의 치료뿐만이 아니라 신경세포 발생과정에서의 중요성 때문에 최근에 커다란 관심의 대상이 되고 있다. 중추신경계의 발생과정 동안에, 중뇌의 도파민 신경세포의 형성은 두 가지의 분자생물학적인 기작에 의해서 결정된다. 첫째로, FGF-8, sonic hedgehog 그리고 전사조절인자 인 Nurr1이 도파민 신경세포의 형질을 결정짓는다. 또 다른 기작으로는, 전사조절인자 인 $Lm{\times}lb$와 $Pt{\times}3$가 중요하게 관련되어있다. 특히 Nurr1이 결핍된 생쥐에서, 타이로신수산화효소 (Tyrosine bydroxylase, TH) 면역양성 세포들이 중뇌흑색질에서 발견되지 않으므로 Nurr1이 도파민 신경세포의 발생에 필수적임을 알 수 있다. 본 연구에서는 도파민 신경세포의 형질을 유도하는데 있어서 Nurr1이 매개하는 기작을 연구하기 위해서 레트로 바이러스를 이용하여 Nurr1을 도입한 무한증식 신경간세포를 사용하였다. Nurr1 유전자의 과발현 만으로는 신경간세포에서 도파민 신경세포의 형질을 유도하지는 못하지만, 레티노이드 (retinoid, RA)와 폴스콜린 (forskolin, FK)을 처리하여 TH와 방향성 L-아미노산 탈카르복시화효소 (aromatic L-amino acid decarboxylase, AADC) mRNA의 발현을 유도하였다. 또한, Nurr1 과발현 신경간세포를 사람 별아교세포와 공동배양 하여 TH 발현량을 많이 증가시켰다. 이러한 공동배양실험에서, RA와 FK를 처리하면 TH의 발현수준이 더욱 더 증가함을 발견하였다. 이러한 결과들은 Nurr1 유전자를 도입한 사람 신경간세포가 파킨슨병 환자들에게 세포이식을 통한 유전자 치료의 유용성을 시사하고 있다.

• Animal cells and systems
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• 제13권4호
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• pp.381-389
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• 2009

Using the DDRT-PCR, a series of differentially expressed genes in human primary cervical cancer was isolated. Among the 250 PCR amplimers, 88 gene fragments were confirmed by reverse Northern hybridization. Homology searches indicated that 26 out of 88 were previously known genes including calmodulin, human BBC1, histone H3.3, a series of ribosomal proteins (RPL19, RPS19, and RPS12), translation initiation factor (eIF-4AI), lactoferrin, integrin ${\alpha}6$, cell-surface antigens (CD9 and CD59), transcription factor (mbp-1), and mitochondrial proteins. Several unknown clones showed sequence homology with known genes. Furthermore, six of the unknown genes showed identical sequence with expressed sequence tags (EST) of unknown function. Differential expression patterns of identified genes were further examined and confirmed with multiple pairs of cervical cancer samples using Northern hybridization. Our profiling of differentially expressed genes may provide useful information about the underlying genetic alterations in human cervical carcinoma and diagnostic markers for this disease. The precise roles of these genes in cancer development remain to be elucidated.

• Molecules and Cells
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• 제39권7호
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• pp.543-549
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• 2016

MicroRNAs (miRNAs) have been reported to be involved in many neurodegenerative diseases. The present study focused on the role of hsa-miR-144-3p in one of the neuro-degenerative diseases, Parkinson's disease (PD). Our study showed a remarkable down-regulation of miR-144-3p expression in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated SH-SY5Y cells. MiR-144-3p was then overexpressed and silenced in human SH-SY5Y cells by miRNA-mimics and miRNA-inhibitor transfections, respectively. Furthermore, ${\beta}$-amyloid precursor protein (APP) was identified as a target gene of miR-144-3p via a luciferase reporter assay. We found that miR-144-3p overexpression significantly inhibited the protein expression of APP. Since mitochondrial dysfunction has been shown to be one of the major pathological events in PD, we also focused on the role of miR-144-3p and APP in regulating mitochondrial functions. Our study demonstrated that up-regulation of miR-144-3p increased expression of the key genes involved in maintaining mitochondrial function, including peroxisome proliferator-activated receptor ${\gamma}$ coactivator-$1{\alpha}$(PGC-$1{\alpha}$), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM). Moreover, there was also a significant increase in cellular ATP, cell viability and the relative copy number of mtDNA in the presence of miR-144-3p overexpression. In contrast, miR-144-3p silencing showed opposite effects. We also found that APP overexpression significantly decreased ATP level, cell viability, the relative copy number of mtDNA and the expression of these three genes, which reversed the effects of miR-144-3p overexpression. Taken together, these results show that miR-144-3p plays an important role in maintaining mitochondrial function, and its target gene APP is also involved in this process.

• Journal of Preventive Medicine and Public Health
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• 제45권4호
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• pp.235-243
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• 2012

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.