• 생명과학회지
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• 제27권12호
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• pp.1393-1402
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• 2017

식물에서, 칼슘-의존적 단백질 카이네즈(CDPKs)는 $Ca^{2+}$ 신호전달에서 중요한 $Ca^{2+}$ 수용체이다. 벼(Oryza sativa L.)의 CDPKs인 3개의 OsCPKs는 생물정보에 대한 분석이 이루어졌으나, OsCPK11 유전자는 연구가 완전히 수행되지 않았다. 다양한 조직에서 OsCPK11 유전자가 전사수준에서 발현한다는 것은 알려져 있으나, 단백질 수준에서 발현과 생화학적인 특징은 잘 알려져 있지 않다. 이 연구는 OsCPK11의 몇 가지 생화학적 특징을 알아보기 위해 이루어졌다. 먼저 in vitro에서 E. coli를 이용하여 GST-OsCPK11를 발현시키고, 카이네즈 활성 측정과 칼슘-의존적 단백질 카이네즈로서 OsCPK11의 생화학적 분석도 수행하였다. OsCPK11은 스스로 자가인산화하며, $Ca^{2+}$의 존재 하에서 기질로서 histone III-s와 MBP로 인산기 전달 작용을 수행한다. 재조합 OsCPK11의 활성은 $Mg^{2+}$에 의해 영향을 받으며, pH 7.0-7.5에서 최적의 활성을 보인다. 또한 OsCPK11의 활성은 높은 수준의 $Ca^{2+}$가 존재하는 조건에서는 $Mg^{2+}$, $Mn^{2+}$, $Na^+$의 영향을 받지 않는다. 또한 OsCPK11의 자가인산화는 OsCPK11의 $Ca^{2+}$ 민감도를 감소시키는 것으로 밝혀졌다. 마지막으로, OsCPK11의 N-말단 다양화 지역으로 토끼 항체를 만들었고, immunoblot을 기초로 polyclonal antibody는 95.5 kD의 GST-OsCPK11를 인식하는 것으로 나타났다. 이 결과는 벼의 $Ca^{2+}$ 매개 신호전달에서 OsCPK11의 기능을 더 잘 이해하는데 도움을 줄 것이며, 심화 연구를 위해 다양한 OsCPKs의 단백질 정보를 결정하는 것이 필요할 것이다.

• 대한약침학회지
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• 제19권1호
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• pp.59-69
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• 2016

Objectives: The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, $H_2O_2$) and elucidated the underlying mechanisms in a C2C12 myoblast cell line. Methods: Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS) generation and deoxyribonucleic acid (DNA) damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and $H_2O_2$ in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering ribonucleic acid (siRNA) was conducted to understand the relationship between Nrf2 expression and $H_2O_2$-induced growth inhibition. Results: The results showed that EGL effectively inhibited $H_2O_2$-induced growth and the generation of ROS. EGL markedly suppressed $H_2O_2$-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 ($p-{\gamma}H2AX$), a widely used marker of DNA damage, suggesting that EGL prevented $H_2O_2$-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1), with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against $H_2O_2$-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner. Conclusion: Collectively, these results demonstrate that EGL augments the cellular anti-oxidant defense capacity through activation of Nrf2/HO-1, thereby protecting C2C12 myoblasts from $H_2O_2$-induced oxidative cytotoxicity.

• 한국동물생명공학회지
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• 제34권3호
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• pp.197-204
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• 2019

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

• 대한의생명과학회지
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• 제19권3호
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• pp.266-269
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• 2013

Hox genes encode transcription factors important for anterior-posterior body patterning at early stages of embryonic development. However, the precise mechanisms by which signal pathways are stimulated to regulate Hox gene expression are not clear. In the previous study, protein kinase B alpha (Akt1) has been identified as a putative upstream regulator of Hox genes, and Akt1 has shown to regulate Gcn5, a prototypical histone acetyltransferase (HAT), in a negative way in mouse embryonic fibroblast (MEF) cells. Since the activity of HAT such as the CBP/p300, and PCAF (a Gcn5 homolog), was down-regulated by Akt through a phosphorylation at the Akt consensus substrate motif (RXRXXS/T), the amino acid sequence of Gcn5 protein was analyzed. Mouse Gcn5 contains an Akt consensus substrate motif as RQRSQS sequence while human Gcn5 does not have it. In order to see whether Akt1 directly binds to Gcn5, immunoprecipitation with anti-Akt1 antibody was carried out in wild-type (WT) mouse embryonic fibroblast (MEF) cells, and then western blot analysis was performed with anti-Akt1 and anti-Gcn5 antibodies. Gcn5 protein was detected in the Akt1 immunoprecipitated samples of MEFs. This result demonstrates that Akt1 directly binds to Gcn5, which might have contributed the down regulation of the 5' Hoxc gene expressions in wild type MEF cells.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 춘계학술발표회
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• pp.68-68
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• 2018

In this study, we investigated whether S. baicalensis rhizome ethanol extract (SBRE) has antioxidant capacities against oxidative stress induced cellular damage in the HaCaT keratinocytes. Our results revealed that treatment with SBRE prior to hydrogen peroxide ($H_2O_2$) exposure significantly increased the HaCaT cell viability. SBRE also effectively attenuated $H_2O_2$ induced comet tail formation, and inhibited the $H_2O_2$ induced phosphorylation levels of the histone ${\gamma}H2AX$, as well as the number of apoptotic bodies and Annexin V positive cells. In addition, SBRE exhibited scavenging activity against intracellular ROS generation and restored the mitochondria membrane potential loss induced by $H_2O_2$. Moreover, $H_2O_2$ enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with SBRE. Furthermore, SBRE increased the levels of HO-1 associated with the induction of Nrf2. Therefore, we believed that SBRE may potentially serve as an agent for the treatment and prevention of neurodegenerative diseases caused by oxidative stress.

• Journal of Ginseng Research
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• 제46권3호
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• pp.481-488
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• 2022

Background: Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods: Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results: Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion: Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

• 생명과학회지
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• 제30권12호
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• pp.1092-1100
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• 2020

Six-transmembrane epithelial antigen of prostate 4 (Steap4)는 철과 구리를 환원하여 철과 구리의 세포내 유입에 관여하는 금속 환원효소로, 구리 철의 항상성 뿐만 아니라 염증, 포도당 대사, 지질 대사에도 중요한 역할을 한다. 최근에 Steap4가 지방세포의 분화를 촉진한다는 보고가 발표되었으나, 이에 관련된 분자적 기전에 대해서는 알려지지 않았다. 그래서, 본 연구에서는 Steap4에 의한 지방세포분화 촉진에 관련된 기전을 연구하였다. 이를 위해 3T3-L1 백색지방세포, 불멸화된 갈색지방세포(iBA) 및 생쥐의 배아 섬유아 세포인 C3H10T1/3 세포에서 Steap4을 감소시킨 후 지방세포분화 초기단계에 관련된 신호들을 분석하였다. Steap4을 shRNA로 감소시켰을 때 지방세포분화 초기 단계에서 3종류 지방세포의 세포 증식이 억제되었으며, 세포주기 관련 단백질인 cyclin A, cyclin D 그리고 cdk2의 발현은 감소하는 반면 세포주기 저해 단백질인 p21과 p27의 발현은 증가하였다. 또한 세포주기 관련 신호인 p38, ERK 그리고 Akt의 활성화는 억제되었다. 한편 지방세포분화 초기 단계에 관여하는 지방세포분화 전사인자들을 분석하였을 때, Steap4의 감소는 지방세포분화 활성 전사 인자인 C/EBPβ, KLF4의 발현을 저해하는 반면, 지방세포분화 억제 전사 인자인 KLF2, KLF3 그리고 GATA2의 발현은 증가시켰다. 또한 Steap4의 과발현은 C/EBPβ promoter에 존재하는 전사억제 히스톤 표지자인 H3K9me2과 H3K27me3을 감소시켰다. 따라서, 이상의 결과를 종합하면 Steap4는 지방세포분화 초기단계인 mitotic clonal expansion을 촉진하고 지방세포분화 전사인자들의 발현을 조절함으로써, 지방세포분화를 촉진시킴을 알 수 있었다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.103-103
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• 2018

Oenothera biennis, commonly known as evening primrose, a potential source of natural bioactive substances: flavonoids, steroids, tannins, fatty acids and terpenoids responsible for a diverse range of pharmacological functions. However, whether extract prepared from aerial part of O. biennis (APOB) protects skin against oxidative stress remains unknown. To investigate the protective effects of APOB against oxidative stress-induced cellular damage and elucidated the underlying mechanisms in the HaCaT human skin keratinocytes. Our results revealed that treatment with APOB prior to hydrogen peroxide ($H_2O_2$) exposure significantly increased viability, and the highest DPPH radical-scavenging activities and reducing power of HaCaT cells. APOB also effectively attenuated H2O2-induced comet tail formation and inhibited the $H_2O_2$-induced phosphorylation levels of the histone ${\gamma}H2AX$, as well as the number of apoptotic bodies and Annexin V-positive cells. In addition, APOB exhibited scavenging activity against intracellular reactive oxygen species (ROS) accumulation and restored the mitochondrial membrane potential loss by $H_2O_2$. Moreover, $H_2O_2$ enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase (PARP), a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with APOB. Furthermore, APOB increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, APOB is able to protect HaCaT cells from $H_2O_2$-induced DNA damage and cell death through blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating the Nri2/HO-1 signaling pathway.

• 대한한의학방제학회지
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• 제25권1호
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• pp.51-68
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• 2017

Objectives : Oxidative stress due to excessive accumulation of reactive oxygen species (ROS) is one of the risk factors for the development of several chronic diseases, including neurodegenerative diseases. Methods : In the present study, we investigated the protective effects of cheongnoemyeongsin-hwan (CNMSH) against oxidative stress‑induced cellular damage and elucidated the underlying mechanisms in neuronal-derived SH-SY5Y cells. Results : Our results revealed that treatment with CNMSH prior to hydrogen peroxide (H2O2) exposure significantly increased the SH-SY5Y cell viability, indicating that the exposure of the SH-SY5Y cells to CNMSH conferred a protective effect against oxidative stress. CNMSH also effectively attenuated H2O2‑induced comet tail formation, and decreased the phosphorylation levels of the histone ${\gamma}H2AX$, as well as the number of apoptotic bodies and Annexin V‑positive cells. In addition, CNMSH exhibited scavenging activity against intracellular ROS generation and restored the mitochondria membrane potential (MMP) loss that were induced by H2O2, suggesting that CNMSH prevents H2O2‑induced DNA damage and cell apoptosis. Moreover, H2O2 enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with CNMSH. Furthermore, CNMSH increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, CNMSH is able to protect SH-SY5Y cells from H2O2-induced apoptosis throughout blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating Nrf2/HO-1 signaling pathway. Conclusions : Therefore, we believed that CNMSH may potentially serve as an agent for the treatment and prevention of neurodegenerative diseases caused by oxidative stress.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.73-81
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• 2023

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.