• Reproductive and Developmental Biology
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• 제30권2호
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• pp.143-156
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• 2006

Major characteristics of embryonic stem cells (ESCs) are sustaining of sternness and pluripotency by self-renewal. In this report, transcriptional profiles of the molecules in the developmentally important signaling pathways including Wnt, BMP4, $TGF-\beta$, RTK, Hh, Notch, and JAK/STAT signaling pathways were investigated to understand the self-renewal of mouse ESCs (mESCs), J1 line, and compared with the NIH3T3 cell line and mouse embryonic fibroblast (MEF) cells as controls. In the Wnt signaling pathway, the expression of Wnt3 was seen widely in mESCs, suggesting that the ligand may be an important regulator for self-renewal in mESCs. In the Hh signaling pathway, the expression of Gli and N-myc were observed extensively in mESCs, whereas the expression levels of in a Shh was low, suggesting that intracellular molecules may be essential for the self-renewal of mESCs. IGF-I, IGF-II, IGF-IR and IGF-IIR of RTK signaling showed a lower expression in mESCs, these molecules related to embryo development may be restrained in mESCs. The expression levels of the Delta and HESS in Notch signaling were enriched in mESCs. The expression of the molecules related to BMP and JAK-STAT signaling pathways were similar or at a slightly lower level in mESCs compared to those in MEF and NIH3T3 cells. It is suggested that the observed differences in gene expression profiles among the signaling pathways may contribute to the self-renewal and differentiation of mESCs in a signaling-specific manner.

• Reproductive and Developmental Biology
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• 제31권3호
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• pp.215-220
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• 2007

본 연구는 소 배반포의 내부 세포괴로부터 다능성(pluripotency)을 지닌 배아 줄기 세포(embryonic stem cell) 또는 그 유사 세포를 분리 및 배양함으로써 줄기 세포 관련 분야의 기반 기술을 확립하고자 하였다. 소 체외수정란을 $10{\sim}12$일간 체외배양하여 생산된 부화 배반포를 세포분열이 불활성화된 생쥐 태아 섬유아 세포(mouse embryonic fibroblast, MEF) 위에서 배양하여 콜로니 형성을 유도하였으며, 이들로부터 내부 세포괴 유래의 형태를 지닌 것만을 광학현미경 하에서 물리적으로 분리하여 약 $5{\sim}7$일 간격으로 계대배양을 실시하였다. 이러한 방법을 통하여 배아 줄기 유사 세포의 특성을 40계대 이상 유지하는 2개의 세포주를 확립하였다. 각각의 세포주들은 높은 alkaline phosphatase(AP) 활성을 지니고 있었으며, 형광 면역 염색법과 PCR 기법을 사용하여 Oct-4, Nanog, STAT3, SSEA3 및 SSEA4의 발현을 관찰할 수 있었다. 이러한 결과를 종합하여 볼 때, 본 연구에서는 소 배반포로부터 배아 줄기 세포주를 확립하는 제반 기술이 확립되었다고 판단되며, 향후 관련 분야 연구에 활용될 수 있을 것으로 기대된다.

• The Korean Journal of Physiology and Pharmacology
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• 제8권1호
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• pp.33-41
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• 2004

We developed a cardiac cell model to explain the phenomenon of mechano-electric feedback (MEF), based on the experimental data with rat atrial myocytes. It incorporated the activity of ion channels, pumps, exchangers, and changes of intracellular ion concentration. Changes in membrane excitability and $Ca^{2+}$ transients could then be calculated. In the model, the major ion channels responsible for the stretch-induced changes in electrical activity were the stretch-activated channels (SACs). The relationship between the extent of stretch and activation of SACs was formulated based on the experimental findings. Then, the effects of mechanical stretch on the electrical activity were reproduced. The shape of the action potential (AP) was significantly changed by stretch in the model simulation. The duration was decreased at initial fast phase of repolarization (AP duration at 20% repolarization level from 3.7 to 2.5 ms) and increased at late slow phase of repolarization (AP duration at 90% repolarization level from 62 to 178 ms). The resting potential was depolarized from -75 to -61 mV. This mathematical model of SACs may quantitatively predict changes in cardiomyocytes by mechanical stretch.

• Toxicological Research
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• 제24권3호
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• pp.175-182
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• 2008

DNA-dependent protein kinase(DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be $2^{nd}$ upstream kinase for protein kinase B(PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells(MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK(M059J) and a wild-type of DNA-PK(M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

• 한국동물학회지
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• 제30권3호
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• pp.311-323
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• 1987

생쥐의 섬 유아세포(MEP)와 종양세포(SCK)를 이용하여 정상세포와 종양세포 사이에 열 감수성의 차이가 있는지의 여부 및 환경의 pH가 이 세포들의 열감수성과 heat shock protein(HSP) 합성에 미치는 영향을 생존곡선과 HSP합성 kinetics등을 써서 검토하였다. MEF와 SCK 세포를 정상 pH(7.4) 또는 산성 pH(6.7)에서 42"C에서 2시간 온열처리 후 3일간에 걸쳐 생존을을 비교해 븐 결과, ME선와 SCK세포 사이에 생득적 열강수성의 차이는 없었고 산성 P광에서는 세포의 종류에 관계없이 열감수성 이 증감되었다. 온열처리의 결과 유도되는 내일성이 conditioning Leat의 크기와 어떤 관계가 있는지를 보기 위해서 45"C에서 5분 또는 20분을 주어본 결과 체은 conditioning heat를 주었을 때 내일성이 신속히 그리고 높은 수준으로 발생하였고, 이러한 열 감수성의 kinetics는 HSP의 합성 kinetics와 잘 일치하였다. 단백질, 특히 HSP 합성에 미치는 PH의 영 향을 알아보기 위해서 46"C에서 6분간의 heat shock를 주어 본 바 전반적인 단백질 및 major HSP의 합성양상에는 별로 차이를 보이지 않았다. 그러나 SCK 세포에 43"C에서 30분의 온열처리를 주고 새로 합성되는 HSPSP의 kinetics를 검토해 본 결과 정상 P반에서는 0-5시간에 합성이 일어나나 산성 PH에서는 3-9시간에 합성이 일어나서 몇시간의 합성지연이 관찰되었다. 아울러 HSP68, HSPTC, HSP87을 Peptidemapping하여 본 결과 HSP68과 HSP70 은 유사한 peptide fragment pattern을 보여 amino acid sequence는 유사하고 기능도 같을 것으로 추론되었으나 HSP87은 전혀 다른 pattern을 보였다. 전혀 다른 pattern을 보였다.

• 대한의생명과학회지
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• 제25권2호
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• pp.177-184
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• 2019

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). LRRK2 contains a functional kinase and GTPase domains. A pathogenic G2019S mutation that is the most prevalent among the LRRK2 mutations and is also found in sporadic cases, increases its kinase activity. Therefore, identification of LRRK2 kinase substrates and the development of kinase inhibitors are under intensive investigation to find PD therapeutics. Several recent studies have suggested members of Rab proteins, a branch of the GTPase superfamily, as LRRK2 kinase substrates. Rab proteins are key regulators of cellular vesicle trafficking. Among more than 60 members of human Rab proteins, Rab3, Rab5, Rab8, Rab10, Rab12, Rab29, Rab35, and Rab43 have been identified as LRRK2 kinase substrates. However, most studies have used human embryonic kidney (HEK) 293T cells overexpressing LRRK2/Rab proteins or murine embryonic fibroblast (MEF) cells which are not relevant to PD, rather than neuronal cells. In this study, we tested whether Rab proteins are phosphorylated by LRRK2 in astroglia in addition to neurons. Among the various Rab substrates, we tested phosphorylation of Rab10, because of the commercial availability and credibility of the phospho-Rab10 (pRab10) antibody, in combination with a specific LRRK2 kinase inhibitor. Based on the results of specific LRRK2 kinase inhibitor treatment, we concluded that LRRK2 phosphorylates Rab10 in the tested brain cells such as primary neurons, astrocytes and BV2 microglial cells.

• Genomics & Informatics
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• 제10권1호
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• pp.16-22
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• 2012

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of multiple fluid-filled cysts that expand over time and destroy renal architecture. The proteins encoded by the $PKD1$ and $PKD2$ genes, mutations in which account for nearly all cases of ADPKD, may help guard against cystogenesis. Previously developed mouse models of $PKD1$ and $PKD2$ demonstrated an embryonic lethal phenotype and massive cyst formation in the kidney, indicating that $PKD1$ and $PKD2$ probably play important roles during normal renal tubular development. However, their precise role in development and the cellular mechanisms of cyst formation induced by $PKD1$ and $PKD2$ mutations are not fully understood. To address this question, we presently created $Pkd2$ knockout and $PKD2$ transgenic mouse embryo fibroblasts. We used a mouse oligonucleotide microarray to identify messenger RNAs whose expression was altered by the overexpression of the $PKD2$ or knockout of the $Pkd2$. The majority of identified mutations was involved in critical biological processes, such as metabolism, transcription, cell adhesion, cell cycle, and signal transduction. Herein, we confirmed differential expressions of several genes including aquaporin-1, according to different $PKD2$ expression levels in ADPKD mouse models, through microarray analysis. These data may be helpful in $PKD2$-related mechanisms of ADPKD pathogenesis.

• 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.

• 대한한의학방제학회지
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• 제29권1호
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• pp.45-55
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• 2021

Objectives : This study was conducted to evaluate the anti-atrophic effect of polycan in dexamethasone-induced skeletal muscle atrophy in vitro model. Methods : C2C12 myoblast were differentiated into myotube by 2% horese serum medium for 6 days, and then treated polycan extract at different concentrations for 24h. The effect of dexamethasone on the induction of muscle atrophy and expression of atrophy-related genes in differentiated C2C12 myotubes using a GSH, ROS, real-time PCR, western blots analysis. Results : The results showed that Treatment with polycan (100 and 200 ㎍/㎖) noncytotoxic levels on both myoblast and myotube. Polycan decreased the ROS level overproduced with dexamethasone and improved the depletion of GSH level. Dexamethasone showed a decrease in myotube diameter, which was associated with up-regulation muscle-specific ubiquitin ligases markers, such as atrogin-1, FoxO3, myostatin and muscle RING finger-1 (MuRF1), and down-regulation of myogenin, MEF2, Myogenic regulatory factor 5, 6 and MyoD. The results showed that polycan treatment significantly dose-dependently inhibited it. Furthermore, decreased expressions of PI3K/Akt signal pathway by dexamethasone were reversed by treatment with polycan. Conclusions : Thus, polycan suppresses dexamethasone induced muscle atrophy in C2C12 myotube in vitro model through activation of PI3K/Akt pathway and protective effect of improve skeletal muscle function.

• Journal of Veterinary Science
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• 제21권4호
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• pp.57.1-57.11
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• 2020

Background: Streptococcus dysgalactiae subspecies equisimilis (SDSE) acts as an etiological agent for lameness, neurological signs, and high mortality in pigs. Despite its importance in pig industries and zoonotic potential, little is known about the effects of this pathogen. Objectives: This study aimed to determine the molecular characteristics and antimicrobial resistance of SDSE strains isolated from diseased pigs. Methods: A total 11 SDSE isolates were obtained from diseased pigs. Bacterial identification, PCR for virulence genes, emm typing, and antimicrobial resistance genes, multilocus sequence typing, and antimicrobial susceptibility test were performed. Results: Nine isolates were from piglets, and 8 showed lameness, sudden death, or neurological signs. The isolates were PCR-positive for sla (100%), sagA (100%), and scpA (45.5%), and only 1 isolate amplified the emm gene (stL2764). Eight different sequence types were detected, categorized into 2 clonal complexes and 4 singletons. All the isolates in this study were included in a small cluster, which also contained other strains derived from humans and horses. The minimum inhibitory concentrations for the tested beta-lactams were low, while those for macrolides, tetracyclines, and fluoroquinolones were relatively high. PCR analysis of the macrolide and tetracycline resistance genes demonstrated that the isolates carried erm(B) (18.2%, n = 2), mef(A/E) (9.1%, n = 1), tet(M) (18.2%, n = 2), and tet(O) (90.2%, n = 10). Two isolates presented a mutation in parC, which is associated with fluoroquinolone resistance. Conclusion: This study provided insight into swine-derived SDSE, as it is related to veterinary medicine, and elucidated its zoonotic potential, in the context of molecular epidemiology and antimicrobial resistance in public health.