• The Korean Journal of Physiology and Pharmacology
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• 제27권3호
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• pp.277-287
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• 2023

Actin dynamics play an essential role in myogenesis through multiple mechanisms, such as mechanotransduction, cell proliferation, and myogenic differentiation. Twinfilin-1 (TWF1), an actin-depolymerizing protein, is known to be required for the myogenic differentiation of progenitor cells. However, the mechanisms by which they epigenetically regulate TWF1 by microRNAs under muscle wasting conditions related to obesity are almost unknown. Here, we investigated the role of miR-103-3p in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation of progenitor cells. Palmitic acid, the most abundant saturated fatty acid (SFA) in the diet, reduced TWF1 expression and impeded myogenic differentiation of C2C12 myoblasts, while elevating miR-103-3p levels in myoblasts. Interestingly, miR-103-3p inhibited TWF1 expression by directly targeting its 3'UTR. Furthermore, ectopic expression of miR-103-3p reduced the expression of myogenic factors, i.e., MyoD and MyoG, and subsequently impaired myoblast differentiation. We demonstrated that miR-103-3p induction increased filamentous actin (F-actin) and facilitated the nuclear translocation of Yes-associated protein 1 (YAP1), thereby stimulating cell cycle progression and cell proliferation. Hence, this study suggests that epigenetic suppression of TWF1 by SFA-inducible miR-103-3p impairs myogenesis by enhancing the cell proliferation triggered by F-actin/YAP1.

• Journal of Korean Dental Science
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• 제8권2호
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• pp.74-81
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• 2015

Purpose: In osteoclast differentiation, actin-rich membrane protrusions play a crucial role in cell adhesion. Odontogenic ameloblast-associated protein (Odam) contributes to cell adhesion by inducing actin rearrangement. Odam-mediated RhoA activity may play a significant role in multinucleation of osteoclasts. However, the precise function of Odam in osteoclast cell adhesion and differentiation remains largely unknown. Here, we identify a critical role for Odam in inducing osteoclast adhesion and differentiation. Materials and Methods: The expression of Odam in osteoclasts was evaluated by immunohistochemistry. Primary mouse bone marrow and RAW264.7 cells were used to test the cell adhesion and actin ring formation induced by Odam. Result: Odam was expressed in osteoclasts around alveolar bone. Odam transfection induced actin filament rearrangement and cell adhesion compared with the control or collagen groups. Overexpression of Odam promoted actin stress fiber remodeling and cell adhesion, resulting in increased osteoclast fusion. Conclusion: These results suggest that Odam expression in primary mouse osteoclasts and RAW264.7 cells promotes their adhesion, resulting in the induction of osteoclast differentiation.

• Applied Microscopy
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• 제29권4호
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• pp.437-450
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• 1999

간세포의 기능적 연구를 위한 배양계를 확립하기 위하여 Sprague-Dawley계 흰쥐의 간장에서 collagenase와 hyaluronidase의 혼합액을 이용하여 간세포를 분리하고 배양하여, 배양중인 간세포의 구조적인 변화와 담세관의 형성 과정을 확인하고, cytochalasin D를 배양계에 첨가한 경우 발생되는 간세포 및 담세관의 구조적인 변화를 살펴보아 다음과 같은 결과를 얻었다. 분리 배양한 흰쥐의 간세포는 원형이었고, 표면에 미세융모를 가지고 있었으며. 배양중 서로 부착되어 세포띠를 형성하였다. Cytochalasin D처리후 간세포의 표면은 미세융모가 소실되어 편평하게 변화되었으며, 소포성 돌출물이 자주 관찰되었다. 담세관은 부착된 간세포의 사이에서 형성되었으며, 간세포 표면의 작은 융기에서 기시하는 다양한 길이 및 형태의 미세융모로 채워져 있었고, 양단에는 치밀결합 및 부착만 등으로 구성된 연접복합체가 존재하였다. Cytochalasin D 처리후 당세관의 내강은 팽창되었으며 미세융모는 소실되어 거의 존재하지 많았고, 양단에 존재하는 연접복합체는 파괴되어 간격이 벌어진 곳이 많았다. 담세관내에 존재하는 미세융모 속에 존재하는 액틴미세섬유심은 완전하게 형성되어 있는 경우, 불완전하게 적은 양만 존재하는 경우, 그리고 전혀 존재하지 않는 경우가 있었다. 담세관주변세포질에 존재하는 액틴미세섬유얼기의 형성은 불완전하여 부위에 따라 없는 곳도 있었다. Cytochalasin D처리후 담세관주변세포질의 액면미세섬유얼기는 존재하지 많았다. 이상의 결과로 흰쥐의 간장에서 분리한 간세포는 배양중 성장하면서 정상적인 담세관을 형성함을 알 수 있었으며, 담세관의 형성은 접착부위의 연접복합체의 형성 및 미세융모의 형성,담세관 내 액틴미세섬유심 및 담세관주변세포질내 액틴미세섬유얼기의 형성 등을 특정으로 하는 것으로 판단된다.

• Applied Microscopy
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• 제21권1호
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• pp.46-62
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• 1991

The intermediate filament is one of the most important constituents of the intracytoplasmic cytoskeleton microtubule, actin, myosin and intermediate filament. It is composed of keratin, desmin, vimentin, neurofilament and glial filament, and has important role as a cellular marker, epithelial or mesenchymal origin. So it will be important to differentiated from some poorly or undifferentiated neoplasm to provide adequate therapeutic modalities. This study was performed by using immunohistochemical staining and electron microscopic observation to find out intermediate filaments of epithelial and non-epithelial tumor cells evaluate the degree of differentiation in tumors and therefore to provide some diagnostic and therapeutic modalities. The materials consisted of 83 epithelial and non-epithelial elements bearing 23 normal control, 28 epithelial tumors, and 32 non-epithelial tumors, that are resected for definite treatment at Chosun University Hospital from June, 1988 to June, 1990. Immunohistochemical stain for keratin, desmin and vimentin, and electron microscopic study were performed in all cases. The results obtained were as follows. 1. Immunohistochemical stain for intermediate filament were very useful diagnostic aid for differentiated epithelial tumor to non-epithelial tumor in diagnostic neoplasia. 2. In the electron microscopic finding, the size of intermediate filaments were possible differentiated to cell components of epithelial tumor and non-epithelial tumors.

• KSBB Journal
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• 제27권5호
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• pp.308-312
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• 2012

Argon-plasma jet (Ar-PJ) is generated by ionizing Ar gas, and the resulting Ar-PJ consists of a mixture of neutral particles, positive ions, negative electrons, and various reactive species. Although Ar-PJ has been used in various biomedical applications, little is known about the biological effects on cells located near the plasma-exposed region. Here, we investigated the effects of the Ar-PJ on actin cytoskeleton of mouse embryonic fibroblasts (MEFs) in response to indirect as well as direct exposure to Ar-PJ. This Ar-PJ was generated at 500 mL/min of flow rate and 100 V electric power by our device mainly consisting of electrodes, dielectrics, and a high-voltage power supply. Because actin cytoskeleton is the key cellular machinery involved in cellular movement and is implicated in regulation of cancer metastasis and thus resulting in a highly desirable cancer therapeutic target, we examined the actin filament architectures in Ar-PJ-treated MEFs by staining with an actin-specific phalloidin labeled with fluorescent dye. Interestingly, the Ar-PJ treatment causes destabilization of actin filament architectures in the regions indirectly exposed to Ar-PJ, but no differences in MEFs treated with Ar gas alone and in untreated cell control, indicating that this phenomenon is a specific cellular response against Ar-PJ in the live cells, which are indirectly exposed to Ar-PJ. Collectively, our study raises the possibility that Ar-PJ may have potential as anti-cancer drug effect through direct destabilization of the actin cytoskeleton.

• International Journal of Oral Biology
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• 제40권1호
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• pp.27-33
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• 2015

In the present study, we investigated the effect of staurosporine on the formation of cellular processes in human gingival fibroblasts and rat astrocytes. Staurosporine caused a rapid induction of process formation in human gingival fibroblasts and rat astrocytes in a concentration dependent manner. The process formation of human gingival fibroblasts and rat astrocytes was prevented by the pretreatment with N-acetylcysteine, suggesting that staurosporine-induced ROS production was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited the staurosporine-induced process formation, whereas cytochalasin D, an actin filament breakdown agent, failed to suppress the formation of cellular processes. This result indicated that polymerization of microtubule, and not actin filament, was responsible for the formation of cellular processes induced by staurosporine. In support of this hypothesis, Western blot analysis was conducted using anti-tubulin antibody, and the results showed that the amount of polymerized microtubule was increased by the treatment with staurosporine while that of depolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROS-mediated, microtubule-dependent formation of cellular processes in human gingival fibroblasts and rat astrocytes.

• Toxicological Research
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• 제14권4호
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• pp.577-585
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• 1998

Cadmium is an important industrial and environmental pollutant and has adverse effects on cell growth and metabolism, although the mechanisms of its cellular toxicity are still unclear. This study was performed to elucidate the cytotoxic mechanism of cadmium in the viewpoint of oxidative stress and cytoskeleton alterations in WB-F344 rat liver epithelial cells. 200 $\mu\textrm{M}$ $CdCl_2$ caused a severe disassembling of microtubule and micro filament and an apparent cell retraction under an observation with fluorescence micoscope. (equation omitted)-tubulin and F-actin protein were highly thiolated at 20 min and then disappeared from 1 hour after the treatment of 200 $\mu$M CdCl$_2$in the immunoblot analysis. Intracellular GSH was decreased from 1hr to 24 hrs by 66.6 or 200 $\mu\textrm{M}$ of $CdCl_2$. Intracellular protein thiol was also decreased by 22.2, 66.6 and 200 $\mu\textrm{M}$ of $CdCl_2$ at 1 hour after its treatment. The product of lipid peroxidation (malondialdehyde) was increased from 4 hrs by 66.6 and 200$\mu\textrm{M}$ of $CdCl_2$. These data indicate that cadmium induces oxidative stress involving disassembling of microtubule and micro filament, thiolation of (equation omitted)-tubulin and actin protein, depletion of GSH and protein thiol, and increase of lipid peroxidation.

• 생명과학회지
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• 제23권9호
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• pp.1163-1169
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• 2013

인슐린은 근육세포 표면으로 포도당 수송체 4(glucose transporter 4, GLUT4)를 유도하여 혈액 속의 포도당을 세포 내로 유입시키도록 작용한다고 알려져 있다. Fagopyritol은 인슐린과 유사한 작용을 하는 것으로 알려져 있으므로, 본 연구에서는 혈당강하 효과가 있다고 알려진 fagopyritol을 랫드의 근육세포주(L6GLUT4myc 세포)에 처리하여, 아직 명확하게 밝혀지지 않은 fagopyritol의 혈당강하 기전을 규명하고자 수행하였다. Fagopyritol의 혈당강하 기전을 규명하기 위하여 근원세포(myoblast)와 근관세포(myotube)에 fagopyritol을 처리하여 액틴 필라멘트의 구조와 GLUT4에 미치는 영향을 분석하였다. Fagopyritol을 myoblast에 처리하였을 때, GLUT4가 처리군에서 대조군과 비교하여 유의 있게 원형질막 쪽으로 유도되는 것을 확인하였고, 액틴 필라멘트의 구조가 재조정되면서 GLUT4의 이동을 돕는 것으로 생각된다. 또한 fagopyritol이 인슐린과 유사한 작용 경로를 가지는지 확인하기 위하여, 인슐린 작용 경로에서 중요한 역할을 하는 것으로 알려진 phosphatidylinositol 3-kinase (PI3K)의 억제제인 LY294002를 fagopyritol과 함께 처리하였을 때 GLUT4가 원형질막 쪽으로 유도되지 않는 것을 확인하였다. Fagopyritol을 myotube에 처리하였을 때, myoblast에 처리하였을 때와 유사한 결과를 나타내었다. 이러한 결과를 종합하면 fagopyritol이 인슐린과 유사한 작용을 하여 액틴 필라멘트의 구조 변경과 GLUT4의 이동을 촉진시키는 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제23권3호
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• pp.285-295
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• 2019

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonic development. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

• Asian-Australasian Journal of Animal Sciences
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• 제33권10호
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• pp.1579-1589
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• 2020

Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.