• BMB Reports
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• v.29 no.5
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• pp.405-410
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• 1996

Previous studies have shown that transforming growth factor beta ($TGF-{\beta}$) is a potent regulator of cell growth and differentiation. To study the effects of $TGF-{\beta}$ on cell morphology and cytoskeleton reorganization, we conducted a survey using Mv1Lu mink lung epithelial cells with antibodies to cytoskeletal proteins and an extracellular matrix protein. While the untreated cells showed a cuboidal shape of typical epithelia, the Mv1Lu cells displayed a drastic shape change in the presence of $TGF-{\beta}$. This alteration was most prominent when near-confluent cells were treated with $TGF-{\beta}$. Since the morphology alteration is known to be accompanied by the reorganization of cytoskeletal proteins in other cell types, we investigated the intracellular distribution of the three major cytoskeletal structures: microfilaments, microtubules, and intermediate filaments. In the microfilament system, $TGF-{\beta}$ induced new stress fiber formation, which was caused primarily by the polymerization of cytoplasmic G-actin. However, $TGF-{\beta}$ appeared not to induce any significant changes in microtubular structures and vimentin filaments as determined by indirect fluorescence microscopy. Finally we confirmed the rapid accumulation of fibronectin by immunoblot analysis and chased the protein locations by immunofluorescence microscopy.

• Journal of Acupuncture Research
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• v.32 no.3
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• pp.27-40
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• 2015

Objectives : This study was performed to evaluate the effects of water extract of Cervi Parvum Cornu(CPC), Aconiti Lateralis Radix Preparata(ALR), and Yongbu-tang(YBT) on suppression of the receptor activator of nuclear factor kappa-B ligand(RANKL)-induced osteoclast differentiation and bone resorption. Methods : The effects of CPC, ALR, YBT extracts on osteoclast differentiation were determined by culture of bone marrow macrophage(BMM). The mRNA expression levels of the nuclear factor of activated T-cells cytoplasmic 1(NFATc1), c-Fos and tartrate-resistant acid phosphatase(TRAP) in BMMs were analyzed by reverse transcriptase polymerase chain reaction(RT-PCR). Similarly, the protein expression levels of NFATc1, c-Fos, mitogen-activated protein kinase(MAPK)s and ${\beta}$-actin in cell lysates were measured by western blotting. In addition, effects of CPC, ALR and YBT extracts were determined by means of Lipopolysaccharide(LPS)-induced bone-loss with mice. Results : CPC, ALR and YBT extracts showed remarkable inhibition on RANKL-induced osteoclast differentiation without cytotoxicity. CPC and ALR extracts significantly reduced the protein expression level of NFATc1. YBT extract significantly reduced the mRNA expression levels of c-Fos, NFATc1 and the protein expression levels of c-Fos, NFATc1, AKT, p38, c-Jun N-terminal kinase(JNK). Further, YBT extract suppressed degradation of$ I-{\kappa}B$. And ALR extract significantly restored the bone erosion by LPS treatment in mice. Conclusions : YBT extract showed more remarkable inhibition on osteoclast differentiation than CPC and ALR extracts in vitro. ALR extract showed remarkable inhibition on bone resorption in vivo. Thus, YBT extract can be a useful treatment for bone-loss diseases such as osteoporosis.

• Animal cells and systems
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• v.1 no.4
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• pp.657-663
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• 1997

The CD4 and CDS coreceptors, in conjunction with the T cell receptor (TCR) , make important contributions to the differentiation of thymocytes. They have been shown to be involved in the clonal deletion and positive selection processes during T cell development in thymus. To further analyze the role of CD4 and CDS proteins during T cell differentiation, we have generated transgenic mice constitutively expressing high levels of a native CD4 and a CD4{CDSa hybrid protein. The hybrid protein is composed of CD4 extracellular domain linked to the CD8a transmembrane region and cytoplasmic tail. The transgenes were driven by human beta-actin promoter, and therefore, they were expressed in all tissues examined including thymus, spleen, and lymph nodes. The resulting CD4 and CD4{CD8${\alpha}$transgenic mice were found to express the CD4 and CD4{CD8${\alpha}$ respectively, in developing thymocytes and peripheral T cells. The expression levels of transgenic proteins were 5-10 times higher than that of endogenous CD4 in thymus. However, total surface CD4 expression (CD4 or CD4{CD8${\alpha}$ transgenic protein plus endogenous CD4) of the transgenic mice were main. tained at similar levels compared to control littermates. Surface CD4 expression on CDS T cells, however, was significantly lower than that on cells expressing endogenous CD4. These results suggest that a total avidity between developing thymocytes and thymic stromal cells is impor. tant for differentiation of thymocytes.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2002.05a
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• pp.25-35
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• 2002

Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

• The Journal of Korean Academy of Prosthodontics
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• v.44 no.1
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• pp.73-84
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• 2006

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality fir the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts adhesions to certain substrata can be explained by the 'focal adhesion' theory. On the other hand, epithelial cells adhere tn the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions' defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.

• International Journal of Industrial Entomology and Biomaterials
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• v.20 no.2
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• pp.107-114
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• 2010

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Journal of Acupuncture Research
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• v.32 no.3
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• pp.69-81
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• 2015

Purpose : The first purpose of this study is to find out whether the water extract of Rehmanniae Radix Preparat(RRP), Cuscutae Semen(CS) and their combination(Ssangbohwan, SBH) have the effect of suppressing Receptor activator of nuclear factor kappa-B ligand(RANKL)-induced osteoclast differentiation. The second purpose of this study is to find out whether the water extract of RRP, CS and SBH have the effect of inhibiting osteoporosis in an osteoporosis model induced by lipopolysaccharide(LPS). Methods : After promoting differentiation of osteoclasts by treating the RANKL, we observed the effect by the administration of RRP, CS and SBH. In addition, by means of Reverse transcription polymerase chain reaction(RT-PCR), we assayed mRNA expression levels of NFATc1, c-Fos, TRAP and GAPDHS(Glyceraldehyde-3-phosphate dehydrogenase, spermatogeni) from bone marrow macrophages(BMMs). Similarly, the protein expression levels of NFATc1 (Nuclear factor of activated T-cells, cytoplasmic1), C-Fos, MAPKs(Mitogen-activated protein kinases) and ${\beta}$-actin in cell lysates were analyzed by means of Western Blotting. Finally, we determined the anti-osteoporotic effects of RRP, CS and SBH, through the use of Lipopolysaccharide-induced bone-loss mouse. Results : RRP, CS and SBH showed remarkable inhibitive effect on RANKL-treated osteoclast differentiation without cytotoxicity. SBH inhibited the phosphorylation of p38, Jun N-terminal kinases(JNK), and I-${\kappa}B$ and down-regulated the induction of c-Fos and NFATc1 by RANKL. RRP, CS suppressed degradation of I-${\kappa}B$, but it did not affect c-Fos and NFATc1 by RANKL. Lastly, in vivo data showed that RRP and SBH prevented bone erosion by LPS treatment. Conclusions : These results demonstrate SBH can be effective remedy for bone-loss diseases such as osteoporosis.

• Journal of Acupuncture Research
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• v.34 no.2
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• pp.1-18
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• 2017

Objectives : The purpose of this study was to evaluate the effects of water extracts of Eucommiae cortex (EC), Psoraleae semen (PS), and their combination on receptor activator of nuclear factor-kappa-B ligand (RANKL)-induced osteoclast differentiation. Methods : We assayed the protein expression levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-Fos, mitogen-activated protein kinases (MAPKs), and ${\beta}-actin$ in cell lysates using western blotting. Similarly, mRNA expression levels of NFATc1, c-Fos, tartrateresistant acid phosphate (TRAP), and glyceraldehyde-3-phosphate dehydrogenase, spermatogeni (GAPDHS) from bone marrow macrophages (BMMs) were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, we determined the anti-osteoporotic effects of the water extracts of EC, PS, and their combination in a lipopolysaccharide (LPS)-induced bone-loss mouse model. Results : The in vitro data revealed showed that the combination of EC and PS extract showed a more remarkable inhibition of osteoclast differentiation than each herb did alone. The combination downregulated the induction of c-Fos, NFATc1, and TRAP by suppressing the phosphorylation of p38 and c-Jun N-terminal kinases (JNKs) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$). Lastly, the in vivo data showed that PS reduced the LPS-induced bone erosion. Conclusion : The result of this study suggests that EC and PS could be potential therapeutic agents for bone loss diseases such as osteoporosis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.2
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• pp.81-93
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• 2006

Obstructive sialadenitis is one of common disease in salivary gland, and most common histologic features are loss of acinar cell and ductal dilatation associated with fibrosis, and infiltration of inflammatory cells. Although many experimental studies has been accomplished for the salivary acinar cell change in obstructive salivary gland disease, studies for myoepithelial cell were deficient. This study is designed for salivary gland tissue change, especially myoepithelial cell when nonspecific chronic sialadenitis or salivary duct injury by duct obstruction or cut can be occurred that is common encounted clinically. After ligation and cutting of submandibular gland of rabbit, groups of aminmal were sacrificed at 1, 2, 4 weeks postoperatively, submandibular gland were removed. The histopathologic evaluation was done with light microscopy. And, with immunohistochemical staining with ${\alpha}$-smooth muscle actin, characteristics of myoepithelial cell were examined. With transmission electron microscopy, ultrastructure of myoepithelial cell were examined for distribution and ultrastructure of myoepithelial cell. The results were obtained as follows: 1. In the histopathologic evaluation, ligation and cutting group of 1 week, linkage of myoepithelial cell associated with acinar atrophy and degeneration were disappeared in both group. 2. More prominent squamous metaplasia was seen in acinar cells of ligation group of 2 weeks experimental rabbit than cutting group. 3. Acinar cells are nearly disappeared in both ligation and cutting group of 4 weeks, and myoepithelial cell also disappeared associated with acinar cell atrophy, and duct-like structure composed by squamous cells by squamous metaplasia in acinar cells were distributed. 4. In immunohistochemical study, both ligation and cutting group ${\alpha}$-SMA distribution were diminished at 1 week experimental rabbits, but myoepithelial cell was more diminished in ligation group than cutting group, which were distributed around cells of squamous metaplasia. 5. Nuclear condensation, chromosome margination, and cytoplasmic vaculoation were appeared in myoepithelial cell of both cutting and ligation group after 1 week with transmission electron microscopy. But degenerative substance were seen in cytoplasm of myoepithelial cell of ligation group of 4 weeks. From the results obtained in this study, atrophy and degeneration of myoepithelial cell was more prominent in duct ligation group than duct cutting group, and myoepithelial cells were seen around cells squamous metaplasia of acinar cell.

• Childhood Kidney Diseases
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• v.8 no.2
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• pp.138-148
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• 2004

Purpose: Regardless of the underlying diseases, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. We examined the molecular basis for this alteration of the podocyte phenotypes, including quantitative and distributional changes of ZO-1 protein as a candidate contributing to the pathogenic changes in the barrier to protein filtration. Methods: To investigate whether high glucose and advanced glycosylation endproduct(AGE) induce podocyte cytoskeletal changes, we cultured rat GEpC under 1) normal glucose(5 mM=control) or 2) high glucose(30 mM) or 3) AGE-added or 4) high glucose plus AGE-added conditions. The distribution of ZO-1 was observed by confocal microscope and the change of ZO-1 expression was measured by Western blotting and RT-PCR. Results: By confocal microscopy, we observed that ZO-1 moves from peripheral cytoplasm to inner actin filaments complexes in both AGE-added and high glucose condition. In Western blotting, high glucose or AGE-added condition decreased the ZO-1 protein expression by 11.1%(P>0.05) and 2.3%(P>0.05), respectively compared to the normal glucose condition. High glucose plus AGE-added condition further decreased ZO-1 protein expression to statistically significant level(12%, P<0.05). No significant change was seen in the osmotic control. In RT-PCR, high glucose plus AGE-added condition significantly decreased the expression of ZO-1 mRNA by 12% compared to normal glucose condition. Conclusion: We suggest that both high glucose and AGE-added condition induce the cytoplasmic translocation and suppresses the production of ZO-1 at transcriptional level and these changes may explain the functional changes of podocytes in diabetic conditions.