• Toxicological Research
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• 제30권4호
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• pp.261-266
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• 2014

Environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) have been implicated in cancer development and progression. However, the effects of PAHs on carcinogenesis are still poorly understood. Here, we characterized a mouse cancer cell line BNL 1ME A. 7R.1 (1MEA) derived by transformation of non-tumorigenic liver cell line BNL CL.2 (BNL) using 3-methylcholanthrene (3MC), a carcinogenic PAH. RT-PCR and immunoblot analysis were used to determine the expression level of mRNA and proteins, respectively. To determine functionality, cell motility was assessed in vitro using a transwell migration assay. Both mRNA and protein levels of E-cadherin were significantly decreased in 1MEA cells in comparison with BNL cells. While the expression levels of mesenchymal markers and related transcription factors were enhanced in 1MEA cells, which could lead to increase in cell motility. Indeed, we found that 7-day exposure of BNL cells to 3-MC reduced the level of the adhesion molecule and epithelial marker E-cadherin and increased reciprocally the level of the mesenchymal marker vimentin in a dose-dependent manner. Taken together, these results indicate that the process of epithelial-mesenchymal transition (EMT) may be activated during premalignant transformation induced by 3-MC. A mechanism study to elucidate the relation between 3-MC exposure and EMT is underway in our laboratory.

• Journal of Animal Science and Technology
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• 제63권5호
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• pp.977-983
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• 2021

Closely correlated expression patterns between ubiquitin specific peptidase 9X-linked (USP9X) and adherens junction formation factor (Afadin) in mouse testis development suggests that Usp9x regulates the deubiquitination of Af-6 (also known as Afadin, AFDN), and subsequently, the cell adhesion dynamics during gametogenesis. However, this relationship has not yet been tested in other domestic animals. The study was examined the temporal and spatial expression patterns of porcine USP9X and AFDN from the pre-pubertal to adult stages using real time-PCR and immunohistochemistry. Furthermore, we detected the transcripts of USP9X and AFDN in the testis of 1-, 6- and 12-months old boar, respectively. USP9X and AFDN were found to have similar expressions patterns, with basal expression after 1 month followed by a significant up-regulation from 6 months (puberty) onwards. In addition, neither the AFDN or USP9X proteins were detected in spermatogenic cells but they were expressed in the leydig cells and sertoli cells. USP9X was detected around the basal lamina during pre-puberty, and predominantly expressed in the leydig cells at puberty. Finally, in adult testis, USP9X was increased at the sertoli cell-cell interface and the sertoli cell-spermatid interface. In summary, closely correlated expression patterns between USP9X and AFDN in boar testis supports the previous findings in mice. Furthermore, the junction connections between the sertoli cells may be regulated by the ubiquitination process mediated via USP9X.

• Clinical and Experimental Reproductive Medicine
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• 제33권2호
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• pp.115-123
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• 2006

목 적: 본 연구를 통해 GnRH 의한 세포 분열의 억제는 integrin, FAK 빛 ERK를 통한 세포 내 신호전달 기전을 통하여 일어남을 규명하고자 하였다. 연구방법: 연구에 사용된 인간자궁내막암 세포주는 DMEM/F12 (10% FBS)의 조건에서 배양 하였다. GnRH-I과 -II는 실험 목적에 따라 100 nM 농도로 0, 5, 10, 15, 20, 30분간 또는 10 nM or 100 nM의 농도로 20분간 처리 하였다. 세포의 분열 정도는 [$^3H$] thymidine incorporation assay를 이용하여 정량적으로 측정 하였으며, Immunoblotting 방법을 이용하여 단백질의 발현을 확인 하였다. 결 과: GnRH-I과 -II 모두 HEC1A 세포의 세보분열을 억제하였으며 integrin ${\beta}3$의 발현을 증가 시켰다. GnRH-I과 -II를 처리 후 FAK 및 ERK의 안산화가 증가됨을 관찰할 수 있었다. 결 론: GnRH에 의한 세포분열의 억제는 integrin의 발현과 FAK 및 ERK의 인산화 과정을 통하여 일어남을 알 수 있었다.

• Molecules and Cells
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• 제45권12호
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• pp.950-962
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• 2022

Aging is a major risk factor for common neurodegenerative diseases. Although multiple molecular, cellular, structural, and functional changes occur in the brain during aging, the involvement of caveolin-2 (Cav-2) in brain ageing remains unknown. We investigated Cav-2 expression in brains of aged mice and its effects on endothelial cells. The human umbilical vein endothelial cells (HUVECs) showed decreased THP-1 adhesion and infiltration when treated with Cav-2 siRNA compared to control siRNA. In contrast, Cav-2 overexpression increased THP-1 adhesion and infiltration in HUVECs. Increased expression of Cav-2 and iba-1 was observed in brains of old mice. Moreover, there were fewer iba-1-positive cells in the brains of aged Cav-2 knockout (KO) mice than of wild-type aged mice. The levels of several chemokines were higher in brains of aged wild-type mice than in young wild-type mice; moreover, chemokine levels were significantly lower in brains of young mice as well as aged Cav-2 KO mice than in their wild-type counterparts. Expression of PECAM1 and VE-cadherin proteins increased in brains of old wild-type mice but was barely detected in brains of young wild-type and Cav-2 KO mice. Collectively, our results suggest that Cav-2 expression increases in the endothelial cells of aged brain, and promotes leukocyte infiltration and age-associated neuroinflammation.

• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.141-149
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• 2008

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for $2{\sim}4$ months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

• 대한치과보철학회지
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• 제45권3호
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• pp.375-381
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• 2007

Statement of problem. Prior to determining an optimal width of micropatterned grooves provided on titanium substrata, we have done a pilot study using surface topographies in combined microm and submicrom levels. Purpose. The purpose of this study was twofold 1) to assess the proliferation and 2) to analyze the expression of genes encoding the intracellular signaling proteins involved in cell-substratum adhesions and adhesion-dependent G1 phase cell cycle progression of human gingival fibroblasts plated on smooth and microgrooved/acid-etched titanium substrata. Material and methods. Three groups of titanium discs as NE0 (smooth Ti substrata), E15 (Ti substrata with microgrooves of $15{\mu}m$ of spacing and $3.5{\mu}m$ in depth and with further acidetching), and E30 (Ti substrata with microgrooves of $30{\mu}m$ spacing and $3.5{\mu}m$ in depth and with further acid-etching) served as the human gingival fibroblasts' substrata. Viability and proliferation of fibroblasts were determined using an XTT assay. Gene expressions of fibronectin, ${\alpha}5$ integrin, CDK4, and $p27^{kip}$ were analyzed in RT-PCR. Cell-substratum interactions were analyzed in SEM. Results. From the XTT assay at 24 h incubation, the mean optical density (OD) value of E15 was significantly greater than the values of E30 and NE0. At 48 and 96 h however, the mean OD values of E30 were significantly greater than the values of E15 and NE0. No differences in the expression of PCR transcripts at 96 h incubations were noted between groups, whereas at 48 h, an unexpected increase in the expression of all the transcripts were noted in E15 compared with other two groups. Fibroblasts were observed to orient and adhere inside the microgrooves. Conclusion. Micropatterned grooves and acid-etching on Ti substrata alter viability and gene expression of adhered human gingival fibroblasts.

• International Journal of Oral Biology
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• 제41권2호
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• pp.89-96
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• 2016

Tooth development shows dynamic morphological changes from the stages of cap to hard tissue formation and is strictly regulated during development. In the present study, we compared expression and localization of 3 major enamel matrix proteins in rats: amelogenin, enamel and ameloblastin. DD-PCR and RT-PCR revealed differential expression of the major proteins from the cap stage to root stage. Immunofluorescence staining results indicated that amelogenin was not detected in either inner enamel epithelium or reduced enamel epithelium, but highly immunoreactive in preameloblasts and ameloblasts; in addition, it was sporadically expressed in preodontoblasts abutting preameloblasts. Ameloblastin expression was also observed in not only differentiated ameloblasts but also osteoblasts. Immunoreactivity to ameloblastin in ameloblasts was strong in Tomes' processes. Enamelin was exclusively localized along the entire newly formed and maturing enamel. Enamelin was largely localized in near Tomes' processes and enamel rods in maturing enamel. Alendronate treatment resulted in down-regulation of amelogenin and ameloblastin at both transcription and translation levels; whereas, enamelin expression was unchanged in response to the treatment. These results suggested that amelogenin, ameloblastin and enamelin might be implicated in cell differentiation, adhesion of ameloblasts to enamel and enamel crystallization during enamel matrix formation, respectively.

• 생명과학회지
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• 제9권2호
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• pp.160-168
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• 1999

Butyrate는 탄소사슬이 짧은 지방산 중 하나로 소화되지 않은 식이성 섬유의 혐기적 발효결과 포유동물의 위장관내에 millimolar 농도로 유지되며 대장의 상피세포에서 흡수되어 energy원으로 사용된다. 70%정도 confluent하게 자란 사람의 대장암세포주인 HT29 cell에 5mM sodium butyrate를 시간별로 처리하고 세포의 생존율, 암세포의 분화정도의 biomarker로 알려진 alkaline phosphatase 및 PLC-rl의 발현정도를 측정하였으며 sphingolipid의 생합성 및 ceramidase 활성도 측정하였다. Sodium butyrate 처리는 성장중인 HT29 cell의 부착을 저해하여 처리 1일째부터 세포수가 감소하였고 형질막 효소인 alkaline phosphatase의 발현을 증가시켰으며 PLC-${\gamma}$의 발현을 감소시켰다. 또한, 복합 sphingolipid들의 생합성을 측정한 결과, 세포성장의 저해와 함께 sphingomyelin은 2일째부터 감소하였으며, galactosyl ceramide는 1일째부터 급속히 감소하였다. 그러나 ceramide의 경우, 1일째는 처리하기 전보다 680dpm/mg protein정도 증가하였으며 2일째에는 다시 급속히 감소하였다. 또한 butyrate처리에 의하여 HT29 cell의 acid ceramidase와 neutral ceramidase활성이 저해됨을 관찰하였는데 그 결과 ceramide함량이 초기에 증가된 것으로 생각된다. 본 실험결과들로부터 HT29 cell의 sodium butyrate처리는 세포분화 또는 세포성장저해를 가져오는데 이와 함께 초기의 ceramide함량 및 alkaline phosphatase활성의 증가와 galactosylceramide함량 및 LC-rl 발현의 감소현상이 동반됨을 알 수 있다.

• 대한의생명과학회지
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• 제7권2호
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• pp.59-64
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• 2001

Nebulin is an unusually large actin-binding protein specific to the skeletal muscle of vertebrates. The correlation of nebulin size with thin filament length have led to the suggestion that nebulin acts as a molecular ruler for the length of thin filaments. An SH3 domain occupies the C terminus of nebulin, in the sarcomeric Z-disk and is preceded by a 120-residue stretch containing multiple putative phosphorylation sites. SH3 domain mediates protein-protein interaction involved in the subcellular localization of proteins, cytoskeletal organization and signal transduction. However the binding partner and physiological role of nebulin SH3 domains remains unknown. Using the yeast two-hybrid system, we identified supervillin, an actin-binding protein, as a nebulin SH3 domain-interacting protein. The SH3 domain of nebulin binds to the sequence encoding amino acids 977 to 1335 of supervillin. But the sequence encoding amino acids 977 to 1335 displays weaker binding than the sequence encoding amino acids 977 to 1788.

• Journal of Ginseng Research
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• 제45권3호
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• pp.433-441
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• 2021

Background: Multiple sclerosis (MS) and its animal model, the experimental autoimmune encephalomyelitis (EAE), are primarily characterized as dysfunction of the blood-brain barrier (BBB). Ginsenoside-Rg3-enriched Korean Red Ginseng extract (Rg3-KRGE) is known to exert neuroprotective, anti-inflammatory, and anti-oxidative effects on neurological disorders. However, effects of Rg3-KRGE in EAE remain unclear. Methods: Here, we investigated whether Rg3-KRGE may improve the symptoms and pathological features of myelin oligodendroglial glycoprotein (MOG)_35-55 peptide - induced chronic EAE mice through improving the integrity of the BBB. Results: Rg3-KRGE decreased EAE score and spinal demyelination. Rg3-KRGE inhibited Evan's blue dye leakage in spinal cord, suppressed increases of adhesion molecule platelet endothelial cell adhesion molecule-1, extracellular matrix proteins fibronection, and matrix metallopeptidase-9, and prevented decreases of tight junction proteins zonula occludens-1, claudin-3, and claudin-5 in spinal cord following EAE induction. Rg3-KRGE repressed increases of proinflammatory transcripts cyclooxygenase-2, inducible nitric oxide synthase, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha, but enhanced expression levels of anti-inflammatory transcripts arginase-1 and IL-10 in the spinal cord following EAE induction. Rg3-KRGE inhibited the expression of oxidative stress markers (MitoSOX and 4-hydroxynonenal), the enhancement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and NOX4, and NADPH activity in the spinal cord of chronic EAE mice. Furthermore, apocynin, a NOX inhibitor, mimicked beneficial effects of Rg3-KRGE in chronic EAE mice. Conclusion: Our findings suggest that Rg3-KRGE might alleviate behavioral symptoms and pathological features of MS by improving BBB integrity through modulation of NOX2/4 expression.