• IMMUNE NETWORK
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• 제9권2호
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• pp.58-63
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• 2009

Background: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on $CD4^+CD25^+$T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to $CD4^+CD25^+$T cells. Methods: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to $CD4^+CD25^+$T cells was analyzed using flow cytometry. Results: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to $CD4^+CD25^+$T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. Conclusion: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on $CD4^+CD25^+$T cells.

• IMMUNE NETWORK
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• 제11권1호
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• pp.50-58
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• 2011

Background: Epstein-Barr virus associated gastric lymphoepithelioma-like carcinoma (LELC) is characterized by the intensive infiltration of lymphoid cells, the presence of EBV, and the better prognosis over typical adenocarcinoma. Thus, it was assumable that viral latent proteins may be responsible for the recruitment of a certain T cell repertoire to EBV-associated gastric carcinoma. Methods: To examine above possibility, EBV gene expression in gastric carcinoma tissues and usage of TCR among the tumor infiltrating lymphocytes were analyzed. Results: EBV specific DNA and EBERs RNA were detected in 4 out of 30 patients. RT-PCR analysis revealed that all 4 of EBV-positive tumor tissues expressed EBNA1 mRNA and BARTs and LMP2a was detected only one sample out of 4. However, the EBNA2 and LMP-1 transcripts were not detected in these tissues. $CD8^+$ T cells were the predominant population of infiltrating lymphocytes in the EBV-positive gastric carcinoma. According to spectra type analysis of infiltrating T cells, 10 predominant bands were detected by TCR $V{\beta}$ CDR3 specific RT-PCR from 4 EBV-positive tumor tissues. Sequence analysis of these bands revealed oligoclonal expansion of T cells. Conclusion: These findings suggest that clonally expanded T cells in vivo might be a population of cytotoxic T cells reactive to EBV-associated gastric carcinoma.

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1055-1059
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• 2003

Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue and development specific manner. There are two types of keratin in bovine. The type I is acidic keratin and the type II is neutral/basic keratin. 1.5 kb of 5' flanking sequence of Korean cattle Keratin IV gene, type II keratin (59 kDa), was cloned and sequenced. A symmetrical motif AApuCCAAA are located in a defined region upstream of the TATA box. Proximal SP1, AP1, E-box and CACC elements as the major determinants of transcription are identified. When it was compared to the bovine sequence from -600 bp to ATG upstream, the homology was 97% in nucleotide sequence. Several A and T sequences, located in the promoter region, are deleted in the Korean cattle. An expression vector consisted of Korean cattle Keratin IV gene promoter/SV40 large T antigen was transfected to HaCaT cell (Epithelial keratinocyte). The transformed HaCaT cells showed active proliferation when treated with PDGF (Platelet-derived growth factor) in 0.3% soft agar compared to control cells. These results indicate that Korean cattle Keratin IVgene promoter can be used as a promoter for transfection into epithelial cell.

• Journal of Periodontal and Implant Science
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• 제30권2호
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• pp.347-360
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• 2000

Bone remodeling results from the combined process of bone resorption and new bone formation which is regulated in part by some of the polypeptide growth factors such as platelet derived growth factor(PDGF), which has been known to be an important local regulator of bone cell activity and participate in normal bone remodeling. This process includes strictly regulated gene expression of several bone matrix proteins such as type I collagen and osteopontin, a 44 kDa phosphorylated glycoprotein, which has important roles in bone formation. The purpose of this study is to evaluate the effecs of PDGF-BB on the mRNA expression of bone matrix protein, type I collagen and osteopontin, in MC3T3- E1 cell culture. Cells were seeded at $5{\times}10^5$ cells in 10 ml of minimum essential medium alpha(${\alpha}-MEM$) containig 10% fetal bovine serum, 10 mM beta glycerophosphate. 0.1, 1, 10 ng/ml PDGF-BB were added to the cells for the day 3, 7, 14, 21, 28 and cultured for 24 hours. Type I collagen cDNA, Hf677, and osteopontin cDNA were used as probes for northern blot analysis. Total cellular RNA was purified at indicated day and northern blot analysis was performed. The results were as follows : Type I collagen mRNA expressions were higher at the day 3 and 7, and lower in the day 14, 21 in the control groups. In the experimental groups, mRNA expressions were increased when 0.1 ng/ml PDGF-BB were added on the day 3, 7, 21, and decreased in dose-dependent manner on the day 14, decreased at all added dose on the day 28. Osteopontin mRNA expressions were highest in the day 21 groups and lowest in the day 14 groups in the control groups. Interesting results were shown in the day 14 and 21 groups. We found that osteopontin mRNA level was increased in dose dependent manner in the day 14 groups, and decreased dose dependent manner in the day 21 groups. In conclusion, PDGF-BB may have various control effects on type I mRNA expression in the growth and differentiation process of MC3T3-E1 cells and may have contrary regulatory effects on osteopontin mRNA expression. For examples, when the baseline level of osteopontin mRNA was low, as in the day 14, PDGF-BB up-regulated osteopontin mRNA expression in dose dependent manner, and when the baseline level was high as in the day 21, PDGF-BB down-regulated dose dependent manner. Thus, it may be useful for clinical application in periodontal regeneration procedure if further study were performed.

• 대한세포병리학회지
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• 제6권2호
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• pp.199-203
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• 1995

Chordoma is relatively uncommon tumor comprising $1\sim4%$ of primary malignant bone tumors, and believed to arise from the remnants of notochordal tissue. Because of its rare occurrence in the thoracic spine, we report a case of chordoma involving the thoracic spine. A 45-year-old male was sufferred from chest pain radiating to the back. Chest CT showed a well marginated, round huge mass with multiseptated enhancement at the thoracic spine from T5 to T8 level. After percutaneous needle aspiration, piecemeal resection of the tumor was done. On cytologic smears, two types of neoplastic cells were arranged in sheets and cords in mucinous background. One type of cells consisted of medium sized cells with pink cytoplasm and round nuclei. The other type had voluminous bubbly or clear cytoplasm divided by intracytoplasmic septae imparting a feathery or basket-like appearance. Histologically, the tumor showed lobulated feature divided by fibrous septae and the tumor cells were pink eosinophilic or physaliphorous in morphology. Immunohistochemically, the tumor cells revealed strong positivity for low(AE1) and high (AE3) molecular weight cytokeratins.

• Current Photovoltaic Research
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• 제7권3호
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• pp.55-60
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• 2019

Polymer:nonfullerene solar cells with an inverted-type device structure were fabricated by employing the bulk heterojunction (BHJ) active layers, which are composed of poly[(2,6-(4,8-bis(5-(2-ethylhexyl)thiophene-2-yl)-benzo[1,2-b:4,5-b']dithiophene))-alt-(5,5-(1',3'-di-2-thienyl-5',7-bis(2-ethylhexyl)benzo[1',2'-c:4',5'-c']dithiophene-4,8-dione))] (PBDB-T) and 3,9-bis(6-methyl-2-methylene-(3-(1,1-dicyanomethylene)-indanone))-5,5,11,11-tetrakis(4-hexylphenyl)-dithieno[2,3-d:2',3-d']-s-indaceno[1,2-b:5,6-b']dithiophene (IT-M). The BHJ layers were formed on a pre-patterned indium-tin oxide (ITO)-coated glass substrate by spin-coating using the blend solutions of PBDB-T and IT-M. The solar cell performances were investigated with respect to the cell position on the ITO-glass substrates. In addition, the short-term shelf lifetime of solar cells was tested by storing the PBDB-T:IT-M solar cells in a glovebox filled with inert gas. The results showed that the performance of solar cells was relatively higher for the cells close to the center of substrates, which was maintained even after storage for 24 h. In particular, the PCE of PBDB-T:IT-M solar cells was marginally decreased after storage for 24 h owing to the slightly reduced fill factor, even though the open circuit voltage was unchanged after 24 h.

• The Korean Journal of Physiology and Pharmacology
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• 제28권1호
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• pp.1-10
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• 2024

Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia and dyslipidemia. Carvacrol (CAR) has demonstrated the potential to mitigate dyslipidemia. This study aims to investigate whether CAR can modulate blood glucose and lipid levels in a T2DM rat model by regulating short-chain fatty acids (SCFAs) and the GPR41/43 pathway. The T2DM rat model was induced by a high-fat diet combined with low-dose streptozocin injection and treated with oral CAR and/or mixed antibiotics. Fasting blood glucose, oral glucose tolerance, and insulin tolerance tests were assessed. Serum lipid parameters, hepatic and renal function indicators, tissue morphology, and SCFAs were measured. In vitro, high glucose (HG)-induced IEC-6 cells were treated with CAR, and optimal CAR concentration was determined. HG-induced IEC-6 cells were treated with SCFAs or/and GPR41/43 agonists. CAR significantly reduced blood lipid and glucose levels, improved tissue damage, and increased SCFA levels in feces and GPR41/43 expression in colonic tissues of T2DM rats. CAR also attenuated HG-induced apoptosis of IEC-6 cells and enhanced GPR41/43 expression. Overall, these findings suggest that CAR alleviates blood lipid and glucose abnormalities in T2DM rats by modulating SCFAs and the GPR41/43 pathway.

• Bulletin of the Korean Chemical Society
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• 제31권9호
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• pp.2607-2612
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• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

• Imaging Science in Dentistry
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• 제33권1호
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• pp.51-57
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• 2003

Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly an the expression of type I collagen and alkaline phosphatase mRNA. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1, 2, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. The specimens were then harvested and RNA extraction was carried out at 1 and 3 days after irradiation. The extracted RNA strands were reverse-transcribed and the resulting cDNA fragments were amplified by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in type I collagen mRNA expression relative to the control group, with a maximum level of type I collagen mRNA expression occurring at 8 Gy. The degree of type I collagen mRNA expression increased significantly at 1 day after irradiation, but little differences were found between the control group and at the 3rd day. The amount of alkaline phosphatase mRNA expression increased significantly at land 3 days after irradiation in the 1 Gy exposed group compared with the control group. Conclusion: The amount of type I collagen and alkaline phosphatase mRNA expression increased significantly 1 day after irradiation when compared with the control group.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2439-2445
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• 2014

Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salvia miotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibit tumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Using a mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo with polarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation, and also increased IFN-${\gamma}$ and perforin production of CD4+ T cells in response to tumor-activated splenocytes. Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogated by concanamycin A(CMA), a perforin inhibitor, but not IFN-${\gamma}$ Ab. On the other hand, after depletion of CD4+ T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumor growth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggested that CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin. We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4 phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, and demonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.