• IMMUNE NETWORK
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• v.1 no.2
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• pp.151-161
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• 2001

Background: Inactivation in p53 tumor suppressor gene through a point mutation and deletion is one of the most frequent genetic changes found in human cancer, with 50% of an incidence. This high rate of mutation mostly suggests that the gene plays a central role in the development of cancer and the mutations detected so far were found in exons 5 to 8. Mutation of p53 locus produced accumulation of abnormal p53 protein, and negative regulation of cell proliferation and transcriptional activation as a suppressor of transformation were lost. In addition, inhibition of its normal cellular function of wild-type by mutant is an important step in tumorigenesis. Method: 4 colon cancer cell lines (SNU C1, C2A, C4, C5) were examined for mutation in exons 5 to 8 of the p53 tumor suppressor gene by PCR-SSCP analysis and expression pattern by western blotting and immunoprecipitation. p53-mediated transactivation ability were examined by CAT assay and base substitution of p53 in SNU C2A cell were detected by DNA sequencing. Results: 1) SNU C2A cell and SNU C5 cell were detected mobility shifts each in exon 5 and exon 7 of p53 gene by the PCR-SSCP method, implicating being of p53 mutation. 2) 3 colon cancer cell lines (SNU C1, SNU C2A, SNU C5) expressed wild type and mutant type p53 protein. 3) In northern blot experiment, SNU C2A and SNU C5 cell expressed high level of p53 mRNA. 4) Results of p53-mediated transactivation in colon cancer cell lines by CAT assay represented only SNU C2A cell has transcriptional activity. 5) DNA sequencing in SNU C2A cell showed missense mutation in codon 179 of one allele, histidine to arginine and wild type p53 in the other allele. Conclusion: Colon cancer cell lines showed correlation with mutation in p53 gene and accumulation of abnormal p53 protein. Colon cancer cell SNU C2A retained p53-mediated transactivation as heterozygous p53 with one mutant allele in 179 codon and the other wild-type allele.

• IMMUNE NETWORK
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• v.4 no.4
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• pp.216-223
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• 2004

Background: It is well known that IgA isotype switching is induced by $TGF-{\beta}1$. LPS-activated mouse normal B cells well differentiate into IgA secreting plasma cells under the influence of $TGF-{\beta}1$. Nevertheless, there are lots of difficulties in studying normal B cells in detail because it is not simple to obtain highly purified B cells, showing low reproducibility and transfection efficacy, moreover impossible to keep continuous culture. To overcome these obstacles, it is desperately needed to develop B cell line which acts like normal B cells. In the present study, we investigated whether CH12F3-2A lymphoma cells are appropriate for studying IgA isotype switching event. Methods: CH12F3-2A B cell line was treated with LPS and $TGF-{\beta}1$, then levels of germ-line (GL) transcripts were measured by RT-PCR, and $GL{\alpha}$ promoter activity was measured by luciferase assay. In addition, membrane IgA (mIgA) expression and IgA secretion were determined by FACS and ELISA, respectively. Results: $TGF-{\beta}1$, regardless of the presence of LPS, increased level of $GL{\alpha}$ transcripts but not $GL{\gamma}2b$ transcripts. However, IgA secretion was increased dramatically by co-stimulation of LPS and $TGF-{\beta}1$. Both mIgA and IgA secretion in the presence of $TGF-{\beta}1$ were further increased by over-expression of Smad3/4. Finally, $GL{\alpha}$ promoter activity was increased by $TGF-{\beta}1$. Conclusion: CH12F3-2A cell line acts quite similarly to the normal B cells which have been previously reported regarding IgA expression. Thus, CH12F3-2A lymphoma cell line appears to be adequate for the investigation of the mechanism(s) of IgA isotype switching at the cellular and molecular levels.

• The Journal of Pediatrics of Korean Medicine
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• v.29 no.4
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• pp.39-66
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• 2015

Objectives The purpose of this study is to investigate the effects of Sesamum indicum extracted (SEI) on atopic dermatitis in an in-vitro and in-vivo experiment using a MC/9 murine mast cells and a NC/Nga mouse. Methods In-vitro experiment, IL-4, IL-5, IL-6, IL-13, TNF-${\alpha}$ and GM-CSF mRNA expression were evaluated by Real-time PCR, IL-13, MIP-$1{\alpha}$ production by ELISA and manifestations of NFAT-1, NFAT-2, c-jun, c-fos, NF-${\kappa}B$ p65 transcription factors by western blotting. In-vivo experiment, we measured WBC, Eosinophil, Neutrophil, and serum IL-5, IL-13 in NC/Nga atopic dermatitis mouse, IL-5, IL-13, IFN-${\gamma}$, IL-4 in the spleenocyte culture supernatant by ELISA, the absolute cell numbers of CD4+, CD8+, +Gr-1+CD11b, B220+CD23+ in the axillary lymph node (ALN), peripheral blood mononuclear cells (PBMCs) and dorsal skin tissue, IL-5, IL-13 by Real-time PCR, the distribution of tissue inflammation and cellular infiltration by H&E and toluidine blue. Results SEI decreased IL-4, IL-5, IL-6, IL-13, GM-CSF, TNF-${\alpha}$ mRNA expression, IL-13, MIP-$1{\alpha}$ production and the expression of transcription factors including NFAT-1, c-jun, NF-${\kappa}B$ p65 in MC/9 murine mast cells. SEI orally administration decreased cell number of WBC, Eosinophil, the level of serum IgE, total cell number of ALN and dorsal skin tissue, absolute cell number of CD4+, CD8+, B220+CD23+ in the ALN. SEI orally administration also increased absolute cell number of CD8+/CD3+ and decreased Gr-1+/CD11b+ in PBMCs, decreased CD4+ in dorsal skin tissue, inhibited IL-5, IL-13 mRNA expression. Infiltration levels of inflammatory immune cells, mast cells and thickness of epidermis decreased in dorsal skin tissue. Conclusions SEI can regulate allergic inflammatory response suppressed the gene expression and production of cytokines that mediate allergic reactions, and will be able to be effectively utilized in the treatment of atopic dermatitis future.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.69-77
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• 2003

Background: Celecoxib, a COX-2 specific inhibitor, has recently been used for the treatment of rheumatoid arthritis. However, the molecular and cellular mechanisms of celecoxib against RA inflammation remain to be defined. To elucidate the action mechanism of celecoxib on inflammatory cells, we investigated the effect of celecoxib on the production of two important mediators of inflammation, nitric oxide and PGE2 Methods: RAW 264.7 cells stimulated with LPS were preincubated with various concentrations of celecoxib (from $10^{-8}$ to $10^{-5}$ M) and $10{\mu}M$ hydrocortisone, respectively. The production of NO and PGE2, the end products of iNOS and COX-2 genes, were estimated in culture supernatants by Greiss method and EIA, respectively. The expression of iNOS gene, COX-2 gene, $NF-{\kappa}B$, and $I-{\kappa}B$ were determined by RT-PCR and western blot analysis. Results: Celecoxib and hydrocortisone inhibited the production of NO and PGE2 in dose dependent manner, when RAW 264.7 cells were stimulated with LPS. The expression of iNOS was also down-regulated by celecoxib and hydrocortisone. Interestingly, COX-2 gene differentially expressed according to the dose of celecoxib, a decrease with lower dose ($10^{-8}$ M) but an increase with higher dose ($10^{-5}$ M). $NF-{\kappa}B$ binding activity was decreased by lower dose of celecoxib, whereas was not affected by higher dose of it. The expression of $I-{\kappa}B$ was suppressed by higher dose of celecoxib. Conclusion: The celecoxib strongly suppressed the production of NO and PGE2 in LPS-stimulated RAW264.7 cells. The decrease of NO seems to be linked to the inhibition of iNOS by celecoxib. The lower and higher dose of celecoxib differentially regulated the COX-2 expression and $NF-{\kappa}B$ activity.

• Journal of Life Science
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• v.19 no.2
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• pp.284-288
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• 2009

The innate immune system is important for the first line of host defence against infectious agents, which have penetrated the mechanical barriers. Mannose-binding lectin (MBL or mannan-binding protein, MBP) is a serum protein that is synthesized in the liver as a part of the acute phase response. MBL binds to carbohydrate structures presented by a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL is synthesized as a monomer that has a carboxy-terminal carbohydrate recognition domain, a neck region and a collagen region. Low MBL level was reported to be the most frequent immuno-deficiency syndrome. Although extensive studies have yielded detailed information on the structure of MBL, functions of the MBL complex are not fully understood yet. We, here, present cloning process of MBL cDNA from the rat liver and production of truncated recombinant MBL protein using a bacterial expression system in order to produce anti-MBL polyclonal antibody. Anti-MBL polyclonal antibody was raised in a New Zealand rabbit and its affinity was tested against recombinant protein using western blot technique. MBL cDNA, recombinant protein and anti-MBL antibody could be used as great arsenals to dissect cellular biochemistry of MBL.

• Molecules and Cells
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• v.41 no.4
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• pp.271-281
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• 2018

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on $IFN-{\alpha}/{\beta}$ production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of $IFN-{\alpha}/{\beta}$ also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.4
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• pp.161-167
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• 2015

These commensal intestinal bacteria can enhance the immune system and aid in nutrient absorption but can also act as opportunistic pathogens. Among these intestinal bacteria, the anaerobic Bacteroides fragilis are divided into enterotoxigenic B. fragilis (ETBF) which secrete the B. fragilis toxin (BFT) and non-enterotoxigenic B. fragilis (NTBF) which do not secrete BFT. ETBF can cause diarrhea and colitis in both humans and livestock but can also be found in asymptomatic individuals. ETBF is predominantly found in patients with inflammatory diarrheal diseases and traveller's diarrhea. Several clinical studies have also reported an increased prevalence of ETBF in human patients with inflammatory bowel disease (IBD), colitis and colorectal cancer. In small animal models (C57BL/6 wild-type mice, germ-free mice, multiple intestinal neoplasia (Min) mice, rabbits and Mongolian gerbils), ETBF have been found to initiate and/or aggravate IBD, colitis and colorectal cancer. BFT induces E-cadherin cleavage in intestinal epithelial cells resulting in loss of epithelial cell integrity. Subsequent activation of the ${\beta}$-catenin pathway leads to increased cellular proliferation. In addition, ETBF causes acute and chronic colitis in wild-type mice as well as enhances tumorigenesis in Min mice via activation of the Stat3/Th17 pathway. Currently, ETBF can be detected using a BFT toxin bioassay and by PCR. Advances in molecular biological techniques such as real-time PCR have allowed both researchers as well as clinicians to rapidly detect ETBF in clinical samples. The emergence of more sensitive techniques will likely advance molecular insight into the role of ETBF in colitis and cancer.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.19-29
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• 1996

The neurological manifestations of AIDS include dementia, encountered even in the absence of opportunistic superinfection or malignancy. The AIDS Dementia Complex appears to be associated with several neuropathological abnormalities, including astrogliosis and neuronal injury or loss. How can HIV-1 result in neuronal damage if neurons themselves are only rarely, if ever, infected by the vitus\ulcorner In vitro experiments from several different laboratiories have lent support to the existence of HIV- and immune-related toxins. In one recently defined pathway to neuronal injury, HIV-infected macrophages/microglia as well as macrophages activated by HIV-1 envelope protein gp120 appear to secrete excitants/neurotoxins. These substances may include arachidonic acid, platelet-activating factor, free radicals (NO - and O$_2$), glutamate, quinolinate, cysteine, cytokines (TNF-${\alpha}$, IL1-B, IL-6), and as yet unidentified factors emanating from stimulated macrophages and possibly reactive astrocytes. A final common pathway for newonal suscepubility appears to be operative, similar to that observed in stroke, trauma, epilepsy, and several neurodegenerative diseases, including Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. This mechanism involves excessive activation of N-methyl-D-aspartate (NMDA) receptor-operated channels, with resultant excessive influx of Ca$\^$2+/ leading to neuronal damage, and thus offers hope for future pharmacological intervention. This chapter reviews two clinically-tolerated NMDA antagonists, memantine and nitroglycerin; (ⅰ) Memantine is an open-channel blocker of the NMDA-associated ion channel and a close congener of the anti-viral and anti-parkinsonian drug amantadine. Memantine blocks the effects of escalating levels of excitotoxins to a greater degree than lower (piysiological) levels of these excitatory amino acids, thus sparing to some extent normal neuronal function. (ⅱ) Niuoglycerin acts at a redox modulatory site of the NMDA receptor/complex to downregulate its activity. The neuroprotective action of nitroglycerin at this site is mediated by n chemical species related to nitric oxide, but in a higher oxidation state, resulting in transfer of an NO group to a critical cysteine on the NMDA receptor. Because of the clinical safety of these drugs, they have the potential for trials in humans. As the structural basis for redox modulation is further elucidated, it may become possible to design even better redox reactive reagents of chinical value. To this end, redox modulatory sites of NMDA receptors have begun to be characterized at a molecular level using site-directed mutagenesis of recombinant subunits (NMDAR1, NMDAR2A-D). Two types of redox modulation can be distinguished. The first type gives rise to a persistent change in the functional activity of the receptor, and we have identified two cysteine residues on the NMDARI subunit (#744 and #798) that are responsible for this action. A second site, presumably also a cysteine(s) because <1 mM N-ethylmaleimide can block its effect in native neurons, underlies the other, more transient redox action. It appears to be at this, as yet unidentified, site on the NMDA receptor that the NO group acts, at least in recombinant receptors.

• The Korean Journal of Pharmacology
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• v.31 no.3
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• pp.323-331
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• 1995

Monocyte/macrophage infiltration is the well known initial features associated with the development of glomerular disease including non-immune mediated nephropathy. Tumor necrosis factor ${\alpha}(TNF{\alpha})$, a cytokine produced primarily by monocyte/macrophage, exhibits similar effects as observed at the initial stages and during the progression of glomerular injury. Because the mesangial cells are target cells for glomerular injury, the present study examined the effect of $TNF{\alpha}$ on glomerular mesangial cell membrane lipid peroxidation as an index of cytotoxicity attributing to $TNF{\alpha}$. Primary culture of rat mesangial cell was established by incubation of glomeruli isolated from male Sprague-Dawley rat kidneys utilizing a standard sieving method. The levels of lipid peroxides in the mesangial cells were quantitated by malondialdehyde- thiobarbituric acid adduct formation. During an 8 hour incubation at $37^{\circ}C$, $TNF{\alpha}$ at 10 to 10,000 units/ml increased the levels of lipid peroxides dose dependently. Western blot analysis demonstrated that a short thermal stress induced heat shock response and the synthesis of heat shock protein 70(hsp70) in this mesangial cells. Further, this induction of hsp 70 prevented increase of lipid peroxides in the mesangial cells exposed to $TNF{\alpha}$. These data suggest that $TNF{\alpha}-induced$ lipid peroxidation in the mesangial cells may have pathophysiological relevance to glomerular injury and prior induction of heat shock response may play a role in the cellular resistance against $TNF{\alpha}-induced$ glomerular injury.

• Journal of Biomedical Engineering Research
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• v.38 no.1
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• pp.43-48
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• 2017

Chemotherapy, radiation therapy, and surgery are major methods to treat cancer. However, current cancer treatments report severe side effects and high recurrences. Recent studies about engineering nanoparticles as a drug carrier suggest possibilities in terms of specific targeting and spatiotemporal release of drugs. While many nanoparticles demonstrate lower toxicity and better targeting results than free drugs, they still need to improve their performance dramatically in terms of targeting accuracy, immune responses, and non-specific accumulation at organs. One possible way to overcome the challenges is to make precisely controlled nanoparticles with respect to size, shape, surface properties, and mechanical stiffness. Here, we demonstrate $500{\times}200nm$ discoidal polymeric nanoconstructs (DPNs) as a drug delivery carrier. DPNs were prepared by using a top-down fabrication method that we previously reported to control shape as well as size. Moreover, DPNs have multiple payloads, poly lactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), lipid-Rhodamine B dye (RhB) and Salinomycin. In this study, we demonstrated a potential of DPNs as a drug carrier to treat cancer.