• Journal of Plant Biotechnology
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• 제32권4호
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• pp.269-273
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• 2005

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제33권6호
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.

• 한국임상수의학회지
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• 제26권6호
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• pp.520-523
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• 2009

본 연구는 말의 정상안과 재발성 포도막염이 있는 안구에서의 TLR-2, -4, -9 mRNA 발현의 정량적 차이를 비교하기 위해 수행되었다. 정상 및 재발성 포도막염이 있는 말 각 6두 에서 안구를 적출하여 모양체, 홍채, 망막 및 맥락막을 수집하였다. Real-time PCR assay 통해 정상안과 재발성 포도막염이 있는 안구에서의 TLR-2, -4, -9의 mRNA 발현 차이를 정량적으로 비교하였다. 말의 재발성 포도막염 시에는 모양체, 홍채에서 정상인 경우에 비해 4-12 배의 TLR-2 와 TLR-9 mRNA 발현증가를 보였으며, 맥락막 및 망막에서는 2-6 배의 TLR-2, -4, -9 mRNA 발현 증가를 보였다. 본 연구 결과는 Toll-like receptor 2, -4, -9이 말의 재발성 포도막염의 병리기전에 영향을 미치고 있음을 시사한다. 하지만 재발성 포도막염 시의 Toll-like receptor 2, -4, -9의 구체적 역할을 밝히기 위해서는 다양한 후속 연구가 요구된다.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2008년도 춘계 학술대회
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• pp.76-76
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• 2008

• Parasites, Hosts and Diseases
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• 제53권4호
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• pp.431-438
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• 2015

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T ($T_{reg}$) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and $T_{reg}$ cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/$TIRAP^{-/-}$ MEF cells, and quite substantially decreased in $TRIF^{-/-}$ MEF cells. In contrast, IL-10 and $TGF-{\beta}$ expression levels were not elevated in MyD88/$TIRAP^{-/-}$ MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and $T_{reg}$ cell mediated immune responses, although additional data are needed to convincingly prove this observation.

• 한국패류학회지
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• 제31권3호
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• pp.233-241
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• 2015

Toll-like receptors (TLRs) are a major pattern recognition receptor that recognize the structure of invading pathogen and play key roles by triggering immune response. In this study, we identified a sequence of TLR homolog and characterized at molecular level from the abalone (Haliotis discus hannai). Multiple alignments and phylogenetic analysis of abalone TLR protein belongs to the TLR 2/6. Expression level of abalone TLR 2/6 in the tissue was comparatively high in the mantle, gill, digestive duct, and hemocytes, but lowest in the muscle. Expression level of abalone TLR 2/6 mRNA in the mantle, gill, digestive duct, and hemocytes was 20-fold, 60-fold, 115-fold, 112-fold higher than in the muscle, respectively. Expression level of abalone TLR 2/6 mRNA in the mantle was steadily increased until 12 h and decreased post-infection with Vibrio parahemolyticus. While the expression level of abalone TLR 2/6 mRNA in the gill and hemocytes was drastically increased at 6 and 9 h post-infection with Vibrio parahemolyticus, respectively. These results suggest that abalone TLR 2/6 is conserved through evolution and may play roles similar to its mammalian counterparts.

• Bulletin of the Korean Chemical Society
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• 제33권9호
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• pp.3091-3094
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• 2012

• Journal of Yeungnam Medical Science
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• 제30권1호
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• pp.10-16
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• 2013

Background: Toll-like receptors (TLRs) are well-known pattern recognition receptors. Among the 13 TLRs, TLR2 is the most known receptor for immune response. It activates mitogen-activated protein kinases (MAPKs), which are counterbalanced by MAPK phosphatases [MKPs or dual-specificity phosphatases (DUSPs)]. However, the regulatory mechanism of DUSPs is still unclear. In this study, the effect of a TLR2 ligand (TLR2L, Pam3CSK4) on DUSP4 expression in Raw264.7 cells was demonstrated. Methods: A Raw264.7 mouse macrophage cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at $37^{\circ}C$ in 5% $CO_2$. TLR2L (Pam3CSK4)-mediated DUSP4 expressions were confirmed with RT-PCR and western blot analysis. In addition, the detection of reactive oxygen species (ROS) was measured with lucigenin assay. Results: Pam3CSK4 induced the expression of DUSP1, 2, 4, 5 and 16. The DUSP4 expression was also increased by TLR4 and 9 agonists (lipopolysaccharide and CpG ODN, respectively). Pam3CSK4 also induced ERK1/2 phosphorylation and ROS production, and the Pam3CSK4-induced DUSP4 expression was decreased by ERK1/2 (U0126) and ROS (DPI) inhibitors. U0126 suppressed the ROS production by Pam3CSK4. Conclusion: Pam3CSK4-mediated DUSP4 expression is regulated by ERK1/2 and ROS. This finding suggests the physiological importance of DUSP4 in TLR2-mediated immune response.

• BMB Reports
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• 제51권1호
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• pp.27-32
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• 2018

Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells.

• Molecular & Cellular Toxicology
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• 제3권1호
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• pp.46-52
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• 2007

In the course of novel TLR (Toll like receptor) 9 ligand, we found novel CpG ODN (Oligodeoxynucleotide) was active in augmenting antibody in mice. However, immune mechanism of new CpG ODNs is unclear. To clarify this, we examined immunoadjuvanticity by employing in vitro and in vivo immune profiles. In brief, in vitro treatment of novel CpG ODN upregulated the expression of TNF-$\alpha$, IL-6, and IL-12 mRNA in macrophages as well as that of IFN-$gamma$ mPNA in mouse splenocytes. In parallel, in vivo injection of novel CpG ODN directly activates macrophages and splenocytess, consequently upregulating MHC class II and CD86. Finally, we demonstrated anti-HBs antibody augmentation of novel CpG ODN. Collectively, this data indicates that novel CpG ODN is immunoadjuvant armed with Th1 typed immune machinery.