• Molecules and Cells
• /
• v.41 no.10
• /
• pp.917-922
• /
• 2018

The CRISPR-Cas system is a well-established RNA-guided DNA editing technique widely used to modify genomic DNA sequences. I used the CRISPR-Cas9 system to change the second and third nucleotides of the triplet $T{\underline{CT}}$ of human TNSFSF9 in HepG2 cells to $T{\underline{AG}}$ to create an amber stop codon. The $T{\underline{CT}}$ triplet is the codon for Ser at the $172^{nd}$ position of TNSFSF9. The two substituted nucleotides, AG, were confirmed by DNA sequencing of the PCR product followed by PCR amplification of the genomic TNFSF9 gene. Interestingly, sequencing of the cDNA of transcripts of the edited TNFSF9 gene revealed that the $T{\underline{AG}}$ had been re-edited to the wild type triplet $T{\underline{CT}}$, and 1 or 2 bases just before the triplet had been deleted. These observations indicate that CRISPR-Cas9-mediated editing of bases in target genomic DNA can be followed by spontaneous re-editing (correcting) of the bases during transcription.

• IMMUNE NETWORK
• /
• v.4 no.2
• /
• pp.116-122
• /
• 2004

Background: LIGHT (TNFSF14) is a member of tumor necrosis factor superfamily and is the ligand for TR2 (TNFRSF14/HVEM). LIGHT is known to have proinflammatory roles in atherosclerosis. Methods: To find out the expression pattern of LIGHT in atherosclerotic plaques, immunohistochemical analysis was performed on human carotid atherosclerotic plaque specimens. LIGHT induced atherogenic events using human monocytic cell line THP-1 were also investigated. Results: Immunohistochemical analysis revealed expression of LIGHT and TR2 in foam cell rich regions in the atherosclerotic plaques. Double immunohistochemical analysis further confirmed the expression of LIGHT in foam cells. Stimulation of THP-1 cells, which express TR2, with either recombinant LIGHT or immobilized anti-TR2 monoclonal antibody induced interleukin-8 and matrix metalloproteinase(MMP)-9. Electrophoretic mobility shift assay demonstrated that LIGHT induces nuclear localization of transcription factor, nuclear factor $(NF)-{\kappa}B$. LIGHT induced activation of MMP-9 is mediated by $NF-{\kappa}B$, since treatment of THP-1 cells with the $NF-{\kappa}B$ inhibitor PDTC (pyrrolidine dithiocarbamate) completely blocked the activation of MMP-9. Conclusion: These data indicate that LIGHT is expressed in foam cells in atherosclerotic plaques and is involved in atherogenesis through activation of pro-atherogenic cytokine IL-8 and destabilization of plaque by inducing matrix degrading enzyme.

• The Journal of Internal Korean Medicine
• /
• v.29 no.3
• /
• pp.543-553
• /
• 2008

Objective: The goal of this study was to determine the anti-asthma mechanism of SF on TNF-${\alpha}$ induced activation on A549 (human type II-like epithelial) cells. Using oligonucleotide microarray, we sought to establish the molecular mechanism of the protective effects of SF on A549 cells. Material & Methods : Cells were cultured in three different conditions: 1) negative control group was cultured in normal condition of DMEM, 2) positive control group was activated with TNF-${\alpha}$, IL-4. and IL-1${\beta}$, and 3) SF treated group was previously treated with 0.1${\mu}g/ml$ SF after TNF-${\alpha}$, IL-4. and IL-1 activation. Cells of positive control and SF treated groups were cultured for 30 min, 1hr, 3hr and 6hr. Results : The comparative analysis of the gene expression profile revealed that proinflammatory cytokines such as IL1F8, IL1F9, IL1R1. IL1RN, IL1RAPL1, IL8, TNFRSF4, TNFSF10c, TNFSF13, TRAF5, and TRAF7 and inflammation-related genes including MMP2, MMP11, MMP14, MMP15, MMP16, MMP19, MMP25, and MMP27 were down regulated with SF treatment. Cell adhesion molecule genes such as ITGB1, ITGBL1, selectin P ligand, selectin E, ICAM2, ICAM3, VCAM1, PECAM, FCER1G and MMP28 genes were also down-regulated in SF treated A549 cells. Conclusion : These results suggest that the anti-asthmatic effects of SF could be mediated by regulating specific genes related with cell adhesion, proinflammatory cytokine and inflammation-related genes in A549 cells.

• BMB Reports
• /
• v.51 no.9
• /
• pp.468-473
• /
• 2018

Purinergic receptor signaling is increasingly recognized as an important regulator of inflammation. The P2X family purinergic receptors P2X5 and P2X7 have both been implicated in bone biology, and it has been suggested recently that P2X5 may be a significant regulator of inflammatory bone loss. However, a role for P2X5 in periodontitis is unknown. The present study aimed to evaluate the functional role of P2X5 in ligature-induced periodontitis in mice. Five days after placement of ligature, analysis of alveolar bone revealed decreased bone loss in $P2rx5^{-/-}$ mice compared to $P2rx7^{-/-}$ and WT control mice. Gene expression analysis of the gingival tissue of ligated mice showed that IL1b, IL6, IL17a and Tnfsf11 expression levels were significantly reduced in $P2rx5^{-/-}$ compared to WT mice. These results suggest the P2X5 receptor may regulate bone loss related to periodontitis and it may thus be a novel therapeutic target in this oral disease.

• Korean Journal of Microbiology
• /
• v.42 no.4
• /
• pp.319-325
• /
• 2006

[ $\beta$ ]-Glucans (AG) were prepared from Agaricus blazei cultured in the medium fortified with the roots of Pueraria spp. by repeated extraction with hot water, gel filtration chromatography and DEAE ion exchange chromatography. Oligosaccharides (AO) were derived from the hydrolysis of AG by an endo-$\beta$-(1$\rightarrow$6)-glucanase from Bacillus megaterium. The anti-HT-29 human colon cancer activity of AG or AO was investigated using MTT assay, apoptosis assay, cell cycle analysis, and cDNA microairay. AG and AO both inhibited proliferation and growth of HT-29 cells, and stimulated apoptosis of the cells in a dose-dependent manner. In cell cycle analysis, treating HT-29 cells with AG or AO resulted in the increase of cells in the G0 (sub-G1) and G1 phase. Especially, AO was more effective in inducing G0/G1 cell cycle arrest than AG. To screen the genes involved in the increase of apoptosis, the gene expression profile of the HT-29 cells treated with AO was examined by cDNA microarray. While several genes involved in cell cycle progression (CCND2 and CDK2) were down-regulated, many genes involved in apoptosis (TNFSF9, TNFRSF9, FADD, CASP8, BAD, CRADD, CASP9 etc), cell cycle inhibitor (CDKN2A), immune response (IL6, IL18, IL6R etc), and tumor suppressor (CEACAM1, TP53BP2, IRF1, and PHB) were up-regulated. These results suggest that AO could inhibit the proliferation and growth of HT-29 cells by G0/G1 cell cycle arrest and induction of apoptosis.