• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2004년도 생명공학 실용화를 위한 비젼
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• pp.81-90
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• 2004

Sialic acid containing glycosphingolipids (gangliosides) have been implicated in the regulation of various biological phenomena such as atherosclerosis. Recent report suggeststhat exogenously supplied disialoganglioside (GD3) serves a dual role in vascular smooth muscle cells (VSMC) proliferation and apoptosis. However, the role of the GD3 synthase gene in VSMC responses has not yet been elucidated. To determine whether a ganglioside is able to modulate VSMC growth. the effect of overexpression of the GD3 synthase gene on DNA synthesis was examined. The results show that the overexpression of this gene has a potent inhibitory effect on DNA synthesis and ERK phosphorylation in cultured VSMC in the presence of PDGF. The suppression of the GD3 synthase gene was correlated with the down-regulation of cyclinE/CDK2. the up-regulation of the CDK inhibitor p21 and blocking of the p27 inhibition,whereas up-regulation of p53 as the result of GD3 synthase gene expression was not observed. Consistently, blockade of GD3 function with anti-GD3 antibody reversed VSMC proliferation and cell cycle proteins. The expression of the CD3 synthase gene also led to the inhibition of TNF--induced matrix metalloproteinase-9 (MMP-9) expression in VSMC as determined by zymography and immunoblot. Furthermore, GD3 synthase gene expression strongly decreased MMP-9 promoteractivlty in response to TNF-. This inhibition was characterized by the down-regulation of MMP-9,which was Iranscriptionally regulated at NF-B and activation protein-1 (AP-1) sites in the MMP-9promoter Finally, the overexpression of MMP-9 in GD3 synthase transfectant cells rescued VSMC proliferation. However MMP-2 overexpression was not affected the cell proliferation. These findings suggest that the fl13 synthase gene represents a physiological modulator of VSMC responses that may contribute to plaque instability in atherosclerosis.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2007년도 제48회 학술심포지움 및 추계국제학술대회
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• pp.106.1-106.1
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• 2007

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• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2008년도 제49회 학술심포지움 및 국제학술대회
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• pp.53.2-53.2
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• 2008

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.300.3-301
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• 2003

The activation of the hepatic stellate cell (HSC) is a key step in liver fibrogenesis. Utilizing large scale sequencing of a 3' -directed cDNA library, we investigated expression profiles of quiescent and activated rat HSCs. During the activation process, O-acetyl disialoganglioside synthase (OAcGD3S) was identified as one of the significant upregulated factors. Upregulation of OAcGD3S in cultured HSCs was confirmed by both northern and western blot analyses. (omitted)

• Molecules and Cells
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• 제27권1호
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• pp.113-118
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• 2009

In this study, we have shown the transcriptional regulation of the human GD3 synthase (hST8Sia I) induced by valproic acid (VPA) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the regulation of hST8Sia I gene expression in VPA-stimulated SK-N-BE(2)-C cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5'-flanking region of the hST8Sia I gene by the transient expression method showed that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-${\kappa}B$, functions as the VPA-inducible promoter of hST8Sia I in SK-N-BE(2)-C cells. Site-directed mutagenesis and electrophoretic mobility shift assay indicated that the NF-${\kappa}B$ binding site at -731 to -722 was crucial for the VPA-induced expression of hST8Sia I in SK-N-BE(2)-C cells. In addition, the transcriptional activity of hST8Sia I induced by VPA in SK-N-BE(2)-C cells was strongly inhibited by SP600125, which is a c-Jun N-terminal kinase (JNK) inhibitor, and $G{\ddot{O}}6976$, which is a protein kinase C (PKC) inhibitor, as determined by RT-PCR (reverse transcription-polymerase chain reaction) and luciferase assays. These results suggest that VPA markedly modulated transcriptional regulation of hST8Sia I gene expression through PKC/JNK signal pathways in SK-N-BE(2)-C cells.

• Archives of Pharmacal Research
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• 제27권1호
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• pp.111-117
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• 2004

Hepatic microcirculatory failure is a major component of reperfusion injury in the liver. Recent data provided some evidence that endothelium-derived vasoconstrictors and vasodilators may be functionally important to the control of the total hepatic blood flow under these conditions of circulatory failure. Since Kupffer cells provide signals that regulate the hepatic response in ischemia/reperfusion (I/R), the aim of this study was to investigate the role of Kupffer cells in the I/R-induced imbalance of vasoregulatory gene expression. Rats were subjected to 60 min hepatic ischemia, followed by 5 h of reperfusion. The Kupffer cells were inactivated by gadolinium chloride ($GdCl_3$, 7.5 mg/kg body weight, intravenously) 1 day prior to ischemia. Liver samples were obtained 5 hrs after reperfusion for RT-PCR analysis of the mRNA for genes of interest: endothelin-1 (ET-1), its receptors $ET_A and ET_B$, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). ET-1 mRNA expression was increased by I/R. mRNA levels for $ET_A$ receptors showed no change, whereas $ET_B$ receptor transcripts increased in the I/R group. The increases in ET-1 and $ET_B$ mRNA were not prevented by the $GdCI_3$ pretreatment. The mRNA levels for iNOS and eNOS significantly increased within the I/R group with no significant difference between the I/R group and the $GdCl_3$-treated I/R group. HO-1 mRNA expression significantly increased in the I/R group and this increase was attenuated by $GdCI_3$. In conclusion, we have demonstrated that an imbalance in hepatic vasoregulatory gene expression occurs during I/R. Our findings suggest that the activation of Kupffer cells is not required for I/R-induced hepatic microvascular dysfunction.

• Biomolecules & Therapeutics
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• 제16권4호
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• pp.306-311
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• 2008

Although hepatic microcirculatory dysfunction occurs during endotoxemia, the mechanism responsible for this remains unclear. Since Kupffer cells provide signals that regulate hepatic response in inflammation, this study was designed to investigate the role of Kupffer cells in the imbalance in the expression of vasoactive mediators. Endotoxemia was induced by intraperitoneal E. coli endotoxin (LPS, 1 mg/kg body weight). Kupffer cells were inactivated with gadolinium chloride ($GdCl_3$, 7.5 mg/kg body weight, intravenously) 2 days prior to LPS exposure. Liver samples were taken 6 h following LPS exposure for RT-PCR analysis of mRNA for genes of interest: endothelin (ET-1), its receptors $ET_A$ and $ET_B$, inducible nitric oxide synthase (iNOS), heme oxygenase (HO-1), and tumor necrosis factor-$\alpha$ (TNF-$\alpha$). mRNA levels for iNOS and TNF-$\alpha$ were significantly increased 31.8-fold and 26.7-fold in LPS-treated animals, respectively. This increase was markedly attenuated by $GdCl_3$, HO-1 expression significantly increased in LPS-treated animals, with no significant difference between saline and $GdCl_3$ groups. ET-1 was increased by LPS. mRNA levels for $ET_A$ receptor showed no change, whereas $ET_B$ transcripts increased in LPS-treated animals. The increase in $ET_B$ transcripts was potentiated by $GdCl_3$. We conclude that activation of Kupffer cells plays an important role in the imbalanced hepatic vasoregulatory gene expression induced by endotoxin.

• Archives of Pharmacal Research
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• 제26권12호
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• pp.1079-1086
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• 2003

The aim of this study was to investigate the effect of trauma on cytochrome P450 (CYP) gene expression and to determine the role of Kupffer cells in trauma-induced alteration of CYP isozymes. Rats underwent closed femur fracture (FFx) with associated soft-tissue injury under anesthesia. To deplete Kupffer cells in vivo, gadolinium chloride ($GdCl_3$) was intravenously injected at 7.5 mg/kg body wt., 1 and 2 days prior to FFx surgery. At 72 h of FFx, liver tissues were isolated to determine the mRNA and protein expression of CYP isozymes and NADPH-P450 reductase by reverse transcription-polymerase chain reaction and Western immunoblotting, respectively. In addition, the mRNA levels of tumor necrosis factor alpha (TNF-$\alpha$), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were evaluated. FFx increased the mRNA level of CYP1A1; an increase that was not prevented by $GdCl_3$. There were no significant differences in the mRNA expression of CYP1A2, 2B1 and 2E1 among any of the experimental groups. The protein levels of CYP2B1 and 2E1 were significantly decreased by FFx; a decrease that was not prevented by $GdCl_3$ treatment. The gene expression of NADPH-P450 reductase was unchanged by FFx. FFx significantly increased the expression of TNF-$\alpha$ mRNA; an increase that was attenuated by $GdCl_3$. The mRNA expression of HO-1 was increased by FFx, but not by $GdCl_3$ . Our findings suggest that FFx differentially regulates the expression of CYP isozyme through Kupffer cell-independent mechanisms.

• Bulletin of the Korean Chemical Society
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• 제28권12호
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• 생명과학회지
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• 제20권9호
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• pp.1332-1338
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• 2010

사람 신경모세포종 세포주 SH-SY5Y에서 Fenretinide (FenR)에 의한 GD3합성효소(hST8Sia I)의 발현증가기작을 규명하게 위하여 hST8Sia I의 프로모터 활성을 조사해 본 결과 -1146에서 -646영역에서 FenR에 의한 활성증가를 나타내었다. 또한 부위특이적 변이의 분석은 -731에서 -722영역에 위치한 전사인자 NF-kB 결합부위가 hST8Sia I의 FenR에 의한 활성증가에 중요하게 관여하고 있음을 나타내었다. FenR에 의한 hST8Sia I 유전자의 발현유도에 포함된 신호전달기작을 전사인자 단백질의 항체를 이용하여 조사해 본 결과 FenR처리에 의해 세포질에서는 인산화된 AKT단백질 수준의 증가가 관찰되었고 핵내에서는 NF-kB의 p65단백질의 증가가 관찰되었다. 이러한 결과들은 FenR에 의한 hST8Sia I 유전자의 발현증가는 AKT신호전달경로에 의해 활성화된 NF-kB의 핵내로 이동하여 hST8Sia I 유전자의 프로모터에 결합함으로서 전사가 촉진되어 일어난다는 것을 나타낸다.