• Archives of Pharmacal Research
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• 제28권3호
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• pp.274-279
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• 2005

Pharmacological inhibition of interleukin-12 (IL-12) production may be a therapeutic strategy for preventing the development and progression of disease in experimental models of autoimmunity. In this study, the effects of myricetin, a naturally occurring flavonoid present in fruits, vegetables and medicinal herbs, on the production of IL-12 were investigated in mouse macrophages stimulated with lipopolysaccharide (LPS). Myricetin significantly inhibited the LPS­induced IL-12 production from both primary macrophages and the RAW264.7 monocytic cell-line in a dose-dependent manner. The effect of myricetin on IL-12 gene promoter activation was analyzed by transfecting RAW264.7 cells with IL-12 gene promoter/luciferase constructs. The repressive effect was mapped to a region in the IL-12 gene promoter containing a binding site for NF-${\kappa}B$. Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the NF-${\kappa}B$ site, which significantly decreased upon addition of myricetin, indicating that myricetin inhibited IL-12 production in LPS-activated macrophages via the down­regulation of NF-KB binding activity.

• BMB Reports
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• 제40권3호
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• pp.358-367
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• 2007

A novel mannose-binding tuber lectin with in vitro antiproliferative activity towards human cancer cell lines and antiviral activity against HSV-II was isolated from fresh tubers of a traditional Chinese medicinal herb, Typhonium divaricatum (L.) Decne by a combined procedure involving extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-SEPHAROSE, CM-SEPHAROSE and gel-filtration on sephacryl S-200. The apparent molecular mass of the purified Typhonium divaricatum lectin (TDL) was 48 kDa. TDL exhibits hemagglutinating activity toward rabbit erythrocytes at 0.95 $\mu$g/ml, and its activity could be strongly inhibited by mannan, ovomucoid, asialofetuin and thyroglobulin. TDL showed antiproliferative activity towards some well established human cancer cell lines, e.g. Pro-01 (56.7 $\pm$ 6.8), Bre-04 (41.5 $\pm$ 4.8), and Lu-04 (11.4 $\pm$ 0.3). The anti-HSV-II activity of TDL was elucidated by testing its HSV-II infection inhibitory activity in Vero cells with $TC_50$ and $EC_50$ of 5.176 mg/ml and 3.054 $\mu$g/ml respectively. The full-length cDNA sequence of TDL was 1145 bp and contained an 813-bp open reading frame (ORF) encoding a 271 amino acid precursor of 29-kDa. Homology analysis showed that TDL had high homology with many other mannose-binding lectins. Secondary and three-dimensional structures analyses showed that TDL is heterotetramer and similar with lectins from mannose-binding lectin superfamily, especially those from family Araceae.

• 미생물학회지
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• 제43권4호
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• pp.250-255
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• 2007

Deinococcus radiodurans recA는 이 미생물의 방사선 저항성을 나타내는 표현형에 필수적이며 재조합성 DNA 수선 과정에 관여한다. 이 과정에서 RecA 단백질은DNA와 결합하여 반응의 활성 종인 RecA nucleoprotein 필라멘트를 형성한다. DNA-의존성 ATPase 활성과 함께, RecA 단배질의 외가닥 DNA 혹은 이중가닥 DNA와의 상호작용은 RecA 단백질이 관여하는 반응의 중심과정으로 이에 관한 분석을 시도하였다. D. radiodurans RecA 단배질은 DNA에 결합한 DNA-단백질 복합체만이 ATPase 활성을 나타내므로, ATP (혹은 dATP) 가수분해를 측정함으로써 RecA와 외가닥 DNA와의 상호작용 정도를 분석하였다. D. radiodurans RecA 단백질은 외가닥 DNA의 염기 구성의 이질성에 영향을 받았으며, homopolymer인 poly(dT)와의 상호작용에서 가장 높은 가수분해 활성을 보였다. Homopolymer인 합성 DNA-의존성 ATP 및 dATP의 가수분해는 pH 6.0과 9.0의 범위에서 다소 일정한속도로 일어났으며 최적 pH는 7.0과 7.5 사이였다. 외가닥 DNA-의존성 ATPase 활성은 염의 존재에 영향을 받아 KCl이 존재하면 다소 억제되나, K-glutamate가 존재하면 오히려 촉진되었다. RecA 단백질과 외가닥 DNA의 상호작용을 ATP 가수분해로 분석하였을 때 2 mM 이상의 magnesium 이온이 DNA 결합반응에 필요하였으며, 비교적 넓은 범위의 pH에서 외가닥 DNA와의 결합반응이 일어나며, 이러한 결합반응은 당량적인 비(1:3, RecA protein: DNA nucleotide)로 일어났다.

• 생명과학회지
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• 제12권3호
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• pp.250-255
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• 2002

독성이 있는 특이한 편모충인 Alexandrium tamarense의 구별하기 위한 종특이적인 형광 DNA탐침 AT1이 서로 다른 3가지의 고정액에서, 그리고 배양기간의 차이와 whole cell hybridization을 사용하는 광학 현미경 또는 형광 현미경에 의한 세포밀도 측정에 있어서 hiding activity를 비교하는 방법으로 여러 다른 종의 실험에 사용되어졌고, 그의 결과를 보고하는 바이다. 형광 DNA탐침 AT1은 특이적으로, A. tamarense에 결합했지만, 형태학적으로 유사한 다른 편모충에는 결합하지 않았다. 특히 형태적으로 A. tamarense에 유사한 A. catenella는 형광 DNN탐침 AT1에 결합하지 않았다. A. tamarense은 다른 세가지 고정액을 처리하였을 때, 고정액과 상관없이 강한 형광신호를 발산하였다. 추가해서 말하면, 배양기간에 관계없이 형광 DNA탐침 AT1의 biding activity는 강했다. 광학 현미경에 의해서 측정 되어지기 보다는 형광현미경에 의해서 세포밀도가 측정되었다. 형광 DNA탐침 AT1을 이용한 A. tamarense의 동정과 계산은 이 분야에서 새로운 생물독성 감시와 예측 시스템에 기여 할 것이다.

• Biomolecules & Therapeutics
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• 제25권5호
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• pp.535-544
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• 2017

Carnosol is a phenolic antioxidant present in rosemary (Rosmarinus officinalis). It is known for anti-inflammatory effects, analgesic activity and anti-cancer effects. However, no study has been dedicated yet to its effect on atopic dermatitis (AD). Here, we show that carnosol effectively inhibited LPS-induced nitric oxide (NO) generation and expression of inflammatory marker proteins (iNOS and COX-2) in RAW 264.7 cells. In addition, carnosol effectively inhibits the phosphorylation of STAT3 and DNA binding activity in RAW 264.7 cells. Pull down assay and docking model analysis showed that carnosol directly binds to the DNA binding domain (DBD) of STAT3. We next examined the anti-atopic activity of carnosol ($0.05{\mu}g/cm^2$) using 5% Phthalic anhydride (PA)-induced AD model in HR1 mice. Carnosol treatment significantly reduced 5% PA-induced AD like skin inflammation in skin tissues compared with control mice. Moreover, carnosol treatment inhibits the expression of iNOS and COX-2 in skin tissue. In addition, the levels of $TNF-{\alpha}$, $IL-1{\beta}$, and Immunoglobulin-E in blood serum was significantly decreased in carnosol treated mice compared with those of 5% PA treated group. Furthermore, the activation of STAT3 in skin tissue was decreased in carnosol treated mice compared with control mice. In conclusion, these findings suggest that carnosol exhibited a potential anti-AD activity by inhibiting pro-inflammatory mediators through suppression of STAT3 activation via direct binding to DBD of STAT3.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.180-180
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• 1996

Previous studies have shown that kainic acid (KA) causes an elevation of hippocampal proenkephalin mRNA level. However, the role of proto-oncogene products, such as c-Fos, c-Jun and Fra proteins in the regulation of KA-induced proenkephalin mRNA increase in the hippocampus has not been well characterized. Thus, in the present study, the effect of cycloheximide (CHX) on KA-induced proenkephalin mRNA and immediate early gene products induction was examined. After pretreating with either vehicle or CHX (20 mg/kg, s.c.) for 30 min, KA (10 mg/kg) was administered s.c. The animals were sacrificed 1,2, or 8 hrs after KA administration. Total RNA and were isolated for Northern blot assay, and proteins were isolated for Western and electrophoretic gel-shift assays. First, we found that CHX inhibited KA-induced proenkephalin mRNA increase without altering intracellular proenkephalin protein level. Secondly, Western blot assays showed that KA increased c-Fos, c-Jun and Fra proteins at 1,2, and 8 hrs and CHX inhibited these immediate early gene products. Finally, electrophoretic gel shift assays revealed that KA increased both AP-1 and ENKCRE-2 DNA binding activities. Furthermore, CHX attenuated KA-induced AP-1 and ENKCRE-2 DNA binding activities. Both AP-1 and ENKCRE-2 DNA binding activities were abolished by cold AP-1 or ENKCRE-2 oligonucleotides, and further reduced by antibodies against c-Fos or c-Jun. Antibody against CREB reduced ENKCRE-2, but not AP-1, DNA binding activity. Our results suggest that on-going protein synthesis is required for elevation of hippocampal proenkephalin mRNA level induced by KA. All c-Fos, c-Jun, and Fra proteins appears to be involved in the regulation of hippocampal proenkephalin mRNA level induced by KA (This study was supported by a grant from KOSEF).

• 상하수도학회지
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• 제20권6호
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• pp.893-904
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• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.

• BMB Reports
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• 제38권6호
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• pp.676-682
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• 2005

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of $NaBH_4$, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1557-1564
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• 2009

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

• IMMUNE NETWORK
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• 제24권1호
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• pp.1.1-1.6
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• 2024

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.