• The Plant Pathology Journal
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• 제30권4호
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• pp.343-354
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• 2014

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

• 한국약용작물학회지
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• 제26권1호
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• pp.19-25
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• 2018

Background: Inula japonica Thunb. is a plant belonging to the family compositae. Inulae flos (flower of I. britannica var. chinensis Regal.) is the dried flower of I. japonica Thunb. and contains various flavonoids (patulitrin, nepitrin and kaempferol), which have been utilized in traditional oriental medicine to treat nausea, phlegm, and coughs. However, ethanol extract of I. britannica (IJE) has not been previously studied for its use in cancer treatment, and its effects on oxidative stress, or inflammation. Thus, the present study investigated the anti-oxidant, anti-inflammatory, and anti-colorectal cancer effects of IJE using RAW264.7 and HCT-116 cells, which are human colorectal cancer cell line. Methods and Results: IJE contained flavonoids ($80.95{\pm}5.3mg/g$) and polyphenols ($310.53{\pm}10.6mg/g$). Moreover, it reduced lipopolysaccharide (LPS)-induced nitric oxide (NO) production and $H_2O_2$-induced oxidative stress by decreasing reactive oxygen species (ROS) levels. Additionally, the $500{\mu}g/m{\ell}$ IJE treatment increased caspase-3 activity and apoptotic cell death in HCT-116 cells. Conclusions: These results demonstrate that the anti-cancer effect of IJE against human colorectal cancer cells involves caspase-3 activation and apoptotic cell death. IJE also inhibited LPS-induced NO production, and $H_2O_2$-induced oxidative stress in RAW264.7 cells. However, further studies are required to explore how IJE treatment regulates signal transduction in NO and ROS production.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• BMB Reports
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• 제53권9호
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• pp.478-483
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• 2020

Kudoa septempunctata is a myxozoan parasite that causes food poisoning in individuals consuming olive flounder. The present study aimed to investigate the currently insufficiently elucidated early molecular mechanisms of inflammatory responses in the intestine owing to parasite ingestion. After Kudoa spores were isolated from olive flounder, HT29 cells were exposed to spores identified to be alive using SYTO-9 and propidium iodide staining or to antigens of Kudoa spores (KsAg). IL-1β, IL-8, TNF-α and NFKB1 expression and NF-κB activation were assessed using real-time PCR, cytokine array and western blotting. The immunofluorescence of FITC-conjugated lectins, results of ligand binding assays using Mincle-Fc and IgG-Fc, CLEC4E expressions in response to KsAg stimulation, and Mincle-dependent NF-κB activation were assessed to clarify the early immune-triggering mechanism. Inflammatory cytokines (IL-1β, GM-CSF and TNF-α), chemokines (IL-8, CCL2, CCL5 and CXCL1) and NF-κB activation (pNF-κB/NF-κB) in HT29 cells increased following stimulation by KsAg. The immunofluorescence results of spores and lectins (concanavalin A and wheat germ agglutinin) suggested the importance of Mincle in molecular recognition between Kudoa spores and intestinal cells. Practically, data for Mincle-Fc and KsAg binding affinity, CLEC4E mRNA expression, Mincle immunofluorescence staining and hMincle-dependent NF-κB activation demonstrated the involvement of Mincle in the early immune-triggering mechanism. The present study newly elucidated that the molecular recognition and immune-triggering mechanism of K. septempunctata are associated with Mincle on human intestinal epithelial cells.

• 한국의학물리학회지:의학물리
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• 제11권1호
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• pp.29-38
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• 2000

지금까지 각종 신경전달물질의 칼슘통로 억제 효과는 일반적으로 protein kinase 의 관여없이 G-protein mediated, membrane-delimited mechanism$^{1)}$ 으로 설명되어왔다. 그러나 최근들어 protein kinase C (PKC)의 활성화가 몇몇 신경전달물질에 의한 칼슘통로 억제효과를 야기하는 중요한 세포내 기전으로 보고되고 있다 그러므로 본 연구에서는 흰쥐 교감신경뉴론을 대상으로 하여 whole cell patch clamp technique을 사용하여 칼슘전류를 기록하고, 세포밖에 norepinephrine (NE)과 함께 PKC agonist 인 phorbol-12, 13-dibutyrate (PDBu)을 투여하면서 PDBu 전 처치로 인하여 NE 에 의한 칼슘전류 억제에 어떤 변화가 초래되는 지를 분석함으로써, 신경전달물질의 칼슘전류 억제효과시 PKC의 역할을 밝히고자 하였다. PDBu (500 nM) 처치는 칼슘전류의 크기를 증가시켰으며 이는 막전압 의존성을 보여 -10 mV ~ +10 mV 의 저분극 자극시 가장 크게 전류크기가 증가하였다. 또한 PDBu 처치는 tail current 의 deactivation을 느리게 하였다. PDBu 는 NE 에 의하여 활성화되는 pertussis toxin 예민성 G protein pathway를 통한 칼슘전류 억제를 감소시켰다. 비특이적인 protein kinase 길항제인 staurosporine (1 $\mu$M) 을 전처치 하고 PDBu를 투여하면 PDBu의 칼슘전류 크기 증가 효과가 소실되었으며 또한 NE에 의한 칼슘전류 억제를 해제하는 PDBu 의 조절효과도 소실되었다. 이상의 결과로부터 Protein Kinase C 가 활성되면 G protein을 경유하여 나타나는 칼슘전류 억제 효과가 소실된다고 결론지을 수 있다. Protein Kinase C 에 의하여 인산화되는 부위가 G-protein 인지 혹은 칼슘통로인 지에 관한 해답을 얻기 위하여는 추후 연구가 진행되어야 한다.

• Toxicological Research
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• 제17권
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• pp.47-57
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• 2001

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, and its pathology is characterized by the presence of numerous numbers of senile plaques and neurofibrillary tangles. Several genetic and transgenic studies have indicated that excess amount of $\beta$-amyloid protein (A$\beta$) is produced by mutations of $\beta$TEX>$\beta$-amyloid precursor protein and causes learning impairment. Moreover, $A\beta$ has a toxic effect on cultured nerve cells. To prepare AD model animals, we have examined continuous (2 weeks) infusion of $A\beta$ into the cerebral ventricle of rats. Continuous infusion of $A\beta$ induces learning impairment in water maze and passive avoidance tasks, and decreases choline acetyltransferase activity in the frontal cortex and hippocampus. Immunohistochemical analysis revealed diffuse depositions of $A\beta$ in the cerebral cortex and hippocampus around the ventricle. Furthermore, the nicotine-evoked release of acetylcholine and dopamine in the frontal cortex/hippocampus and striatum, respectively, is decreased in the $A\beta$-infused group. Perfusion of nicotine (50 $\mu\textrm{M}$) reduced the amplitude of electrically evoked population spikes in the CA1 pyramidal cells of the control group, but not in those of the $A\beta$-infused group, suggesting the impairment of nicotinic signaling in the $A\beta$-infused group. In fact, Kd, but not Bmax, values for ［$^3H$］ cytisine binding in the hippocampus significantly increased in the $A\beta$-infused rats. suggesting the decrease in affinity of nicotinic acetylcholine receptors. Long-term potentiation (LTP) induced by tetanic stimulations in CA1 pyramidal cells, which is thought to be an essential mechanism underlying learning and memory, was readily observed in the control group, whereas it was impaired in the $A\beta$-infused group. Taken together, these results suggest that $A\beta$ infusion impairs the signal transduction mechanisms via nicotinic acetylcholine receptors. This dysfunction may be responsible, at least in part, for the impairment of LTP induction and may lead to learning and memory impairment. We also found the reduction of glutathione- and Mn-superoxide dismutase-like immunoreactivity in the brains of $A\beta$-infused rats. Administration of antioxidants or nootropics alleviated learning and memory impairment induced by $A\beta$ infusion. We believe that investigation of currently available transgenic and non-transgenic animal models for AD will help to clarify the pathogenic mechanisms and allow assessment of new therapeutic strategies.

• Journal of Microbiology and Biotechnology
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• 제21권4호
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• pp.366-373
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• 2011

We characterized the proteomes of murine N2a cells following infection with three rabies virus (RV) strains, characterized by distinct virulence phenotypes (i.e., virulent BD06, fixed CVS-11, and attenuated SRV9 strains), and identified 35 changes to protein expression using two-dimensional gel electrophoresis in whole-cell lysates. The annotated functions of these proteins are involved in various cytoskeletal, signal transduction, stress response, and metabolic processes. Specifically, a-enolase, prx-4, vimentin, cytokine-induced apoptosis inhibitor 1 (CIAPIN1) and prx-6 were significantly up-regulated, whereas Trx like-1 and galectin-1 were down-regulated following infection of N2a cells with all three rabies virus strains. However, comparing expressions of all 35 proteins affected between BD06-, CVS-11-, and SRV9-infected cells, specific changes in expression were also observed. The up-regulation of vimentin, CIAPIN1, prx-4, and 14-3-3 ${\theta}/{\delta}$, and down-regulation of NDPK-B and HSP-1 with CVS and SRV9 infection were ${\geq}2$ times greater than with BD06. Meanwhile, Zfp12 protein, splicing factor, and arginine/serine-rich 1 were unaltered in the cells infected with BD06 and CVS-11, but were up-regulated in the group infected with SRV9. The proteomic alterations described here may suggest that these changes to protein expression correlate with the rabies virus' adaptability and virulence in N2a cells, and hence provides new clues as to the response of N2a host cells to rabies virus infections, and may also aid in uncovering new pathways in these cells that are involved in rabies infections. Further characterization of the functions of the affected proteins may contribute to our understanding of the mechanisms of RV infection and pathogenesis.

• Applied Biological Chemistry
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• 제48권4호
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• pp.339-344
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• 2005

도열병은 곰팡이 균(Magnaporthe grisea)에 의해 발병되는 것으로, 벼 수확량에 가장 큰 손실을 일으킨다. 본 연구에서는 벼 도열병 저항성 신호전달 체계에 관여하는 유전자를 분리하기 위하여 DEB(1, 3-Butadiene diepoxide) 처리를 통하여 벼 도열병 저항성 품종인 RIl260의 돌연변이 2,000여 종을 생산하고, 이들로부터 병 저항성 변이 개체를 조사하였다. 돌연변이 집단에서 백변종 돌연변이의 비율은 6.7%로 매우 높았으며, 이것은 DEB 처리에 의해서 생산된 집단 내에 돌연변이가 높은 빈도로 발생하였음을 보여준다. 돌연변이 집단의 병 저항성 분석을 통하여 완전히 혹은 부분적으로 벼 도열병에 저항성을 상실한 29개의 돌연변이체를 분리하였다. 이들 중에서 가장 심한 이병성 라인으로 확인된 M5465는 DNA 혼성화 반응 분석을 사용하여 분석하였으며, RIL260 품종에서 도열병 병 저항성과 밀접한 관련을 갖고 있는 것으로 보고된 Pi5(t) 유전좌위의 DNA 표지들이 실험에 사용되었다. 이 결과들은 M5465에서 Pi5(t) 유전좌위 내부에 DNA의 큰 결손 및 재배열이 있었음을 보여준다. 분리된 병 저항성 돌연변이 라인들은 Pi5(t)에 의해 매개된 도열병 저항성의 신호 전달 과정을 이해하는 데 유용하게 사용 될 수 있을 것이다.

• Journal of Nutrition and Health
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• 제44권3호
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• pp.196-202
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• 2011

본 연구는 EGCG의 항 당뇨 활성기전으로 췌장종양 선세포 AR42J의 분화 및 내분비기능 개선에 미치는 영향과 그 세포 신호전달 기전을 확인하였다. 그 결과 첫째, AR42J 세포에 EGCG 처리 시 췌장종양 선세포의 세포증식이 농도 의존적으로 감소되었다. 둘째, 세포사멸 유도가 유의적으로 일어나지 않는 농도인 1uM EGCG를 AR42J 세포에 처리한 결과 ngn 3, ${\alpha}$-amylase, insulin은 EGCG처리 24시간에 mRNA, 단백질 발현증가를 나타내었고 48시간에 유의적 증가를 나타내었다. 셋째, EGCG 처리 시 ERK, JNK MAP Kinase 기전은 인산화 억제를 나타내었고 반면에 p38 기전의 인산화는 48시간에 유의적 증가를 하였다. 넷째, p38기전 저해제인 SB203580을 처리하여 EGCG가 MAP Kinase 기전중의 하나인 p38 기전 인산화 활성의 회복을 나타내어 ngn 3 발현을 위한 전사 신호전달 기전임을 다시 확인하였다. 따라서 녹차 생리활성 성분인 EGCG의 췌장종양 선 세포 AR42J 처리 결과 EGCG는 p38 MAP Kinase 기전 활성을 통해 췌장 선세포의 분화지표인 ngn 3 발현을 증가시키며 췌장내분비 기능 지표인 ${\alpha}$-amylase, insulin 발현증가를 나타내어 세포의 내분비기능 개선에도 영향을 미치는 것으로 사료된다.

• 한국산학기술학회논문지
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• 제12권1호
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• pp.312-317
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• 2011

캡사이신 채널로 알려진 바닐로이드 수용체 TRPV1 (캡사이신채널, Transient Receptor Potential Vanilloid 1)은 통증발현에서 중요한 역할을 하는 것으로 알려져 있다. 하지만 TRPV1의 활성조절에 관여하는 단백질에 대하여는 알려진 바가 많지 않다. 최근 rat TRPV1과 직접적으로 결합하는 단백질을 탐색하여 mouse Rab11-FIP3 (rab11-family interaction protein 3)가 rat TRPV1과 직접적으로 결합한다는 것이 보고되었다. Rab11은 여러 가지의 세포내 이동에 관여하는 것으로 보고되었다. 그러므로 Rab11-FIP3과의 결합을 통해 TRPV1의 세포막으로의 이동에 관여할 것으로 추측할 수 있다. 본 연구에서는 전에 보고된 연구가 mouse와 rat 이라는 다른 종의 단백질끼리의 결합이기 때문에 같은 종에서의 상호작용을 확인하고 Rab11-FIP3의 TRPV1의 세포막으로의 이동에서의 역할을 알아보고자 현재까지 동정되지 않은 rat의 Rab11-FIP3의 유전자를 GenBank 서열을 바탕으로 rat 뇌의 RNA 로부터 cDNA 를 클로닝하여 유전자를 분리하고 TRPV1 과의 관계를 세포생물학적으로 알아보았다. 연구결과 rat의 Rab11-FIP3는 489개의 아미노산 서열을 가지고 있으며 human과는 80%, mouse와는 90% 이상 아미노산 서열의 상동성을 보였다. 조직별 분포는 심장, 뇌, 간, 콩팥, 정소에서 발현되고 있는 것을 northern blot assay와 western blot assay 로 확인하였다. rat 의 뇌조직에서 TRPV1 과 Rab11-FIP3 단백질이 결합하여 colocalize 하는 것을 면역화학방법으로 확인하였다. 이 결합은 같은 family 의 TRPV2 와는 결합하지 않는 특이적 결합이므로 Rab11-FIP3 가 TRPV1 과 상호작용하여 세포막으로의 이동에 관여할 것이라는 것을 시사한다.