• 생명과학회지
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• 제20권2호
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• pp.275-280
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• 2010

Sulforaphane은 십자가화 채소에 존재하는 화합물로 항염증, 항암 및 신생혈관 생성의 억제 효과가 알려짐으로써 최근 많은 연구가 활발히 이루어지고 있으나, LPS에 의한 MMP-9 활성 조절에 대한 연구는 매우 미흡한 편이다. 따라서 본 연구에서 sulforaphane이 LPS 유도에 의한 MMP-9 활성에 미치는 영향에 대해서 조사해 보았다. Raw 264.7 세포에 sulforaphane을 전처리 한 후 LPS를 처리하여 gelatin zymography를 실시해 본 결과, LPS에 의해 유도된 MMP-9 활성 증가가 sulforaphane 농도 의존적으로 감소됨을 확인 하였다. 또한 RT-PCR과 MMP-9의 luciferase assay를 통한 실험에서 sulforaphane의 MMP-9 억제효과가 전사단계에서 조절됨을 추측 할 수 있었다. MMP-9 promoter 부위에 여러 가지의 전사조절인자 결합부위가 존재한다. 특히 AP-1과 NF-${\kappa}B$가 중요 전사조절인자로 작용하여 MMP-9 발현조절에 관여한다. 본 실험에서 sulforaphane에 의한 MMP-9 억제효과 기전에 이들 전사조절인자들의 중요한 역할을 조사하였다. AP-1과 NF-${\kappa}B$ 결합부위를 변형 시킨 vector를 transfection하여 MMP-9의 promoter 활성을 측정한 결과, 정상 vector에 비해 그 활성도가 현저히 떨어짐을 확인하였고, LPS에 의해 증가되는 AP-1과 NF-${\kappa}B$의 basal promoter 활성 또한 sulforaphane에 의해 감소됨을 관찰 할 수 있었다. 이상의 결과에서 sulforaphane의 MMP-9 활성억제효과는 AP-1과 NF-${\kappa}B$와 같은 전사인자들이 MMP-9의 전사를 조절함으로써 일어나는 것임을 알 수 있었다. 그리고 sulforaphane은 세포의 invasion능력 또한 효과적으로 억제시킴을 관찰 할 수 있었는데 이는 MMP-9 활성억제효과와 밀접한 관련이 있음을 추측 할 수 있었다.

• The Plant Pathology Journal
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• 제36권4호
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• pp.323-334
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• 2020

Lysin motif (LysM) proteins are reported to be necessary for the virulence and immune response suppression in many herbaceous plant pathogens, while far less is documented in woody plant pathogens. In this study, we preliminarily characterized the molecular function of a LysM protein LtLysM1 in woody plant pathogen Lasiodiplodia theobromae. Transcriptional profiles revealed that LtLysM1 is highly expressed at infectious stages, especially at 36 and 48 hours post inoculation. Amino acid sequence analyses revealed that LtLysM1 was a putative glycoprotein with 10 predicted N-glycosylation sites and one LysM domain. Pathogenicity tests showed that overexpressed transformants of LtLysM1 displayed increased virulence on grapevine shoots in comparison with that of wild type CSS-01s, and RNAi transformants of LtLysM1 exhibited significantly decreased lesion length when compared with that of wild type CSS-01s. Moreover, LtLysM1 was confirmed to be a secreted protein by a yeast signal peptide trap assay. Transient expression in Nicotiana benthamiana together with protein immunoblotting confirmed that LtLysM1 was an N-glycosylated protein. In contrast to previously reported LysM protein Slp1 and OsCEBiP, LtLysM1 molecule did not interact with itself based on yeast two hybrid and co-immunoprecipitation assays. These results indicate that LtLysM1 is a secreted protein and functions as a critical virulence factor during the disease symptom development in woody plants.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.149-156
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• 1999

포유류의 자궁조직은 발정주기를 통하여 역동적으로 변화하고 있으며 이러한 자궁조직의 분화는 시상하부-뇌하수체-생식소를 잇는 축에 의해 조절되는 스테로이드 호르몬에 의해 이루어진다. 그러나 에스트로겐 (E)이 어떤 유전자를 발현하여 자궁 내의 변화를 일으키는지는 아직 자세히 알려지지 않고 있다. 본 연구는 난소를 절제한 성숙한 흰쥐에 E을 처리한 후 자궁조직 내에서 c-fos, c-jun 및 hsp25 mRNA의 발현 변화를 Northern blot analysis방법을 사용하여 연구한 것이다. c-fos및 c-jun 암원유전자의 mRNA발현은 E처리 후 1시간 이전부터 증가하기 시작하며, 3시간 이내에 최고치에 도달한 후 급격히 감소하여 기저수준으로 되돌아갔다. 반면에 hsp25 mRNA수준은 E처리 후 3시간 대에서 최고치를 나타내나 증가된 발현량이 서서히 감소하며 12시간이 지난 후 까지도 정상대조군에 비해 높은 수준으로 유지되었다. 이러한 E의 영향이 선별적인지를 검증하기 위하여 E의 길항제인 tamoxifen을 사전처리하고 E을 추가로 처리하여 c-fos, c-jun및 hsp25 mRNA의 발현이 최고치에 이르렀던 3시간대에 자궁조직을 얻어 각각의 유전자 발현량을 조사한 결과 E에 의해 증가되었던 c-fos, c-jun 및 hsp25 mRNA의 수준이 억제됨을 확인하였다. 이러한 결과는 E이 자궁조직에 영향을 미치는데 초기의 일시적인 변화를 보이는 암원유전자인 c-fos 및 c-jun이 중요한 역할을 하리라는 것을 시사하며 hsp25의 경우는 좀더 늦은 반응에 관여하거나 c-fos및 c-jun에 의하여 간접적으로 조절을 받을 수도 있음을 보여 주는 것으로 사료된다.

• BMB Reports
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• 제37권5호
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• pp.538-545
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• 2004

A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants.

• 대한한의학회지
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• 제23권4호
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• pp.161-168
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• 2002

Objectives: Hwangryunhaedok-tang (Huang-lian-jie-du-tang, HRHDT, 黃連解毒湯) is a traditional Korean herbal medicine that is formulated with Coptidis Rhizoma, Phellodendri Cortex, Scutellariae Radix and Gardeniae Fructus. HRHDT is cold (寒) and bitter (苦) in nature and has general properties of clearing heat and detoxifying (淸熱解毒), strengthening the stomach and settling the liver (健胃平肝), and reducing inflammation, fever and swelling. This formula can prevent and treat artherosclerosis, hyperplasia of the endothelium, cerebral fluid circulation, cerebral vascular deterioration through aging, impairment of neurotransmitters, or disruption of the functioning of the cerebral cortex following infection or trauma. The purpose of the study reported here was to determine the neuroprotective effect of HRHDT on global ischemia induced by 4-vessel occlusion in Wistar rats. Methods: HRHDT extract was lyophilized after extraction with 85% methanol and 100% water. Rats were induced to 10 minutes of forebrain ischemia by 4-vessel occlusion (4-VO) and reperfused again. HRHDT was administered with a dose of 100 mg/kg, and 500 mg/kg of 85% methanol extracts and 100 mg/kg of 100% water extracts, respectively, at 0 min and 90 min after 4-VO. Rats were killed at 7 days after ischemia and the number of CA1 pyramidal neurons was counted in hippocampal sections stained with cresyl violet. Results: Body temperature of animals showed no significant difference between saline-treated groups and HRHDT extracts-treated groups until 5 hours of reperfusion. This result indicated that neuroprotective effects of HRHDT extracts were not due to hypothermic effects. The administration of HRHDT showed a significant neuroprotective effect on hippocampal CA1 neurons at 7 days after ischemia compared to the saline-treated group (P<0.001). HRHDT methanol extracts of 100 mg/kg, 500 mg/kg and HRHDT water extracts of 100 mg/kg showed 88.5%, 98.3% and 95.1 % neuroprotection, respectively. Conclusions: The results of this study demonstrate that administration of HRHDT is highly effective in reducing neuronal damage in response to transient global cerebral ischemia. HRHDT may involve many mechanisms that might account for its high degree of efficacy. A number of factors including free radicals, glutamate, calcium overload, NO, and various cytokines have been proposed to have an important role in causing neuronal death after short periods of global ischemia. Further studies are needed to know the neuroprotective mechanisms of HRHDT.

• 생명과학회지
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• 제25권1호
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• pp.1-7
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• 2015

칼슘 의존적으로 활성화되는 neutral protease calpain에 의한 단백질 분해는 세포의 성장을 조절하는데 중요한 단백질들의 역할에 매우 중요한 역할을 한다. Cyclin의 분해는 세포주기의 진행을 위한 필연적인 과정이다. D-type cyclins는 외부자극이나 신호에 의하여 세포주기의 G1 초기에 합성이 된 후 cyclin-dependent kinases (cdk4 및 cdk6)와의 결합하여 세포주기 S기 진입을 촉진하는 역할을 한다. 본 연구에서는 cyclin D3 단백질이 calpain protease에 의하여 번역 후 수준에서 조절 받고 있음을 제시하였다. 본 실험의 조건에서 lovastatin과 actinomycin D가 처리된 PC-3-M 전립선 암세포에서 cyclin D3 단백질의 발현이 완전히 사라졌지만, calpain inhibitor인 LLnL의 처리에 의하여 정상 수준으로 회복되었음을 알 수 있었다. 그러나 26S proteasome의 선택적 억제제인 lactacystin, lysosome 억제제인 ammonium chloride 및 chloroquine, serine protease 억제제인 PMSF는 동일 조건에서 lovastatin과 actinomycin D 처리에 의한 cyclin D3 단백질의 발현저하를 억제하지는 못하였다. In vitro 조건에서 순수 분리된 calpain은 cyclin D3 단백질을 칼슘 농도 의존적으로 분해하였으며, cyclin D3 단백질의 반감기는 LLnL 처리에 의하여 매우 유의적으로 증가되었다. 또한 calpain 저해인자인 calpastatin의 과발현은 PC-3-M 세포에서 뿐만 아니라 NIH 3T3 섬유아세포에서도 cyclin D3 단백질의 반감기 및 안전성을 증대시켰다. 이러한 결과는 cyclin D3 단백질이 칼슘에 의해 활성화 되는 protease calpain에 의해 조절됨을 보여주는 것이다.

• Journal of Plant Biotechnology
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• 제37권1호
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• pp.77-83
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• 2010

국립원예특작과학원에서 분양받은 토마토 18계통을 공시하여 재분화가 잘되는 적정 품종을 탐색한 결과, 계통번호 2001-58에서의 재분화율이 93%로 양호하였다. 또한 식물체로의 재분화 과정에서 보여 지는 갈변현상과 phenolic compound에 의한 식물조직의 괴사현상을 막기 위하여 항산화제인 ascorbic acid와 cystein을 단용 또는 혼용으로 첨가한 후 토마토 재분화에 미치는 영향을 살펴 본 결과, ascorbic acid $200{\sim}300\;{\mu}M/L$ 처리구에서 줄기형성율 및 생체중이 증가되는 현상을 관찰할 수 있었다. 토마토 엽록체 형질전환체 선발을 위해 spectinomycin의 적정 농도를 살펴본 결과, 재분화배지에 spectinomycin 20~25 mg/L 농도가 첨가되어진 처리구에서 재분화가 거의 이루어지지 않았다. 토마토 엽록체 형질전환을 위해 토마토 엽록체 게놈 일부를 분리하여 염기서열을 분석하여 담배와 비교 분석한 결과, homology가 매우 높음을 알 수 있었다. Homologous recombination에 의한 엽록체 형질전환이 되기 위해서 분리한 토마토 엽록체 게놈 일부를 border sequence로 이용하였고, transient assay를 위해 GFP 유전자가 포함된 토마토 엽록체 형질전환용 운반체 pKRT22-AG를 제작하였다. Bombardment을 한 후 원형질체를 나출하여 공초점 현미경하에서 관찰한 결과 엽록체 내에서만 GFP가 발현됨을 알 수 있었으며, DNA 농도 $1\;{\mu}g$, $0.6\;{\mu}m$ gold particle 1 mg, target distance 9 cm 조건이 가장 좋았다.

• Journal of Crop Science and Biotechnology
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• 제10권1호
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• pp.50-56
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• 2007

Barley S-adenosylmethionine synthetase1 gene, which was differentially expressed in seed development of extra early barley, was regulated by the phytohormones and abiotic stresses. In order to identify the regulation regions which were involved in transcriptional control of the phytohormones and abiotic stresses, we isolated 1459 bp fragment of HvSAMS1 gene promoter using genome walking strategy and deletion series were constructed. Deleted upstream fragments(-1459, -1223, -999, -766, -545, -301 bp) were fused to the GUS reporter gene and evaluated via Agrobacterium-mediated transient expression assay. Increased GUS activity of HvSMAS1 promoter -301/GUS construct under each of NaCl, $GA_3$, ABA and ethylene application was found. However, GUS activity was negligible in the leaves transformed with the HvSMAS1 promoter(-1459, -1223, -999, -766 and -545)/GUS constructs. No significant induction of GUS activity was observed for the ethionine and spermidine treatments. In order to locate promoter sequence of the HvSAMS1 gene that was critical for the activation of gene expression, deletion and addition promoter derivatives(+, includes 43 bp of 5' ORF) of the HvSAMS1 gene fused to the GUS reporter gene were applied. The tobacco leaves which harbored the additional HvSAMS1 promoter(-1459+, -1459 to -546, -545+ and -301+)/GUS construct did not significantly induce GUS activity as compared to the HvSAMS1 promoter(-1459, -545 and -301)/GUS constructs under each of NaCl, ABA and $GA_3$ treatment. However, the GUS activity was high in the tobacco leaves which harboring the -211 to -141 regions of the HvSAMS1 promoter. This result suggested that HvSAMS1 gene expression might be regulated by this region(from -211 to -141).

• Molecules and Cells
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• 제20권3호
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• pp.401-408
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• 2005

Reactive oxygen species (ROS) contribute to the development of various human diseases. Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS. SOD activity is dependent upon bound copper ions supplied by its partner metallochaperone protein, copper chaperone for SOD (CCS). In the present study, we investigated the protective effects of PEP-1-CCS against neuronal cell death and ischemic insults. When PEP-1-CCS was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Moreover, transduced PEP-1-CCS markedly increased endogenous SOD activity in the cells. Immunohistochemical analysis revealed that it prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that CCS is essential to activate SOD, and that transduction of PEP-1-CCS provides a potential strategy for therapeutic delivery in various human diseases including stroke related to SOD or ROS.

• The Korean Journal of Physiology and Pharmacology
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• 제11권5호
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• pp.175-182
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• 2007

Members of prostaglandin(PG) E-series elicit cellular effects mainly through adenylyl cyclase-cAMP signaling. The role of $PGE_2$-induced increase in cAMP has been shown to be compartmentalized in the cardiac myocytes: $PGE_2$-induced increase of cAMP is not involved in the control of cardiomyocytic contraction. The purpose of the present study was to define the effect of $PGE_1$ on the cGMP levels and the role of $PGE_1$ in the atrial secretory function. Experiments were performed in perfused beating rabbit atria and atrial contractile responses, cGMP and cAMP efflux, and atrial natriuretic peptide(ANP) secretion were measured. $PGE_1$ increased cGMP as well as cAMP efflux concentration in a concentration-dependent manner, however, no significant changes in atrial secretory responses were observed(with $1.0{\mu}M\;PGE_1$; for cGMP, $144.76{\pm}37.5%$, n=11 versus $-16.81{\pm}4.76%$, n=6, control, p<0.01; for cAMP, $187.60{\pm}41.52%$, n=11 versus $7.38{\pm}19.44%$, n=6, control, p<0.01). $PGE_1$ decreased atrial dynamics slightly but transiently, whereas $PGE_2$ showed similar effects but with lower potency. Isoproterenol increased atrial cAMP efflux(with 2.0 nM; $145.71{\pm}41.89$, n=5 versus $7.38{\pm}19.44%$, n=6, control, p<0.05) and mechanical dynamics and decreased ANP secretion. The $PGE_1$-induced increase in cGMP efflux showed a bell-shaped concentration-response curve. $PGE_1$-induced increase of cGMP efflux was not observed in the presence of L-NAME, an inhibitor of nitric oxide(NO) synthase, or ODQ, an inhibitor of NO-sensitive guanylyl cyclase. L-NAME and ODQ showed no significant effect on the $PGE_1$-induced transient decrease of atrial dynamics. These data indicate that $PGE_1$ increases cGMP levels via NO-soluble GC signaling in the cardiac atrium and also show that $PGE_1$-induced increases in cGMP and cAMP levels are not involved in the regulation of atrial secretory and contractile functions.