환경오염원인 구리 , 셀렌에 의한 Mouse 조직내 스트레스 반응

이정채,임계택 ( Jeong Chae Lee,Kye Taek Lim ) 한국환경생물학회 1996 환경생물 : 환경생물학회지 Vol.14 No.2

In order to test the response of stress (inducible protein) by copper or selenium which is toxic mineral, as environmental pollutant, we measured the amounts of the inducible protein from muscle, spleen, liver and brian tissues of mouse in vivo. Mice were fed at 0.5, 1 or 1.5mM copper or 0.25, 0.5 or 1mM selenium with the oral administration. The results of these experiments were summarized as follows; 1. The inducible protein was mainly produced at 1.0∼1.5mM after 18hrs treatment of copper in the spleen and muscle. 2. In selenium case, the amounts of the inducible protein was increased even at 0.5mM after 12hrs in the spleen and muscle, at 1mM after 6hrs in the liver. 3. Generally, the amounts of the inducible protein was arised between 6hrs and 18hrs after treatment of copper in the muscle, whereas it was decreased at 24hrs. The molecular weight of the inducible protein was about 18 KDa and its life span is about 12hrs. 4. In the spleen and muscle, the inducible protein was produced chiefly from 12hrs to 24hrs after treatment of selenium. The molecular weight of the inducible protein was about 20 KDa in spleen, about 35 KDa in muscle respectively and its life span was at least 6hrs. 5. The molecular weight of DNA was shown differently at 0.5 and 1mM after 12hrs administration of copper in the spleen. Commonly, the difference of DNA molecular weight by copper was about 0.5∼2Kb, comparing with control. 6. The changes of DNA molecular weight was distinctively different at 1∼12hrs after treatment of 0.25∼1mM selenium in the spleen. 7. The weight of mouse tissues was increased largely after 12hrs on treatment of 1.5 mM copper or 1mM selenium in both spleen and liver.

Epstein-Barr Virus-infected Akata Cells Are Sensitive to HistoneDeacetylase Inhibitor TSA-provoked Apoptosis

이정채,Hyung-Soon Lee,최기춘,국성호,장용석,한성규,김범태,손영옥,임지영,전영미,Keun-Soo Lee,김정기,김소순 한국생화학분자생물학회 2005 BMB Reports Vol.38 No.6

Epstein-Barr virus (EBV) infects more than 90% of the world’s population and has a potential oncogenic nature. A histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), has shown potential ability in cancer chemoprevention and treatment, but its effect on EBV-infected Akata cells has not been examined. This study investigated the effect of TSA on the proliferation and apoptosis of the cells. TSA inhibited cell growth and induced cytotoxicity in the EBV-infected Akata cells. TSA treatment sensitively induced apoptosis in the cell, which was demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to the sub-G/G phase in flow cytometric analysis, and the ladder formation of genomic DNA. Western blot analysis showed that caspasedependent pathways are involved in the TSA-induced apoptosis of EBV-infected Akata cells. Overall, this study shows that EBV-infected B lymphomas are quite sensitive to TSA-provoked apoptosis.

쥐 전뇌세포 배양에 있어서 천연 생리활성물질이 하이드록실 라디칼에 의한 세포독성에 미치는 영향

이정채,임계택 한국독성학회 1998 Toxicological Research Vol.14 No.2

The biological effects of the water extracts of Rhus Verniciflua Stokes (RVS) were evaluated by protection against hydroxyl radicals. Antioxidative activities were measured using both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and thiocyanate method. Also we used the Glucose oxidase (GO) 20 mU/$\textrm{m}{\ell}$ hydroxyl radical generating system in mouse forebrain cell culture. Water was used for ex-traction from RVS as a solvent which has high polarity especially. In DPPH method, the antioxidative activities of the crude water extract were stronger than any other extracts of low polar-solvents. In the antioxidative effects of mouse forebrain culture using 20 mU/$\textrm{m}{\ell}$ GO, cell viabilities were evaluated 65.6%, 68.8% at 1 $\mu\textrm{g}$. 5 $\mu\textrm{g}$ addition of crude water extracts (30 mg/$\textrm{m}{\ell}$) respectively. 10 $\mu\textrm{g}$ addition of crude water extracts had more than 86.1% cell viabilities, P<0.0l significantly, compared with the group treated with GO alone. In comparison with the antioxidative activities of several commercial antioxidants (ascorbic acid, $\alpha$-tocopherol, catalase, serum), 273 $\mu\textrm{g}$/$\textrm{m}{\ell}$ addition of crude water extracts (300 $\mu\textrm{g}$/$\textrm{m}{\ell}$) showed equivalent antioxidative effect to 25 uM ascorbic acid.

Flavonoid Fraction Purified from Rhus verniciflua Stoke Actively Inhibits Cell Growth Via Induction of Apoptosis in Mouse Tumorigenic Hepatocytes

이정채 한국생약학회 2004 Natural Product Sciences Vol.10 No.2

Dietary flavonoids are currently receiving considerable attention in developing novel cancerpreventive approaches because of their potential capacities to actively induce apoptosis of cancer cells. In our previous report, a flavonoid fraction, which consisted mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein and named RCMF (RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS) that is traditionally used as food additive and herbal medicine. In this study, we evaluated the effects of the RCMF on cell proliferation and apoptosis using SV40-transformed tumorigenic hepatocytes, BNL SV A.8. Tritium uptake assay showing the proliferative capacity of the cells was strongly suppressed in the presence of RCMF. This anti-proliferative effect was further confirmed through trypan blue exclusion. RCMF-mediated suppression of cell growth was verified to be apoptotic, based on the increase in DNA fragmentation, low fluorescence intensity in nuclei after propidium iodide staining, and the appearance of DNA laddering. Collectively, this study demonstrated that RCMF can be approached as a potential agent that is capable of significantly inhibiting cell growth of hepatic cancer cells.

Inhibitory Effects of Glycoprotein-120 (G-120) from Ulmus davidiana Nakai on Cell Growth and Activation of Matrix Metalloproteinases

이정채,손영옥,Kyung-Yeol Lee,Ki-Choon Choi,Youngji Chung,Jong-Ghee Kim,Young-Mi Jeon,Yong-Suk Jang 한국분자세포생물학회 2004 Molecules and cells Vol.18 No.2

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions. Consequently, methods for inhibiting MMP activity have therapeutic potential. In this study, we investigated the effect of G-120, a 120 kDa glycoprotein purified from the Oriental herbal plant, Ulmus davidiana Nakai (UDN), on the activity and production of several MMPs by evaluating its growth inhibitory effect on NIH 3T3 cells. Tritium uptake assays showed that proliferation of NIH 3T3 cells was strongly suppressed, and G-120-mediated inhibition of DNA synthesis proved to involve a cytostatic, rather than a cytotoxic, effect, as shown by cytotoxicity and apoptosis assays. More importantly, G-120 strongly reduced the gelatinolytic and collagenase activities of MMP proteins, as well as expression of MMP-2 and MMP-9. Electrophoretic mobility shift assays revealed that it suppressed the DNA binding activity of NF-κB. Collectively, our observations show that G-120 strongly inhibits the activation of MMPs and NF-κB.

Mouse 조직에 있어서 셀렌에 의해 유도되는 단백질

이정채,임계택,최민규,박승택,정연태 원광대학교 환경과학연구소 1997 環境科學硏究誌 Vol.6 No.1

MPC 11 cell에 TPA 처리로 유도되는 DNA 결합 단백질의 특성

임계택,이정채 全南大學校 農業科學技術硏究所 1996 農業科學技術硏究 Vol.31 No.-

Vimentin의 전사인자(transcriptional factor)에 대한 기초자료로소 그 발현정도를 알아보기 위해 MPC11 cell에 Phorbol ester, 12-0-tetradecanoy phorbol 13-acetate (TPA)를 처리한 다음 DNA 결합 단백질을 세포질과 핵으로부터 분리하여 추출 한 후 gel mobility shift assay 방법으로서 그 성격을 연구했다. TPA를 처리한 MPC11 cell에서 처리 후 3시간에 단백질이 발현되었으며 그 단백질은 vimentin으로 추측된다. 한편 DNA 결합 단백질은 NF1과 TFN보다는 AP2에 대해 더 큰 결합력을 가지고 있는데 이는 AP2가 vimentin 발현과정에 있어 어떤 specific promotor element와 상호작용을 하는 것으로 여겨지며, TPA 의 처리에 의해서 vimentin의 발현이 촉진됨을 추측할 수 있었다.

척수신경세포와 신경아세포종의 Hybrid 세포에 있어서 active oxygen radicals의 cytotoxicity

이정채,임계택 全南大學校 農業科學技術硏究所 1995 農業科學技術硏究 Vol.30 No.-

Oxygen radical의 neurotoxicity에 대한 mechanism을 mouse spinal cord-neuroblastoma hybrid neuron cell line (NSC-19)에서 연구하였다. Oxygen radical과 hydrogen peroxide 및 hydroxyl radical 들은 세포배양용액에 glucose oxidase를 첨가하여 생성시켜 hybrid neuron cells 를 배양액속에서 4시간 동안 반응시켰을 때 대구조 세포수포다 약 50% 이상이 사멸되어지는 것이 관찰되었다. Oxygen radical에 의한 neurotoxicity는 catalase와 glutathione 같은 Oxygen radical scavenger에 의해 block 되어졌으나 superoxide dismutase (SOD) 및 vitamin E에 의해선 block 되지 않았다. 그리고, excitotoxic amino acid를 blocking하는 agents에 있어서 2-amino-5-phosphovaleric acid (APV, competitive NMDA antagonist), MK 801 (non-competitive NMDA antagonist), 7-chlorokynurenic acid (CKA, NMDA receptor glycine site antagonist) 및 N-methyl-D-aspartate (NMDA) receptor의 대한 antagonist도 oxygen radical 에 의한 neurotoxicity를 효율적으로 억제 한다는 것을 발견할 수 있었다. Iron은 hydroxyl radical의 생성을 촉진시키기 때문에 metal chelator에 의한 항산화작용 관계도 연구 되어졌다. 여기에서 deferoxamine(DFX)은 세포막에 대한 투과성이 없기 때문에 cytotoxicity를 block하지 못했다. 반면에 세포막을 투과할 수 있는 tetrakis(2-pyridyimethyl) ethylenediamine (TPEN)은 neurotoxicity를 억제함에 있어 효율적임이 역시 밝혀졌다. 또 다른 oxygen radical scavenger로서 spin trapping agent인 α-phenyl-t-butylnitrone(PBN)의 경우에 있어선 oxygen radical에 의해 유리되는 neurotoxicity를 block 하지 못했다. 이러한 결과들은 active oxygen radical 및 excitotoxic acid를 blocking 하는 antioxidants 및 agents가 monse spinal cord-neuroblstoma hybrid neuron의 neurotoxicity에 관여한다는 것을 의미한다.

옻나무 추출물의 생리활성 이용에 대한 연구 : 옻나무 추출물의 생물학적 기능

임계택,이정채,Lim, Kye-Taek,Lee, Jeong-Chae 한국식품과학회 1999 한국식품과학회지 Vol.31 No.1

옻나무에서 극성이 큰 물과 에탄올로 추출한 물질을 생쥐 뇌세포를 배양하여 glucose oxidase에 의해 생성되는 hydroxyl radical에 대한 항산화 효과와 암세포에 미치는 영향을 알아보았다. 먼저 항산화 효과에 있어서, $7{\sim}10$일 정도 배양된 생쥐 뇌세포에 20 mU/mL GO system을 처리한 후 물 및 에탄올 추출물 (30 mg/mL)을 일정량 첨가하여 hydroxyl radical에 대한 옻나무 추출물의 항산화 효과를 측정하였다. 그 결과 GO 20 mU/mL만을 처리한 구에서는 생쥐 뇌세포의 생존률이 52.0%인데 반하여, 옻나무 물 추출물을 1, 2, 4, 7, $10\;{\mu}L$ 첨가시 각각 60.0, 66.0, 72.0, 84.0 및 90.1%로서 첨가량이 증가할수록 매우 높은 생존률을 보였다. 이러한 경향은 에탄올 추출물의 첨가시에도 유사하였는데, $1\;{\mu}L$과 $2\;{\mu}L$ 첨가시 생존률은 55.0%와 64.0% 였고, 4, 7, $10\;{\mu}L$에서는 각각 70.0, 79.0, 91.0%로서 나타났다. 항산화력을 비교하기 위하여 잘 알려진 항산화제인 ascorbic acid를 50, $100\;{\mu}M$ 첨가시 쥐의 뇌세포의 생존률은 대조군에 대해 각각 87.0%와 90.0%이었는데 이것은 각각의 옻나무 추출물 10% $(10\;{\mu}L/well)$ 첨가시 나타났던 항산화 효과와 비슷한 결과였다. 따라서 이러한 항산화 효과에 관여하는 주요 성분을 전기영동과 작용기 분석을 통해 알아본 결과 laccase라는 물질이 주성분이라는 것과 그것은 구리를 함유한 당단백질로서 크기는 약 210 KDa과 230 KDa으로서 dimer로 되어 있다는 것을 알 수 있었다. 한편 옻나무 추출물의 HeLa cell에 미치는 영향을 보기 위해 in vitro 방법으로서 HeLa cell에 대해 물 및 에탄올 추출물(30 mg/mL)을 최고 10% 농도까지 첨가하여 시간별로 측정한 결과 $10\;{\mu}L$(10%) 첨가 후 12시간에는 40.0%가, 48시간에는 60.0% 정도의 HeLa cell이 사멸되는 것을 알 수 있었다. 암세포 성장 억제 효과에 대한 결과는 in vivo 방법에 있어서도 유사한 결과를 얻었다. 즉 BALB/c의 복강에 CT-26 $(1{\times}10^6\;cells/mL)$을 접종한 후 종양을 발생케 한 후 옻나무 추출물을 주입시 주입 7일 후 대조군에 비해 종양크기가 현저하게 작아지는 것을 볼 수 있었다. Antioxidative effects of the water or ethanol extracts from Rhus verniciflua Stokes (RVS) were measured by protection against hydroxyl radicals in mouse brain tissue culture. In the water extracts from RVS, cell viabilities were estimated 60.0, 66.0, 72.0, 84.0 and 90.0% at addition of 1, 2, 4, 7 and $10{\mu}L$, respectively, compared with GO (20 mU/mL) alone. The cell viability in the ethanol extracts was similarly with water extracts. In the antitumor effects, the results showed that percentages of the HeLa cell death were approximately 24% for 12 hrs, 57% for 48 hrs at addition of 10%/well ethanol extracts respectively. To know inhibition of tumor growth, in vivo, mice (BALB/c) were inoculated with 0.25 mL CT-26 $(1{\times}10^6\;cells/mL)$ subcutaneously. After the generation of tumor, the results of RVS extracts (ethanol, water) injection showed generally that the tumor size in BALB/c was reduced. For physicochemical characterization of the RVS extracts, purified substances of water or ethanol extracts were analized with SDS-PAGE and ICP spectrometer. In electrophoresis, gel showed 2 bands (210, 230 KDa). The results of ICP verified that RVS extracts contain $Cu^{2+}$ in both samples. Conclusively, this substance might be a laccase which has a biological effective function, as a natural bioactive substance.

척수신경세포와 신경아세포종의 Hybrid 세포에 있어서 active oxygen radicals의 cytotoxiciity

임계택,이정채 전남대학교 한국농어촌개발연구소 1995 농산어촌개발연구 Vol.30 No.-

Oxygen radical 의 neurotoxicity에 대한 mechanism을 mouse spinal cord-neuroblas- toma hybrid neuron cell line (NSC-19)에서 연구하였다. Oxygen radical과. hydrogen peroxide 및 hydroxyl radical 들은 세포배양용액에 glucose oxidase를 첨가하여 생성시켜 hybrid neuron cells 을 배양액속에서 4시간 동안 반응시켰을 때 대조구 세포수보다 약 50% 이상이 사멸되어지는 것이 관찰되었다. Oxygen radical에 의한 neurotoxicity는 catalase와 glutathione 같은 oxygen radical scavenger에 의해 block 되어졌으나 superoxide dismutase (SOD) 및 vitamin E에 의해선 block 되지 않았다. 그리고 excitotoxic amino acid를 blocking하는 agents에 있어서 2-amino-5- phosphovaleric acid (APV, competitive NMDA antagonist) , MK 801 (non-competitive NMDA antagonist), 7-chlorokynurenic acid (CKA, NMDA receptor glycine site anta- gonist) 및 N-methyl-D-aspartate (NMDA) receptor 의 대한 antagonist도 oxygen radical 에 의한 neurotoxicity를 효율적으로 억제 한다는 것을 발견할 수 있었다. Iron은 hydroxyl radical의 생성을 촉진시키기 때문에 metal chelator에 의한 항산화작 용 관계도 연구 되어졌다. 여기에서 deferoxarnine (DFX) 은 세포막에 대한 투과성이 없기 때문에 cytotoxicity를 block 하지 못했다. 반면에 세포막을 투과할 수 있는 tetrakis (2-pyridyimethyl) ethylenediamine (TPEN) 은 neurotoxicity를 억제함에 있어 효율적임 이 역시 밝혀졌다. 또, 다른 oxygen radical scavenger로서 spin trapping agent 인 α- phenyl-t-butylnitrone (PBN) 의 경우에 있어선 oxygen radical 에 의해 유리되는 neuro- toxicity를 block 하지 못했다. 이러한 결과들은 active oxygen radical 및 excitotoxic arnino acid를 blocking 하는 antioxidants 및 agents가 monse spinal cord-neuroblstorna hybrid neuron의 neuro- toxicity에 관여한다는 것을 의미한다.