Proteomic Analysis of Thiol Specific‐Protein Oxidation

Tae‐Hoon Lee,Hee‐Young Yang,Hoon‐In Choi,Gia‐Buu Tran,Ung Yang 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1

Peroxiredoxin II (Prdx II; a typical 2‐Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice absent to Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol‐modifying reagent biotinylated iodoacetamide (BIAM) in Prdx I‒/‒ mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation‐sensitive proteins in Prdx II‒/‒ mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano‐UPLC‐MSE shotgun proteomics. We found oxidation‐sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II‒/‒ mice, and abnormal RBCs of Prdx II‒/‒ mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress‐induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome may provide clues to understand redox‐imbalanced diseases.

정신분열병에 대한 리스페리돈의 효과 및 안정성

이민수,김용구,김영훈,연병길,오병훈,윤도준,윤진상,이철,정희연,강병조,김광수,김동언,김명정,김상훈,김희철,나철,노승호,민경준,박기창,박두병,백기청,백인호,손봉기,손진욱,양병환,양창국,우행원,이정호,이종범,이홍식,임기영,전태연,정영조,정영철,정인과,정인원,지익성,채정호,한상익,한선호,한진희,서광윤 大韓神經精神醫學會 1998 신경정신의학 Vol.37 No.1

연구목적 : 본 시험의 목적은 임상시험 시작전에 연구자들을 대상으로 PANSS Workshop을 통하여 PANSS, ESRS에 대한 국내에서의 표준화 작업을 구축하고 새로운 정신병 치료제인 리스페리돈의 효과와 안정성을 재확인하여 리스페리돈 사용에 대한 적정화를 이루는데 있다. 연구방법 : 1996년 4월부터 1996년 9월까지 국내 39개 대학병원 정신과에 입원중인 혹은 증상이 악화되어 입원하는 정신분열병 환자 377명을 대상으로 다시설 개방 연구를 시행하였다. 1주일간의 약물 배설기간을 가진후, 리스페리돈을 8주간 투여하였고, 기준점, 1주, 2주, 4주, 그리고 8주후에 평가되었다. 용량은 제1일에는 리스페리돈 1mg씩 1일 2회, 제2일에는 2mg씩 1일 2회, 제3∼7일에는 3mg씩 1일 2회 투여하였다. 이후 환자의 임상상태에 따라 임의로 증량할 수 있으며, 최대 일일 16mg을 초과하지 않도록 하였다. 추체외로 증상을 조절하기 위한 투약을 허용하였다. 임상증상 및 부작용의 평가는 PANSS(Positive and Negative Syndrome Scale), CGI(Clinical Global Impression) 그리고 ESRS(Extrapyramidal Symptom Rating Scale)을 사용하였다. 연구결과 : 377명중 343명(91%)이 8주간의 연구를 완결하였다. 치료 종결시점인 8주후 PANSS 총점수가 20% 이상 호전된 경우를 약물 반응군으로 정의할때, 약물반응군은 81.3%였다. 리스페리돈에 반응하는 예측인자로는 발병연령, 이전의 입원 횟수, 유병기간이 관련 있었다. 리스페리돈은 1주후부터 PANSS양성, 음성, 및 일반정신병리 점수상에 유의한 호전을 보여 효과가 빨랐다. CGI의 경우도 기준점에 비해 1주후부터 유의한 감소를 나타내었다. ESRS의 경우, 파킨슨 평가점수는 기준점과 비교해 투여 1주, 2주, 4주후 유의하게 증가되었다가 8주후 기준점과 차이가 없었다. Dystonia 평가점수는 1주후만 유의한 증가를 보였으며, dyskinesia 평가점수는 유의한 차이가 없었다. 혈압, 맥박수의 생명징후 및 일반 혈액학 검사, 생화학적 검사, 심전도 검사에서 유의한 변화는 없었다. 결 론 : 이상의 다시설 개방 임상 연구를 통해 리스페리돈은 정신분열병 환자에서 양성증상뿐만 아니라 음성증상 및 전반적인 증상에도 효과적인 것으로 사료된다. 보다 명확한 평가를 위해서는 다른 항정신병약물과의 이중맹검 연구가 필요할 것으로 생각되며, 또한 장기적 치료에 대한 평가도 함께 이루어져야 하겠다. Objective : The purpose of this study was to investigate the efficacy and safety of risperidone in the treatment of Korean schizophrenic patients. Method : This multicenter open study included 377 schizophrenic patients drawn from 39 university hospitals. After a wash-out period of 1 week, the schizophrenic patients were treated with risperidone for 8 weeks and evaluated at 5 points ; at baseline, and 1, 2, 4 and 8 weeks of treatment. The dose was increased from 2mg/day(1mg twice daily) to 6mg/day(3mg twice daily) during the first week and adjusted to a maximum of 16mg/day over the next 7 weeks according to the patient's clinical response. Medication to control extrapyramidal symptoms was permitted. The psychiatric and neurological status of the patients was assessed by PANSS, CGI, and ESRS scales. Results : 343(91%) of 377 patients completed the 8-week trial period. Clinical improvement, as defined by a 20% or more reduction in total PANSS score at end point, was shown by 81.3% of patients. The predictors of response to risperidone were associated older age, shorter duration of illness, fewer previous hospitalization. Risperidone had rapid onset of action ; a significant decrease of the total PANSS and three PANSS factor(positive, negative, general), and CGI was already noticed at the end of first week. For the ESRS, parkinsonism rating scores were significantly increased until week 4 comparing with baseline. Dystonia rating scores were significantly increased until week 1, and dyskinesia rating scores were not significantly changed during the study. Laboratory parameters including vital sign, EKG, hematological, and biochemical values showed no significant changes during the trial. Conclusions : This study suggests that risperidone is generally safe and effective against both the positive and negative symptoms in our group of patients.

Identification of Redox Sensitive Proteins in Prdx II Deficient Red Blood Cells

Tae‐Hoon Lee,Hee‐Young Yang,Hoon‐In Choi,Gia‐Buu Tran,Ung Yang 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1

Red blood cells (RBCs) have been studied as models for infectious diseases, various symptoms of anemia, hemolysis, and erythrocyte aging. Although do not directly affect RBCs, other diseases may cause RBC physiological alterations that could be advanced for diagnostic aim or to convince better understanding of a certain pathological pattern. In this study, comparative RBC proteomics between healthy and abnormal conditions involve to promote aging related‐biomarker discovery. Peroxiredoxin II (Prdx II; a typical 2‐Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice absent to Prdx II proteins had heinz bodies in their peripheral blood, and morphologically aged cells were detected in the dense RBC fractions, which contained markedly higher levels of reactive oxygen species (ROS). In addition, a labeling experiment with the thiol‐modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II‒/‒ mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation‐sensitive proteins in Prdx II‒/‒ mice, we performed RBC comparative proteome analysis by nano‐UPLC‐MSE shotgun proteomics with relative protein quantitative analysis. We found oxidation‐sensitive 18 membrane and 41 cytosol proteins from 32 and 85 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice (W1), healthy RBCs of Prdx II‒/‒ mice (K1), and abnormal RBCs of Prdx II‒/‒ mice (K2). These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress‐induced proteins, amino acid/nucleic acid metabolic enzymes, signal transduction, and molecular transporters. Furthermore, protein networks among identified oxidation sensitive proteins were analyzed to associate with aging consequence. Consequently, we expected that RBC proteome may provide clues to understand redox‐imbalanced diseases.

Targeting Multiple Myeloma Cell Death with Polyphenol EGCG

Tae‐Hoon Lee,Hee‐Young Yang,Hoon‐In Choi,Ung Yang,Kyoung‐Jin Chung,Lina Ren 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1

(‐)‐Epigallocatechin 3‐gallate (EGCG) is a potent antioxidant polyphenol in green tea that acts as an anticancer agent via both direct and indirect pathways. Although the relationship between EGCG’s anticancer effects and its antioxidant activity is not fully understood, it is known that EGCG stimulates production of reactive oxygen species (ROS), which induce oxidative stress leading to cell death. In IM9 multiple myeloma cells, EGCG acted in a dose‐ and time‐dependent manner to induce apoptotic cell death. Among the antioxidant enzymes expressed in IM9 cells, levels of peroxiredoxin (Prdx) V were selectively and significantly reduced by EGCG. Moreover, the ROS scavenger NAC completely inhibited EGCG‐induced apoptosis and PrdxV reduction, while overexpression of PrdxV, but not a PrdxVC48S mutant, protected IM9 cells from EGCG‐induced apoptosis. EGCG‐induced reductions in cell viability and PrdxV levels were also observed in primary CD138+ multiple myeloma cells from patients. These results suggest that PrdxV is a key target via which EGCG mediates its anticancer effects.

Peroxiredoxin V Regulates Aco2, Acadm, and Acox1 Activity in Hypoxic Kidney

Tae‐Hoon Lee,Hee‐Young Yang,Joseph Kwon,Hoon‐In Choi,Lina Ren,Ung Yang,Byung‐Ju Park,Zae Young Ryoo 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1

Peroxiredoxin V, an atypical thioredoxin peroxidase, is widely expressed in mammalian tissues. In addition, Prdx V is localized in mitochondria, peroxisome, cytosol, and nucleus. Prdx V has been reported to protect a wide range of cellular environments as antioxidant enzyme, and its dysfunctions may be implicated in several diseases, such as cancer, inflammation, and neurodegenerative disease. Identification and relative quantification of proteins affected by Prdx V may help identify novel signaling mechanisms that are important for oxidative stress response. However, the role of Prdx V in the modulation of hypoxia‐related cellular response is not studied yet. In order to examine the function of endogenous Prdx V in hypoxic condition in vivo, we generated a transgenic mouse model with Prdx V siRNA expression controlled by U6 promoter. Of many tissues, the knockdown of Prdx V expression was displayed in kidney, lung, and liver, but not spleen and skin. We conducted on the basis of nano‐UPLC‐MSE proteomic study to identify the Prdx V‐affected protein networks in hypoxic kidneys. In this study, we identified protein networks associated with oxidative stress, fatty acid metabolism, and mitochondrial dysfunction. Our results indicated that Prdx V affected to regulation of kidney homeostasis under hypoxia stress.

Effect of Developmental Lead Exposure on the Expression of Hippocampal NMDA Receptor Subunit mRNA

Kim, Tae-Wan,Chung, In-Sung,Bae, Jae-Hoon,Shin, Dong-Hoon,Lee, Mi-Young,Kim, Joon-Sik 大韓産業醫學會 2005 대한직업환경의학회지 Vol.17 No.4

목적: in vivo 및 vitro에서 해마 신경세포의 발생단계별 NMDA 수용체 아단위 mRNA 발현에 대한 연 폭로 영향을 알아보고자 하였다. 방법: 흰쥐 해마 신경세포의 발생단계별 NMDA 수용체 NR2A, NR2B 아단위 mRNA 발현에 대한 연의 영향은 정상군과 연 폭로군의 출생 후 7일, 14일, 22일 흰쥐의 해마에서 in situ hybridization으로 mRNA 발현 정도를 densitometer로 측정하여 비교하였고, 연과 NMDA 의 세포독성은 해마 신경세포 일차배양 후 도립현미경을 이용한 형태학적인 관찰과 LDH 활성도를 이용하여 측정하였다. 결과: 연 과 NMDA 에 의한 세포독성에 대한 in vitro 실험에서 형태학적 소견과 LDH 활성도에서 해마 미성숙 신경세포와 성숙 신경세포사이의 차이가 있었으므로, 연과 NMDA 독성효과는 해마 신경세포의 발달 단계에 따라 차이가 있다. 정상군의 해마에서의 NR2A mRNA 발현은 출생 후 연령이 증가함에 따라 점진적으로 증가하였으나, NR2B mRNA 발현은 출생 후 연령이 증가함에 따라 점진적으로 증가하였으나 NR2B mRNA 발현은 연령의 증가에 따른 변화가 없었다. 연 폭로에 희한 NR2A mRNA 발현은 유의하게 감소하였으나(p<0.05), NR2B mRNA 발현은 변화가 나타나지 않았다. 만성적 연 폭로는 NR2A를 포함하는 NMDA 수용체를 감소시킬 수 있음을 알 수 있다. 결론: 연은 해마신경세포의 발생단계에서 NMDA 수용체 아단위 특히 NR2A mRNA 발현의 변화를 야기하여 시냅스 신호 전달에 영향을 나타냄을 알 수 있었다. Hippocampus

한국인 관상동맥의 변화양상에 대한 형태학적 연구

권태정,이상용,이한영,고영창,권기석,조재홍,김태공,김유훈,조갑래,서중석,이원태 內務部 國立科學搜査硏究所 2001 國立 科學 搜査 硏究所 年報 Vol.33 No.-

Special Issue of Professor Kiehwa Lee`s Retirement: Geophysical Environments of the Korean Peninsula : Articles ; Geoelectrical Structure of the Kyongsang Basin from Magnetotelluric Sounding

( Choon Ki Lee ),( Heui Soon Lee ),( Byung Doo Kwon ),( In Ky Cho ),( Seok Hoon Oh ),( Yoon Ho Song ),( Tae Jong Lee ) 대한지구물리학회 2006 지구물리 Vol.9 No.3

한국산 약용식물의 화장품천연소재로서 응용에 관한연구

안봉전,이진태,이순애,곽재훈,박정미,이진영,박태순,손준호 경산대학교 생명자원개발연구소 2003 생명자원과 산업 Vol.7 No.-

Biological activities and application of sanguisorbae officinalis L. were investigated. In the enzymological physiological activities, the electron donating ability(EDA) was 54.92% in 10 ppm and it was over 90% over 50ppm and SOD-like activity was high as 65.36% in 1000 ppm, it was gradual increased. As inhibitory effect of xanthine oxidase, it was 17.90% in 200 ppm and a little low as 36.89% in 500 ppm and inhibitory effect of tyrosinase, it was a little low as 20.45% below 1000 ppm. As the result of measuring the lipid oxidation, all the concentrations of medical ion treatments had the ability to keep it from acidification and metal ion blocking effects about the lipid oxidation promoting factors(Fe^(2+) and Cu^(2+)), Fe^(2+) was better than Cu^(2+) and all concentrations of medical ion treatments was 40% in 50ppm. When it was applied into normal skin-softener it showed safe effect so that we can expect that as the natural material of cosmetics.

단계적 온도 하강법을 이용한 췌도세포 냉동보존법

정인경,오승훈,김병준,양태영,이병완,하창영,노정현,정재훈,민용기,이명식,이문규,김광원 대한당뇨병학회 2002 Diabetes and Metabolism Journal Vol.26 No.1

연구배경:최근 당뇨병의 새로운 치료법으로 시도되고 있는 췌도이식은 충분한 췌도수의 확보와 췌도생존율을 높이기 위한 면역억제제 사용이 제한점이 되고 있다. 본 연구의 목적은 이식 전 충분한 췌도 수의 확보를 위해 분리한 췌도를 냉동보존하는 방법을 확립하고 냉동보존한 백서 췌도세포의 시험관내 그리고 생체내 기능을 조사하였다. 방법:분리한 백서의 췌장소도를 48시간 배양한 후 한 시험관당 췌도세포 1000개씩 나누었다. 냉동보존은 6개의 시험관에 DMSO를 첨가한 후 초 냉각(supercooling), 핵화(nucleation)단계를 거친 후 99% isopropanol과 액체질소가 들어있는 dewer를 이용하여-0.25℃/분의 냉각속도로 -40℃까지 단계적으로 얼린후-70℃ 액체질소 탱크에 보관했다. 해동은 냉동시킨 vial들을 액체 질소 태으에서 꺼내 37℃ 항온조에 담가 급격히 해동시킨 후, 원심분리하여 상층액을 제거하고 각 vial에 0.75M sucrose 용액을 가한 후, 10% fetal calf serum이 함유된 RPMI 1640 media에서 배양하였다 각각 6개의 시험관에서 해동한 췌도들을 광학현미경 및 형광현미경하에서 췌도의 모양과 생존율에 대해 조사하고 인슐린 정적반응을 알아보았다. 또한 분리한 췌도를 냉동보존하지 않고 이식한 경우를 대조군(6마리)과 생체내 기능을 비교하였다. 결과:① 냉동보존후 획득한 췌도의 수와 생존율 해동후 획득한 췌도의 수는 해동시킨 당일날이 902±21, 24시간 배양 후에는 857±16, 72시간 후에는 817±18개로 점차 감소되었다. AO/PI 염색상 각 췌도의 생존율은 냉동 전을 100으로 하였을 때 해동당일, 24시간 후, 72시간 후가 각각 60±5, 80±5, 90±5%로 해동후 3일간 배양하였을 때 냉동전의 수준으로 회복하였다. ② 냉동보존후 췌도의 포도당에 대한 정적 인슐린 분비능:냉동직후 감소된 경향을 보였으나 해동후 3일간 배양한 췌도의 인슐린 분비는 냉동전과 통계적으로 의미있는 차이가 없이 냉동보존 전의 수준으로 회복되었다. ③ 냉동보존후 췌도의 포도당에 대한 동적 인슐린 분비능:냉동보존한 췌도를 해동후 3일째의 인슐린 동적 분비능은 냉동 전과 마찬가지로 자극 인슐린의 반응의 제1기와 2기가 잘 관찰되었다. ④ 냉동보존한 췌도세포 이식 후 혈당 변화:스트렙토조토신으로 유도된 당뇨병 쥐에 췌도이식 후 혈당은 냉동보존한 췌도이식군에서 대조군에 비해 혈당의 조절효과가 더 오래 지속되었다. 결론:소동물에서 단계적 온도 하강법을 이용한 췌도세포 냉동보존법을 확립하였으며 이는 기능, 구조 및 생존율에 큰 이상을 보이지 않았으므로 장차 사람의 췌도세포 동종이식시 부족한 췌도세포수를 극복하고 면역반응을 줄일 수 있는 매우 유용한 방법이 될 것으로 판단된다. Background : Although islet transplantation has been attempted to reverse the state of diabetes, achieving a critical number of islets and modulating the immune response limit the success of islet transplantation. Cryo-preservation of islets offers many important benefits for islet transplantation by collecting islets with a wide variety of HLA phenotypes and islet MHC expression. The aims of this study was to determine the optimal conditions for cryo-preservation by using a controlled cooling method and to evaluate in vitro and in vivo functional properties of the cryo-preserved islets. Methods : Collagenase-isolated, Ficoll-purified islets were cultured for 48 hours. They were aliquoted into freezing tubes (1000 islets per tube), equilibrated with 2M dimethyl sulfoxide (DMSO) in three steps, supercooled, nucleated, and controll-cooled at rate of 0.25℃/min to - 40℃ prior to storage at - 196℃. Rapid thawing and removal of DMSO with 0.75 M sucrose preceded 48 hour of culture and the morphology, viability, glucose-induced insulin secretion, and in vivo function of rats transplanted with cryopreserved islets was reexamined. Results : ① Recovery was 90.2±0.2%, 85.7±0.1% and 81.7±0.1% immendiately after, 24 hours and 72 hours after thawing respectively. The viability was 60±5%, 80±5%, 90±5% immediately after, 24 hours and 72 hours after thawing respectively. ② The glucose-stimulated-insulin secretion (GSIS) tended to decrease immediately after thawing, but GSIS increase to the level of pre-cryopreservation 72 hours after thawing. ③ The in dynamic GSIS, the first and the second phase of insulin secretion were well preserved in islets cultured for 72 hours after thawing. ④ The cryopreserved islets were cultured for 3 days and transplanted into renal sub-capsular space of streptozotocin (STZ) induced diabetic rats. The duration of normoglycemia in the STZ-induced diabetic rats transplanted with cryopreserved islets was significantly longer than of the fresh islets. Conclusion : The optimal condition of cryopreservation using the controlled cooling method was established in rat pancreatic islets. This cryopreservation method can be a feasible approach for human islet transplantation (J Kor Diabetes Asso 26:64~74, 2002).