백년초(Opuntia ficus-indica) 열매 유래 물질의 항궤양 효과, 독성 및 작용 기전

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 협동과정 농업생물공학전공, 2021. 2. 양태진.위염은 주로 짠 음식과 매운 음식을 주로 먹는 한국 성인들에게 흔한 질병이 다. 일반적으로 증상에는 상복부 통증, 메스꺼움, 구토, 복통, 소화 불량 및 복부 팽 만감이 있다. 위염은 병태생리학적으로 위 공격 인자 (산, 펩신, 헬리코박터 파일로 리)와 위 점막 방어 인자 (위 점액, 중탄산염 분비, 프로스타글란딘 및 점막 세포의 선천적인 저항성) 사이의 불균형으로 발병한다. 위궤양 치료제로 여러 약물이 사용 되고 있다. 그러나 이러한 치료제에는 부작용이 있다. 부작용이 적은 새로운 위염 치료제 개발이 절실히 요구되고 있다. 식물은 잠재적인 치료제를 가지고 있을 것으 로 생각되어 약용 식물에서 새로운 화합물을 찾기 위한 노력이 계속되고 있다. Opuntia ficus-indica (선인장과) (백년초)는 많은 국가에서 전통 의약품으로 사용 되고 있다. 대한민국의 제주도에서는 건강 식품 제조 용도로 널리 재배되고 있다. 본 연구는 백년초 열매 추출물을 위염 치료 천연물의약품으로 개발하기 위해 수행 되었다. OF-80E (백년초 열매 80% 에탄올 추출물)의 항궤양 활성은 에탄올, 비스테로 이드성 항염증제 (인도메타신, 아스피린 및 디클로페낙) 및 스트레스 유발 위염 랫 트 모델을 사용하여 평가하였다. 그 결과는, 시판된 약물인 스티렌정 (Stillen® tablet) 및 뮤코스타정 (Mucosta® tablet)과 비교하였다. 또한, OF-80E의 급성독성, 아만성독성, 유전독성 및 안전성 약리 연구는 인간의 안전한 섭취를 위해 경제협력 개발기구 지 침 및 우수실험실관리 규정에 따라 평가하였다. 마지막으로, 위염 모델에서 OF-80E 의 항궤양작용의 가능한 메커니즘은 생화학 및 분자 분석을 사용하여 설명하였다. AGS 세포를 이용한 아스피린 유도 세포독성 억제 시험을 기반으로 성분 분리 를 통하여 백년초 열매로부터 분리하고, 전자이온화 질량분석법과 핵자기 공명 분 광법을 포함한 분광 분석을 통해 두 종의 활성 화합물을 동정하였다. 두 종의 항궤 양 성분은 플라보노이드인 aromadendrin과 narcissin으로 확인되었다. IC50 값으로 보 면, flavone인 aromadendrin (<0.5 μM)과 flavonol인 narcissin (<0.5 μM)은 다른 flavones인 naringenin (5.9 μM), eriodictyol (>10 μM), taxifolin (1.1 μM) 및 다른 flavonols인 kaempferol, quercetin 및 isokaempfride (>10 μM) 보다 AGS 세포를 이용한 아스피린 유도 세포독 성 억제 효과가 우수하였다. OF-80E는 시판된 약물보다 공격인자에 대해서 위 점막 손상을 보호하는 효과 가 우수하였다. 에탄올, 비스테로이드성 항염증제 및 스트레스 유발 위염 랫트 모델 에서, OF-80E는 시판된 약물에 비해 효과적으로 위 출혈성 병변과 조직학적 조직 손상을 억제하였다. 단회 경구투여 독성연구에서 OF-80E의 개략적인 치사량은 SD 랫트의 암, 수 모두에서 10000 mg/kg 이상으로 확인되었다. 13주 반복 경구투여 독성연구에서 OF- 80E의 무독성량은 SD 랫트의 암, 수 모두에서 2000 mg/kg/day로 확인되었다. 4주 반 복 경구투여 독성 연구에서 OF-80E의 최대내성용량은 비글견 암, 수 모두에서 1500 mg/kg/day로 확인되었다. Salmonella typhimurium과 Escherichia coli를 이용한 복귀돌연 변이 시험에서 OF-80E는 돌연변이를 유발하지 않았다. Chinese hamster lung 세포를 이용한 염색체 이상시험에서 OF-80E는 염색체 이상을 유발하지 않았다. 소핵시험에 서 OF-80E는 동물 골수세포에서 소핵을 유발하지 않았다. OF-80E를 5000 mg/kg 이하 로 설치류에 단회 경구 투여했을 때, ICR 마우스의 중추신경계에 영향을 미치지 않 았고, SD 랫트의 호흡기계에 영향을 미치지 않았다. OF-80E는 500 μg/mL 농도까지 human ether-a-go-go related gene 채널에 영향을 미치지 않는 것으로 보아, OF-80E는 심 혈관계에 미치는 영향이 낮을 것으로 확인되었다. OF-80E는 AGS 세포에서 아스피린에 의해 감소되고, 랫트에서 인도메타신에 의해 감소한 glutathione을 증가시켰다. OF-80E는 AGS 세포에서 아스피린에 의해 감 소한 prostaglandin E2의 농도를 증가시켰다. 인도메타신 유도 위염 랫트에서 감소된 부착성 위점액은 OF-80E의 처리에 의해 합성되고 분비되었다. OF-80E는 인도메타신 유도 랫트의 위 점막에서 myeloperoxidase의 활성을 억제하고, 스트레스 유도 위염 랫트의 위 점막에서 tumor necrosis factor-α를 감소시켰다.Gastritis is a common disease among Korean adults who take mainly very salty and spicy foods. Usually, symptoms include epigastric pain, nausea, vomiting, abdominal pain, indigestion, and bloating. Pathophysiology of gastritis is due to a lack of equilibrium between the gastric aggressive factor (acid, pepsin, and Helicobacter pylori) and the mucosal defense factor (gastric mucus, bicarbonate secretion, prostaglandins, and innate resistance of the mucosal cells). There are several types of medicines used to treat a gastric ulcer. However, these treatments have side effects. There is a pressing need to develop a new gastritis treatment with fewer side effects. Plants are regarded to represent a reservoir of potential therapeutics and therefore the efforts to search for novel compounds from medicinal plants have been continued. Opuntia ficus-indica (Cactaceae) has been used in traditional medicine of many countries. It is widely cultivated in Jeju Island, Korea, for use in the manufacture of health foods. The aim of this study was to develop O. ficus-indica fruits extract as an antiulcer botanical drug. The antiulcer activity of OF-80E (80% ethanol extract of O. ficus-indica fruits) was assessed using the ethanol-, non-steroidal anti-inflammatory drugs (indomethacin, aspirin, and diclofenac)-, and stress-induced gastritis rat models. The results were compared with those of commercially available drugs, Stillen® tablet and Mucosta® tablet. In addition, the acute toxicity, sub-chronic toxicity, genotoxicity, and safety pharmacology studies of OF-80E were analyzed under Organization for Economic Cooperation and Development guideline and Good Laboratory Practice regulations for human safe consumption. Finally, the possible mechanism underlying the antiulcer actions of OF-80E in gastritis models were elucidated using biochemical and molecular analyses. Inhibition of aspirin-induced cytotoxicity in AGS cells assay-guided fractionation of the O. ficus-indica fruits led to the identification of two active compounds through spectroscopic analyses, including electron ionization mass spectrometry and nuclear magnetic resonance spectroscopy. The two antiulcer constituents were the flavonoids aromadendrin and narcissin. Based on the IC50 values, the flavone, aromadendrin (10 μM) and taxifolin (1.1 μM), and flavonols, kaempferol, quercetin, and isokaempfride (>10 μM). OF-80E was more effective than commercially available drugs to protect gastric mucosal damage against aggressive factors. In ethanol-, non-steroidal anti-inflammatory drugs-, and stress-induced gastritis rat models, OF-80E inhibited gastric hemorrhagic lesions and histological tissue damage effectively comparing than commercially available drugs. In a single dose oral toxicity study, the approximate lethal dose of OF-80E in both male and female of Sprague Dawley (SD) rats was higher than 10000 mg/kg. In a 13-week repeated oral toxicity study, the no observed adverse effect level of OF-80E was 2000 mg/kg/day for both sexes of SD rats. In a 4-week repeated oral toxicity study, the maximum tolerance dose of OF-80E was 1500 mg/kg/day for both sexes of beagle dogs. In Salmonella typhimurium and Escherichia coli reverse mutation studies, OF-80E did not cause mutation. In a chromosome aberration test, OF-80E did not cause chromosomal aberration in Chinese hamster lung cells. In micronucleus assay, OF-80E did not induce micronuclei in the mammalian bone marrow cells. Single oral administration of OF-80E to rodent at below 5000 mg/kg did not affect the central nervous system of ICR mice and did not induce adverse effects on the respiratory system of SD rats. OF-80E did not effect on the human ether-a-go-go related gene channel up to the concentration of 500 μg/mL, indicating that the effect of OF-80E on cardiovascular system was to be low. OF-80E increased glutathione reduced by aspirin in AGS cells and decreased by indomethacin in rats. OF-80E increased prostaglandin E2 levels reduced by aspirin in AGS cells. Decreased adherent mucus was synthesized and stimulated, by OF-80E pretreatment in indomethacin-induced gastritis rats. OF-80E inhibited myeloperoxidase activity in indomethacin-induced rat gastric mucosal and reduced tumor necrosis factor-α in stress-induced rat gastric mucosal.Abstract i Contents iv List of Abbreviations xi List of Figures xiv List of Tables xvi Ⅰ. Introduction １ Ⅱ. Literature reviews ４ 1. Gastritis (Gastric ulcer) ４ 1.1. Pathophysiology and risk factors ５ 1.1.1. Helicobacter pylori ６ 1.1.2. Nonsteroidal anti-inflammatory drugs ８ 1.2. Morbidity and mortality ９ 1.3. Diagnosis １０ 1.4. Prevention １２ 2. Therapeutic agents of gastritis １２ 2.1. Histamine H2 antagonists １５ 2.2. Proton pump inhibitors １５ 2.3. Antacids １７ 2.4. Prostaglandin derivatives １８ 2.5. Antimuscarinic agents １８ 2.6. Anti-Helicobacter pylori therapy １９ 2.6.1. Combination therapy ２０ 2.6.2. Anti-Helicobacter pylori vaccines ２１ 2.7. CCKB antagonists ２２ 3. Study of new drug from plant extracts to treat gastritis ２３ 3.1. Plant extracts with antigastritis activity ２３ 3.2. Phytochemicals with antigastritis activity ２７ 3.3. Herbal medicines tested in clinical trials ３０ 4. Opuntia ficus-indica (L.) Miller ３４ 4.1. Nutritional contents and bioactive constituents of Opuntia ficus-indica ３５ 4.2. Biological activities of Opuntia ficus-indica ３９ 4.2.1. Flower ４０ 4.2.2. Fruit/pulp ４１ 4.2.3. Seed ４２ 4.2.4. Peel/skin ４３ 4.2.5. Cladode ４４ Ⅲ. Materials and Methods ４５ 1. Preparation of test materials ４５ 1.1. Instrumental analysis ４５ 1.2. Chemicals and reagents ４６ 1.3. Preparation of O. ficus-indica fruit ethanol extracts ４６ 1.4. Bioassay-guided fractionation and isolation of O. ficus-indica fruits ４７ 1.5. High-performance liquid chromatography with diode array detector and electrospray ionization mass spectrometry chemical analysis ４９ 1.6. High-performance liquid chromatography analysis of narcissin and aromadendrin ５０ 1.7. High-performance liquid chromatography analysis of betanin ５０ 1.8. Mass production of OF-80E (O. ficus-indica fruits 80% ethanol extract) ５１ 2. Evaluation of gastro-protective activity in an in vitro model ５１ 2.1. Gastric AGS cell cultures ５１ 2.2. Ethanol-induced cytotoxicity in AGS cells ５２ 2.3. Aspirin-induced cytotoxicity in AGS cells ５２ 2.4. Determination of reduced glutathione level in AGS cells ５３ 2.5. Determination prostaglandin E2 level in AGS cells ５３ 2.6. Data analysis ５４ 3. Evaluation of gastro-protective activity in an in vivo model ５４ 3.1. Animals ５４ 3.2. Ethanol-induced gastritis rats ５５ 3.3. Indomethacin-induced gastritis rats ５５ 3.4. Aspirin-induced gastritis rats ５５ 3.5. Stress-induced gastritis rats ５６ 3.6. Diclofenac-induced gastritis rats ５６ 3.7. Determination of gastric lesion index ５７ 3.8. Gastric adherent mucus assay ５７ 3.9. Measurement of mucosal myeloperoxidase and tumor necrosis factor-alpha ５８ 3.10. Measurement of reduced glutathione level of mucosa ５８ 3.11. Measurement of histological index of gastric tissue ５８ 3.12. Data analysis ５９ 4. Toxicity studies of OF-80E ５９ 4.1. Single dose oral toxicity study in Sprague Dawley rats ５９ 4.1.1. Animals ５９ 4.1.2. Experimental design ６０ 4.1.3. Data analysis ６１ 4.2. Thirteen-week repeated-dose oral toxicity study with a four-week recovery in Sprague Dawley rats ６１ 4.2.1. Experimental design ６１ 4.2.2. Urine collection and blood sampling ６１ 4.2.3. Urinalysis ６２ 4.2.4. Hematological test ６２ 4.2.5. Clinical biochemistry test ６３ 4.2.6. Histopathology ６３ 4.2.7. Data analysis ６４ 4.3. Four-week repeated-dose oral toxicity study in beagle dogs ６５ 4.3.1. Animals ６５ 4.3.2. Experimental design ６６ 4.3.3. Urine collection and blood sampling ６６ 4.3.4. Urinalysis ６７ 4.3.5. Hematological test ６７ 4.3.6. Clinical biochemistry test ６７ 4.3.7. Histopathology ６８ 4.4. Bacterial reverse mutation test ６８ 4.4.1. Test strains and materials preparation ６８ 4.4.2. Experimental procedures ６９ 4.5. Chromosome aberration test in CHL cells ７０ 4.5.1. Test system ７０ 4.5.2. Experimental procedure ７１ 4.5.3. Evaluation of chromosomal aberration ７２ 4.6. In vivo micronucleus assay ７３ 4.6.1. Test system ７３ 4.6.2. Observations and examinations ７４ 4.7. Effect of OF-80E on the central nervous system in ICR mice ７５ 4.7.1. Test system ７５ 4.7.2. Observations and examinations ７６ 4.8. Effect of OF-80E on the respiratory rate and tidal volume in Sprague Dawley rats ７９ 4.8.1. Test system ７９ 4.8.2. Observations and examinations ７９ 4.9. Effect of OF-80E on human ether-a-go-go-related gene potassium channel expressed in Chinese hamster ovary cells ８０ 4.9.1. Cells cultures ８０ 4.9.2. Preparation of test substances and test solution ８１ 4.9.3. Measurement and analysis ８１ 4.9.4. Data analysis ８２ Ⅳ. Results ８４ 1. Gastro-protective activity of O. ficus-indica fruits in an in vitro model ８４ 1.1. Activity comparisons of various O. ficus-indica fruit ethanol extracts on aspirin-induced cytotoxicity in AGS cells ８４ 1.2. Chemical constituent of O. ficus-indica fruit ethanol extracts ８５ 1.3. Bioassay-guided fractionation and identification of O. ficus-indica fruits ８５ 1.4. Effect of the isolated flavonoids on aspirin-induced cytotoxicity in AGS cells ９１ 1.5. Effect of OF-80E on ethanol-induced cytotoxicity in AGS cells ９２ 1.7. Effect of OF-80E on aspirin-induced cytotoxicity in AGS cells ９３ 1.8. Reduced glutathione and prostaglandin E2 levels in AGS cells treated with OF-80E ９４ 2. Gastro-protective activity of O. ficus-indica fruits in an in vivo model ９６ 2.1. Gastro-protective activity of OF-80E in an ethanol-induced gastritis rat ９６ 2.2. Gastro-protective activity of OF-80E in an indomethacin-induced gastritis rat １００ 2.3. Gastro-protective activity of OF-80E in an aspirin-induced gastritis rat １０２ 2.4. Gastro-protective activity of OF-80E in a stress-induced gastritis rat １０４ 2.5. Gastro-protective activity of OF-80E in a diclofenac-induced gastritis rat １０６ 2.6. Anti-inflammatory and antioxidative activity of OF-80E １０８ 2.6.1. Effect of OF-80E on membrane-bound myeloperoxidase activity １０８ 2.6.2. Effect of OF-80E on tumor necrosis factor-α level １０９ 2.6.3. Effect of OF-80E on reduced glutathione level １０９ 2.7. Effect of OF-80E on adherent mucus level １１０ 3. Toxicity studies of OF-80E １１１ 3.1. Single dose oral toxicity study in Sprague Dawley rats １１１ 3.2. Thirteen-week repeated-dose oral toxicity study in Sprague Dawley rats １１４ 3.2.1. General clinical signs １１４ 3.2.2. Body weights １１６ 3.2.3. Food consumption １１６ 3.2.4. Water consumption １２０ 3.2.5. Urinalysis １２３ 3.2.6. Hematological test １２９ 3.2.7. Clinical blood biochemistry test １３０ 3.2.8. Organ weights １３０ 3.2.9. Necropsy finding １３１ 3.2.10. Histopathological examination １３２ 3.3. Four-week repeated-dose oral toxicity study in beagle dogs １３３ 3.4. Bacterial reverse mutation study １３６ 3.5. Chromosome aberration test in CHL cells １３８ 3.5.1. Results in the presence of S9 mixture １３８ 3.5.2. Results in the absence of S9 mixture １３８ 3.6. Frequency of micronucleated polychromatic erythrocyte and cytotoxicity in an in vivo micronucleus assay １４１ 3.7. Effect of OF-80E on the central nervous system in ICR mice １４３ 3.8. Effect of OF-80E on the respiratory rate and tidal volume in Sprague Dawley rats １４６ 3.9. Effect of OF-80E on human ether-a-go-go-related gene potassium channel expressed in Chinese hamster ovary cells １４８ Ⅴ. Discussion １５０ References １５８ Abstract in Korean １８９Docto

심장 분할을 위한 윤곽선 및 거리 변환 기반 모양 인식 어텐션 네트워크

학위논문 (석사) -- 서울대학교 대학원 : 공과대학 컴퓨터공학부, 2021. 2. 신영길.Cardiac image segmentation is an important task in the development of clinical cardiac applications. Many recent studies came up with deep learning models, mostly composed of convolutional neural networks(CNN), and showed significant outcomes on the segmentation of target organs in medical images. Unlike other major biological structures such as lungs and liver, the cardiac organ consists of multiple substructures. Those cardiac substructures are intimately adjacent to each other, which means that the segmentation network should concentrate on the boundaries of the substructures. In this paper, to increase the performance of cardiac image segmentation, we introduce a novel model to learn shape-aware and boundary-aware features using the distance transformation and the contour image of the labeled data. We present a shape-aware attention module that can guide a model to focus on edges between structures. we also propose the regularization for refining the contour probabilistic map. The experimental results show that the proposed network produces more accurate results compared to state-of-the-art networks by obtaining 4.97\% more in terms of dice similarity coefficient(DSC) score. We used 20 CT cardiac images for training and validation, and 40 CT cardiac images for the test. Moreover, our segmentation results describe that learning precise contour and distance transform features can help improve model performance. The ablations studies are presented to emphasize the importance of our proposed shape-aware attention mechanism.심장 영상 분할은 심장 의료 수술의 설계에 있어 중요한 과정이다. 최근에는 의료 영상에서 장기 분할을 위해 딥러닝을 이용한 CNN들이 많이 제안되었다. 다른 주요 인체의 장기들에 비해, 심장은 여러 개의 부분 장기들로 구성되어있다. 그리고 심장의 부분 장기들은 매우 인접하여 존재하기 떄문에 CNN은 장기들의 경계선 부분에 집중하여 학습되어야한다. 본 논문에서는 심장 분할 모델의 성능을 높이기 위하여 distance transform과 윤곽선 영상을 활용하여 모양 인식 특징맵 그리고 경계선 인식 특징맵을 학습할 수 있는 네트워크 구조를 제안하였다. 또한 부분 장기들의 경계선 특징을 모델 학습에 도와줄 수 있는 모양 인식 그리고 경계선 인식 특징 attention 기술을 제안한다. 또한 정확한 경계선 확률맵을 찾기 위한 정규화 방법도 제안한다. 실험 결과들은 제안한 네트워크가 다른 최신의 영상 분할 네트워크들에 비해 4.97\% 높은 DSC 성능을 보여줌으로 영상 분할 결과가 더욱 정확함을 알려준다. 영상 분할 네트워크의 학습과 검증에 있어 20개의 심장 CT를 사용하였고, 40개의 영상을 실험에 사용하였다. 이에 더해, 제안한 네트워크의 분할 결과는 정확한 경계면과 distance transform을 얻는 것이 모델의 성능을 높여줄 수 있음을 보여준다. 본 논문에서는 제안한 모양 인식 attention 방법의 검증과 중요성을 강조하기 위해 ablation study를 진행하였다.Chapter 1 Introduction 1 Chapter 2 Backgrounds 5 2.1 Deep Neural Networks for Image Segmentation 5 2.1.1 Overview 5 2.1.2 Convolutional Layer 6 2.1.3 Residual Connection 8 2.2 Visual Attention Mechanism 9 Chapter 3 Related Works 11 3.1 CNNs for Biomedical Image Segmentation 11 3.2 Distance Transformation for CNN Image Segmentation 14 3.2.1 Distance Transformation(DT) 14 3.2.2 DT-based Image Segmentation 15 Chapter 4 Methodology 18 4.1 Overview 18 4.2 CDA-Net Architecture 18 4.2.1 DT Regression for Image Segmentation19 4.2.2 Shape-aware Attention 21 4.2.3 Regularization with the Penalty Term to Refine Feature 22 4.3 Overall Loss Function 23 4.4 Data Preparation 25 Chapter 5 Experimental Results 28 5.1 Dataset 28 5.2 Evaluation Metrics 29 5.3 Experiments Detail 30 5.4 Comparison with State-of-the-Art 30 5.5 Ablation Study 31 Chapter 6 Conclusion 37 Bibliography 39 초록 45Maste

Piceatannol reduces resistance to statins in hypercholesterolemia by reducing PCSK9 expression through p300 acetyltransferase inhibition

The purpose of this study was to investigate the role of piceatannol (PT) in statin (rosuvastatin and simvastatin) resistance and tolerance and its association with PCSK9 expression via its p300 inhibitory (p300i) activity. An in vitro study was performed using HepG2 cells that were exposed to statins (rosuvastatin or simvastatin) with or without PT in delipidated serum (DLPS) medium. In the statin exposed conditions, PCSK9 expression was reduced following PT treatment when compared to HepG2 cells w/o PT treatment. Furthermore, no significant difference was observed in the expression of the transcription factors SREBP2 and HNF1α, which regulate PCSK9 expression. This resulted in low density lipoprotein receptor (LDLR) stabilization and reduced cellular cholesterol levels. This indicates that PT epigenetically controls statin-induced PCSK9 expression. Interestingly, PT attenuated p300 histone acetyltransferase (HAT) activity. Moreover, simulation of PT-p300 binding suggested that PT inhibits p300 as PT could be docked in the p300 HAT domain. Furthermore, inhibition of p300 HAT activity using C-646, a selective p300 inhibitor, or through an siRNA system effectively reduced PCSK9 induction upon statin exposure in HepG2 cells. The chromatin immunoprecipitation (ChIP) assays revealed that PT blocked the recruitment of p300 to the PCSK9 promoter region. In summary, PT attenuated statin-induced PCSK9 expression by inhibiting p300 HAT activity. Finally, co-administration of simvastatin and PT for 10 weeks further reduced plasma low-density lipoprotein-cholesterol (LDL-C) levels and stabilized the hepatic LDLR protein level compared with those resulting from single treatment of simvastatin in a high-fat diet-induced hypercholesterolemia mouse model. Our findings indicate that PT is a new nutraceutical candidate to reduce the statin resistance and tolerance that occurs in patients with hypercholesterolemia.ope

Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver

Lipid homeostasis is transcriptionally regulated by three DNA-binding proteins, designated sterol regulatory element-binding protein (SREBP)-1a, -1c, and -2. Oligonucleotide arrays hybridized with RNA made from livers of transgenic SREBP-1a, transgenic SREBP-2, and SREBP cleavage-activating protein knockout mice recently identified 33 genes regulated by SREBPs in liver, four of which had no known connection to lipid metabolism. One of the four genes was PCSK9, which encodes proprotein convertase subtilisin/kexin type 9a, a protein that belongs to the proteinase K subfamily of subtilases. Mutations in PCSK9 are associated with an autosomal dominant form of hypercholesterolemia. Here, we demonstrate that hepatic overexpression of either wild-type or mutant PCSK9 in mice results in hypercholesterolemia. The hypercholesterolemia is due to a post-transcriptional event causing a reduction in low density lipoprotein (LDL) receptor protein prior to the internalization and recycling of the receptor. Overexpression of PCSK9 in primary hepatocytes and in mice lacking the LDL receptor does not alter apolipoprotein B secretion. These data are consistent with PCSK9 affecting plasma LDL cholesterol levels by altering LDL receptor protein levels via a post-transcriptional mechanism.ope

Implementation of multi-dimensional flow analysis capability in MARS-KS motion model for marine reactor safety analysis

학위논문 (석사) -- 서울대학교 대학원 : 공과대학 에너지시스템공학부, 2021. 2. 조형규.In 2018, International Maritime Organization(IMO) adopted an initial strategy on the reduction of greenhouse gas emissions from ships to reduce the total annual greenhouse gas emissions by at least 50% by 2050 compared to 2008 levels. Thus, researches for marine reactor development as eco-friendly energy and safety analysis of marine reactor are actively being conducted. It is expected that new regulatory demand arises, and fundamental research is required to secure independent verification capability at the level of regulatory agencies. In this background, an improvement study for the MARS-KS motion model was conducted for marine reactor safety analysis. The goal of the study is to implement the multi-dimensional flow analysis capability in MARS-KS motion model. The research was conducted in the order of code improvement, verification, and application study. First, the 7 code improvements were performed. Improvement of volume direction unit vector generation method for user-friendly code implementation, correction of linear acceleration term considering inertial force, correction of the formula for the volumes position under the rotational condition, update of junction property, calculation of pressure drop along each axis and improvement of connection information formula between connected volumes to realize multi-dimensional flow analysis. And improvement study was conducted to extend the MARS-KS motion model to the MULTID component. The second is the verification of the improved code. First, 1D component and cross-flow verification was performed by solving the conceptual problem used in the previous research for the MARS-KS motion model at Seoul National University(H.K. Beom et al., 2019). For the verification of the 1D component, the manometer and vertical pipe conceptual problem were analyzed under various motion conditions, and analysis results were compared quantitatively with the analytical solution. Next, for cross-flow verification, a conceptual problem simulating the downcomer in the reactor was selected by connecting 1D vertical pipes with a cross-junction. When simulating the downcomer in the reactor, the six-pipe and eight-pipe conceptual problems that could be implemented through this study were verified with the existing conceptual problem consisting of four-pipe. Following the verification of 1D component and cross-flow, the conceptual problems for a 3D slab and an annular 3D cylinder were selected for the verification of the MULTID component. Likewise, it was analyzed under various motion conditions. In addition, analysis capability of the motion model for MULTID component was confirmed by applying the motion conditions in which various external forces act on the 3D slab. Finally, the modified code was used to predict the thermal-hydraulic phenomena, and the change of flow instability and critical heat flux under motion conditions was examined. First, the results of predicting the flow instability under two-phase conditions through the RELAP5 code(M. Colombo et al., 2012) were compared with the MARS-KS analysis results. Next, utilizing the MARS-KS motion model, the unstable region that appears due to flow instability in stationary and motion conditions was predicted and compared. The change of critical heat flux due to dynamic motion under the same heat flux condition was also examined using the same analytical model. When the critical heat flux appears, the heat transfer efficiency to the working fluid greatly decreases, thus the wall temperature increases rapidly. From this, it was confirmed whether the critical heat flux occurs in the stationary and motion conditions. Through this application study, the usability of the improved code was verified.2018년 국제해사기구(IMO)는 IMO 선박 온실가스 감축 초기전략을 채택하여 선박에서 배출되는 온실가스를 2050년까지 2008년 대비 50% 감축하기 위한 계획을 수립하였다. 국제해사기구의 조치에 따라 친환경 에너지로써 해양원전을 개발하기 위한 국내·외 연구가 활발히 진행되고 있다. 산학연에서 해양원전 개발과 안전성 평가를 위한 연구가 활발히 진행됨에 따라 신규 규제수요의 발생이 예상되며, 규제기관 차원의 독자적인 검증능력 확보를 위한 기반 연구가 요구된다. 이러한 배경에서 해양원전의 안전성 평가를 위한 MARS-KS motion model의 개선연구를 수행하였다. 연구목표는 MARS-KS motion model의 다차원 유동 해석능력의 구현이며, 이를 위해 코드 개선, 개념문제를 통한 검증, 그리고 개선된 코드를 활용한 응용 순으로 연구를 수행하였다. 첫 번째로 MARS-KS motion model에 대해 다음과 같은 7가지 사항을 개선하였다. 사용자 친화적인 코드 구현을 위한 Volume direction unit vector 생성방법의 개선, 관성력을 고려한 직선가속도 항 수정, 회전 중심에 관한 오류 수정, 매시간 단계마다 정션(Junction) 정보 업데이트, 다차원 유동을 구현하기 위한 축별 압력 수두 계산 및 연결정보 계산식의 개선, 그리고 MARS-KS motion model을 MULTID 컴포넌트로 확장하기 위한 개선연구를 수행하였다. 두 번째는 개선된 MARS-KS motion model에 대한 검증이다. 먼저, 서울대학교에서 MARS-KS motion model의 물리적 모델 검증(H.K. Beom et al., 2019) 시 사용하였던 개념문제 풀이를 통해 1D 컴포넌트와 교차유동(Cross-flow)에 대한 검증을 수행하였다. 1D 컴포넌트의 검증을 위해 1차원 마노미터와 수직 파이프 개념문제를 다양한 운동 조건에서 해석하였고, 이론해와 정량적으로 비교하여 코드의 적절성을 확인하였다. 다음으로 교차유동 검증을 위해 단상 조건의 1차원 수직 파이프를 교차정션(Cross-junction)으로 연결하여 강수부를 모사한 개념문제를 선정하였다. 강수부를 모사할 때, 4개 파이프로 구성된 기존 개념문제와 함께 본 연구를 통해 구현할 수 있게 된 6개 파이프와 8개 파이프 개념문제에 대한 검증을 수행하였다. 1D 컴포넌트와 교차유동 검증에 이어 MULTID 컴포넌트 검증을 위해 3차원 슬래브와 환형의 3차원 실린더 개념문제를 선정하였다. 마찬가지로 다양한 운동 조건에 따른 해석결과가 이론해를 적절하게 예측하는지 확인하였다. 또한, 3차원 슬래브를 다양한 외력이 복합적으로 작용하는 운동 조건을 적용하여 Motion model의 MULTID 컴포넌트 해석능력을 확인하였다. 마지막 세 번째로 개선된 코드를 열수력 현상 예측에 활용하여 운동 조건에서 유동 불안정성(Flow instability)과 임계열유속(Critical Heat Flux)의 변화 거동을 검토하였다. 먼저, RELAP5 코드를 통해 2상 조건에서 유동 불안정성을 예측한 결과(M. Colombo et al., 2012)를 MARS-KS 해석결과와 비교하였다. 다음으로 MARS-KS motion model을 통해 정지 상태와 운동 조건에서 유동 불안정성으로 인해 나타나는 불안정 영역(Unstable region)을 예측·비교하였다. 유동 불안정성에 이어, 동일한 해석모델을 사용하여 같은 열속 조건에서 동적운동에 의한 임계열유속의 변화를 확인하였다. 임계열유속이 발생하면 작동 유체에 대한 열전달 효율이 크게 감소하여 벽면 온도가 급격히 증가한다. 따라서 정지 상태와 운동 조건에서 벽면 온도를 비교하여 임계열유속의 발생 여부를 확인하였다. 이와 같은 응용연구를 통해 개선된 코드의 활용성을 입증하였다.제 1 장 서 론 1 제 1 절 연구배경 및 목적 1 제 2 절 연구 범위 4 제 2 장 MARS-KS motion model 개선연구 6 제 1 절 Motion model 개요 6 제 2 절 Motion model 개선연구 현황 8 제 3 절 Motion model 개선 13 1. Volume direction unit vector 생성방법 개선 13 2. 관성력을 고려한 직선가속도 항 수정 16 3. 회전 중심에 관한 오류 수정 17 4. 정션 정보의 업데이트 18 5. 다차원 유동 해석능력 구현 19 6. Motion model의 MULTID 컴포넌트 확장 23 제 3 장 MARS-KS motion model 검증연구 37 제 1 절 1D 컴포넌트 검증 37 1. 마노미터의 운동 38 2. 수직 파이프의 수직요동 41 제 2 절 교차유동 검증 43 1. 4개 파이프의 운동 44 2. 6개 파이프의 운동 45 3. 8개 파이프의 운동 45 제 3 절 MULTID 컴포넌트 검증 47 1. 3차원 슬래브의 운동 47 2. 3차원 실린더의 운동 52 3. MULTID 컴포넌트 해석능력 확인 55 제 4 장 MARS-KS motion model 응용연구 82 제 1 절 Motion model을 통한 유동 불안정성 예측 83 제 2 절 Motion model을 통한 임계열유속 예측 87 제 5 장 결론 99 용어설명 101 참고문헌 103 부록 1. MARS-KS motion model 매뉴얼 106 Abstract 110Maste

Organization of the 5' region of the rat ATP-citrate lyase gene

A genomic clone, encompassing the 5' flanking region and the first seven exons of rat ATP citrate lyase gene, was isolated from a rat genomic library and sequenced. Primer-extension analysis showed that mRNA is transcribed at 4407 nucleotides upstream from the translation start site. Primer-extension analysis and sequencing of ATP citrate lyase cDNA amplified by PCR showed that the promoter used for transcription is identical in mammary gland, lung, liver, brain and kidney. Southern-blot analysis showed that the ATP citrate lyase gene exists as a single copy. The 5' flanking region contains several consensus sequences defined as promoter elements. These include a CAAT box and Sp1-binding sites. However, a TATA box lacks this promoter. The expression of the chloramphenicol acetyltransferase gene was induced by the 5' flanking region (-2370 to -1) in the CHO cell line. The 5' flanking region also contains several sequence elements that may be involved in the transcriptional regulation of the gene.ope

Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cell-surface LDLRs in a dose- and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.ope

Cyclase-associated Protein 1 Is a Binding Partner of Proprotein Convertase Subtilisin/Kexin type-9 and Is Required for the Degradation of Low-Density Lipoprotein Receptors by Proprotein Convertase Subtilisin/Kexin type-9

Aims: Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. Methods and results: The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. Conclusion: We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.ope

The roles of sterol regulatory element-binding proteins in the transactivation of the rat ATP citrate-lyase promoter

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from -114 to -60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the -102/-94 region, immediately upstream of the inverted Y-box, and the -70/-61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter.ope

A Hidden Pressure Gradient That can be Easily Passed Over in Prosthetic Mitral Valve

ope