1,720,973 research outputs found
Investigation of Anticancer Mechanism of a Flavonoid Derivative in Human Androgen-Independent Prostate Cancer Cells
No full text前列腺癌一直是西方國家高罹患率及高死亡率的癌症之一，而隨著生活習慣及飲食的西化，前列腺癌的罹患率及死亡率也在亞洲地區快速的攀升。造成前列腺癌高死亡率的主要原因來自於癌症的轉移及惡化，但目前並沒有有效治療的藥物，極需要相關研究的投入。本次研究之化合物WJ9708012 (6-(3-Hydroxy-3-methylbutyl)-2’-(7-hydroxy-3,7-dimethyloctyl)-3’,4’,5,7-Tetramethoxy
-flavanone) 為類黃酮類結構，與一般類黃酮類化合物不同，其不具有強效的抗氧化能力，但對於人類賀爾蒙抗性前列腺癌細胞PC-3及DU-145，則展現抑制細胞生長及促進細胞凋亡之能力。於抑制細胞生長功能上，WJ9708012藉由抑制mTOR相關訊息傳遞路徑，使cyclin D1等G1細胞週期調控之重要分子表現量降低，造成G1細胞週期停滯現象。使用西方點墨法觀察發現，WJ9708012造成p-mTORser2448、p-P70S6KThr389及p-4E-BP1Thr37/46位置的磷酸化現象明顯減少，使eIF4E調控的7-methylguanosine 5’-triphosphate (m7GTP) cap-dependent translation無法進行，下游cyclin D1等G1細胞週期相關蛋白在mRNA沒有明顯減少的情況下蛋白表現量皆大幅降低，使細胞無法順利通過G1細胞週期，產生停滯生長現象。此現象可藉由給予胎牛血清使mTOR相關訊息傳遞路徑活化的方式降低，間接證明抑制mTOR相關訊息傳遞在此化合物抑制細胞生長之功能中扮演重要角色。在引起細胞凋亡方面，短時間處理WJ9708012可產生一個由細胞外進入胞內的鈣離子流，造成胞內鈣離子濃度迅速上升並活化鈣離子相關訊息分子，如：μ-calpain、PKC-α及calmodulin，使細胞內粒線體及內質網兩個調控鈣離子之重要胞器產生壓力或傷害等影響。粒線體部分，WJ9708012造成粒線體損傷，使膜電位流失及下游內生性細胞凋亡訊息途徑啟動。以西方點墨法觀察，可發現化合物造成粒線體凋亡因子釋出、caspase-9、-3、-7活化及晚期Bcl-2家族蛋白表現量改變及斷裂現象。另外，化合物也會造成細胞內能量恆定失調，使在此扮演細胞保護功能之AMPK蛋白活化，調控下游訊息分子。於內質網部分，則可觀察到內質網壓力產生，使GRP78、GADD153及Caspase12表現量增加或斷裂活化的現象。WJ9708012引起的粒線體損傷和內質網壓力在密切的訊息傳遞後，會造成細胞凋亡途徑的啟動。使用流式細胞儀可觀察到subG1細胞增加和具有PI-Annexin X雙重螢光訊號細胞增加的現象。此外，使用TUNEL assay亦可觀察到具有DNA斷點的螢光訊號之細胞增加情形。總體而言，WJ9708012引起人類賀爾蒙抗性前列腺癌細胞PC-3細胞G1細胞週期停滯及細胞凋亡反應，主要是藉由抑制mTOR相關訊息傳遞路徑及引起鈣離子流入細胞質，影響相關訊息傳遞分子之活性，並造成粒線體及內質網損傷，而啟動細胞凋亡途徑來達成。Prostate cancer is one of the leading causes of cancer death among men in the United States and Western Europe. Following the transition of lifestyle to Western type, the mortality of prostate cancer is rapidly increased in Asian countries. Most prostate cancer-related deaths are due to advanced disease and, until recently, the hormone therapy was the only well-acceptable treatment approved for advanced prostate cancer. However, many patients were resistant to hormonal therapies after two to three years of treatment and no effective therapy currently exists for advanced prostate cancers that are resistant to hormonal therapy. In this thesis, we reported that a methoxyflavanone – WJ9708012 (6-(3-Hydroxy-3-methylbutyl)-2’-(7-hydroxy-3,7-dimethyloctyl)-3’,4’,5,7-
Tetramethoxyflavanone) inhibited growth and produced apoptosis of human hormone-resistant prostate cancer PC-3 and DU-145 cells. WJ9708012 inhibited cell proliferation and induced G1-phase cell-cycle arrest. By using Western blotting analysis, we found that WJ9708012 induced a rapid and time-dependent decrease of phosphorylation level of mTORSer2448, P70S6KThr389 and 4E-BP1Thr37/Thr46, which would suppress 7-methylguanosine 5’-triphosphate (m7GTP) cap-dependent translation through inhibiting eIF4E release from the translational repressor 4E-BP1 and down-regulating the expression of cyclin D1. The stimulation of mTOR-related signaling pathway by fetal bovine serum could rescue the anti-proliferative effect of WJ9708012, indicating an important role of mTOR-related signaling pathway to WJ9708012 action. The treatment of PC-3 prostate cancer cells with WJ9708012 also induced a prompt calcium influx from the extracellular space. The increase in [Ca2+]i was associated with the activation of calcium-related signaling molecules, including calpain, PKC-α and calmodulin. Subsequently, these events caused mitochondria damage and resulted in the induction of cell apoptosis. By using Western blotting analysis, we found that WJ9708012 induced a mitochondria-regulated apoptosis, which involves the release of cytochrome c, activation of caspase-9, -3, and -7 and alteration of protein expression of Bcl-2 family members. Moreover, because of the damage of mitochondria function, WJ9708012 also caused the activation of AMPK, which served as a protective signal and regulated the downstream signal molecules. The endoplasmic reticulum (ER) stress was also induced by WJ9708012, which is associated with the cleavage of pro-caspase12 and the up-regulation of protein levels of GADD153 and GRP-78. A crosstalk between ER stress and mitochondrial insult was observed, leading to the apoptosis events in PC-3 cells. The apoptosis triggered by WJ9708012 in PC-3 cells was assessed using annexin V/ PI labeling and the TUNEL assay. Taken together, the data suggest that WJ9708012 causes a G1-phase cell cycle arrest by inhibiting mTOR-related pathways and induces the cell death through the activation of calcium influx from extracellular space matrix followed by ER stress and mitochondria damage, which would trigger the intrinsic apoptotic pathways
Purification and Characterization of Candida rugosa Lipase 3 Produced by Pichia pastoris and Its Application in Deinking of Waste Newspaper
No full text廢紙回收利用具有確保資源、節約能源、延長纖維資源使用年限及減少污染與CO2 排放等重大意義。尤其台灣地狹人稠，教育普及，紙張消耗甚巨，廢紙之回收再利用益顯重要。本研究乃探討中研院植物暨微生物學研究所蕭介夫教授研究室構築之 Pichia pastoris 轉形株所生產 Candida rugosa 脂肪酶第三型 (CRL3) 在印刷廢報紙脫墨上之應用。經由硫酸銨分劃法、膠體過濾法及疏水性層析法可成功獲得純化之 CRL3，酵素性質分析結果顯示在 pH 5~8，37oC~60oC 的範圍內，酵素活性皆保持穩定，接著針對國內最大宗之新聞廢紙，使用黑白印刷報紙製備紙漿，比較化學及酵素脫墨之脫墨效率，建立具體可行之酵素脫墨流程。當以化學脫墨劑，Sinopol 964H (polyoxyethylene nonylphenyl ether)，於 50 mL 離心管，在 pH 11.5, 50oC, 150 rpm 下震盪 16 小時可測得每克紙漿 (乾重) 內含化學可脫油墨量為 5 mg，以此作為每克紙漿之油墨含量，三小時之脫墨效率可達 86%，在紙漿 (乾重) 與脫墨劑為 10:1 之最適條件下，脫墨速率為 2.4 mg/g．hr。比較 CRL3 之粗酵素液、純化酵素及市售商品脂肪酶 (Sigma) 之脫墨效果，在 pH 7 與 60oC 之最適反應條件下，脫墨效率分別為化學脫墨之 55%、54% 及 73%，每單位酵素活性之脫墨速率分別為 13 μg/g．hr、13 μg/g．hr 及 18 μg/g．hr；或每毫升培養液之脫墨速率為 2.7 mg/g．hr。結果顯示 CRL3 每毫升培養液之脫墨速率，已相當於使用化學脫墨劑之脫墨效果，且無須使用純化後酵素，故在工業應用上極具發展潛力。Recycling of waste paper has great significance, including economizing energy, reducing pollution and CO2 exhaustion, etc. Because of the high education rate and large paper consumption, recycling of waste paper is fairly important in the especially populated Taiwan. The objective of this study is to explore Candida rugosa lipase 3 (CRL3), produced by recombinant Pichia pastoris, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, in deinking effect of waste newspaper. By ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography, simple band of CRL3 on a SDS-PAGE was obtained. Purified enzyme is stable at pH 5~8 and 37oC~60oC. Then comparing the deinking efficiency of chemical and enzymatic method for domestic black and white waste newspaper, our goal is to build the optimal enzymatic deinking procedure. The results show that when chemical agent 964 and pulp react at pH 11.5, 50oC and shake for 16 hours at 150 rpm, the maximum chemical deinking quantity is 5 mg per dry weight and the deinking efficiency can reach to 86 % in 3 hours. Under the optimal condition of mixing pulp and chemical agent in the ratio of 10:1, the deinking rate is 2.4 mg/g．hr. Compared to CRL3 crude extract, purified CRL3 and commercial lipase (Sigma), under the optimal condition of pH 7 and 60oC, the deinking efficiency are 55%, 54% and 73% of chemical deinking, respectively. Moreover, the deinking rate is 13 μg/g．hr, 13 μg/g．hr and 18 μg/g．hr per unit, respectively; or 2.7 mg/g．hr per milliliter culture medium.第一章 前言 1
一、 脂肪酶 1
1. 生化特性 1
2. 催化反應 4
3. 結構特性 4
4. 應用性 8
二、 Candida rugosa 脂肪酶 (CRL) 11
三、 廢紙脫墨技術 12
1. 使用廢紙脫墨漿的好處 14
2. 油墨組成 15
3. 脫墨機制及方法 15
4. 界面活性劑 17
5. 酵素脫墨法的優點 19
6. 酵素脫墨文獻回顧 19
四、 研究動機與目的 21
五、 研究架構 23
第二章 材料與方法 24
I. CRL3 之純化與性質分析 24
一、 使用菌種、培養基及培養方法 24
二、 藥品與試劑 24
三、 分析方法 24
1. 蛋白質定量 24
2. 酵素活性分析 26
3. 蛋白質電泳分析 28
四、 蛋白質純化 30
1. 粗酵素液 30
2. 硫酸銨分劃 30
3. 膠體過濾法 30
4. 疏水性層析法 31
五、 純化酵素之性質分析 31
1. 最適反應 pH 值 31
2. pH 穩定性 32
3. 最適作用溫度 32
4. 熱穩定性分析 32
II. CRL3 之脫墨試驗 32
一、 試驗材料 32
二、 脫墨方法之建立 33
1. 樣品前處理 33
2. 油墨濃度之標準曲線 33
3. 化學脫墨 33
4. 酵素脫墨 35
三、 脫墨動力學 37
1. 脫墨劑濃度與反應時間 37
2. 酵素反應時間與添加量 37
3. 脫墨劑與酵素脫墨之動力學研究 39
四、 酵素脫墨法之最適化 40
1. 最適反應 pH 值 40
2. 最適反應溫度 40
3. 化學法與酵素法之比較 40
III. 儀器設備 41
第三章 結果 42
一、 CRL3 之純化 42
二、 CRL3 之性質分析 49
三、 脫墨方法之建立 54
四、 脫墨動力學 62
五、 酵素脫墨法之最適化 66
第四章 討論 72
一、 CRL3 之純化流程 72
二、 CRL3 之最適反應 pH 值與 pH 穩定性 72
三、 CRL3 之最適反應溫度與熱穩定性 73
四、 脫墨方法之建立 73
五、 脫墨動力學 74
六、 酵素脫墨法之最適化 75
第五章 結論 76
參考文獻 7
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Phytoplankton community reorganization driven by eutrophication and warming in Lake Biwa
No full textVariations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Obstructive jaundice as a complication of a right hepatic artery pseudoaneurysm after laparoscopic cholecystectomy
No full textA hepatic artery pseudoaneurysm is a rare, but a potentially life-threatening complication after laparoscopic cholecystectomy (LC). Obstructive jaundice owing to a hepatic artery pseudoaneurysm after LC has never been reported. We report a patient with a hepatic artery pseudoaneurysm after LC who presented with tarry stools, bloody drainage and obstructive jaundice
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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