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The effect of phosphorylation and Tyr 55 mutation on the catalytic activity of phytochelatin synthase from Arabidopsis thaliana
No full text阿拉伯芥 (Arabidopsis thaliana) 中含有植物螯合素合成酶 (phytochelatin synthase, PCS, EC 2.3.2.15)，會利用 glutathione (GSH) 為基質來合成植物螯合素 (phytochelatin, PC) 以結合入侵的重金屬，降低對植物的傷害。由於本實驗室以前表現出的PCS重組蛋白質N端帶有容易與金屬螫合的His-Tag，因此本論文試著將重組蛋白質上的Tag以thrombin去除。結果顯示不論是否帶有His-Tag，PCS的活性均無明顯改變。這可能是我們給予的鎘離子濃度和基質GSH超過活性催化所需，所以His-Tag的有無對PCS重組蛋白質來說沒有太大的差異。利用點突變技術將PCS的 N端保守性區域的Tyr 55進行點突變之後，測定不同突變株之表現蛋白質活性，發現同為芳香族的Y55F和Y55W突變株的活性和野生種比起來，僅稍微下降一些，而Y55H和Y55A突變株的活性則劇烈下降。進一步利用酵素動力學計算各突變株的Km和kcat，發現當側基失去芳香基團，酵素的整體催化能力也隨之下降50% 左右。由以上結果我們推測Tyr 55的苯環可以經由cation-pi 作用力，與帶著鎘的GSH複合物結合，以此將第二個基質GSH帶進催化區合成PC。另一方面，本論文以二次元電泳分析在植物體中PCS是否有磷酸化現象，發現未經過鎘逆境處理的阿拉伯芥樣本中，若加入phosphatase會使PCS蛋白質色點位移至pI較大之處，這可能是PCS被去除磷酸化所造成的結果。分別以不同濃度的鎘逆境處理後的阿拉伯芥樣本則無色點位移現象，但是各色點的比例有些不同。推測阿拉伯芥受到鎘逆境時PCS的磷酸化程度會隨之改變，所以二維電泳圖譜上才會呈現出色點比例的差異。分析阿拉伯芥粗抽取液的PCS活性，發現加以phosphatase inhibitor處理會提升PCS的活性，顯示磷酸化的確會對PCS的活性造成影響。本論文也使用免疫沉澱法來純化內生性PCS，將利用質譜儀 (LC-MS/MS) 來分析抗體辨認到的蛋白質身分，以及有無磷酸化修飾的現象。Phytochelatin synthase (PCS, EC 2.3.2.15) in Arabidopsis thaliana uses glutathione (GSH) as its substrate for the synthesis of phytochelatins (PCs) which could bind to heavy metals to reduce damages to cells. We have expressed AtPCS1 with His-Tag on its N-terminal sequence using E. coli expression system. In this study, His-Tag on AtPCS1 construct was removed by thrombin and the activity was then analyzed. There is no change on PCS catalytic activities whether the recombinant proteins contained His-Tag or not. This might be caused by using high concentration of GSH and Cd in the activity assay solution. To explore possible functions of the putative second substrate binding site, we tested several mutants at Tyr 55. Results showed that the activities of the mutants Y55F and Y55W were slightly lower than that of the wild-type. However, activities of Y55H and Y55A were dramatically decreased. Furthermore, changes on enzyme kinetic parameters of Tyr 55 mutants indicated that the aromatic group on Tyr 55 was important to PCS catalytic activity. These results suggested that Tyr 55 might bind GSH through cation-pi interaction. On the other hand, the two-dimensional electrophoresis (2-DE) analysis revealed that the native PCS in Arabidopsis showed several spots with different pI values. Samples treated with calf intestinal alkaline phosphatase (CIP) showed shifts of PCS spots on 2-DE, which might resulted from dephosphorylation of PCS. Samples treated with various Cd stress were also tested; the pI values of PCS spots were not affected by CIP, but the propotion of spots were slightly changed. These results suggested that phosphorylation of PCS might be enhanced in plants under Cd stress. In addition, PCS activity in Arabidopsis was decreased when sample was treated with CIP, and the activity could be recovered by adding phosphatase inhibitors. The above observations showed that the PCS activity might be regulated by phosphorylation. We also purified the endogenous PCS from Arabidopsis plants by immunoprecipitation and will identify the protein spots and its phosphorylation by LC-MS/MS
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
Variations 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
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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