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Cloning and Characterization of 1-Aminocyclopropane-1-carboxylate Oxidase cDNAs from Guava（Pisdium guajava L.） Varieties with Different Ripening-behaviour
No full text番石榴（Psidium guajava L.）為常綠熱帶果樹，果實富含維生素、礦物質及具抗氧化能力之特點，為臺灣極具發展潛力之經濟果樹。本島栽培番石榴品種依果實後熟特性可分為更年型與非更年型。試驗分析目前臺灣常見的非更年型番石榴栽培品種：‘珍珠拔’、‘水晶拔’、‘帝王拔’、‘二十世紀拔’及大村‘紅心拔’，果實採收後於20℃貯藏204 hr過程之乙烯生成率皆低於 0.20 μL C2H4‧kg-1‧hr-1，試驗期間皆沒有顯著乙烯生成的變化；相較於更年型品種‘梨仔拔’果實於20℃環境貯藏，其乙烯生成率可達71.02 μL C2H4‧kg-1‧hr-1，明顯高於非更年之品種。非更年型番石榴品種於自然環境下果實缺少大量的自生乙烯，而無劇烈軟化、轉色、濃郁香味生成的後熟反應，因此具有較長的儲運壽命與商品價值；而另以外施丙烯處理的結果顯示，丙烯可誘導非更年型番石榴果實進行後熟相關的生理變化，卻無法產生大量系統Ⅱ內生乙烯，顯示其後熟機制應與乙烯之生合成能力有著密切的關係，而非乙烯感受能力的差異所致。
以RT-PCR（reverse transcriptase- polymerase chain reaction）策略針對‘珍珠拔’及‘梨仔拔’兩品種番石榴組織進行ACO cDNA之選殖，兩品種果實皆可分離出兩個相同的番石榴cDNA選殖系，分別具有1,259及1,241個核苷酸，推估可轉譯出321個胺基酸，具有ACO活性相關的12個胺基酸與 4 個白胺酸拉鍊（leucine zipper）的結構位，其估算分子量為36.21及36.28 kDa，等電點（pI）為5.39 及 5.78。進一步將兩cDNA選殖系由大腸菌異源表現系統表達，皆能產生具有ACO活性的融合蛋白質，確認其為番石榴 ACO cDNA選殖系，分別命名為Pg-ACO1及Pg-ACO2。南方氏墨點法分析結果推估ACO基因在番石榴組基因的拷貝數約為4至5個。
北方墨點分析及RT-PCR分析Pg-ACO1及Pg-ACO2的表現結果，番石榴果實及枝條具有Pg-ACO1及Pg-ACO2轉錄產物的累積，而花朵及葉片無顯著轉錄產物的累積。Pg-ACO1於‘珍珠拔’與‘梨仔拔’分別於花後第60及120天持續表現至採後第12天，轉錄產物累積於不同天數並無明顯變化；Pg-ACO2於‘珍珠拔’與‘梨仔拔’皆於花後第15天幼果可偵測到轉錄產物的累積，隨後表現降低，分別於花後第60及120天才又恢復mRNA累積的情形。‘梨仔拔’果實Pg-ACO1及Pg-ACO2轉錄產物累積量於後熟過程皆有顯著上升趨勢，‘珍珠拔’則於採後貯藏期間持續表達。以100 μL‧L-1乙烯及10 mg‧L-1 1-MCP處理‘梨仔拔’番石榴綠熟果實，結果顯示Pg-ACO1及Pg-ACO2皆可受到乙烯誘導而增加轉錄產物的累積量；而1-MCP處理則具有壓抑兩者表達的效果，因而推估兩者皆具“系統Ⅱ”乙烯生成特性的ACO cDNA選殖系，兩選殖系皆可於‘珍珠拔’果實發育第60天即可偵測到轉錄產物的累積，又可於果實後熟階段表達，兩者表現的特性類似番茄無明顯系統區分的ACO基因，皆能參與果實乙烯生成系統Ⅰ與系統Ⅱ期間的乙烯生合成。果實組織酵素活性分析的結果得知，‘珍珠拔’果實組織ACO活性於採後第0~10天皆顯著高於‘梨仔拔’果實組織，顯示兩類型品種果實組織皆具催化ACC為乙烯的能力。
由上述結果指出，‘梨仔拔’與‘珍珠拔’之後熟特性差異並非Pg-ACO1及Pg-ACO2無法表達或缺乏ACO活性所造成。Pg-ACO1及Pg-ACO2於番石榴果實乙烯生成並無明顯的系統區分，Pg-ACO2為果實後熟主要表達的ACO。Guava (Psidium guajava L.) is a tropical evergreen fruit crop of Myrtaceae, and its fruit is rich in vitamins and minerals and has antioxidant capacity. The guava cultivars in Taiwan are divided into 2 groups, climacteric and non-climacteric, based on their ripening behavior. Five of the common non-climacteric guava cultivars in Taiwan, ‘Jen-Ju Bar’, ‘Shuei-Jin Bar’, ‘Di-Wang Bar’, ‘Er-Shr-Shr-Ji Bar’ and ‘Hung-Shin Bar’, and one of the climacteric guava cultivar, ‘Li-Tzy Bar’, were used to investigated the postharvest physiology. The ethylene production of non-climacteric fruits remained under 0.20 μL‧kg-1‧L-1 during 204 hours storage at 20℃, while that of climacteic fruits could reach 71.02 μL‧kg-1‧L-1. Treatment of exogenous propylene induced the ripening related response of non-climacteric cultivar guava, indicating that the ripening mechanism in cultivars of this group is closely linked with ethylene synthesis instead of ethylene perception.
The two ACO cDNA clones in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ tissues were isolated by RT-PCR (reverse transcriptase- polymerase chain reaction). The numbers of base pairs, molecular weights of the derived amino acid sequence, and the pI value of these two cDNA clones were 1,259 and 1,241, 36.21 and 36.28 kDa, and 5.39 and 5.78, respectively. It was estimated that both of the cDNA clones were able to translate 321 amino acids, in which with 12 ACO activity related conserved residues and 4 leucine zipper sites. Fusion proteins of the two cDNA clones obtained by using E. coli heterologous expression system demonstrated ACO enzymatic activity, which confirm that the two cDNA clones are guava ACO cDNA clones, and thus named as Pg-ACO1and Pg-ACO2.The result of Southern blotting suggested that the copy number of ACO genes in guava genome is approximately 4 or 5.
The results of northern blotting and RT-PCR assay on Pg-ACO1and Pg-ACO2 showed that there was accumulation of transcripts of Pg-ACO1and Pg-ACO2 in guava fruits and shoots but not significant in flowers and leaves. Pg-ACO1 in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ constitutively expressed from 60 and 120 days after anthesis, respectively, to 12 days during postharvest. Pg-ACO2 transcripts in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ were detectable in the 15th day after anthesis, and then decreased until the 60th and 120th day after anthesis, respectively. Transcripts of Pg-ACO1and Pg-ACO2 in ‘Li-Tzy Bar’ fruit significantly increased during ripening, and that in ‘Jen-Ju Bar’ constitutively expressed during storage. The results of 100 μL‧L-1 ethylene and 10 mg‧L-1 1-MCP treatments showed that the transcripts of Pg-ACO1 and Pg-ACO2 in mature green ‘Li-Tzy Bar’ were both increased by ethylene elicitation and suppressed by 1-MCP. Therefore, it was inferred that two ACO cDNA clones possess the characteristics of “system Ⅱ” ethylene synthesis. However, in ‘Jen-Ju Bar’ fruits, the two ACO transcripts were detected from 60th day after anthesis to 12th day postharvest suggesting that both ACO clones might be involved in ethylene biosynthesis in both fruit development and ripening stage and that the two ACO transcripts possess characteristics of both “system Ⅰ” and “system Ⅱ”. In addition, the ACO activity of ‘Jen-Ju Bar’ was significantly higher than that of ‘Li-Tzy Bar’ during the first 10 days postharvest.
The results shown above indicate that the difference in the ripening behaviors between ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ is neither caused by silence of Pg-ACO1and Pg-ACO2 nor by the lack of ACO activity. Furthermore, according the ethylene and 1-MCP experiment on ACO transcripts in ‘Jen-Ju Bar’ fruits, it is hard to conclude which system of ethylene synthesis that both of Pg-ACO1 and Pg-ACO2 belong to
Effect of Particle Size in Diffusion Efficiency within Nucleus Pulposus and Anulus Fibrosus
No full text背景介紹：擴散作用是椎間盤內部養分與廢物代謝的主要方式之一，因此擴散作用與受傷椎間盤的自我修復能力有著重要的關聯性，而這些養分和廢物的擴散能力亦會受到椎間盤的退化程度與重組結構影響，但是目前對於這方面的研究尚不明朗。在退化性的椎間盤模型方面可注射生化藥劑進行模擬，例如使用胰蛋白酶改變椎間盤內的生物分子組成。另外，近年來許多藥劑被使用於恢復椎間盤的組成與功能，其中胜肽交聯劑的梔子素已被證實可使肽結構間彼此產生交聯反應，並且恢復椎間盤的力學性質。因此研究變性及交聯後的椎間環與椎間核的擴散效能，將有助於了解胜肽交聯劑在椎間盤再生治療方面的粒子傳輸效能。
研究目的：本研究的目的在於使用螢光影像分析技術評估經由變性及交聯後的椎間環與椎間核之粒子擴散效能變化。
材料與方法：本實驗所使用的椎間環與椎間核皆取自六個月大的豬隻腰椎。在實驗上，會使用自製手術刀將椎間環切成立方體形狀，另一方面則使用1c.c.的針筒來收集椎間核，每次實驗會將椎間核(約0.09克)擠入自製夾具中。試樣上，使用72組環向椎間環、72組徑向椎間環與72組椎間核，試樣總數為216組。而這三種形式的試樣又將進一步區分為三種狀態，分別為健康組，降解組與交聯組。為了模擬退化的試樣，將胰蛋白酶(0.25%, 0.5c.c.)注入椎間盤中並且於4℃與室溫環境下各反應12小時，接著將椎間盤剖開取出椎間核。椎間環則是於取下後，浸泡於胰蛋白酶溶液中並且於4℃與室溫環境下各反應12小時，接下來椎間環的準備步驟則是與健康組相同。交聯組方面，所有的試樣將先經由降解組的流程反應，接著將梔子素(0.33%，0.5c.c.)注入椎間盤中於室溫下反應24小時之後剖開取出椎間核，椎間環則是浸泡於梔子素中於室溫下反應24小時。本實驗使用三種不同分子量的螢光染劑進行測試，分別為螢光素鈉(FS，MW=376 Da)、四甲基異硫氰酸羅丹明-多醣共軛物(TRITC-Dextran，MW=4400 Da)、異硫氰酸螢光素-多醣共軛物(FITC-Dextran，MW=40000 Da)。試樣將被置於兩端設有水槽的夾具中，右側的上游槽將注入螢光染劑(100μL，100 μM)，左側的下游槽則注入相同體積的生理食鹽水。實驗中會使用自製的螢光攝影系統來測試60分鐘內每隔5分鐘上、下游槽螢光訊號的變化，再經由Fick’s 第一定律便可計算出擴散係數。
實驗結果：小分子量染劑(FS)的擴散係數都比中分子量(TRITC)與大分子量的染劑(FITC)來得高。環向椎間環的擴散效能比徑向椎間環與椎間核來得好。健康組的椎間環與椎間核之擴散係數都比降解組與交聯組的擴散係數來的高，然而降解組與交聯組之間的擴散係數並無顯著性差異。
結論：(1)染劑的分子量大小與擴散係數成反比關係，即分子量越大的染劑，其擴散係數將越小。(2)由環向椎間環的擴散係數大於徑向椎間環可知試樣的方向性會影響其擴散效能。(3)經過胰蛋白酶降解與梔子素交聯的試樣，兩者的擴散性會比健康組來得低。Background：Diffusion is one of the nutrient and waste particles transportation mechanisms within intervertebral discs, and thus could be crucial for disc self-recovery from destructive injury. Disc diffusion capacity could be affected by the degeneration change and structural remodeling. However, the underlying mechanism is not well investigated yet. Disc degeneration can be modeled by introducing the biochemical stresses, e.g., trypsin, which may denature the biomolecular compositions within disc. Recently, many reagents have been studied in recovering the disc function. The genipin, a peptide crosslinker, is well proved to restore disc mechanical properties by forming crosslinking between the peptide structures. Determining the diffusion capacity of anulus fibrosus and nucleus fibrosus tissue after denaturation and crosslinking will help to verify the feasibility of peptide crosslinkers for disc regeneration therapy from the aspect of particle transportation function.
Purpose：The purpose of this study is to evaluate the diffusion capacity of anulus fibrosus (AF) and nucleus pulposus (NP) after denaturation and crosslinking with fluorescent image analysis techniques
Material and method：Specimens of AF and NP were harvested from healthy 6-month-old juvenile pigs. The AF was dissected by a home-made scalpel into a cubic shape and NP were store in a inject syringe (1cc.), and 0.09 g of NP was measured and squeezed into the home-made cubic slots. Seventy-two AF along the circumferential direction, Seventy-two AF along the radial direction, and Seventy-two NP (total two hundred and sixteen groups) specimens were prepared. The three types of specimens were further assigned into 3 groups; i.e., intact group, denatured group and crosslinked group. To simulate the denaturation, trypsin (0.25%, 0.5cc.) was injected into the disc and stored at 4℃ for 12 hours and at room temperature for 12 hours, and then the NP was obtained by dissecting the disc. The AF were first dissected and immerged in trypsin solution at 4℃ for 12 hours and at room temperature for 12 hours, then the AF were prepared using the same procedure as intact group. For the crosslinked group, all the specimen were prepared first using the denatured group protocol, and then the specimen were treated with genipin for 24 hours at room-temperature. The Fluorescein Sodium Salt (FS), Tetramethylrhodamine Isothiocyanate-Dextran (TRITC-Dextran) and Fluorescein Isothiocyanate-dextran (FITC-Dextran) were used in this study. Specimens were set in a channel with two sink at both end. The source sink at right hand side was filled with 100μL fluorescence dye at 100 μM intensity and the drain sink at left hand side was filled with same amount of saline. An in-house fluorescent photographic system was used to find the signal changes of both source and drain sink every 5 minutes for 60 minutes. The diffusion coefficient can be calculated from these images using Fick’s first law.
Result：Smaller molecule (FS) always has higher diffusion coefficient than the medium (TRITC) and large (FITC) molecule. The diffusion coefficient of AF along the circumferential direction is higher than the AF along radial direction and NP. The diffusion coefficient of intact AF and NP is higher than the coefficient of denatured and crosslinked one. However, the difference between the denatured and crosslinked disc is not significant.
Conclusion：(1) Increasing the particle size lead to decrease the diffusion coefficient. (2) Higher diffusion coefficient through circumferential specimen in comparison with radial one shows the effect of direction. (3) The diffusion coefficient decreased significantly after denaturation and crosslinking
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Full text link“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
Full text linkWe 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
Full text linkWe 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
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