基因突變對線蟲（Caenorhabditis elegans）的神經系統退化突變株的搜尋以及對其
This research is mainly in observation with Caenorhabditis elegans ’s genetic mutation caused via nervous system abnormal character. In the study, I the sample have been cultivated purified and add some chemical material EMS to speed up C.elegans mutation. Then based on the character to further analysis what causeof gene deal with mutation and observe the effects in heredity. The research has two stages, on the first stage of study the mainly target is to both search and purify the mutation of C.elegans. The second stage is based on the exploration of mutation’s searching and purifying. Because the certain mutation bodies aren’t easy to find out, the project is still on progress at the beginning of second stage, and we conclude some heredity special cases in preliminary of study. 這個實驗主要是觀察並針對線蟲因為基因的突變所產生的神經系統異常的變異性狀，在實驗中我先將樣品線蟲培養並純化至一定數量，並加入適當藥劑EMS造成其突變，經篩選並分析此性狀，進而找出造成其突變之基因，以及觀察此性狀對遺傳表現所造成的影響。該計畫分成兩階段，第一階段的實驗重點是在突變株的搜尋以及純化上，第二階段則是在突變基因的探討上，由於特定突變株的搜尋並非容易，所以目前計畫只進展至第二階段的遺傳實驗初期，對於其遺傳特徵與突變形式上已有了初步的分析，但尚未定位出該基因的位置。
澱粉?抑制劑之研究
植物合成澱粉?抑制劑可以對抗動物的取食，國外實驗證明數種澱粉?抑制劑對害蟲防 治具有顯著效果，其中以腰豆(Phaseolus vulgaris)研究最多。我們利用5% T.C.A.進行粗萃，從台灣常見豆類中篩選出四季豆(與腰豆同種不同品系)與菜豆，對麗蠅的澱粉?具有明顯的抑制效果，對豬胰臟與黃豆澱粉?的抑制效果則小或無，此種抑制特異性深具害蟲防治的潛力。經由溫度與pH 的試驗發現粗萃中的澱粉?抑制劑成分應為蛋白質。我們以四季豆作為繼續研究的對象，將粗萃進一步純化，經由陰離子交換與膠體過濾層析，分離出單一蛋白質，經蛋 白質定序比對確認其可能為國外發表的腰豆澱粉?抑制劑—αAI-1。經由測試發現此抑制劑在 85℃時仍能抑制果蠅澱粉?，為一相當穩定的蛋白質；且抑制劑的作用受pH 值影響很大，在偏酸性環境下的效果最好，與昆蟲分泌澱粉?的部位亦為酸性環境有相當密切的關聯；且其 抑制作用具特異性，可明顯抑制果蠅、入侵紅火蟻、白蟻、蟑螂及麵包蟲等昆蟲的澱粉?活性，對人類唾液、豬胰臟、四季豆本身及黃豆澱粉?的抑制效果很小或無，值得繼續深入研究。 Plant amylase inhibitors can fight against predation from plant-eating animals. It has been reported that several amylase inhibitors have an obvious effect on pest control; among them that from Phaseolus vulgaris got the most surveyed. 5% T.C.A was employed to make crude extracts. We have screened the amylase inhibitor activities from crude extract among beans common in Taiwan. The inhibitors from both string beans (the different strain of Phaseolus vulgaris) and cowpea notably inhibited the amylases in Chrysomia megacephala, but little or no inhibition in porcine pancreas and soy bean. This specific inhibition behavior suggested strong potential in pest control. Its activity can be affected by temperature and pH suggested that amylase inhibitors in crude extracts should be proteins. String beans were chosen to be further purified from the crude extracts. A single protein was isolated after ion exchange and gel filtration chromatography. Through protein sequencing, the partial amino acid sequences were highly homologous to that ofαAI-1 from Phaseolus vulgaris, indicating it might beαAI-1. The purified protein still can inhibit the amylase from Drosophila melanogaster at 85℃, suggesting it is thermal-stable. Its activity was affected by pH and reached the peak in weak acidic environment, which might be related to the fact that amylases are secreted in acidic environment of insect’s midgut. It obviously inhibited the amylases from D. melanogaster,Solenopsis invicta, Odontotermes formosanus, Periplaneta Americana Linnaeus, and Alphitobius sp., while not to human saliva, porcine pancreas, soy bean and string beans itself. The unique pattern of inhibition activities of the purified amylase inhibitor was worthy of further anlysis.
榕樹粗萃取液對種子萌發及生長之影響
相剋作用是指植物在代謝過程中釋放出有毒物質以抑制本身或鄰近植物種子萌發及植株發育生長的過程。一般認為，榕樹是具有相剋作用的常見植物，但很少見到相關的文獻探討。本實驗利用榕樹不同組織萃取液，以小白菜、結球白菜與阿拉伯芥作材料，探討該萃取液對上述植物發芽與生長之影響，結果顯示榕樹葉萃取液所造成之影響較顯著，不同榕樹組織的萃取液、不同濃度的萃取液對不同植物所產生的效果不盡相同，針對不同生長期的植物進行處理所造成的影響亦有差異。進一步以管柱層析法與高壓液相層析法（HPLC）分離葉萃取液，結果發現以正己烷和乙酸乙酯75：25為沖提液的條件下，於HPLC的層析圖中分離時間約8分時所流出的物質具最明顯的抑制作用。電泳剖面分析顯示處理與未處理間有些許蛋白質條帶差異，此變化蛋白可能與植物幼苗對該萃取液之耐受性有關。利用核酸微陣列(microarray)對榕樹萃取液處理之阿拉伯芥植物進行基因表現分析，發現榕樹萃取液對植物部分基因表現也有促進和抑制的情形，經由基因體與蛋白質體分析法可推論出一些受影響之相關基因。 Allelopathy was defined as a phenomenon which certain plant species, by secreting metabolites to the environment, can suppress the growth of themselves, seed germination and/or growth of other plants in the same habitats. Banyan （Ficus microcarpa L.f.） is a plant species in our campus likely to have allelopathy effect. However, documents describing such action on allelopathy were rare. In this study, we applied the crude tissue extracts including leaves, stems, roots of banyan onto the germinating seeds of Pai-Tsai （Brassica rapa L.ssp.chinensis（Rupr.（Olsson）））, Chinese cabbage （Brassica pekinensis Rupr.） and Arabidopsis（Arabidopsis thaliana, ecotype Columbia-0） to study the effects of such plant extract on the germination and seedling growth of other plants. It is found that allelopathic effects on seed germination vary among different tissues used for extract preparation. Different concentration of the extract also yield various degree of allelophathic effects. It is also noted that the application of extract onto the post-germinated sprouts has less effects on plant growth. The extract of leaves was subsequently chromatographed over silica gel using hexane/EtOH gradient solvent system and HPLC. The result showed that there was one fraction about 8 min in the chromatography of HPLC eluted with 25% EtOAc in hexane had the most inhibitory effect. SDS-PAGE analysis on the electrophoretic profiles of water soluble proteins has explored a different band pattern between the treated and non-treated sprouts. The observed band difference might provide a clue for exploring proteins which reacted differently upon the application of extract. DNA microarray analysis on the effect of banyan extract on Arabidopsis gene expression has also been employed to characterize genes responsive to the allelophathic treatment. Cross-comparison between the differential transcript and protein profiles will reveal key regulators in plants experiencing allelophathic condition. \r
核醣核酸蛋白粒之K 蛋白基因表現及功能之研究
核糖核酸蛋白粒之K 蛋白具有多種功能，可參與在基因轉錄和蛋白質轉譯等過程。我們發現\r 一新奇的cDNA 較已報告的K 蛋白cDNA 少了72 個核?酸，分別命名為S 形與L 形。由基因\r 組DNA 的分析，S 形可能是經由替代剪接少了第8 個exon 所形成。以西方墨點法分析細胞中\r 的K 蛋白，亦證明有S 形存在，其表現量較少且多存在於細胞核中。為了探究兩種K 蛋白等\r 形的表現和功能，我們以PCR 偵測K 蛋白在不同組織與細胞株和人類乳腺腫瘤組織中的RNA\r 表現，結果顯示主要以L 形為主;我們並將S 形與L 形K 蛋白分別轉染至人類乳癌細胞株MCF7\r 中，以抗生素篩選出穩定表現K 蛋白之細胞株，生長速率分析顯示表現S 形之細胞株生長速\r 率較慢，其分子機轉仍有待研究。另外在比對K 蛋白之基因組DNA 時，除了在第九對色體\r 上有完整之K 蛋白基因組DNA，我們亦發現在第2、3、5 及11 對染色體上有類似K 蛋白基\r 因的序列，以cDNA 的形式存在，這些DNA 序列是否可以表現RNA 及其意義為何，目前尚\r 不清楚。Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be\r involved in the regulation of transcription, translation, nuclear transport and signal transduction. We\r have identified an alternative splicing transcript of hnRNP K lacking exon 8. This novel isoform\r (named S form compares to L form of full-length hnRNP K) encodes a protein that is 24 amino acids\r depletion between RNA binding KH1 and KH2 domain. To explore the functional roles of both isoforms,\r we detect their expression in several tissues (including liver, lung, kidney and heart), cell lines (MCF7,\r 293T, HeLa, NIH3T3, WEHI, P388D1) and specimens of human mammary gland tumor by RT- PCR.\r The results showed that L form was expressed in all samples, whereas S form was only expressed in cell\r lines. Using Western blotting analysis, we found that L form existed in both cytosol and nuclear\r fractions, and little amount of S form was detected in the nuclear extract. Furthermore, we construct the\r MCF7 cell lines that stably expressed S form or L form hnRNP K. Growth rate analysis indicates that\r the overexpression of S form of hnRNP K could decrease cell growth rate. The molecular mechanisms\r of growth inhibition by S form hnRNP K are to be further investigated. On the other hand, when we\r blast the human genomic genebank, we found except the chromosome 9 containing complete hnRNP K\r genomic DNA, there are near complete hnRNP K cDNA sequence appears in chromosome 2, 3, 5, and 11.\r The meaning of these sequences is unclear.
雞胰臟去氧核醣核酸水解？多型性之探討
去氧核糖核酸水解?，(deoxyribonuclease,簡稱DNase)為一種核酸內切?。目前研究得最透徹的去氧核糖核酸水解?為牛胰臟的DNase I，有四種以上的同功?存在。而牛和雞的DNA序列有極高的相似度，由之前的研究中在等電點焦集電泳法顯示雞胰臟DNase I 具A、B 兩種同功?，本實驗則希望能進一步了解兩種同功?的差異原因。在抽取RNA 後，以RT-PCR 方法合成cDNA，將其以限制?切割，再以大腸桿菌作為載體進行轉殖及繁殖培養，最後將其DNA 定序後進行分析比較。目前已完成第一次定序，但因引子接合過程中有部分位置錯亂造成殖體無法進行表現。另外在此次所得之序列中有發現一個胺基酸的轉變，而其是否為多型性之表現則有待進一步的實驗證實。The most advanced research on deoxyribonuclease in current states is on the bovine pancreatic DNase I, more than four of which isoforms have been found. It is shown that the DNA sequences of chicken and bovine have relatively high similarity. In addition, according to the result of isoelectric focusing from previous researches, chicken pancreatic DNaseI has two isoforms (A and B.) In this research, the author expects to establish more knowledge on the differences on the isoforms and the causes. After receiving the RNA, the RT-PCR is preformed to incorporate the cDNA, which is later cleaved by restriction enzyme and inserted into the plasmid DNA of the E. coli host cell to be cloned. So far a polynucleotide sequence has been deduced from clones of the cDNA, but it cannot be expressed successfully in E.coli due to few random mistaken splicing. There is an alternation of one amino acid, and whether it is the actual state of isoform or not still requires further proof.
竹筍老化之謎
本研究是在探討收割後的綠竹筍(Bambusa oldhamii, green bamboo)的老化(aging)現象。一般人說的竹筍老了，通常是指竹筍的質地變硬，口感變差，此即是竹筍硬化的現象，而硬化的主因可能是竹筍受到逆境 (stress) 的刺激後，影響了基因表現的形態，導致纖維素和木質素的增加。竹筍採收後以不同方式處理，觀察切面的變化後發現，以0.2 M蔗糖水浸泡48小時後的竹筍，其切面比浸泡於水中或置於空氣中的竹筍切面較白，筍尖較綠且沒有枯萎的情況。不管是浸泡糖水、水或置於空氣中，都無法防止竹筍的硬化，但浸水和糖水可延緩竹筍硬化的情形，可見要防止竹筍老化，基本上要從抑制合成纖維素與木質素的酵素來著手。抽取竹筍切面處組織中的DNA並以DNA電泳分析之後發現，竹筍的DNA有被降解成小片段的現象，其大小差不多是180 bp的倍數，可見竹筍遇到逆境時也可能會有類似PCD (programmed cell death, 細胞程序性死亡) 的現象。抽取不同處理竹筍的蛋白質進行2D電泳，比較電泳結果發現，三種處理的竹筍的共同點在於減少的蛋白質幾乎都分布在等電點較低的部分。增加的蛋白質大多數分布在等電點較高的區域，這些增加的蛋白質可能和竹筍老現象與PCD有關。本研究還有兩個方向可以繼續延伸研究，第一個是將2D電泳上有明顯差異的蛋白質色點挖出，進行蛋白質定序，再從資料庫中比對，推測可能是何種蛋白質。第二個是研究抑制竹筍合成纖維素和木質素的?的方法，保持竹筍的口感，使竹筍能成為一種能外銷的食品。 The purpose of this study is to investigate and analyze the aging of the harvested green bamboo shoots. The research focused on how to prevent the aging of bamboo shoots and why green bamboo shoots become aging. The term “aging” means that the taste of bamboo shoots becomes hardness post harvest. At first, we tried to find out an anti-aging method, which is not only to keep the green bamboo shoots fresh, but also delicious. The method was to soak bamboo shoots in 0.2 M sucrose, in the water or without any treatment. After 48 hours, the cutting surfaces of bamboo shoots treated with sucrose were whiter, and their outer sheaths were greener than those of shoots soaked in the water or without treatment. The results showed that none of them can stop hardness. But the aging of sucrose- or water-treated shoots was retarded. The results suggest that inhibition of the enzyme activities involving cellulose and lignin synthesis may be required to prevent the aging of bamboo shoot post harvest. To get insight into the reason why bamboo shoots become hardness, the differences of protein patterns and DNA patterns between aging and fresh green bamboo shoots were analyzed and compared. The DNA from the bamboo tissues near cutting surface was isolated and analyzed by agarose gel electrophoresis. The results showed that DNA from shoots was partially degraded. The fragments appear as a ladder of DNA with sizes in multiples of approximately 180 bp. The presence of the olignucleosome-size DNA fragments suggest that the cells may undergo programmed cell death (PCD). The degradation of DNA was not observed in shoots treated with sucrose. By comparing the results of 2-D gel electrophoresis, it was found that some proteins with low pI values decreased or disappeared post harvest, while proteins with increased levels were detected in the high pI area. These changes in these proteins may result in the aging of the bamboo shoots. Prevention of the aging of green bamboo shoots is not easy. However, I found out from this study that soaking the bamboo shoots in the 0.2 M sucrose was a possible way to preserve them. The cutting surface of sucrose-treated shoots remained white, and the sheaths of the shoots was greener than those treated with other methods. Moreover, the degradation of DNA was not observed. However, it still cannot completely stop the aging of bamboo shoots. Reducing the enzyme activities involving cellulose and lignin synthesis may be a direct way to prevent the aging of bamboo shoots. It seems like there are many things to discover in the future.
人類粒腺體蘋果酸.活性中心輔.NAD(P) + 結合位置之探討
人類粒線體蘋果酸.可利用NAD+或NADP+為輔.，幫助腫瘤細胞獲得能量，但一般生理條件較傾向以NAD+為輔.。本研究將K346 修改成偏好NADP+之粒線體蘋果酸.家族中具有高度保留性的絲胺酸、及不具極性之丙胺酸，探討為何此酵素較偏好以NAD+為輔.。天然及突變株酵素的催化常數 (kcat)、基質Km 值、及抑制常數 (Ki) 測定結果顯示K346 之點突變不會影響基質Km 值，但K346S 之kcat明顯上升，繼而改變此酵素對NADP+之選擇性。本研究對於人類粒線體蘋果酸.催化機制的了解，有助設計具專一性的活性抑制劑，未來可應用於抑制腫瘤細胞能量來源，進而抑制腫瘤細胞生長。;Human mitochondrial NAD(P)+-dependent malic enzyme can help tumor cells gain energy, using either NAD+ or NADP+ as the cofactor, but prefers NAD+ as the coenzyme. By mutating the K346 to Ser, conserved in NADP+-dependent ME and to Ala with non-polar, we explore why human mitochondrial NAD(P)+-dependent malic enzyme prefers NAD+ as the coenzyme. We measured the proteins kcat, Km and the Ki values. The experiments showed that mutantions don’t affect the Km values, but K346S increased in the kcatvalue, transferring the coenzyme specificity to NADP+. If we develop deeper understanding of the human mitochondrial NAD(P)+-dependent malic enzyme, we can design a specific drag to inactivate the enzyme activity, and inhibit tumor cell growth.
Mechanisms of Tumor Cell Invasion: The Role of Stat3 in Squamous Cell Carcinoma
Skin cancer, including basal cell and squamous cell skin carcinoma, is known to\r be the most common cancer type. Skin cancer is thought to make up half of all known\r cancers. Over one million non-melanoma skin cancers (NMSC) are reported every year. \r Approximately 300,000 of these cases are squamous cell carcinoma (SCC). It is\r estimated that about 2,000 people die from NMSC each year. Of the two skin cancer\r types, SCC tends to be the more clinically aggressive and likely to spread and invade,\r typically by way of blood or lymphatic vessels. Understanding the signaling pathways\r in SCC cells that regulate invasion will be important for developing improved cancer\r treatments. The signal transducer and activator of transcription 3 (Stat3) protein is a\r central regulator of numerous cellular activities, including proliferation, survival, and\r motility. Stat3 also has enhanced activity in many cancers, including skin SCC. This\r study shows that Stat3 regulates several invasive properties in a human skin SCC cell culture model.\r HGF (hepatocyte growth factor)- induced cell schattering was assessed for\r SRB12-p9 cells (p9WT), a human skin SCC cell line, along with SRB12-p9 cells\r engineered to have reduced Stat3 activity. Next, a cell viability-based adhesion assay\r was performed with these cells. Finally, severe combined immunodeficient (SCID)\r mice were injected subcutaneously with P9WT and S3DN cells and tumors were\r measured twice weekly. Extracted tumors were analyzed by immunohistochemistry and Western blotting for expression of the invasion related enzyme, MMP-2 and MMP-9.\r The suppression of Stat3 activity in S3DN cell lines resulted in reduced motility,\r greater adhesion, and a less invasive phenotype in SCID mice. Immunohistochemistry and Western blotting indicated higher levels of MMPs in the P9WT cells with\r expression localization towards the outer perimeter of the tumors. This data suggests\r that Stat3 plays a role in skin SCC invasion and better understanding of Stat3 function\r could lead to improved treatment and prevention of the disease.