八分鐘快速免疫呈色法檢測市售牛乳中有無摻雜粉
台灣過去40 年來，許多牛乳廠商會因牛乳供應量不足摻雜奶粉以增加利潤，為了解決這問題，本實驗提供一種快速且準確的免疫呈色法檢測牛乳中有無摻雜奶粉。因奶粉的加工過程中加熱是必須的，所以本實驗是利用單株抗體只與牛乳中因加熱而變性的蛋白質反應。實驗步驟非常簡單，首先，用一支玻棒沾附待測鮮乳，經過簡短的清洗及化學處理後，玻棒尖端會與經特殊製備的單株抗體反應。最後，將玻棒放入已製備好的溶劑中呈色。當溶液呈現綠色，表示待測乳中摻有奶粉。整個實驗流程只需短短八分鐘。據我所知，該方法是相當具有新穎性且從來未被應用過。這是個令人興奮的發現，足以解決多年來酪農業中牛乳摻雜奶粉的問題。For the last 40-years, owing to the limited supply of commercial raw milk, the industry sometimes would mix the dry milk into the raw milk to increase their profit. To detect whether or not the milk on the market contains the poor quality’s dry milk, I invented a rapid and sensitive colorimetric immunoassay. The assay essentially utilizes a monoclonal antibody that only reacted with the thermal denatured protein presented in the dry milk. First, a glass tip-stick was dipped in to the milk to be tested. Second, following a brief wash and chemical treatment, the glass tip was reacted with the monoclonal antibody that has been specifically produced. Finally, the glass tip was dipped into a reagent containing developer. When the solution develops a color in green, it indicates the milk definitely contains dry milk. The entire procedure only takes 8 minutes to finish. To the best of my knowledge, this method is novel and has never been shown before. It represents an exciting discovery that solves the malpractice of mixing dry and raw milk in our dairy industry.
水果與DNA
本研究的目的在探討數種水果分解?，對萃取洋蔥DNA 的影響，想了解在什麼情況下，哪一種水果分解?的反應最快。研究結果發現：實驗所選水果都含分解?，以火龍果汁分解?反應最快。沒有加火龍果汁分解?的洋蔥溶液，加入酒精後一樣會分層出像DNA 的白色棉絮團狀物，但分層時間長，超過15 分鐘以上；而加入火龍果汁後，反應時間明顯會增快，快至30 到40 秒內完成，可知火龍果汁分解?有催化作用。在火龍果汁的量方面，由0%逐步加到20%時，以10%反應時間就達到極限，再增加單位果汁量並沒有顯著差異。在不同溫度的火龍果汁方面，除了常溫(22.5℃)外，冷凍(-10℃)、冷藏(4.2℃)和加溫(42.9℃)反應時間都變慢，這是因為每一種?都有一個反應最佳的溫度，溫度太低，?的活性會降低，溫度太高?可能會變性失去催化作用。本研究用的火龍果分解?在常溫下的反應最快。更進一步的研究發現，以最不易被萃取DNA 的鳳梨當分解?，萃取火龍果種子的DNA 時，可以萃取到較純又多的火龍果DNA。In this study, what I notice most about the phenomena of a extraction DNA method by using fruit’s enzyme are stepping progressive researches, that are at first qualitative analysis, then quantitative analysis (volumetric analysis), and finally deep study the interaction (special phenomena) among the onion juice, fruit’s enzymes, salt solution and 95% ethyl alcohol. The enzymes of various kinds of fruits were explored in a kitchen as a laboratory. The compare methods of fruits’ enzymes reaction rate have been proposed. The hand-made experimental systems, six synchronic agitation machines and six synchronic ejectors were developed and have been set up to control the key parameters and find out the phenomena of the reaction process. The key parameters are the quantity of onion slurry, fruit’s enzymes, salt solution, 95% ethyl alcohol, temperature and mixing time. The results show that: (1) The onion DNA phenomena through fruit’s juice as enzyme is visible in kitchen as a laboratory. (2) The hand-made experimental system demonstrated an effective way to control mixing times, therefore compare methods have well been simplified. (3) All fruits’ juice has enzymes function. Normally, the reaction time in process is more than 15 minutes. (4) The enzyme of fruit Pitaya has the highest activity. The reaction time in process of Pitaya juice as enzyme in the reactant onion slurry is less than 40 second. (5)And the optimal quantity of Pitaya juice is 10% of the reactants, since the reaction rate might seem like to the saturated speed, if more than 10%. (6) The reaction rate depends strongly on temperature. The optimal temperature is room temperature, 22.5℃. Finally, the aggregating effect of the DNA and protein was also observed. The reacted products were examined with the optical microscope and the composition of the products was analyzed and identified by using the UV diffraction method. The interaction among DNA, proteins, salt, ethyl alcohol and water was investigated with computer simulation ( the software “Hyper Chem”).
解開神秘果的奧秘-檸檬變柳丁的原因
原產於西非的「神秘果」，嘗了之後，30~200 分內，所有酸苦的東西嘗起來都是甜的。在深入蒐集相關資料後，我們發現神秘果有多種特殊效果，僅擷取以下幾種感興趣之方向來研究。〈1〉使酸苦的感覺變甜〈2〉解酒〈3〉消除蚊蟲叮咬之腫、癢〈4〉抗氧化能力極強。用食鹽水可萃取出miraculin 這種醣蛋白，經由生化實驗，推測使酸味變甜為其cover 舌尖甜味味蕾之結果，分子量約為40000 左右；但在檢測過程中，發現對咖啡、黃連和肉桂，都沒有太顯著的效果，只有酸味有顯著的改變，和以往所閱讀的研究報告有出入，因此懷 疑有氧化還原等其他化學效果，將再做深一層研究。消除蚊蟲叮咬之腫癢的成分確定為小分子所致。經由Prolox 當量測定法檢測神秘果抗氧化能力數值高達4974g/nmol，比一般中草藥及蔬菜多3000 左右。使酸變甜的原因若深入研究對糖尿病患者和減肥者都是一大福音，塗抹蚊蟲叮咬藥膏也可用天然物質製作，而抗氧化能力高更對人體健康有所幫助。當台灣已大量栽植，相對於日本及美國因地寒而無法培育成功，神秘果研究可成為另一項產業發展契機。 "Miracle fruit” is a fruit from West Africa. Though it's not sweet itself, if you eat anything that is sour or bitter after eating miracle fruit, the taste will turn sweet. After researching further material, we discovered that there are many amazing functions in miracle fruit, and decided to pick up some of which to study. (1) Turning the sour and bitter tastes into sweetness (2) Relieving alcohol (3) Relieving the hurt from mosquitoes and bugs (4) An excellent antioxidant. We can extract the miraculin that changes the taste from NaCl (aq), and through the biological experiment, we guess that's because miraculin covers the sweet sensor. The molecular weight of miraculin is about 40000.According to the experiment, we found out that miraculin doesn't have a great effect on the taste other than sourness, such as the bitterness of black coffee, Coptis chinensis, and cinnamon. . It is much more different from the former report we read. So we doubt that there are some other reactions. The thing, which relieves the hurt from mosquitoes and bugs, are sure to be a simple molecule, not a protein. By the Prolox equivalent weight experiment, we found that the ability of antioxidation got to 4974g/nmol, which is much higher than the normal vegetables and fruits. The effect of taste changing is really good news for diabetics and weight reducers. And the medicine can also be made by natural material. The excellent antioxidation is helpful for our health, too. Since Japan and America cannot grow the miracle fruit because of the cold weather, developing the functions of miracle fruit seems to be another chance for Taiwan to stand out in the world.
重金屬錯影響細胞生理功能的研究
儘管鍺在電子工業上被廣泛運用，但對於暴露在鍺化合物所產生的毒害則尚未被詳細的探討。在探討鍺對細胞所產生的生理影響中，我們使用了二氧化鍺 ( GeO2）和有機鍺（ Ge-132 ）。由實驗結果顯示， GeO2，會造成人類子宮上皮癌細胞（ A 431 ) 及巨噬細胞株（ Raw264.7 ）死亡，而 Ge -132 對細胞生長則不造成任何影響，為了進一步了解鍺引起細胞死亡是否是經過細胞凋亡（apoptosis ) ，我們將鍺處理過的細胞進行染色體 D NA 的分析，結果發現細胞中 DNA 染色體沒有斷裂。由先前 Huang 等人於 1999 年的研究結果顯示，砷對細胞所造成的毒性是經由有絲分裂活化酵素（ MAPK ）傳導路徑，所以為了解鍺誘導細胞死亡的路徑，我們亦分析 MAPK 傳導路徑是否亦參與其中，我們發現 GeO2加入 A431 細胞後，會活化有絲分裂活化酵素中的 ERK ，但對JNK 及 p38 皆無影響，在對蛋白質表現方面，轉錄因子 c-Jun 的蛋白質表現也是隨著GeO2加入的時間增加而上升。 GeO2加入 Raw 264 . 7cell 後，會造成 JNK 、 ERK 的活化，同樣的轉錄因子 c- Jun 也會增加，由此一結果得知鍺對細胞的影響會因細胞的不同而有所差異，為了分析自由基是否參與砷及鍺所造成細胞死亡的過程，我們分析在 A431 細胞中可產生的 NO 的可誘導性 nitric oxide synthase ( iNOS ）的表現，我們發現氧化鍺及砷都會誘導 iNOS 的表現量增加。綜合以上結果，可能顯示氧化錯可能會經由 M A PK 訊息傳遞路徑來促使細胞的死亡，並且 iNOS 亦可能參與此過程。就我們所知，這是第一個提出重金屬所造成的毒害可能會經由 iNOS 來誘導產生的研究。
Despite the extensive use of germanium (Ge) in the electronic industry and optical devices, the potential risks of exposure to germanium compounds have not been evaluated. The effects of germanium on cell physiological functions were studied. We first asked if germanium oxide (GeO2) or carboxyethylgermanium (Ge-l32) could affect cell viability. We found that GeO2, but not Ge-l32, reduced cell viability in a dose-dependent manner in epidermoid carcinoma A43 I and macrophage Raw 264.7 cells. In order to test whether apoptosis contributes to germanium cytotoxicity, DNA fragmentation was evaluated in A43 1 and Raw 264.7 cells treated with GeO2 or Ge-132, respectively. We found that neither GeO2 nor Ge- 132 had effect on chromosomal DNA fragmentation. Previous studies by Huang (1999) et al indicated that sodium arsenite (NaAsO2) cytotoxicity is mediated through mitogen-activated protein kinase (MAPK) pathways. In order to study the mechanism(s) by which GeO2 mediates cell death, we analyzed the signal transduction pathways triggered by GeO2 We found that GeO2 stimulated the extracellular signal-regulated kinase (ERK) activity and transcription factor c-Jun in a time-dependent manner, but not c-Jun amino-terminal kinasc (JNK), or p38 MAPK in A431 cells. Treatment of the Raw 264.7 cells with GeO2, induced activities of ERK, JNK and c-Jun in a time-dependent manner. Collectively, these results suggested that GeO2 effects might be cell type specific. To test whether free radicals were involved in NaAsO2 or GeO2 mediated cell death, the expression of inducible nitric oxide synthase (iNOS), which produced the NO free radical, was determined in A431 cells treated with NaAsO2 or GeO2. We found that expression of iNOS was induced in a time-dependent manner in NaAsO2 or GeO2-treted A431 cells. Taken together, our results indicated that GeO2-induccd cell death may be mediated through MAPK signal pathways and that iNOS may contribute to NaAsO2 or GeO2 mediated cell death. To our knowledge, this is the first report that iNOS may contribute to heavy metal mediated cytotoxicity.
豆類澱粉?抑制劑之研究與應用
豆類澱粉?抑制劑具害蟲防治與血糖調節之功效。本研究從台灣五種豆類中進行澱粉?抑制劑活性初篩，篩選出四季豆對麗蠅的澱粉?有明顯的抑制效果，粗萃後分離純化出單一蛋白質，經胺基酸定序結果得到兩個多?片段-VGLDFVLV 與TETSFNIDG ， 與已發表文獻比對推測為腰豆澱粉?抑制劑—αAI-1。經測試發現此抑制劑在85℃時仍具備抑制果蠅澱粉?之活性，為一熱安定性蛋白質，其抑制作用受pH 值影響很大，在偏酸性環境下的效果最好，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。在調節血糖方面，本研究利用豬胰臟澱粉?進行抑制活性測試，篩選出四季豆、花豆與黑豆對豬胰臟澱粉?有明顯的抑制效果，進一步利用Wistar 品系大鼠，進行豆類澱粉?抑制劑降血糖功效之活體試驗，將四季豆、花豆和黑豆粗萃經濃縮乾燥後餵食大鼠，發現花豆、黑豆在第一小時具有顯著抑制血糖增加效果；四季豆、黑豆在第三小時血糖回升，花豆仍可維持較低血糖濃度。此初步結果顯示，花豆粗萃物可能具備減緩消化吸收作用，具應用潛能，其確切分子作用機制值得深入探究。Amylase inhibitors can be applied in pest control and glucose adjustments. The study screens Phaseolus vulgaris from five Taiwan’s beans that has the most significant inhibition towards Chrysomia megacephala. The protein was isolated and sequenced two fragments, -VGLDFVLV and TETSFNIDG, highly homologous to that of αAI-1 from Phaseolus vulgaris. The pure protein still inhibits the amylase from Drosophila melanogaster at 85℃, suggesting it is thermal-stable. Its activity was specific and was affected by pH, reaching the peak in weak acidic condition. It obviously inhibits amylases from D. melanogaster, Solenopsis invicta, Odontotermes formosanus, Periplaneta Americana Linnaeus, and Alphitobius sp. In addiction, the study points out Phaseolus vulgaris, Phaseolus coccineus L. and Glycine max, which have significant inhibition toward pancreatic amylase. The amylase inhibitors’ functions of decreasing in blood glucose were detected by animal experiments on Wistar rats. The rats fed with the extracts of Phaseolus coccineus L. and Glycine max showed the inhibition of glucose increasing at the first hour, while blood glucose concentration after rats fed with Phaseolus vulgaris and Glycine max increase at the third hour, and blood glucose after rats fed with Phaseolus coccineus L. maintains low. Its premier result indicates that the extract from Phaseolus coccineus L. might postpones digestion and has potential to be applied. According to these results, amylase inhibitors are worthy to further analysis.
Poly(ADP-ribose)polymerase-1 對細胞內DNA damage修補的調控
Poly(ADP-ribose) polymerase-1 (PARP-1)是一個細胞核內的酵素，它可以被因DNA damage\r 而形成的DNA片段活化，並將NAD(+)上的ADP-ribose轉載到結合蛋白質。這些結合蛋白質對\r 於DNA的合成、DNA的修補、以及細胞週期的調控都有關係。因此，PARP-1被認為是維持基\r 因完整性的重要角色。根據初步的研究，抑制PARP-1的活性對許多疾病的治療都可能有效，\r 其中包括癌症、心臟病、中風、糖尿病、發炎以及反轉錄病毒的感染。然而，以藥物抑制一\r 個對DNA修補這麼重要的酵素會有什麼潛在的問題呢？為了要得到解答，我們需要進一步了\r 解PARP-1在DNA damage反應的機能。在這一份報告中，我製造了一個失去活性的PARP-1突變\r 種，即E988K。經過對E988K詳細的研究，我將比較及分析PARP-1野生型與E988K之間不一樣\r 的互動蛋白質，希望能對PARP-1所控制的DNA修補有更進一步的了解。\r \r \r Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by DNA strand breaks\r during DNA damage response and catalyzes the transfer of ADP-ribose units from the substrate NAD(+)\r to acceptor proteins. These acceptor proteins involve in modulation of chromatin structure, DNA\r synthesis, DNA repair, transcription, and cell cycle control. Thus, PARP-1 is believed to play an\r important role in maintaining genome integrity through modulation of protein-protein and protein-DNA\r interactions. PARP-1 has been the target for design of inhibitors for over twenty-five years. Inhibitors of\r the activity of PARP-1 have been claimed to have applications in the treatment of many disease states,\r including cancer, cardiac infarct, stroke, diabetes, inflammation and retroviral infection. However, are\r there potential problems associated with inhibition of this DNA-repair enzyme? To answer this question,\r we need to further understand the biological function of PARP-1 during DNA damage response. In this\r report, an enzyme dead mutant (E988K) of PARP-1 was generated. Detailed studies of E988K show that\r E988K could be used in the following studies. Compare and identify the different associated proteins of\r PARP-1 wild-type and E988K will shed light into the molecular mechanism of PARP-1-mediated DNA\r repair.
類固醇對斑馬魚胚胎細胞的影響
Steroid hormones are very important for physiological homeostasis, but some functions of steroids are still unclear during embryonic development. Embryonic cell movements are required forming embryonic body. Recently, there is already known Pregeneolone (P5) which could affect epiboly movement of zebrafish embryos is the first product of the steroidogenesis pathway, but effects of further downstream products on epiboly movement are unknown. In order to know this, we treat embryos with Pregnenolone (P5), 17α-Hydroxypregnenolone (17OH-P5), DHEA, Progesterone (P4), 17α-Hydroxyprogesterone (17OH-P4), 11-Deoxycortisol (D), or Testosterone (T). We found out that P5 can accelerate epiboly movement, 17OH-P5 and D have no significant effects on it, and DHEA, P4, 17OH-P4, and T can decelerate it. These results indicated that steroids play important roles on embryonic epiboly movement in zebrafish. 類固醇荷爾蒙對生理平衡很重要，但其對於胚胎發育的影響仍舊不明。胚胎個體的形成需要胚胎細胞進行不同的移動排列。目前研究已知類固醇荷爾蒙生合成機制的第一個產物pregnenolone (P5)對斑馬魚胚胎epiboly 移動有影響，但其它更下游的類固醇荷爾蒙對epiboly移動的影響仍然未知。為了了解類固醇其他下游產物對魚卵早期細胞移動的影響，將胚胎處理Pregnenolone (P5) 、17α-Hydroxypregnenolone (17OH-P5) 、DHEA 、Progesterone (P4) 、17α-Hydroxyprogesterone (17OH-P4)、11-Deoxycortisol (D)、Testosterone (T)，發現17OH-P5、D對epiboly 的移動沒有影響，而DHEA、P4、17OH-P4、T 會使其變慢。這些結果說明了類固醇對胚胎細胞的移動扮演著重要角色。
彩色蠶繭之研究
近幾年來，蠶繭彩色化已引起廣泛的注意，日本與中國大陸紛紛投入此研究領域。我們用食用色素、酸性染料和活體染色劑中性紅等色素水溶液，以沾附於桑葉餵食、浸泡蠶體、注射入蠶體血腔等方法，使白色繭品系的家蠶生產出多種顏色的彩色蠶繭，其中以附於桑葉餵食最有效率，我們就此法找出投與色素的有效期間，可以比日本、中國的方法更節省色素。同法處理黃色品系的家蠶則產生黃色與所加色素的混合色蠶繭。由於這樣得到的彩色蠶繭放久了都會褪色，我們試用奈米色素餵食家蠶所得彩色蠶繭，與一般食用色素所製成的彩色蠶繭比較，發現對各種光照、清潔劑清洗等處理，用奈米色素所得蠶繭明顯較用一般食用色素所得蠶繭不易褪色。這樣用奈米色素生產的彩色蠶繭，因為解決了褪色的問題而更具有潛在的產業價值。To make silkworm cocoons with different colors has received a great attention recently. Japan and China have invested great resources in this field of the study. In order to let white cocoon silkworms produce cocoons of different colors, we used the aqueous solutions of food dyes, acid dyes and neutral red, and fed the worms with mulberry leaves immersed with such aqueous solutions, or directly soaked or injected them with the solutions. We found that using mulberry leaves immersed in the dye solutions was the best approach. We improved this approach by finding a critical, effective time of applying dyes. It saved the dyes and labor than those of Japan and China. We also found that yellow cocoon silkworms produced yellow and mixing colored cocoons by the mulberry leaf feeding method with the same dyes. Because all colors of the cocoons mentioned above faded easily, we furthermore tested nano-dye and found that colors of the cocoons had better resistant to fading away in washing with detergents under various types and intensities of light illumination. This result suggested that nano-dye has a potential in solving the fading problem of the colored cocoons.