類別:

黑黴菌細胞內α-澱粉水解酵素的純化及特性鑑定

Fri, 01 Jan 1988 00:00:00 GMT

標題: 黑黴菌細胞內α-澱粉水解酵素的純化及特性鑑定; The purification and characterization of Intracelluar α-amylase from Aspergillus niger 作者: Pu-Cheng Chen; 陳浦城摘要: 由黑黴菌(Aspergillus niger)自體消化的溶液中可純化出兩種細胞內α-澱粉水解酵素(E.C. 3.2.1.1.)所使用的純化步驟如下: (l)培養黑黴菌。(2)從細胞自體消化液抽取粗酵素。(3) 以DEAE-cellulose初步分離。(4) Sephadex G-75柱體分離。經上述步驟可得到6.5％的回收率。經純化的酵素依其對基質的特異性和分子量的測定，鑑定出有兩個α-澱粉水解酵素，其中一個分子量為六萬五，在pH2時仍具有活性，在此稱之為抗酸性α-澱粉水解酵素。因抗酸性α－澱粉水解酵素純化時無法避免葡萄糖澱粉水解酵素(glucoamylases, E.C. 3.2.1.3)的汙染，所以抗酸性α－澱粉水解酵素的特性只能做到部分純化。另一個α－澱粉水解酵素分子量為五萬三，在pH<4時易失去活性，在此稱之為酸敏感α－澱粉水解酵素，其最佳反應的pH範圍為pH 4.5－5.5，而穩定性則在 pH 5－9。; Two intracellular α-amylases from Aspergillus niger were purifiei. The processes involve: (a) Cellular autolysis. (b) DEAE-cellulose fractionation, (c) Sephadex G-75 filtration and (d) DEAE-cellulose fractionation. α-amylases have molecular weight of 53,000 and 65,000 as measured by poly-acrylamide gel electrophoresis in the present of sodium dodecyl sulfate. The smaller one is a single homogenous polypeptide and acid sensitive. The larger one is acid-stable and contains gluco-amylase. Both α-amylases catalyze hydrolysis of starch into oligosaccharides.

黃花蜜菜抽出物治療前列腺癌功能與機轉之研究

Wed, 01 Jan 2014 00:00:00 GMT

標題: 黃花蜜菜抽出物治療前列腺癌功能與機轉之研究; Study for therapeutic effects and mechanisms of Wedelia chinensis extract on prostate cancer 作者: Chin-Hsien Tsai; 蔡進賢摘要: 對賀爾蒙療法有抗性的癌症與轉移的癌細胞是前列腺癌治療上的一大挑戰，發展前列腺癌動物模式來研究癌症的惡化過程和有效的治療方式是重要且急迫的任務。在這個研究中，我們利用前列腺癌細胞株(22Rv1) 建立原位腫瘤小鼠模式，以初代培養的方式獲得高轉移性的腫瘤細胞。我們的研究發現，隨著前列腺癌症的惡化過程中癌細胞表現的間質蛋白酶(matriptase)會活化而促進癌細胞轉移。因為，matriptase的抑制蛋白，第二型肝細胞生長因子活化抑制因子(HAI-2)，表現量會隨著癌惡化呈下降的趨勢。此外，在草藥治療前列腺癌的研究中，我們經由PSA啟動子活性的測定，鑑定出黃花蜜菜酒精抽出物中主要的活性成分（ Wedelolectone ，Luteolin，Apigenin），研究發現這些活性成分在抑制前列腺癌細胞生長有協同作用。然而，傳統草藥缺乏詳細、正確的有效成分組成的資料，使其在臨床使用上受到限制。因此，我們使用製備級的液相層析儀截取出富含有效成分的區段，並以LC/MS/MS定性定量分析主要的成分。在比較不同批次之後，通過此定義的草藥精華(WCE)，皆含有一定比例的活性成分且能有效的抑制腫瘤生長。在活性成分間對抑制前列腺癌具有協同作用，此外，我們以藥物動力學分析證明了WCE草藥的組成可以增加活性成分的吸收並減緩活性成分的清除，因而增加在體內的作用時間，進一步增加療效。抗癌機制分析上，我們發現WCE有效地抑制雄性激素受體 (androgen receptor)、 HER2/3和AKT信號路徑與回饋機制，因此增強了荷爾蒙療法的效果，防止初期療效反應之後經常發生的抗藥性。儘管HER2/3和AKT的抑制未能引起對賀爾蒙治療有抗性的前列腺癌細胞(PC-3)體外的凋亡，然而在動物實驗中，WCE卻可以抑制癌細胞引起的骨髓細胞(MDSCs)的趨化作用，進而抑制前列腺腫瘤的生長和轉移。因此，WCE可以結合賀爾蒙療法去提升治療成效，也可以調節癌症免疫而抑制前列腺癌細胞(PC-3)的轉移。; Recurrences of hormone-refractory and metastatic prostate cancer (PCa) both highly affect the treatment outcome. Developing the orthotopic PCa animal model to study the cancer progression and effective therapy is the critical mission. Here, we established the orthotopic xenograft mouse model of PCa cell line, 22Rv1, cultured the different stages of tumor cells, and repeated the same procedure to get highly metastatic cells. In this model, we found that HAI-2, a matriptase inhibitor, is decreased during PCa progression, which enhanced the invasive growth and metastasis capacity of PCa cells. This effect was reversed by either ectopic expression of HAI-2 or matriptase knockdown, indicating that derepression of matriptase from HAI-2 contributed to the cancer metastasis. Besides, we used Prostatic Specific Antigen (PSA) promoter activity assay to identify the principal active compounds (wedelolectone, luteolin, and apigenin) in the herbal ethanol extract of Wedelia chinensis and demonstrated the active compounds synergistically inhibited AR-positive PCa cell growth in vitro and in vivo. To promote the potential clinical use as a botanical drug, we developed a standardized preparation of a W. chinensis extract (WCE) by enrichment and quantification of the principal active compounds and analyses of the in vivo biological activities. Pharmacokinetic analyses of active compounds upon oral administration with WCE indicated that presence of copurifiied other compounds in WCE prolonged the systemic exposure to active compounds over a formula of the pure active compounds. In agreement, the resulting anti-tumor efficacy of WCE was higher than the formula of pure active compounds. Furthermore, WCE effectively disrupted the androgen receptor, HER2/3, and AKT signaling network and therefore enhanced therapeutic efficacy of androgen ablation in PCa. While HER2/3 and AKT inhibition by WCE failed to induce apoptosis in hormone-refractory PCa cells, WCE curbed PCa-induced chemotaxis of myeloid cells and the tumor-microenvironment interactions, thus impairing PCa growth and metastasis. In conclusion, the imbalance between HAI-2 and matriptase expression led to matriptase activation, thereby increasing PCa metastasis. In herbal medicine study, we demonstrated that standardized, characterized preparations of WCE improved the outcome of PCa therapy either as an add-on to hormonal therapy for androgen-dependent disease or as a monotherapy for hormone-refractory disease.

麩胱甘肽-精胺質水解酶在大腸桿菌氧化還原調控中所扮演的角色

Thu, 01 Jan 2009 00:00:00 GMT

標題: 麩胱甘肽-精胺質水解酶在大腸桿菌氧化還原調控中所扮演的角色; The role of glutathionylspermidine amidase in redox regulation of Escherichia coli 作者: Tzu-Chieh Chen; 陳姿潔摘要: Glutathionylspermidne (Gsp) 是由glutathione和spermidine以醯胺鍵 (amide bond) 鍵結而形成的化合物。由於Gsp只存在於單細胞原蟲寄生蟲及大腸桿菌等菌體中，故與Gsp代謝相關的酵素被視為抗寄生蟲藥物發展的目標。Glutathionylspermidine synthetase/amidase (GspSA) 是一個雙功能性的酵素，它同時具有glutathionylspermidine synthetase (Gsp synthetase) 和glutathionylspermidine amidase (Gsp amidase) 兩個功能性區塊 (functional domain)，分別具有生成和降解Gsp的能力 (如下圖所示)。雖然大約13年以前就已經發現在大腸桿菌中具有GspSA的存在，但究竟GspSA在大腸桿菌中是扮演什麼角色？Gsp這個分子在大腸桿菌中具有什麼樣的功能？以及GspSA在菌體中是如何調控它兩個相反活性的功能區塊？一直以來都還沒有定論。在先前的研究中發現在過氧化氫 (hydrogen peroxide, H2O2) 的處理下，GspSA會發生選擇性抑制，即Gsp amidase會因為active site中的cysteine-59受氧化而失去活性，但Gsp synthetase的活性不受影響。為了探討GspSA兩個功能區塊的調控方式，和瞭解如何調控Gsp的濃度，本論文建立了一套能靈敏偵測含硫醇 (thiol) 之分子的分析方法。先使用monobromobimane (mBBr) 與thiol分子進行衍生化，產生具有螢光之產物後，再利用HPLC進行對thiol分子的定性與定量分析。利用此方法，我們發現在in vitro或in vivo的實驗中，過氧化氫的處理下都會造成Gsp amidase的選擇性抑制，並且導致Gsp快速而大量的累積，在此同時菌體中GspSA的蛋白質表現量則維持固定。此外我們也發現在營養不足及厭氧的條件下菌體中的Gsp也會有累積的情況發生。最後，我們提出一個模式 (model) 藉以解釋在氧化壓力之下，大腸桿菌如何透過GspSA 調控菌體內的氧化還原平衡；此模式除了指出Gsp amidase的選擇性抑制會造成Gsp的累積，同時也解釋了Gsp disulfide (一種Gsp的氧化形式) 可以藉由Gsp amidase及GSH reductase兩種酵素的協同作用，而得以回復成GSH及spermidine，進而達成生理環境下thiol的平衡。; Glutathionylspermidine (Gsp), the conjugate of glutathione and spermidine, only appears in some parasitical protozoa or bacteria such as Escherichia coli. Therefore, the enzymes involved in Gsp metabolism are considered as the targets for anti-parasitic drugs. Glutathionylspermidine synthetase/amidase (GspSA) is a bifunctional enzyme to catalyze the biosynthesis and hydrolysis of Gsp shown as follows. Although GspSA was identified in Escharichia coli more than a decade ago, several issues still remain ambiguous. For instance, the physiological functions of Gsp and GspSA in E. coli are not clear. There is no clear answer regarding to how the two opposite activities of GspSA (Gsp synthetase and Gsp amidase) communicate. In the previous study, Gsp amidase was found to be selectively inactivated in the presence of hydrogen peroxide (H2O2) owing to temporary oxidation of cysteine-59 in the active site. In contrast, the activity of Gsp synthetase remained intact. In order to understand the regulation of GspSA and the Gsp turnover, a sensitive method was thus developed for monitoring the amount of small thiols by derivatization with mBBr, followed by HPLC analysis. In the in vitro and in vivo study, the Gsp level was found to be accumulative due to selective inactivation of Gsp amidase by H¬2O2, in contrast to the constant expression level of GspSA. In addition, Gsp was also found to increase under starvation or anaerobic conditions. A GspSA-based model was proposed to interpret how E. coli regulates the intracellular redox balance under oxidative stress. In addition to explaining how selective inactivation of Gsp amidase leading to the accumulation of Gsp, the model also demonstrates that Gsp disulfide, the oxidative form of Gsp, could be regenerated to GSH and spermidine by the Gsp amidase and GSH reductase couple.

鹼蛋白水解酵素之純化及基本性質探討

Sat, 01 Jan 1994 00:00:00 GMT

標題: 鹼蛋白水解酵素之純化及基本性質探討; Purification and General Properties of Alkaline Proteases 作者: Shui一Chyr Duh; 杜水池摘要: 蛋白水解酵素在工業上的大量運用，使得其基本性質的研究益形重要；瞭解其勝?一級結構、水溶液及有機溶劑中的穩定性和反應機制等，可使我們更能有效的掌控這些酵素。本論文使用的五種蛋白水解酵素（ alcalase ，三種從枯草桿茵 CCRC 11594 , CCRC 14353 及 CCRC 15413 發酵而得，另一種從絲狀?菌 Conidiobolus coronatus發酵而得），都能從微生物大量分泌取得。將這些酵素經過 TSK CM－650 ( S )陽離子交換層析及 DEAE 5PW 陰離子交換層析純化後，都能得到大於百分之九十的純度。經由實驗結果，五種酵素都屬於絲氨酸型的鹼蛋白水解酵素，活性pH值 9－10 時達到最高點。其在水溶液中的穩定性， PH 6.2 大於pH 8.0 ；而在不同含水量的乙醇溶液中，並未能看出含水量與穩定性的關係，但可看出在乙醇溶液中的穩定性比在水溶液中好（七天仍能保持大於百分之七十的活性）；因此，本論文中所使用的五種鹼蛋白水解酵素，不僅適用於各種工業用途（清潔劑的添加物、食品製造業. . . . . .等），也是研究酵素在有機溶劑中反應的良好題材。本論文另一主題為研究合成 L－malic acid 的方法，由 fumarase 及 aspartase兩種酵素連續催化，可得到百分之八十四的 L－malic acid 及百分之十五點七的 L－aspartic acid ，而反應基質（ substrate ) fumarate則僅剩百分之零點三；利用丙酮萃取，可完全將 L－malic acid 及 L－aspartic acid 分離，得到純度甚高的 L－malic acid 。