類別:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75
2024-03-06T02:44:25Z鹽碘與尿碘檢驗方法之調查與應用
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58775
標題: 鹽碘與尿碘檢驗方法之調查與應用; Application of Methods to Survey Iodine Content in Salt and Urine
作者: Pin - Chu Chen; 陳品竹
摘要: 甲狀腺腫曾列臺灣十大最常見疾病,食鹽加碘後成功降低罹病率。然臺灣於
2002 年加入 WTO,於 2004 年廢止鹽政條例,許多廠商由國外進口天然鹽品,
民國 93 - 97 年國人尿碘中位數降至臨界值。缺碘導致甲狀腺腫,更造成胎兒智
能低下,此時評估碘營養狀況指標更顯重要,因此本研究建立鹽碘與尿碘檢驗
法,應用於調查鹽品供碘狀況與癌存者碘營養狀況。
市場調查發現進口鹽傾向天然無添加,國產鹽強調有複合礦物質。83 項鹽
品中僅 7 項加碘,平均碘含量約為 13 ppm,檢測值與標示相符。檢測方法為
iodometric titration,回收率標準為 100 ± 15%、精密度為 < 5%,結果碘酸鉀強
化鹽之 CV 與回收率皆符合;未加碘鹽回收率範圍為 -11–120%;CV 範圍由
0–69%,是因為濃度過低導致;碘化鉀強化鹽 CV 為 4%,回收率 < 79%,表
示此部分檢驗方法建立不佳。結果岩鹽之碘酸鉀皆低於偵測極限;海鹽含約 0.5
ppm 碘。
尿碘檢驗方法是Ammonium persulfate digestion on microplate method
(APDM) 法,選用 Quality control (QC)、精密度與回收率做為判斷標準,QC 進
行 12 天的檢驗後,訂定後續檢驗 QC 落於 mean ± 1 SD 為佳,回收率與精密
度依照濃度區分,< 100 ug / L 者 CV 需 < 10%,回收率為 100 ± 15%,>100
ug / L 者則各標準皆減少 5%。三項標準皆達到,才可作為該受試者尿碘檢驗結
果。收尿採用86 位癌存者尿碘之隨機尿液,部分受試者捐尿時不斷喝水幫助排
尿,因此範圍為 7.5 ~ > 143384 ug / L,中位數為 156.5 ug / L,屬於正常範圍。2013-01-01T00:00:00Z鹽方扁平古菌中兩種被預測為細菌視紫紅質之特性研究
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63964
標題: 鹽方扁平古菌中兩種被預測為細菌視紫紅質之特性研究; Characterization of two predicted bacteriorhodopsins in Haloquadratum walsbyi
作者: Hung-Yi Liu; 劉鴻毅
摘要: 微生物視紫紅質自 1973 年開始,在模式生物嗜鹽古細菌Halobacterium salinarum 發現四種利用光源為能量行使生理功能的視紫紅質,其中分為調節離子濃度的離子幫浦型與感應光趨性的感光受器型,細菌視紫紅質即為前者,其為最早且研究最為透徹的古生菌視紫紅質,利用光源為能量將氫離子送出胞外,造成氫離子濃度梯度,進而帶動三磷酸腺苷生成系統產生能量。
古生菌 Haloquadratum walsbyi 在 2006 年基因體解碼資料中,有三種預測會表達視紫紅質的基因,包括兩種類細菌視紫紅質及一種類感光型視紫紅質。其中視紫紅質基因bop1 和 bop2 皆被預測為類細菌視紫紅質,其轉譯後蛋白被分別命名為HwBR 和 MR( middle rhodopsin )。本研究旨在研究上述兩類細菌視紫紅質間之特性,並探討為何 H. walsbyi 內擁有兩細菌視紫紅質系統。經序列比對與演化樹比對,HwBR 和 MR有很高的相似度,且基因經大腸桿菌表達系統表達純化後,兩者之特徵吸收峰分別為552 奈米和 488奈米。HwBR 經氫離子幫浦測試後,擁有如細菌視紫紅質般的能力,惟 MR 即使經其特徵吸收峰波長 470奈米雷射光源刺激亦無明顯反應。在光週期測試方面,HwBR 光週期如細菌視紫紅質般,約為200毫秒,然而MR光週期達2秒,類似感光型視紫紅質。進一步利用點突變研究,分別對兩基因突變兩處細菌視紫紅質保守且重要之序列,其影響結果HwBR 與細菌視紫紅質相同,MR則非。爰此,整體研究可以清楚推論H. walsbyi與H. marismortui之兩細菌視紫紅質系統相異,前者只包含一種細菌視紫紅質和一種稱為MR之獨特視紫紅質,其被獨立分類於離子型與感光型間,其生理功能尚需研究與探討,而Asp96之保守性也不再是判斷為細菌或感光型視紫紅質之依據。; Studies in microbial rhodopsins have been focused on those found in Halobacterium salinarum since 1973. In the past, the studies in halophilic archaea identified four kinds of retinal-binding proteins and they use light as energy source to mediate different physiological functions. Among them, two main functions are identified: light-driven ion transporters and sensory receptor for phototaxis responses. Bacteriorhodopsin belong to ion-transporter type and is the first and best-understood archaeal rhodopsin which can pump protons out of cell upon light illumination to induce a proton gradient, which further lead to
ATP synthesis via F1Fo ATP synthase system.
Haloquadratum walsbyi genome was completed in 2006 and a total of three retinal-binding proteins were predicted, including two bacteriorhodopsins-like and one halorhodopsin-like chloride pumping photoreceptor. The two opsin genes in H. walsbyi, bop1 and bop2, were those that identified and assumed to encode two bacteriorhodopsin-like proteins, and their protein products are named HwBR and MR, respectively. The goal of this study is to compare the features between HwBR and MR to further understand whether H. walsbyi indeed posses
a two-bacretiorhodopsin system.
The protein sequence alignment and phylogenetic tree analysis showed the high identity of HwBR and MR. The genes HwbopI and HwbopII were cloned and expressed in E. Coli C43(DE3) for biochemical property studies, and the maximum absorbance were 552 nm and 488 nm, respectively. The light-driven proton pumping activity showed that HwBR to have light-driven proton transportation function as that found in bacteriorhodopsin, while MR showed no such activity at all, even when being activated with the 470nm laser beam. The photocycle kinetic measurements showed HwBR to have a 200 msec photocycle time just like bacteriorhodopsin, while it was a 2-second for MR, a time course similar to those found in sensory rhodopsins. Further different mutagenesis studies at Asp85 and Asp96, two conserved and functionally critical residues found in bacteriorhodopsin, found only HwBR showed¬- the same impact in photocycle kinetics as those reported in HsBR but not in MR. The overall results in this study generated three conclusions: i) H. walsbyi does not contain a two-bacteriorhodopsin system as that identified in Haloarcula marismortui ; it has one bacteriorhodopsin-like protein and one unique rhodopsin, MR, ii) MR possesses biochemical properties that can only be classified as one that between ion-type and sensory-type. Further experiments are needed to perform to determine the function of MR. iii) The existence of a corresponding resiude Asp96 as in HsBR no longer clearly separates a bacteriorhodopsin-type
retinal-binding protein from a sensory rhodopsin-type.2012-01-01T00:00:00Z鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞能力之探討
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53960
標題: 鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞能力之探討; Investigation on light-driven ion translocation capability of halorhodopsin from Haloquadratum walsbyi
作者: Xiao-Ru Chen; 陳筱儒
摘要: 嗜鹽古細菌 (haloarchaea) 生存在高鹽的嚴苛環境中,菌體中存在許多蛋白質協助抵抗極端環境,其中一類為微生物視紫蛋白質(Microbial rhodopsin),當受到光刺激後會有不同的功能,主要可分為兩大類,光驅動離子幫浦及光感受器。在光驅動離子幫浦中,其中一種為氯視紫蛋白質 (halorhodopsin, HR),目前已知是一種光驅動氯離子幫浦,可以維持古生菌的細胞滲透壓。當受光刺激後,氯離子會由胞外送至胞內,氫離子被動運輸至細胞內。在本研究中,透過對於氫離子敏感的光電流測試,進一步測量光驅動氫離子釋出的活性。結果發現當 Halobacterium salinarum (Hs) 及 Haloarcula marismortui (Hm) 上的氯視紫蛋白質 (HR) 以 E.coli 表現時,質子的確被動進入膜內。但是若將蛋白質純化後,以蛋白質溶液進行光電流測試,照光前後並不會產生質子梯度。然而,由結果發現在 Haloquadratum walsbyi 中的氯視紫蛋白質 (HwHR) 有別於 HsHR 及 HmHR,無論是以表現於 E.coli 的菌液或是純化後的蛋白質溶液,進行光電流測試,皆有質子梯度的產生。為了找出在 HwHR 中,與細菌視紫蛋白質 (bacteriorhodopsin) 相似的質子幫浦活性所涉及的胺基酸,找出帶負電胺基酸進行點突變後,發現 D254N 質子釋出的活性消失。D254N-HwHR在特徵吸收波長上往短波長移動 15 nm,光週期則比 wild type 慢 6 倍。綜合上述結果, Asp254 對於光驅動質子釋出及視黃醛重新異構化的效率上扮演重要角色。; Microbial rhodopsins response to light and function as light-driven ion transportation or light sensor. Halorhodopsin (HR), one of these microbial rhodopsins, is known to be a light-driven inward chloride pump for osmolarity maintenance at least in haloarchaea. During inward transportation, chloride ions are proposed to passively carry protons across the membrane. In this study, we used a proton sensitive assay, photocurrent measurement, to measure light-driven proton releasing activity. Here we found halorhodopsin in Halobacterium salinarum (HsHR) and Haloarcula marismortui (HmHR), the protons indeed passively transported across the membrane when they were expressed in E. coli cells, but in the purified protein level, no light-driven photocurrent was recorded, indicating no proton gradients were formed inside and outside protein upon illumination. However, halorhodopsin in Haloquadratum walsbyi (HwHR) generated positive in both cell-based and protein photocurrent measurement. In order to find the residue(s) in HwHR mediating such bacteriorhodopsin-like proton activity, we mutated several negative residues and found D254N eliminate such activity in both cell-based and protein experiments. D254N-HwHR underwent a 15-nm blue-shifted in maximum absorbance and the recovery time of photocycle was ~6 time slower when compared to wild type. According to these results, we conclude Asp254 as the critical residue for light-driven proton releasing and the efficiency of retinal reisomerlization.2015-01-01T00:00:00Z高親和性標籤在單分子螢光標記和生物巨分子固定上之應用
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52603
標題: 高親和性標籤在單分子螢光標記和生物巨分子固定上之應用; Application of high affinity tag strategies in single-molecule labeling and immobilization for protein complex
作者: Zi-Yun Chen; 陳姿澐
摘要: 單分子螢光共振能量轉移 (Single Molecule Fluorescence Resonance Energy Transfer, smFRET) 顯微術中,需將生物巨分子均勻分散且穩定地固定於樣本槽的玻璃表面上,便於長時間量測 FRET pair 的螢光強度。將 RNA polymerase II(Pol II) 固定於鋪有攜鈣素 (Calmodulin) 的玻璃界面時,注入至樣品槽的 Pol II 的濃度範圍通常為 10-12 M 。但 Pol II 次單元上的 Calmodulin binding peptide (CBP tag) 與攜鈣素的解離平衡常數 (equilibrium dissociation constant) KD 值約為10-9 M,造成 CBP tagged Pol II 與攜鈣素之偶合效率較低,成像範圍內的 FRET pair 較少。
為了提升 CBP tagged Pol II 與攜鈣素之偶合效率,透過定點突變修改 CBP tag 的 DNA 序列,分別獲得胺基酸序列 N9A 和 S14A 突變的 CBP tag (NCBP tag),期許能得到對 Calmodulin 具較高親和力(解離平衡常數約10-12 M) 的 NCBP tag。
藉由生物分子交互作用分析系統,生物薄膜干涉儀 (Biolayer Interferometry) 與微量熱泳動儀 (Microscale Thermophoresis) 兩種方式,驗證 NCBP tagged Pol II 對攜鈣素的親和力改善之情形,然而因為 Pol II 對 NeutrAvidin 的非專一性吸附及Pol II 複合體本身的複雜性,兩種方法均無法有效的獲得正確的 KD 值範圍。除了上述兩方法,亦使用單分子螢光顯微鏡分別對 Pol II 與攜鈣素成像,藉由單分子影像位置之映對 (mapping) ,做 colocalization 分析以測定 Pol II 與攜鈣素的結合率。 CBP tagged Pol II 對 Calmodulin 的結合率最佳會落在 1 nM 的濃度範圍,結合率可達 41% ,而兩種 NCBP tagged Pol II 與攜鈣素的結合率分別為 25% (N9A) 和 12% (S14A) 。
本文找到合適的CBP tagged Pol II濃度範圍,能提供穩定且有效率的單分子成像條件進行後續的量測。Biocytin 與 Biotin-BSA的添加可有效地減緩 RNA polymerase II 對 NeutrAvidin 的非專一性吸附,得以在相對單純的環境中分析 CBP tagged Pol II 與攜鈣素間的生物分子交互作用。; Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to study complex biological interactions and dynamics with sub-nanometer sensitivity. In smFRET technique, biomolecule must be immobilized sparsely and stably on a substrate for measuring the intensity of time trajectories of donors and acceptors. In this study, RNA polymerase II (Pol II) on which a calmodulin binding peptide tag (CBP tag) fused was immobilized on a glass surface with the aid of biotinylated-calmodulin and NeutrAvidin system. In the chamber, CBP tagged Pol II concentration range is usually 10-12 pM , but the dissociation constant KD between CBP tag and calmodulin-Ca2+ complex is about 10-9 M. Because the concentration of CBP tagged Pol II is smaller than KD value, the number of FRET pair is low and the binding between donors and acceptors becomes unstable.
To improve the affinity between CBP tagged Pol II and calmodulin, we used site-directed mutagenesis to modify CBP tag. The alanine substitution within CBP tag resulted Asn9Ala and Ser14Ala (N9A/S14A) mutants which may improve affinity from nano- to pico-molar range. Both modified CBP tags are called as NCBP tags. The improved affinity can stabilize the binding between donors and acceptors and then the measurements of FRET efficiencies is more efficient.
We used Biolayer Interferometry and Microscale Thermophoresis to quantify the affinity between NCBP tagged Pol II and calmodulin. However, the results showed unreliable KD range due to the nonspecific binding between Pol II and NeutrAvidin and maybe the complexity of Pol II complex. Besides, we setup a total internal reflection fluorescence microscope to directly measure the binding ratio between CPB tagged
Pol II and calmodulin by colocalized two channel images of different color. The highest binding ratio of CBP tagged Pol II is 41% as Pol II is at 1 nM ; however, at the same Pol II concentration, the binding ratio of two kinds of NCBP tagged Pol II, N9A and S14A, are 25% and 12%, respectively.
In summary, we tried to improve the affinity between CBP tagged Pol II and calmodulin with the substitution of NCBP tag to get more stable FRET pair and optimize the efficiency of FRET measurements. Although the affinity improvement of NCBP tag is not obvious, the experimental results suggest an optimized Pol II concentration to obtain the best FRET efficiency. Furthermore, buffer additives like Biocytin and Biotin-BSA can effectively reduce the nonspecific binding between Pol II and NeutrAvidin.2015-01-01T00:00:00Z