類別:

龍眼花、殼、種子、葉與枝椏萃取物之降尿酸研究

Sun, 01 Jan 2012 00:00:00 GMT

標題: 龍眼花、殼、種子、葉與枝椏萃取物之降尿酸研究; The study of the extracts of flower, shell, seed, leaves and twig of Dimocarpus longan Lour. on reducing uric acid level. 作者: Yung-Ann Chen; 陳韻安摘要: 人類在演化過程中失去可將尿酸轉化為尿素排出的尿酸酶，故尿酸合成過多或排泄減少是高尿酸血症主要形成的原因，而因營養過剩所造成的高尿酸血症與痛風更是現代人常見的文明病。主要位於肝臟的黃嘌呤酶是人體內嘌呤代謝過程產生尿酸的關鍵酵素，如果能抑制黃嘌呤酶作用便可以減低尿酸的形成，多餘的嘌呤可經由體內其他酵素回收利用，避免尿酸其在體內累積對關節、腎臟和心血管系統造成傷害。近年來天然物萃物和自然保健食品已蔚為風潮，而植物化學成分中之抗氧化劑更是許多研究重心。龍眼除了是常見的熱帶水果更是中國傳統醫學中常見補神健胃的藥方，目前已有研究發現龍眼萃取物含有多種抗氧化成分，而抗氧化劑也被證實對許多酵素會產生影響。目前臨床常用之抗高尿酸血症藥物多為化學合成物質，有許多如：過敏、皮疹和肝腎傷害等不良副作用。由天然物中萃取能夠抗高尿酸血症又無副作用的植物化學成分是許多研究的目標。本實驗為證實民間偏方龍眼花茶之降尿酸療效，對其萃取物進行體外和體內之生物活性試驗，且為了充分利用生物資源，一併對龍眼種子、果殼、枝枒和樹葉進行研究。實驗先以風乾龍眼﹙花、種子、果殼、枝枒和樹葉﹚甲醇萃取物在295nm波長下進行黃嘌呤酶抑制試驗測試龍眼萃取物抑制尿酸形成作用，發現五種龍眼萃取物都具有程度不等之黃嘌呤酶抑制效果，其中以龍眼花效果最佳，其IC50 為115.76 μg/ml。另外測試龍眼花所含已知十種化合物之黃嘌呤酶抑制率，發現以proanthocyanidin A2效果最佳，其抑制率可達70.67%。之後進行生物體內活性試驗，在分別口服五種龍眼萃取物1小時後，以腹腔注射尿酸酶抑制劑oxonic acid，建立囓齒類動物﹙大鼠和小鼠﹚高尿酸血症模式，2小時後測量小鼠血中尿酸值的變化並和對照組比較，發現在100mg/kg劑量下五種龍眼甲醇萃取物都有降低小鼠血中尿酸值的效果﹙p< 0.01﹚。另外以高尿酸血症模式大鼠進行尿酸促進排除試驗，注射oxonic acid 2.5小時後，每30分鐘抽取膀胱尿液連續三小時。尿液分析結果顯示龍眼萃取物對促進尿酸排泄並無影響。綜合本研究之實驗結果，可以推測龍眼萃取物主要是藉由抑制黃嘌呤酶作用來降低血中尿酸值，並以龍眼花萃取物效果最佳，其作用主要是由具強抗氧化活性的proanthocyanidin A2對黃嘌呤酶產生抑制效果。; Hyperuricemia results from over production or/and underexcretion of uric acid. Uric acid production is catalyzed by xanthine oxidase (XO) in the liver, which is a key enzyme in the oxidizing process of xanthine into uric acid. This study investigated anti-hyperuricemia bioactivity of Dimocarpus longan Lour. Longan pulp is a Chinese medicinal plant used for the treatment of amnesia and its flower is folklorically made into drinking tea to lower serum uric acid level. Therefore, the methanol extracts of the flower, seed, shell, stem and leaf of Longan were tested in vitro and in vivo to confirm its anti-hyperuricemic effect. Firstly, xanthine oxidase suppression rates were evaluated by the xanthine oxidase inhibition assay. The results showed that all Longan extracts reduced blood uric acid level as compared to the control group (p < 0.01). The flower extract exibited the best blood uric acid lowering effect and IC50 of XO inhibition was 115.76 μg/ml. 10 compounds have been previously isolated from Longan flower in other studies; in this study, we also examined them by the xanthine oxidase inhibition assay, revealing that proanthocyanidin A2 had the strongest inhibitory effect (70.67%). In the in vivo part of this study, the methanol extracts from 5 different Longan parts were orally administrated to different groups of mice at 50 mg/kg or 100 mg/kg 1 hour before oxonic acid was injected. The blood uric acid levels in mice were then detected 2 h later. Furthermore, we carried out the urine uric acid excretion test in rats to find out whether the Longan extracts could promote the excretion of uric acid; however no significant effect on the excretion of uric acid was observed. According to these results, the mechanism behind the antihyperuricemic effect of Longan methanol extracts was indicated to be associated with the inhibition of xanthine oxidase which results in decreased production of uric acid. This study not only revealed the existence of antihyperuricemic effect of of Dimocarpus longan, but also explored possible mechanisms. Our results showed that Dimocarpus longan has a great potential to be developed into novel therapeutic agents for the treatment of hyperuricemia.

鼠傷寒沙門氏菌FimZ 蛋白質可調控不同生理功能

Sun, 01 Jan 2017 00:00:00 GMT

標題: 鼠傷寒沙門氏菌FimZ 蛋白質可調控不同生理功能; FimZ of Salmonella enterica serovar Typhimurium may mediate different physiological functions 作者: Chieh-Hsiang Chang; 張傑翔摘要: 鼠傷寒沙門氏菌會產生第一型線毛，第一型線毛的產生對於鼠傷寒沙門氏菌在定殖上扮演重要的角色。在鼠傷寒沙門氏菌中第一型線毛的表現，主要是藉由fim 基因組內的 fimZ、 fimY、 stm0551、fimW和fimU作為調控因子。其中FimZ屬於two-component regulatory system的反應調控蛋白，能夠活化第一型線毛主要次單位fimA基因的表現。在細菌體內，這類的調控蛋白並不罕見，他們除了能夠影響同源基因外，也可能會調控其他基因的表現。因此在本研究中想探討鼠傷寒沙門氏菌的FimZ除了參與第一型線毛的調控之外，對於其他生理功能是否也有影響。過去實驗室已在S. Typhimurium LB5010與ATCC 14028菌株，利用同源基因交換方法建構出fimZ基因剔除突變株。而在轉錄基因體學的分析上得知，S. Typhimurium LB5010 fimZ的剔除，會造成氧化緊迫相關基因cpxP、soxS以及第一型線毛相關基因fimA-fimF與 fimW表現下降；卻使plasmid-encoded fimbriae、菌株的趨化性、鞭毛還有毒力相關基因virK表現上提升。利用紙錠擴散試驗的方法來進行氧化緊迫反應的分析，相較於parental strain，fimZ突變株對於 H2O2 及paraquat 更具敏感性。在互補試驗中，利用陰溝腸桿菌的fimZ基因片段，卻無法在fimZ突變株回復氧化緊迫反應和第一型線毛的表現。LB5010及ATCC 14028的fimZ突變株均顯示低於parental strains的生長能力。ATCC 14028的fimZ突變株接種在soft agar中展現較強的移動能力，而接種LB5010 fimZ突變株沒有觀察到移動增加的情形。利用Caco-2 細胞株進行黏附試驗評估，發現LB5010或ATCC14028個別菌株的parental strain吸附細胞的能力都比其fimZ突變株來得強。而在Caco-2細胞株入侵試驗結果顯示，fimZ基因的缺損與否並無法造成顯著性的影響。我們的研究顯示fimZ基因產物可能會調控第一型線毛的表現及其他的生理功能。fimZ與其他標的基因交互作用的機制值得更進一步探討。; Salmonella enterica serovar Typhimurium produces type 1 fimbriae and such appendages play a significant role in colonization for S. Typhimurium. Expression of type 1 fimbriae in S. Typhimurium is primarily mediated by fimZ, fimY, stm0551, fimW, and fimU within the fim gene cluster. FimZ belongs to the response regulator of the two-component regulatory system in bacteria and has been shown to activate the expression of the fimbrial major subunit gene fimA. It is not uncommon that those regulatory proteins in bacteria regulate other target genes besides their cognate ones. Therefore, this study would like to investigate if FimZ affects other physiological functions other than type 1 fimbrial regulation. A fimZ mutant in S. Typhimurium LB5010 and ATCC 14028 were constructed by allelic exchange previously and were subjected to transcriptomic analysis by microarray. Our results revealed that the stress response related gene cpxP, soxS and type 1 fimbriae related genes fimA-fimF, and fimW were down-regulated, whereas plasmid-encoded fimbriae, chemotaxis, flagella associated genes and virulence genes like virK were up-regulated in the fimZ mutant strain. Oxidative stress response assay was performed with disc diffusion method and indicated that the fimZ mutant was more sensitive to H2O2 and paraquat than its parental strain. However, a homologue of fimZ from Enterobacter cloacae could not complement the fimZ mutant in the oxidative stress response and type 1 fimbrial expression. Both fimZ mutants in LB5010 and ATCC14028 exhibited decreased growth rate than their parental strains. Elevated motility phenotype on the soft agar was observed in ATCC 14028 fimZ mutant, but not in LB5010 fimZ mutant. Adhesion assays was evaluated by infecting Caco-2 cells with bacteria. The LB5010 and ATCC 14028 exhibited better adherent ability than their respective fimZ mutant strains. However, the absence or presence of fimZ gene did not affect the invasion ability of those strains to Caco-2 cells. Our study may indicate that fimZ gene product of S. Typhimurium could regulate type 1 fimbrial expression and other physiological functions as well. The mechanisms pertinent to cross-talk between fimZ and other target genes warrant further study.

鼠傷寒沙門氏菌fimU基因的表現

Tue, 01 Jan 2019 00:00:00 GMT

標題: 鼠傷寒沙門氏菌fimU基因的表現; Expression of fimU in Salmonella enterica serovar Typhimurium 作者: Hsing-Jung Wu; 吳幸蓉摘要: 沙門氏菌是常見的食源性病原菌，也是造成全球食物中毒以及人畜共通感染爆發的重要致病原。沙門氏菌的線毛可以通過黏附蛋白與上皮細胞的寡甘露醣甘鏈之醣蛋白結合，吸附在細胞上。細菌合成線毛的調控複雜且涉及許多基因，鼠傷寒沙門氏菌具有13種不同的線毛基因組，而第一型線毛由fim基因所轉譯，包含了6個結構基因fimA、fimI、fimC、fimD、fimH和fimF以及5個調控基因fimZ、fimY、stm0551、fimW和fimU。其中fimU基因產物tRNAArg (UCU)為可以識別稀有精氨酸密碼子AGA或AGG的tRNA。鼠傷寒沙門氏菌的fimU與大腸桿菌argU具有相似性。在一項研究中顯示AGA / AGG可以停止轉譯並使核醣體增加以至於蛋白質折疊。先前的研究顯示，tRNAArg (UCU) 藉由辨識FimY中的稀有精氨酸密碼子來調控第一型線毛的表現。本研究對fim的基因序列進行分析，顯示出FimI、FimD、FimZ、Stm0551和FimW也具有可被tRNAArg (UCU)識別的精氨酸密碼子。雖然fimU可以藉著辨識稀有密碼子而做轉譯上的調控，但不知fimU受何種因素所調控。由反轉錄聚合酵素鏈鎖反應的分析顯示，當鼠傷寒沙門氏菌在靜置的培養液或固態培養基上生長時，fimU的表達是相同的。同樣的現象也在鼠傷寒沙門氏菌中的fimZ、fimY、fimW、phoQ和crp基因突變菌株上發現。研究也仿照了細菌隨著食物到達腸胃，會經歷了不同的pH值變換、細菌到達人體的溫度差，以及高溫條件，發現fimU表現皆一致。但不同的是在pH值轉變測試下，只有在pH 3.5的酸性環境，能誘導第一型線毛表現。而在溫度轉變測試下，唯獨在高溫條件下，不表現第一型線毛。由以上測試得知，fimU基因可能為鼠傷寒沙門氏菌內的管家基因，任何環境條件都會持續表現。為了觀測fimU基因與第一型線毛的直接關係，在鼠傷寒沙門氏菌中轉形一個帶有fimU的質體，可以加速誘導第一型線毛表現，酵母菌的凝集片段也比野生型的沙門氏菌來的大。目前測試的環境因子以及調控蛋白不足以影響fimU的表現，但細菌體內有額外的fimU存在的確會促進第一型線毛的表現。fimU基因仍有許多特性值得後續研究。; Salmonella is a commonly seen causative agent of food poisoning and food-borne zoonotic infections outbreaks worldwide. Fimbriae of Salmonella can bind to the oligomannosidic glycoproteins on the epithelial cells through the adhesion protein and adhere to the cells. Regulation of fimbrial biosynthesis in bacteria is complicate and involves a number of genes. There are 13 different fimbrial gene clusters in S. Typhimurium. Expression of the type 1 fimbriae in S. Typhimurium is encoded by the fim gene cluster, which is composed of six structural genes fimA, fimI, fimC, fimD, fimH and fimF, and five regulatory genes fimZ, fimY, stm0551, fimW, and fimU. The regulatory gene fimU encodes a tRNAArg (UCU) that recognizes the rarely used arginine codon AGA or AGG. The fimU of S. Typhimurium is related to the Escherichia coli argU. In one study, it was shown that AGA /AGG can stop the translation and increase the ribosome for protein folding. Previous studies had revealed that tRNAArg (UCU) controls type 1 fimbrial expression by recognizing the rarely used arginine codon within FimY. Our sequence analysis revealed that FimI, FimD, FimZ, Stm0551, and FimW also possessed such arginine codons that can be recognized by tRNAArg (UCU). Although fimU can regulate the translation of the protein by recognizing the rarely used arginine codons, it is not known what factors may regulate the expression of fimU. Reverse-transcription polymerase-chain reaction indicated that the expression of fimU was identical when S. Typhimurium was grown in either static broth or on the agar plate. This same fimU expression pattern in both culture conditions was also observed when the fimZ, fimY, fimW, phoQ, and crp mutant strains were tested. Our study also simulated the environmental cues that S. Typhimurium may encounter when passing through the gastrointestinal tract with food. These conditions included the pH changes, temperature shift, and high temperature. The expression of fimU is consistent under such conditions. Nevertheless, The acidic condition pH 3.5 induced the expression of type 1 fimbriae. Under the temperature conversion test, only high temperature inhibited type 1 fimbrial expression. From the above tests, it is possible that fimU gene is a housekeeping gene in S. Typhimurium and fimU is constitutively expressed express under different conditions. Transforming a recombinant plasmid possessing the fimU gene into S. Typhimurium accelerated the production of type 1 fimbriae, yielding larger agglutination fragments than the parental strain did. From the above results, the current environmental factors and regulatory proteins tested did not influence the expression of fimU. it is known that environmental factors cannot affect the expression of fimU. However, the presence of extra fimU plasmid did activate the expression of type 1 fimbriae. The fimU gene does possess some characteristics to be further explored.

點帶石斑之介白素-6、介白素-6受體與醣蛋白130間蛋白質交互作用探討及介白素-6受體異構型選殖

Sat, 01 Jan 2022 00:00:00 GMT

標題: 點帶石斑之介白素-6、介白素-6受體與醣蛋白130間蛋白質交互作用探討及介白素-6受體異構型選殖; Protein-Protein Interactions between Interleukin-6, Interleukin-6 Receptor and Glycoprotein 130 as well as Cloning of IL-6R Isoform of Epinephelus coioides 作者: 張皓鈞; Hao-Chun Chang 摘要: 介白素-6 (IL-6)是一種多功能性細胞激素，作為細胞間負責聯絡功能的蛋白質，影響細胞活化、增殖與分化等反應。在哺乳類IL-6機制研究當中，IL-6與介白素-6受體(IL-6R)及傳訊分子醣蛋白130 (gp130)組成六聚體複合物以調控生化反應，然而IL-6可依據與膜上型或游離型IL-6R結合而分為古典路徑及替代路徑，藉此發揮其眾多功能。魚類IL-6研究方面目前則多偏向分子選殖與功能分析，先前研究證實魚類IL-6具抑制病原感染擴散與促進抗體保護的效果，也確實能誘發魚體免疫反應如抗菌胜肽表現並降低魚隻遭受細菌及病毒感染的死亡率，未來有機會應用於水生生物疾病防治並作為疫苗佐劑或免疫促進劑。本篇研究為探討魚類IL-6作用機制與功能，先透過載體重構、訊號胜肽移除、重新篩選單一菌落、基因序列點突變方式突破先前點帶石斑IL-6R與gp130膜外全長蛋白表現與純化之困境，接著透過原核蛋白表現系統、親和性管柱純化法及磁珠純化法取得點帶石斑IL-6、IL-6R、gp130膜外全長及Hyper IL-6純化蛋白，接著透過遠端西方墨點法、親和性層析法與蛋白拉下實驗來測試蛋白質間交互作用，實驗結果顯示點帶石斑IL-6、IL-6R與gp130膜外全長間具有蛋白質交互作用結合關係，此外也從石斑組織中選殖出IL-6R異構型，並進一步探討其可能形成原因為mRNA替代性剪接，另已製備石斑IL-6R及其異構型血清抗體，可用於未來探討及解釋更多石斑IL-6R異構型之生物功能。本研究為第一篇提出魚類IL-6、IL-6R與gp130蛋白質間交互作用及IL-6R異構型的論文，此份結果可串連先前研究中點帶石斑之IL-6、IL-6R及gp130基因選殖、訊息傳遞及免疫功能分析等研究成果，並作為點帶石斑IL-6作用機制探討、水生生物疾病預防及現場應用的參考基礎，期許未來可應用於石斑水產養殖產業，成功降低細菌及病毒感染所帶來的肆虐，有效提升經濟價值。; Interleukin-6 (IL-6) is a multifunctional cytokine. As a protein responsible for communication between cells, it affects responses such as cell activation, proliferation, and differentiation. In the mechanism study of IL-6 in mammals, IL-6 forms a hexameric complex with its receptor IL-6R and the signaling molecule glycoprotein 130 (gp130) to regulate biochemical reactions. This complex performs many functions by activating different signaling mechanisms and biochemical functions such as classical pathway and alternative pathway. However, few studies have explored the biochemical function of fish IL-6. Previous studies have confirmed that fish IL-6 has the effect of inhibiting the spread of infection and promoting antibody protection. IL-6 also induce immune responses such as antimicrobial peptides and reduce the mortality of fish from bacterial and viral infections. Fish IL-6 research will have the opportunity to be practically applied for disease control and become an adjuvant or immune booster in the future. In this study, targeted protein expression and purification were solved using vector reconstruction, signal peptide removal, re-screening of single colonies, point mutation, and then the targeted protein were expressed through the prokaryotic protein expression system, and then the affinity column was used to obtain the purified proteins of grouper IL-6, IL-6R, gp130 and Hyper IL-6. Then, the interaction between proteins were tested by far western blotting, affinity chromatography and protein pull-down assay. The experimental results show that there are protein interactions between IL-6, IL-6R and gp130 of grouper. In addition, IL-6R isoform was also cloned from grouper tissue, and the possible cause of this formation was further explored as mRNA alternative splicing. Serum antibodies of grouper IL-6R and its isoform have been prepared, which can be used to explore and explain more biological functions of grouper IL-6R isoforms in the future. This is the first thesis to propose the protein-protein interactions between IL-6, IL-6R and gp130 and discovery of IL-6R isoform in fish. This result can be linked to previous research of grouper IL-6, IL-6R and gp130 in our laboratory including gene cloning, signal pathway transduction, immune function analysis and other related research results. The result also be a reference basis for the mechanism study of grouper IL-6, disease prevention and field application. It is expected that it can be applied to the grouper aquaculture industry to reduce the spread of diseases that caused by pathogens infection and effectively enhance economic value in the future.