博碩士論文 953204035 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:10 、訪客IP:34.239.179.228
姓名 王常旭(Chang-Hsu Wang)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 鹼性胜肽抗生素indolicidin及其類似物之溶血作用機制探討
(Mechanism of hemolytic action of antimicrobial peptide indolicidin and its analogs)
相關論文
★ 老鼠免疫球蛋白IgG2a之位向性固定法—Fc區域的親和性配體設計★ 量子點表面改質與動物細胞標定
★ 以螢光光譜觀測蛋白質吸附於疏水表面後之構型變化與吸附位向★ 利用雙功能吸附基材進行蛋白復性-蛋白吸附狀態對復性的影響
★ 界面聚合之奈米過濾膜的抗氯性研究★ 以螢光光譜探討Indolicidin及其類似物與微脂粒之交互作用
★ 負電性奈米過濾膜之排鹽特性★ 金奈米粒子親水化及與DNA一對一鍵結之探討
★ 以雙重電性表面改質方式製作抗生物吸附之超過濾與奈米過濾膜★ 以表面修飾之材料控制間葉幹細胞貼附及對其往軟骨分化之影響
★ 金奈米粒子與DNA一對一鍵結及其在檢測單一核苷酸變異的應用★ 以三聚氰氯為單體的抗氯型奈米過濾膜
★ 蛋白質特定方向固定化-以α-amylase為例★ Indolicidin及其類似物與微脂粒交互作用之熱力學研究
★ 位向性固定化葡萄糖氧化酶之新方法★ Indolicidin 及其類似物與微脂粒交互作用之焓測 量
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   [檢視]  [下載]
  1. 本電子論文使用權限為同意立即開放。
  2. 已達開放權限電子全文僅授權使用者為學術研究之目的,進行個人非營利性質之檢索、閱讀、列印。
  3. 請遵守中華民國著作權法之相關規定,切勿任意重製、散佈、改作、轉貼、播送,以免觸法。

摘要(中) ndolicidin有廣泛的抗生活性,可以用來對抗多種細菌、黴菌與病毒,但是少許的溶血活性限制了它的應用。本研究主要探討鹼性胜肽抗生素indolicidin (IL)造成溶血的原因,我們將其第8與第9位置的tryptophan(Trp)置換為phenylalanine而成為ILF89,測量IL與ILF89的抗生與溶血活性,並搭配tryptophan的螢光光譜探究IL及ILF89與膜之作用機制。在生物活性檢測上,發現ILF89不僅維持與IL相當的抗菌性,其溶血活性相較於IL有大幅降低,此證實W8與W9與胜肽的溶血性確實相關。而從IL及ILF89於水相的螢光光譜,發現在低濃度時,具有5個Trp的IL,其螢光強度卻低於僅有3個Trp的ILF89,因此推測IL可能會以寡聚體的形式存在而降低自身螢光。另外,我們也進一步探討胜肽與POPC (1-Palmitoyl-2-Oleoyl-sn- Glycero-3-Phosphocholine)或POPC/POPG (1-Palmitoyl-2-Oleoyl-sn- Glycero-3-[Phospho-rac-(1-glycerol)])微脂粒間的作用,發現IL與磷脂膜共存的系統,單位Trp的螢光值大幅提高,推測此為寡聚體的IL,於吸附於膜面後散開所致;而ILF89雖也有類似的表現,但單位Trp產生的螢光升高有限,因此推測ILF89產生寡聚體較小。配合其他文獻的觀察,我們認為W8、W9與胜肽的聚集很有關係,而胜肽的聚集在其溶血活性上扮演著重要的角色。
摘要(英) Indolicidin has broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and virus. However, a limitation to the expression of indolicidin in therapeutic application is its hemolytic activiry. In this study, we focus on the reason of hemolysis of indolicidin. Here, a new indolicidin’s analog ILF89, where the tryptophan at the 8th, and 9th position replaced by phenylalanine, is designed to decrease hemolytic activity. We measure the antimicrobial and hemolytic activity of indolicidin and its analogs, and investigate the mechanism of peptides with the help of fluorescence spectrum. ILF89 remains good antimicrobial activity and substantially decreases the hemolytic activity in biological assay. It demonstrates that W8 and W9 are related to hemolysis of peptides. In the fluorescence spectrum for IL and ILF89 in aqueous phase, the fluorescence intensity of IL with five tryptophans is lower than that of ILF89 with three tryptophans. We conjecture that IL may aggregate in oligomer form at low concentration in aqueous phase and decrease self-fluorescence. In addition, we research the interaction between peptides and POPC or POPG/POPC liposomes. The unit of tryptophan intensity substantially rises in IL and liposomes coexistence system. We infer that IL aggregates disperse after its adsorption to liposomes. ILF89 has similar exhibition but the unit of tryptophan intensity is lower than that of IL. Thus, we think that the oligomers of ILF89 are smaller than that of IL. According to the observation of other literatures, we consider that W8 and W9 are related to peptide aggregation, which plays an important role in hemolytic activity.
關鍵字(中) ★ 溶血活性
★ 螢光光譜
★ 胜肽聚集
關鍵字(英) ★ hemolytic activity
★ peptide aggregation
★ fluorescence spectrum
★ indolicidin
論文目次 中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vi
表目錄 vii
第一章 緒論 1
1-1 研究動機 1
1-2 研究目的 1
第二章 文獻回顧 2
2-1 鹼性抗生胜肽的發展 2
2-1-1 鹼性抗生胜肽的特色 3
2-1-2 鹼性抗生胜肽的作用機制 4
2-2 Indolicidin的生物活性與作用機制 7
2-2-1 Indolicidin概述 7
2-2-2 Indolicidin的抗菌機制 9
2-2-3 Indolicidin的溶血機制 10
2-3 Indolicidin的類似物對於抗菌與溶血的影響 10
2-3-1 電荷(charge)的改變 11
2-3-1-1 CP-11 12
2-3-2 結構(structure)的改變 13
2-3-2-1 ILA (or CP10A) 13
2-3-2-2 cycloCP-11 14
2-3-3 疏水性(hydrophobicity)的改變 15
2-4 ILF低溶血活性的探討 17
2-4-1 ILF89的設計 18
2-4-2 螢光光譜儀 18
2-4-2-1螢光光譜儀量測原理 19
2-4-2-2 色氨酸的螢光光譜 20
2-4-3 圓二色光譜儀 (circular dichroism) 21
2-4-3-1 圓二色光譜儀量測原理 22
2-4-3-2 胜肽的圓二色光譜 24
第三章 實驗藥品、設備與方法 26
3-1 實驗藥品 26
3-2 實驗設備 28
3-3 實驗方法 29
3-3-1 peptide溶液配製 29
3-3-1-1 紫外-可見光光譜測定 29
3-3-1-2 螢光光譜測定 29
3-3-2 溶血活性測定 30
3-3-3 抗菌活性測試 30
3-3-3-1 單一菌落的培養 30
3-3-3-2 菌液預培養 31
3-3-3-3 抗菌活性 32
3-3-4 peptide與磷脂膜之交互作用 32
3-3-4-1 liposome製備 32
3-3-4-2 peptide與磷脂膜之交互作用 34
第四章 結果與討論 35
4-1 IL及ILF89的基本性質 35
4-1-1 UV280吸收值 35
4-1-2 螢光?max強度 36
4-2 peptide的生物活性 38
4-2-1 溶血活性 38
4-2-2 抗菌活性 39
4-3 peptide與磷脂膜的交互作用 42
4-3-1 CD測定 42
第五章 結論與建議 50
5-1 結論 50
5-2 未來規劃 50
參考文獻 51
參考文獻 1. Hsu, C.H., et al., Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA. Nucleic Acids Research, 2005. 33(13): p. 4053-4064.
2. Marsha, S.H. and G. Arenas, Antimicrobial peptides: A natural alternative to chemical antibiotics and a potential for applied biotechnology. Electronic Journal of Biotechnology 2003. 6.
3. Hancock, R.E.W. and D.S. Chapple, Peptide antibiotics. Antimicrobial Agents and Chemotherapy, 1999. 43(6): p. 1317-1323.
4. Wimley, W.C. and S.H. White, Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nature Structural Biology, 1996. 3(10): p. 842-848.
5. Michl, H. and A. Csordas, Isolation and structure of a haemolytic polypeptide from the defensive secretion of European Bombina species. Chemical Monthly, 1970. 101: p. 182-189.
6. Habermann, E., Bee and Wasp Venoms Science, 1972. 177: p. 314-322
7. Boman, H.G., Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature, 1981. 292: p. 246-248.
8. Lehrer, R., Microbicidal Cationic Proteins in Rabbit Alveolar Macrophages: a Potential Host Defense Mechanism. INFECTION AND IMMUNITY, 1980. 30: p. 180-192.
9. Lehrer, R., Defensins:Natural Peptide Antibiotics of Human Neutrophils. J. Clin. Invest., 1985. 76: p. 1427-1435.
10. Matanic, V.C.A. and V. Castilla, Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus. International Journal of Antimicrobial Agents, 2004. 23(4): p. 382-389.
11. Morikawa, N., K. Hagiwaraa, and T. Nakajima, Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa. 1992. 189: p. 184-90.
12. Zasloff, M., Magainins, a class of antimicrobial peptides from Xenopus skin:Isolation, characterization of two active forms, and partial cDNA sequence of a precursor. PNAS, 1987. 84: p. 5449-5453.
13. Selsted, M.E., et al., Indolicidin, a Novel Bactericidal Tridecapeptide Amide from Neutrophils. Journal of Biological Chemistry, 1992. 267(7): p. 4292-4295.
14. Lawyer, C., et al., Antimicrobial activity of a 13 amino acid tryptophan-rich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides. Febs Letters, 1996. 390(1): p. 95-98.
15. CABIAUX, V., et al., Secondary structure and membrane interaction of PR-39,a Pro+Arg-rich antibacterial peptide. Eur. J. Biochem., 1994. 224: p. 1019-1027.
16. Radermacher, S., V. Schoop, and H. Schluesener, Bactenecin, a leukocytic antimicrobial peptide, is cytotoxic to neuronal and glial cells. J Neurosci Res, 1993. 15: p. 657-662.
17. Ding, B., et al., Correlation of the antibacterial activities of cationic peptide antibiotics and cationic steroid antibiotics. Journal of Medicinal Chemistry, 2002. 45(3): p. 663-669.
18. Falla, T.J., D.N. Karunaratne, and R.E.W. Hancock, Mode of action of the antimicrobial peptide indolicidin. Journal of Biological Chemistry, 1996. 271(32): p. 19298-19303.
19. Yang, L., et al., Barrel-stave model or toroidal model? A case study on melittin pores. Biophysical Journal, 2001. 81(3): p. 1475-1485.
20. Biggin, P.C. and M.S.P. Sansom, Interactions of alpha-helices with lipid bilayers: a review of simulation studies. Biophysical Chemistry, 1999. 76(3): p. 161-183.
21. Miteva, M., et al., Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin. Febs Letters, 1999. 462(1-2): p. 155-158.
22. Pokorny, A. and P.F.F. Almeida, Kinetics of dye efflux and lipid flip-flop induced by delta-lysin in phosphatidylcholine vesicles and the mechanism of graded release by amphipathic, alpha-helical peptides. Biochemistry, 2004. 43(27): p. 8846-8857.
23. Chan, D.I., E.J. Prenner, and H.J. Vogel, Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action. Biochimica Et Biophysica Acta-Biomembranes, 2006. 1758(9): p. 1184-1202.
24. Ludtke, S.J., et al., Membrane pores induced by magainin. Biochemistry, 1996. 35(43): p. 13723-13728.
25. Matsuzaki, K., Why and how are peptide^lipid interactions utilized for self-defense?
Magainins and tachyplesins as archetypes. Biochimica Et Biophysica Acta-Biomembranes, 1999. 1462(1-10).
26. Kawano, K., et al., Antimicrobial Peptide, Tachyplesin-I, Isolated from Hemocytes of the Horseshoe-Crab (Tachypleus-Tridentatus) - Nmr Determination of the Beta-Sheet Structure. Journal of Biological Chemistry, 1990. 265(26): p. 15365-15367.
27. Matsuzaki, K., et al., Membrane permeabilization mechanisms of a cyclic antimicrobial peptide, tachyplesin I, and its linear analog. Biochemistry, 1997. 36(32): p. 9799-9806.
28. Rozek, A., C.L. Friedrich, and R.E.W. Hancock, Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles. Biochemistry, 2000. 39(51): p. 15765-15774.
29. Lee, D.G., et al., Fungicidal effect of indolicidin and its interaction with phospholipid membranes. Biochemical and Biophysical Research Communications, 2003. 305(2): p. 305-310.
30. Robinson, W.E., et al., Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils. Journal of Leukocyte Biology, 1998. 63(1): p. 94-100.
31. Schluesener, H.J., et al., Leukocytic Antimicrobial Peptides Kill Autoimmune T-Cells. Journal of Neuroimmunology, 1993. 47(2): p. 199-202.
32. Ahmad, I., et al., Liposomal Entrapment of the Neutrophil-Derived Peptide Indolicidin Endows It with in-Vivo Antifungal Activity. Biochimica Et Biophysica Acta-Biomembranes, 1995. 1237(2): p. 109-114.
33. Subbalakshmi, C., et al., Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin. Febs Letters, 1996. 395(1): p. 48-52.
34. Halevy, R., et al., Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay. Peptides, 2003. 24(11): p. 1753-1761.
35. Subbalakshmi, C. and N. Sitaram, Mechanism of antimicrobial action of indolicidin. Fems Microbiology Letters, 1998. 160(1): p. 91-96.
36. Marchand, C., et al., Covalent binding of the natural antimicrobial peptide indolicidin to DNA abasic sites. Nucleic Acids Research, 2006. 34(18): p. 5157-5165.
37. Staubitz, P., et al., Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin. Journal of Peptide Science, 2001. 7(10): p. 552-564.
38. Yang, S.T., et al., Conformation-dependent antibiotic activity of tritrpticin, a cathelicidin-derived antimicrobial peptide. Biochemical and Biophysical Research Communications, 2002. 296(5): p. 1044-1050.
39. Falla, T.J. and R.E.W. Hancock, Improved activity of a synthetic indolicidin analog. Antimicrobial Agents and Chemotherapy, 1997. 41(4): p. 771-775.
40. Wu, M.H., et al., Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry, 1999. 38(22): p. 7235-7242.
41. Rozek, A., et al., Structure-based design of an indolicidin peptide analogue with increased protease stability. Biochemistry, 2003. 42(48): p. 14130-14138.
42. Friedrich, C.L., et al., Structure and mechanism of action of an indolicidin peptide derivative with improved activity against gram-positive bacteria. Journal of Biological Chemistry, 2001. 276(26): p. 24015-24022.
43. Friedrich, C.L., et al., Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrobial Agents and Chemotherapy, 2000. 44(8): p. 2086-2092.
44. Yew, W.S. and H.E. Khoo, The role of tryptophan residues in the hemolytic activity of stonustoxin, a lethal factor from stonefish (Synanceja horrida) venom. Biochimie, 2000. 82(3): p. 251-257.
45. Subbalakshmi, C., et al., Antibacterial and hemolytic activities of single tryptophan analogs of indolicidin. Biochemical and Biophysical Research Communications, 2000. 274(3): p. 714-716.
46. Ladokhin, A.S., M.E. Selsted, and S.H. White, Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids. Biophysical Journal, 1997. 72(2): p. 794-805.
47. Zhao, H.X., et al., Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin. Biophysical Journal, 2001. 81(5): p. 2979-2991.
48. Schibli, D.J., et al., Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions. Biochemistry and Cell Biology-Biochimie Et Biologie Cellulaire, 2002. 80(5): p. 667-677.
49. Tsai, C.W., et al. Molecular dynamic simulation of the penetration of indolicidin into lipid bilayer. in 6th Asia-Europe Biorecognition Engineering Society Meeting. 2007. Jong-Li, Taiwan.
50. Skoog, D.A., et al., Principles of instrumental analysis. 1997: Brooks/Cole.
51. Wetlaufer, D.B., Ultraviolet spectra of proteins and amino acids. Advances in Protein Chemistry 1962. 17: p. 303–390.
52. Teale, F.W.J. and G. Weber, Ultraviolet Fluorescence of the Aromatic Amino Acids. Biochem. J., 1957. 65: p. 476-482.
53. Petsko, G.A. and D. Ringe, Protein Structure and Function. January 2004: Sinauer Associates. 180.
54. ChooSmith, L.P. and W.K. Surewicz, The interaction between Alzheimer amyloid beta(1-40) peptide and ganglioside G(M1)-containing membranes. Febs Letters, 1997. 402(2-3): p. 95-98.
55. Ladokhin, A.S., M.E. Selsted, and S.H. White, CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix. Biochemistry, 1999. 38(38): p. 12313-12319.
56. Andrushchenko, V.V., H.J. Vogel, and E.J. Prenner, Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy. Biochimica Et Biophysica Acta-Biomembranes, 2006. 1758(10): p. 1596-1608.
57. Yan, H.S., et al., Individual substitution analogs of Mel(12-26), melittin's C-terminal 15-residue peptide: their antimicrobial and hemolytic actions. Febs Letters, 2003. 554(1-2): p. 100-104.
58. Sun, X.J., et al., Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides, 2005. 26(3): p. 369-375.
59. Dykes, G.A., S. Aimoto, and J.W. Hastings, Modification of a synthetic antimicrobial peptide (ESF1) for improved inhibitory activity. Biochemical and Biophysical Research Communications, 1998. 248(2): p. 268-272.
60. Gazit, E., et al., Mode of Action of the Antibacterial Cecropin B2 - a Spectrofluorometric Study. Biochemistry, 1994. 33(35): p. 10681-10692.
61. Jureti, D., et al., Magainin 2 amide and analogues Antimicrobial activity, membrane depolarization and susceptibility to proteolysis FEBS Letters, 1989. 249: p. 219–23.
62. Groisman, E.A., et al., Resistance to Host Antimicrobial Peptides Is Necessary for Salmonella Virulence. Proceedings of the National Academy of Sciences of the United States of America, 1992. 89(24): p. 11939-11943.
63. Stumpe, S., et al., Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli. Journal of Bacteriology, 1998. 180(15): p. 4002-4006.
64. Ulvatne, H., et al., Proteases in Escherichia coli and Staphylococcus aureus confer reduced susceptibility to lactoferricin B. Journal of Antimicrobial Chemotherapy, 2002. 50(4): p. 461-467.
65. Li, Q.S., et al., A tridecapeptide possesses both antimicrobial and protease-inhibitory activities. Peptides, 2002. 23(1): p. 1-6.
66. McInturff, J.E., et al., Granulysin-derived peptides demonstrate antimicrobial and anti-inflammatory effects against Propionibacterium acnes. Journal of Investigative Dermatology, 2005. 125(2): p. 256-263.
67. Perczel, A., et al., Convex Constraint Analysis - a Natural Deconvolution of Circular-Dichroism Curves of Proteins. Protein Engineering, 1991. 4(6): p. 669-679.
68. Andrade, M.A., et al., Evaluation of Secondary Structure of Proteins from Uv Circular-Dichroism Spectra Using an Unsupervised Learning Neural-Network. Protein Engineering, 1993. 6(4): p. 383-390.
指導教授 阮若屈(Ruoh-Chyu Ruaan) 審核日期 2008-7-16
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明