敗血症一直是各國之間非常重要的死亡原因之一．在美國每年約有75萬人罹患敗血性休克，而其中導致死亡的人數更是高達二十二萬五千人．引發敗血症的原因主要是革蘭氏陰性細菌外膜組成成分之一的內毒素（又稱作脂多醣，LPS），其會在細菌死亡，細胞膜破裂、損壞時，釋放到人體血液或組織中．雖然抗菌肽與細菌細胞膜之間的構效關係(structure-activity relationship)已被研究多年，然而對於抗菌肽與細菌細胞膜上內毒素的作用關係卻很少科學家討論．
在之前我們已經建立一個簡易提升抗鹽性與血清穩定性的方法，藉由抗菌肽的尾端加上非自然性胺基酸－萘基丙胺酸(β-naphthylalanine)作為修飾．在研究中，短鏈富含色胺酸的抗菌肽在高鹽濃度下活性會受到抑制，然而末端以萘基丙胺酸修飾的抗菌肽活性則沒有受到高鹽濃度的影響．萘基丙胺酸末端修飾可以有助提升抗菌肽與細菌或真菌細胞膜作用，而讓抗菌肽更有效破壞細胞膜，並且即使位在高鹽濃度中仍保有抗菌活性．因此，我們希望延續此研究，探討利用萘基丙胺酸尾端修飾的抗菌肽是否也能有效抑制細菌細胞膜上內毒素的活性，這將可以幫助我們設計更符合臨床使用的抗菌肽．

In America, there are about 750,000 cases of septic shock each year. Among these cases, 225,000 of them are fatal. The major cause of sepsis is from endotoxin (also referred as lipopolysaccharide or LPS). Endotoxin constitutes the major component of the outer leaflet of Gram-negative bacteria membrane and is released to blood or tissue upon lysis of bacteria. While structure-activity relationships have been extensively studied with regard to antimicrobial peptide and bacteria membrane interactions, less is known about the anti-endotoxin effects of antimicrobial peptides.
Previously, we have developed an easy strategy to boost salt resistance and serum stability of short antimicrobial peptides by adding the non-nature bulky amino acid β-naphthylalanine to their C-termini. The activities of the short salt-sensitive tryptophan-rich peptide S1 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine end-tagged variants were less affected. β-naphthylalanine end-tagging may help these peptides to penetrate deeper into the bacterial and fungal cell membranes, hence making these peptides more efficient at disrupting the membranes even under high salt conditions. Herein, we have extended this study to characterize the anti-endotoxin effects of β-naphthylalanine end-tagged short antimicrobial peptides. The results will be used to help us design more clinical feasible antimicrobial peptides.

1. Ghadiri, H., Vaez, H., Khosravi, S., and Soleymani, E. (2012) The Antibiotic Resistance Profiles of Bacterial Strains Isolated from Patients with Hospital-Acquired Bloodstream and Urinary Tract Infections. Critical Care Research and Practice 2012, 1-6
2. Silverman, M. H., and Ostro, M. J. (1999) Bacterial Endotoxin in Human Disease.
3. Hoyert, D. L., and Xu, J. (2012) Deaths: Preliminary Data for 2011. National Vital Statistics Reports 61, 1-52
4. GS, M., DM, M., S, E., and M, M. (2003) The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 348, 1546-1554
5. DC, A., WT, L.-Z., J, L., G, C., J, C., and MR, P. (2001) Epidemiology of severe sepsis in the United States: analysis of incidence, outcome and associated costs of care. critical care medicine 29, 1303-1310
6. Quality, A. f. H. R. a. (2008) HCUP facts and figures: Statistics on hospital-based care in the United States, 2008.
7. Brandenburg, K., Schromm, A. B., and Gutsmann, T. (2010) Endotoxins: Relationship Between Structure, Function, and Activity. in Subcell biochem. pp 53-67
8. Wenzel, R. P., Pinsky, M. R., Ulevitch, R. J., and Young, L. (1996) Current Understanding of Sepsis. Clinical Infectious Diseases 22, 407-413
9. Wang, X., and Quinn, P. J. (2010) Endotoxins: Lipopolysaccharides of Gram-Negative Bacteria. in Endotoxins: Structure, Function and Recognition (Subcellular Biochemistry) (Wang, X., and Quinn, P. J. eds.). pp 3-25
10. Mueller, M., Lindner, B., Kusumoto, S., Fukase, K., Schromm, A. B., and Seydel, U. (2004) Aggregates Are the Biologically Active Units of Endotoxin. THE JOURNAL OF BIOLOGICAL CHEMISTRY 279, 26307-26313
11. Wiersinga, W. J. (2011) Current insights in sepsis: from pathogenesis to new treatment targets. Current opinion in critical care 17, 480-486
12. Cross, A. S. (2010) Development of an Anti-Endotoxin Vaccine for Sepsis. in Endotoxins: Structure, Function and Recognition (Subcellular Biochemistry) (Wang, X., and Quinn, P. J. eds.). pp 285-302
13. Rachoin, J.-S., Schorr, C. A., and Dellinger, R. P. (2010) Targeting Endotoxin in the Treatment of Sepsis. in Endotoxins: Structure, Function and Recognition (Subcellular Biochemistry) (Wang, X., and Quinn, P. J. eds.). pp 323-338
14. Yeaman, M. R., and Yount, N. Y. (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacological reviews 55, 27-55
15. Nguyen, L. T., Haney, E. F., and Vogel, H. J. (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends in biotechnology 29, 464-472
16. Santamarı, C., Larios, S., Quiro´s, S., Pizarro-Cerda, J., Gorvel, J.-P., Lomonte, B., and Moreno, E. (2005) Bactericidal and Antiendotoxic Properties of Short Cationic Peptides Derived from a Snake Venom Lys49 Phospholipase A2. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 49, 1340-1345
17. Beamer, L. J., Carroll, S. F., and Eisenberg, D. (1998) The BPI/LBP family of proteins: A structural analysis of conserved regions. Protein Science 7, 906-914
18. Ren, J., Gaob, H., Tang, M., Guc, J., Xia, P., and Xiao, G. (2010) Lipopolysaccharide (LPS) detoxification of analogue peptides derived from limulus anti-LPS factor. Peptides 31, 1853-1859
19. Brandenburg, K., Andrä, J., Garidel, P., and Gutsmann, T. (2011) Peptide-based treatment of sepsis. Applied Microbiology and Biotechnology 90, 799-808
20. ANDRÄ, J., LOHNER, K., BLONDELLE, S. E., JERALA, R., MORIYON, I., KOCH, M. H. J., GARIDEL, P., and BRANDENBURG, K. (2005) Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide. biochemical Journal 385, 135-143
21. Mangoni, M. L., and Shai, Y. (2011) Short native antimicrobial peptides and engineered ultrashort lipopeptides: similarities and differences in cell specificities and modes of action. Cellular and molecular life sciences : CMLS 68, 2267-2280
22. Chu, H. L., Yu, H. Y., Yip, B. S., Chih, Y. H., Liang, C. W., Cheng, H. T., and Cheng, J. W. (2013) Boosting Salt Resistance of Short Antimicrobial Peptides. Antimicrob Agents Chemother
23. Yu, H. Y., Tu, C. H., Yip, B. S., Chen, H. L., Cheng, H. T., Huang, K. C., Lo, H. J., and Cheng, J. W. (2011) Easy strategy to increase salt resistance of antimicrobial peptides. Antimicrob Agents Chemother 55, 4918-4921
24. Malmsten, M., Kasetty, G., Pasupuleti, M., and Jan Alenfall3, A. S. (2011) Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP. plos one 6, 1-13
25. Grimsley, G. R., and Pace, C. N. (2004) Spectrophotometric determination of protein concentration. Current protocols in protein science / editorial board, John E. Coligan ... [et al.] Chapter 3, Unit 3 1
26. Levin, J., and Bang, F. B. (1968) Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin. Thrombosis et diathesis haemorrhagica 19, 186-197
27. Levin, J., and Bang, F. B. (1964) A Description of Cellular Coagulation in the Limulus. Bulletin of the Johns Hopkins Hospital 115, 337-345
28. Levin, J., and Bang, F. B. (1964) The Role of Endotoxin in the Extracellular Coagulation of Limulus Blood. Bulletin of the Johns Hopkins Hospital 115, 265-274
29. Iwanaga, S., Miyata, T., Tokunaga, F., and Muta, T. (1992) Molecular mechanism of hemolymph clotting system in Limulus. Thrombosis research 68, 1-32
30. Tokunaga, F., Miyata, T., Nakamura, T., Morita, T., Kuma, K., Miyata, T., and Iwanaga, S. (1987) Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease. European journal of biochemistry / FEBS 167, 405-416
31. Iwanaga, S., Morita, T., Harada, T., Nakamura, S., Niwa, M., Takada, K., Kimura, T., and Sakakibara, S. (1978) Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostasis 7, 183-188
32. Vivian, J. T., and Callis, P. R. (2001) Mechanisms of tryptophan fluorescence shifts in proteins. Biophys J 80, 2093-2109
33. Eftink, M. R. (1991) Topics in Fluorescence Spectroscopy,
34. Blatt, E., Chatelier, R. C., and Sawyer, W. H. (1986) Effects of quenching mechanism and type of quencher association on stern-volmer plots in compartmentalized systems. Biophys J 50, 349-356
35. Wei, S.-Y., Wu, J.-M., Kuo, Y.-Y., Chen, H.-L., Yip, B.-S., Tzeng, S.-R., and Cheng, J.-W. (2006) Solution Structure of a Novel Tryptophan-Rich Peptide with Bidirectional Antimicrobial Activity. Journal of bacteriology 188, 328-334
36. Domingues, M. M., Santiago, P. S., Castanho, M. A., and Santos, N. C. (2008) What can light scattering spectroscopy do for membrane-active peptide studies? Journal of peptide science : an official publication of the European Peptide Society 14, 394-400
37. Berne, B. J., and Pecora, R. (1990) Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics Melbourne, FL
38. Pierce, M. M., Raman, C. S., and Nall, B. T. (1999) Isothermal titration calorimetry of protein-protein interactions. Methods 19, 213-221
39. Brown, R. K., Brandts, J. M., O'Brien, R., and Peters, W. B. (2009) ITC-derived binding constants: Using microgram quantities of protein,
40. Burrows, S. D., Doyle, M. L., Murphy, K. P., Franklin, S. G., White, J. R., Brooks, I., McNulty, D. E., Scott, M. O., Knutson, J. R., Porter, D., and et al. (1994) Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations. Biochemistry 33, 12741-12745
41. Sisido, M., Egusa, S., and Imanishi, Y. (1983) One-Dimensional Aromatic Crystals in Solution. 2. Synthesis, Conformation, and Spectroscopic Properties of Poly( L-2-naphthylalanine). Journal of the American Chemical Society 105, 4077-4082
42. Kaconis, Y., Kowalski, I., Howe, J., Brauser, A., Richter, W., Razquin-Olazaran, I., Inigo-Pestana, M., Garidel, P., Rossle, M., Martinez de Tejada, G., Gutsmann, T., and Brandenburg, K. (2011) Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides. Biophys J 100, 2652-2661
43. Howe, J., Andrä, J., Conde, R., Iriarte, M., Garidel, P., Koch, M. H. J., Gutsmann, T., Moriyón, I., and Brandenburg, K. (2007) Thermodynamic Analysis of the Lipopolysaccharide-Dependent Resistance of Gram-Negative Bacteria against Polymyxin B. Biophysical Journal 92, 2796-2805
44. Bhunia, A., Domadia, P. N., and Bhattacharjya, S. (2007) Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide. Biochimica et Biophysica Acta 1768, 3282-3291
45. Rosenfeld, Y., Papo, N., and Shai, Y. (2006) Endotoxin (Lipopolysaccharide) Neutralization by Innate Immunity Host-Defense Peptides. THE JOURNAL OF BIOLOGICAL CHEMISTRY 281, 1636-1643
46. Rosenfeld, Y., Sahl, H.-G., and Shai, Y. (2008) Parameters Involved in Antimicrobial and Endotoxin Detoxification Activities of Antimicrobial Peptides. Biochemistry 47, 6468-6478
47. Brandenburg, K., David, A., Howe, J., Koch, M. H., Andra, J., and Garidel, P. (2005) Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide. Biophys J 88, 1845-1858
48. Wieprecht, T., and Seelig, J. (2002) Peptide-Lipid interactions, Academic Press, San Diego
49. Garcia-Verdugo, I., Sanchez-Barbero, F., Soldau, K., Tobias, P. S., and Casals, C. (2005) Interaction of SP-A (surfactant protein A) with bacterial rough lipopolysaccharide (Re-LPS), and effects of SP-A on the binding of Re-LPS to CD14 and LPS-binding protein. The Biochemical journal 391, 115-124
50. Tobias, P. S., Soldau, K., Iovine, N. M., Elsbach, P., and Weiss, J. (1997) Lipopolysaccharide (LPS)-binding proteins BPI and LBP form different types of complexes with LPS. J Biol Chem 272, 18682-18685
51. Garcia-Verdugo, I., Canadas, O., Taneva, S. G., Keough, K. M., and Casals, C. (2007) Surfactant protein A forms extensive lattice-like structures on 1,2-dipalmitoylphosphatidylcholine/rough-lipopolysaccharide-mixed monolayers. Biophys J 93, 3529-3540
52. Ding, J. L., and Ho, B. (2010) Endotoxins: Structure, Function and Recognition (Subcellular Biochemistry), Springer Science+Business Media B.V.
53. 行政院衛生署. (2013) 民國101年死因結果摘要表 http://www2.cdc.gov.tw/mp.asp?mp=1