特定细菌胁迫下东北林蛙（Rana dybowskii）皮肤抗菌肽分泌的动态变化
|
摘要
两栖类作为由水生过渡到陆生的物种,其生境中微生物区系十分复杂,在长期进化过程中两栖类皮肤逐步建立了通过皮肤分泌抗菌肽等物质以有效抵御环境中病原微生物侵袭的天然免疫防御体系。但近年来发现两栖类种群数量呈全球性急剧减少或消失趋势,认为导致这一现象的重要原因之一是两栖类感染性疾病。然而目前对于两栖类皮肤抗菌肽与环境微生物之间的关系尚不清楚。
本研究以东北地区两栖类代表物种东北林蛙(Rana dybowskii)为试验对象,实验分6～72h、1～23d两个观察时段。6～72h时段,设4组：E.coli菌浴组、E.coli淋巴囊注射组、S.aureus菌浴组、S.aureus淋巴囊注射组。1～23d时段,采用温和电刺激法排空腺体内的分泌物后,设E.coli菌浴组和LB培养基组。通过对不同细菌胁迫下的不同时段东北林蛙皮肤分泌物的蛋白多肽浓度测定、RP-HPLC多肽图谱分析、抑菌活性和溶血活性分析等,观察其皮肤分泌物的特征和抗菌活性变化,以期揭示两栖类动物抵御环境微生物时皮肤分泌抗菌肽的动态变化规律。
结果显示,胁迫前东北林蛙皮肤分泌物(正常对照组)RP-HPLC分离获得26个洗脱峰,显示出较强的广谱抗菌活性,尤以对G+细菌活性抑制更显著,低溶血活性。
胁迫6～72h后,RP-HPLC多肽图谱显示,洗脱峰数量无明显变化,但峰C～K呈特异性变化,除峰C、D和E呈现波动变化外,峰F、G和H在胁迫后峰面积值均明显上升,峰I和K在胁迫后峰面积值均明显下降。东北林蛙皮肤分泌物蛋白浓度和抑菌活性均呈时间依赖性增强趋势。
胁迫1-23d后,RP-HPLC多肽图谱显示,1～18d皮肤分泌物洗脱峰数量明显减少至4-10个,至23d基本恢复至胁迫前水平,其中A、B、C和D这4个洗脱峰持续出现,并具有较强的抑制G-和G+菌活性。蛋白多肽浓度第1天显著下降,1～7dE.coli菌浴组显著高于LB组,而13～18d则相反。东北林蛙分泌物排空后至少需要23天才可恢复到胁迫前水平,在此期间分泌物仍具有抗菌活性。
本研究结果表明,东北林蛙蛙皮肤分泌物中抗菌肽作为皮肤免疫防御体系的重要组成,对环境中不同微生物胁迫刺激可提高其抗菌肽抗菌活性,并表现出非特异性应答反应,但对致病性较强的S.aureus更为敏感。该研究结果为进一步揭示东北林蛙皮肤分泌物的作用方式及变化规律提供了重要的理论和实验依据。
Amphibians are the transitional species from the aquatic to the terrestrial. Their skin has gradually established a defensive natural immune defense system in the long-term evolution, to remove pathogenic microorganisms in the environment. But it has been found that amphibian populations were drastically reduced or disappeared global trend in recent years, and one of the important reasons of this phenomenon is infectious disease of amphibians. However, the relationship between environmental microorganisms and amphibian skin antimicrobial peptides is not clear.
To reveal the changes of amphibian skin secretions under microbial challenges, the brown frog (Rana dybowskii) was selected as the test object, and the test subjects were divided into 6～72h,1～23d two observation periods.6～72h period, with 4 groups:E.coli bath group, E.coli sac injection group, S.aureus bacteria bath group and S.aureus sac injection group.1～23d period, after using a mild electrical stimulation emptying the secretion of skin gland, divided into E.coli bath group and LB medium group. Results showed by RP-HPLC analysis, protein concentration and antibacterial activity comparison. Observe the features and antimicrobial activity of skin secretions to reveal dynamics of secretion of amphibian skin against environmental microorganisms.
The RP-HPLC results showed that normal skin secretions of R. dybowskii (control group) has isolated 26 peaks, indicating a strong broad-spectrum antimicrobial activity, particularly against G+ bacteria, and had low hemolytic activity.
After challenged 6～72h, RP-HPLC peptide profiles showed no significant changes in the number of elution peaks, but peak C-K showed a specific change, in addition to peaks C, D and E showed fluctuation, the peak area of F, G and H significantly increased, peak I and K were significantly decreased. The antibacterial activity and protein concentration of skin secretions of R. dybowskii showed a time-dependent increasing trend.
After challenged 1～23d, RP-HPLC peptide profiles displayed that 1～18d skin secretions significantly reduced and the number of elution peaks down to 4-10, restored to the level before challenged to 23d. And peaks A, B, C and D were persistent and had strong inhibition of G- and G+ bacteria. Peptide concentrations decreased significantly on day 1, and 1～7d E.coli bath groups were significantly higher than the LB bath groups, but 13-18d were opposite. Restoring the secretions of R. dybowskii to the level of unchallenged needed at least 23 days, as secretion still had antibacterial activity.
This study indicated that antimicrobial peptides of skin secretions of R. dybowskii as the major components of skin immune defense system can induce non-specific immunity when facing different environmental stress, especially more sensitive to S.aureus. The results provide important theoretical and experimental basis for mode of skin secretions of R. dybowskii changes in further.
本研究以东北地区两栖类代表物种东北林蛙(Rana dybowskii)为试验对象,实验分6～72h、1～23d两个观察时段。6～72h时段,设4组：E.coli菌浴组、E.coli淋巴囊注射组、S.aureus菌浴组、S.aureus淋巴囊注射组。1～23d时段,采用温和电刺激法排空腺体内的分泌物后,设E.coli菌浴组和LB培养基组。通过对不同细菌胁迫下的不同时段东北林蛙皮肤分泌物的蛋白多肽浓度测定、RP-HPLC多肽图谱分析、抑菌活性和溶血活性分析等,观察其皮肤分泌物的特征和抗菌活性变化,以期揭示两栖类动物抵御环境微生物时皮肤分泌抗菌肽的动态变化规律。
结果显示,胁迫前东北林蛙皮肤分泌物(正常对照组)RP-HPLC分离获得26个洗脱峰,显示出较强的广谱抗菌活性,尤以对G+细菌活性抑制更显著,低溶血活性。
胁迫6～72h后,RP-HPLC多肽图谱显示,洗脱峰数量无明显变化,但峰C～K呈特异性变化,除峰C、D和E呈现波动变化外,峰F、G和H在胁迫后峰面积值均明显上升,峰I和K在胁迫后峰面积值均明显下降。东北林蛙皮肤分泌物蛋白浓度和抑菌活性均呈时间依赖性增强趋势。
胁迫1-23d后,RP-HPLC多肽图谱显示,1～18d皮肤分泌物洗脱峰数量明显减少至4-10个,至23d基本恢复至胁迫前水平,其中A、B、C和D这4个洗脱峰持续出现,并具有较强的抑制G-和G+菌活性。蛋白多肽浓度第1天显著下降,1～7dE.coli菌浴组显著高于LB组,而13～18d则相反。东北林蛙分泌物排空后至少需要23天才可恢复到胁迫前水平,在此期间分泌物仍具有抗菌活性。
本研究结果表明,东北林蛙蛙皮肤分泌物中抗菌肽作为皮肤免疫防御体系的重要组成,对环境中不同微生物胁迫刺激可提高其抗菌肽抗菌活性,并表现出非特异性应答反应,但对致病性较强的S.aureus更为敏感。该研究结果为进一步揭示东北林蛙皮肤分泌物的作用方式及变化规律提供了重要的理论和实验依据。
Amphibians are the transitional species from the aquatic to the terrestrial. Their skin has gradually established a defensive natural immune defense system in the long-term evolution, to remove pathogenic microorganisms in the environment. But it has been found that amphibian populations were drastically reduced or disappeared global trend in recent years, and one of the important reasons of this phenomenon is infectious disease of amphibians. However, the relationship between environmental microorganisms and amphibian skin antimicrobial peptides is not clear.
To reveal the changes of amphibian skin secretions under microbial challenges, the brown frog (Rana dybowskii) was selected as the test object, and the test subjects were divided into 6～72h,1～23d two observation periods.6～72h period, with 4 groups:E.coli bath group, E.coli sac injection group, S.aureus bacteria bath group and S.aureus sac injection group.1～23d period, after using a mild electrical stimulation emptying the secretion of skin gland, divided into E.coli bath group and LB medium group. Results showed by RP-HPLC analysis, protein concentration and antibacterial activity comparison. Observe the features and antimicrobial activity of skin secretions to reveal dynamics of secretion of amphibian skin against environmental microorganisms.
The RP-HPLC results showed that normal skin secretions of R. dybowskii (control group) has isolated 26 peaks, indicating a strong broad-spectrum antimicrobial activity, particularly against G+ bacteria, and had low hemolytic activity.
After challenged 6～72h, RP-HPLC peptide profiles showed no significant changes in the number of elution peaks, but peak C-K showed a specific change, in addition to peaks C, D and E showed fluctuation, the peak area of F, G and H significantly increased, peak I and K were significantly decreased. The antibacterial activity and protein concentration of skin secretions of R. dybowskii showed a time-dependent increasing trend.
After challenged 1～23d, RP-HPLC peptide profiles displayed that 1～18d skin secretions significantly reduced and the number of elution peaks down to 4-10, restored to the level before challenged to 23d. And peaks A, B, C and D were persistent and had strong inhibition of G- and G+ bacteria. Peptide concentrations decreased significantly on day 1, and 1～7d E.coli bath groups were significantly higher than the LB bath groups, but 13-18d were opposite. Restoring the secretions of R. dybowskii to the level of unchallenged needed at least 23 days, as secretion still had antibacterial activity.
This study indicated that antimicrobial peptides of skin secretions of R. dybowskii as the major components of skin immune defense system can induce non-specific immunity when facing different environmental stress, especially more sensitive to S.aureus. The results provide important theoretical and experimental basis for mode of skin secretions of R. dybowskii changes in further.
引文
[1]GH Boman, I Nisson, B Rasmuson. Inducible Antibacterial Defense System in Droophial. Nature.1972,237 (5):232～235
[2]GL Dockray, CR Hopkins. Caerulein Secretion by Dermal Glands in Xenopus laevis. J Cell Biol.1975,64:724～733
[3]D Hultmark, H Steiner, GH Boman. Insect Immunity Purification and Properties of Three Inducible Bactericidal Proteins from Hemolymph of Immunized Pupae of Hyalophora cecropia. Eur Biochem.1980,106:7～16
[4]S Seebah, A Suresh, S Zhuo, et al. Defensins Knowledge Base:A Manually Curated Database and Information Source Focused on the Defensins Family of Antimicrobial Peptides. Nucleic Acids Res.2007,35:D262～D268
[5]W Kamysz. Are Antimicrobial Peptides an Alternative for Conventional Antibiotics? Nucl Med Rev Cent East Eur.2005,8(1):78～86
[6]Z Wang, G Wang. APD:the Antimicrobial Peptide Database. Nucleic Acids Res.2004, 32:D590～D592
[7]BT Clarke. The Natural History of Amphibian Skin Secretions, their Normal Function and Potential Medical Applications. Biol Rev.1997,72(3):365～379
[8]K Tsuruda, O Arakawa, K Kawatsu, et al. Secretary Glands of Tetrodotoxin in the Skin of the Japanese Newt Cynops Pyrrhogadter. Toxicon.2002,40(2):131～136
[9]H Greven, K Zanger, G Schwinger. Mechanical Properties of the Skin of Xenopus laevis (Anura, Amphibia). J Morphol.1995,224:15～22
[10]C Lacombe, C Cifuentes-Diaz, I Dunia, et al. Peptide Secretion in the Cutaneous Glands of South American Tree Frog Phyllomedusa bicolor: an Ultrastructural Study. European Journal of Cell Biology.2000,79:631～641
[11]AE Levashina, S Ohresser, B Lemaitre, et al. Two Distinct Pathways can Control Expression of the Gene Encoding the Drosophila Antimicrobial Peptide Metchnikowin. J Mol Biol.1998,278:515～527
[12]M Zasloff. Antimicrobial Peptides of Multicellular Organisms. Nature.2002,415:389～395
[13]R Miele, G Bjorklund, D Barra, et al. Involvement of Rel Factors in the Expression of Antimicrobial Peptide Genes in Amphibia. Eur J Biochem.2001,268:443～449
[14]O Froy. Regulation of Mammalian Defensin Expression by Toll-like receptor Dependent and Independent Signaling Pathways. Cell Microbiol.2005,7(10):1387～1397
[15]M Zasloff. Fighting Infections with Vitamin D. Nat Med.2006,12(4):388～390
[16]宋珊珊,胡国斌,董先智.抗菌肽基因异源表达的研究进展.生物医学工程学杂志. 2009,26(6):1372～1375
[17]UniProtKB/SWISS-PROT at EBI. http://www.expasy.org/sprot/relnotes/relstat.html
[18]岳昌武,莫宁萍,刘坤祥等.抗菌肽的结构特点作用机理及应用前景.安徽农业科学.2008,36(5)：1736～1739
[19]SY Choi, YM Choo, KS Lee, et al. Cloning and Expression Profiling of Four Antibacterial Peptide Genes from the Bumblebee Bombus ignites. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology.2008,150(2):141～146
[20]RI Lehrer, T Ganz. Defensins of Vertebrate Animals. Curr Opin Immunol.2002, 14:96～102
[21]KVR Reddy, RD Yedery, C Aranha. Antimicrobial peptides:Premises and Promises. Int J Antimicrob Agents.2004,24 (6):536～573
[22]CB Park, KS Yi, K Matsuzaki, et al. Structure-Activity Analysis of Buforin Ⅱ, a Histone H2 Aderived Antimicrobial Peptide:The Proline Hinge is Responsible for the cell-Penetrating Aability of Buforin Ⅱ. Proc Natl Acad Sci.2000,97:8245～8250
[23]CV Kalfa. Congeners of SMAP29 kill Ovine Pathogens and Induce Ultrastructural Damage in Bacterial Cells. Antimicrob Agents Chemother.2001,45:3256～3261
[24]SA Ladokhin, EM Selsted, HS White. Sizing Membrane Pores in Lipid Vesicles by Leakage of Coencapsulated Markers:Pore Formation by Melittin Biophys J.1997, 72:1762～1766
[25]K Matsuzaki, S Yoneyama, K Miyajima. Pore Formation and Translocation of Melittin. Biophys J.1997,73:831～838
[26]HJ Kang, YS Shin, YS Jang, et al. Release of Aqueous Contents from Phospholipid Vesicles Induced by Cecropin A (1-8) Magainin 2 (1-12) Hybrid and its Analogues. J Peptide Res.1998,52:45～50
[27]K Hristova, EM Selsted, HS White. Critical Role of Lipid Composition in Membrane Permeabilization by Rabbit Neutrophil Defensins. J Biol Chem.1997,272:24224～24233
[28]H Zhao, PJ Mattila, MJ Holopainen, et al. Comparison of the Membrane Association of Two Antimicrobial Peptides, Magainin 2 and Indolicidin. Biophys J.2001,81:2979～2991
[29]B Christensen, J Fink, BR Merrifield, et al. Channel-Forming Properties of Cecropins and Related Model Compounds Incorporated into Planar Lipid Membranes. Proc Natl Acad Sci USA.1988,85:5072～5076
[30]TM Lee, YF Chen, WH Huang. Energetics of Pore Formation Induced by Membrane Active Peptides. Biochemistry.2004,43:3590～3599
[31]Y Wu, WH Huang, AG Olah. Method of Oriented Circular Dichroism. Biophys J.1990, 57:797～806
[32]B Bechinger. The Structure, Dynamics and Orientation of Antimicrobial Peptides in Membranes by Multidimensional Solid-State NMR Spectroscopy. Biochim Biophys Acta. 1999,1462:157～183
[33]S Yamaguchi, T Hong, A Waring, et al. Solid-State NMR Investigations of Peptide-lipid Interaction and Orientation of a β-sheet Antimicrobial Peptide, Protegrin. Biochemistry. 2002,41:9852～9862
[34]B Bechinger, M Zasloff, SJ Opella. Structure and Orientation of the Antibiotic Peptide Magainin in Membranes by Solid-State Nuclear Magnetic Resonance Spectroscopy. Protein Sci.1993,2:2077～2084
[35]S Yamaguchi, et al. Orientation and Dynamics of an Antimicrobial Peptide in the Lipid Bilayer by Solid-State NMR Spectroscopy. Biophys J.2001,81:2203～2214
[36]A Spaar, C Munster, T Salditt. Conformation of Peptides in Lipid Membranes Studied by X-ray Grazing Incidence Scattering. Biophys J.2004,87:396～407
[37]K He, SJ Ludtke, HW Huang, et al. Antimicrobial Peptide Pores in Membranes Detected by Neutron in-plane Scattering. Biochemistry.1995,34:15614～15618
[38]SJ Ludtke, K He, WT Heller, et al. Membrane Pores Induced by Magainin. Biochemistry. 1996,35:13723～13728
[39]L Yang, TA Harroun, WT Heller, et al. Neutron Off-plane Scattering of Aligned Membranes. I. Method of Measurement. Biophys J.1998,75:641～645
[40]L Yang, TM Weiss, TA Harroun, et al. Supramolecular Structures of Peptide Assemblies in Membranes by Neutron Off-plane Scattering:Method of Analysis. Biophys J.1999, 77:2648～2656
[41]H Zhao, JP Mattila, JM Holopainen, et al. Comparison of the Membrane Association of Two Antimicrobial Peptides, Magainin 2 and Indolicidin. Biophys J.2001,81:2979～2991
[42]L Silvestro, K Gupta, JN Weiser, et al. The Concentration-dependent Membrane Activity of Cecropin A. Biochemistry.1997,36:11452～11460
[43]A Shizuo, U Satoshi, T Osamu. Pathogen Recognition and Innate Immunity. Cell.2006, 124:783～801
[44]KA Brogden. Antimicrobial Peptides:Pore Formers or Metabolic Inhibitors in Bacteria? Nature Reviews.2005,3:238～250
[45]HW Huang. Action of Antimicrobial Peptides:Two-state Model. Biochemistry.2000, 39:8347～8352
[46]FY Chen, MT Lee, HW Huang. Evidence for Membrane Thinning Effect as the Mechanism for Peptide Induced Pore Formation. Biophys J.2003,84:3751～3758
[47]L Yang, TA Harroun, TM Weiss, et al. Barrel-stave Model or Toroidal Model? A Case Study on Melittin Pores. Biophys J.2001,81:1475～1485
[48]G Ehrenstein, H Lecar. Electrically Gated Ionic Channels in Lipid Bilayers. Rev Biophys. 1977,10:1～34
[49]Y Pouny, D Rapaport, A Mor, et al. Interaction of Antimicrobial Dermaseptin and its Fluorescently Labeled Analogues with Phospholipid Membranes. Biochemistry.1992, 31:12416～12423
[50]A Spaar, C Munster, T Salditt. Conformation of Peptides in Lipid Membranes Studied by X-ray Grazing Incidence Scattering. Biophys J.2004,87:396～407
[51]Y Shai. Mechanism of the Binding, Insertion and Destabilization of Phospholipid Bilayer Membranes by Alpha-helical Antimicrobial and Cell Non-selective Membrane-lytic peptides. Biochim Biophys Acta.1999,1462:55～70
[52]AS Ladokhin, SH White.'Detergent-like' Permeabilization of Anionic Lipid Vesicles by Melittin. Biochim Biophys Acta.2001,1514:253～260
[53]K Matsuzaki, O Murase, N Fujii, et al. An Antimicrobial Peptide, Magainin 2, Induced Rapid Flip-flop of Phospholipids Coupled with Pore Formation and Peptide Translocation. Biochemistry.1996,35:11361～11368
[54]KJ Hallock, DK Lee, A Ramamoorthy. MSI-78, an Analogue of the Magainin Antimicrobial Peptides, Disrupts Lipid Bilayer Structure via Positive Curvature Strain. Biophys J.2003,84:3052～3060
[55]K Matsuzaki, K Sugishita, N Ishibe, et al. Relationship of Membrane Curvature to the Formation of Pores by Magainin 2. Biochemistry.1998,37(34):11856～11863
[56]K Matsuzaki, K Sugishita, M Harada, et al. Interactions of an Antimicrobial Peptide, Magainin 2, with Outer and Inner Membranes of Gram-negative Bacteria. Biochim Biophys Acta.1997,1327:119～130
[57]S Yamaguchi, T Hong, A Waring, et al. Solid-state NMR Investigations of Peptide-lipid Interaction and Orientation of a β-sheet Antimicrobial Peptide, Protegrin. Biochemistry. 2002,41:9852～9862
[58]R Gennaro, M Zanetti. Structural Features and Biological Activities of the Cathelicidin-derived Antimicrobial Peptides. Biopolymers.2000,55:31～49
[59]J Shi, CR Ross, MM Chengappa, et al. Antibacterial Activity of a Synthetic Peptide (PR-26) Derived from PR-39, a Proline-arginine-rich Neutrophil Antimicrobial Peptide. Antimicrob Agents Chemother.1996,40(1):115～121
[60]C Subbalakshmi, N Sitaram. Mechanism of Antimicrobial Action of Indolicidin. FEMS Microbiol Lett.1998,160:91～96
[61]CB Park, KS Yi, K Matsuzaki, et al. Structure-activity Analysis of Buforin Ⅱ, a Histone H Aderived Antimicrobial Peptide:the Proline Hinge is Responsible for the Cell-penetrating Ability of Buforin Ⅱ. Proc Natl Acad Sci USA.2000,97:8245～8250
[62]WS Home, CM Wiethoff, C Cui, et al. Antiviral Cyclic D,L-a-peptides:Targeting a General Biochemical Pathway in Virus Infections. Bioorganic and Medicinal Chemistry. 2005,13:5145～5153
[63]GJ Kotwal, JN Kaczmarek, S Leivers, et al. Anti-HIV, Anti-poxvirus, and Anti-SARS Activity of a Nontoxic, Acidic Plant Extract from the Trifollium Species Secomet-V/anti-Vac Suggests that it Contains a Novel Broad-spectrum Antiviral. Annals New York Academy of Sciences.2005,1056:293～302
[64]Y Yu, X Huang, Q Wang, et al. A Novel Peptide that Inhibits HIV-1 Entry. Chinese Science Bulletin.2004,49 (19):2110～2112
[65]C Lorin, H Saidi, A Belaid, et al. The Antimicrobial Peptide Dermaseptin S4 Inhibits HIV-1 Infectivity in Vitro.Virology.2005,334:264～275
[66]M Wachinger, A Kleinschmidt, D Winder. Antimicrobial Peptides Melttin and Cecropin Inhibit Replication of Human Immunodeficiency Virus I by Suppressing Viral Gene Expression. Journal of General Virology.1998,79:731～740
[67]MA Clark, TM Conway, RGL Shorr.Identification and Isolation of a Mammalian Protein which is Antigenically and Functionally Related to the Phospholipase A2 Stimulatory Peptide Melittin. Journal of Biological Chemistry.1987,262:4402～4406
[68]牛国琴.空间稳定免疫脂质体体内过程及其对策的研究进展.国外医学药学分册.2002,29(5)：301～306
[69]SV Sharma. Melittin-induced Hyperactivation of Phospholipase A2 Activity and Calcium Influx in Rastransformed Cells. Oncogene.1993,8:939～947
[70]PJ Fisher, FG Prendergast, MR Ehrhardt. Calmodulin Interacts with Amphiphilic Peptides Composed of all Amino Acids. Nature.1994,368:651～653
[71]KR Gravitt, NE Ward, CA Brian. Inhibition of Protein Kinase C by Melittin:Antagonism of Binding Interactions between Melittin and the Catalytic Domain by Active Site Binding of Mg ATP. Biochemical Pharmacology.1994,47:425～427
[72]D Andreu, L Rivas. Animal Antimicrobial Peptides:an Overview. Biopolymers.1999, 47:413～433
[73]Y Aboudy, E Mendelson, I Shalit. Activity of Two Synthetic Amphiphilic Peptides and Magainin-2 against Herpes Simplex Virus Types 1 and 2. International Journal of Peptide and Protein Research.1994,43(6):573～582
[74]EJ Helmeerhorst, P Breeuwer, WV Hof, et al. The Cellular Target of Histatin5 on Candida Albicans is the Energized Mitochondrion. J oil Chem.1999,274(11):7286～7291
[75]EJ Helmeerhorst, W van't Hof, P Breeuwer, et al. Characterization of Histatin 5 with Respect to Amphipathicity and effects on Cell and Mitochondrial Membrane Integrity Excludes a Candidacidal Mechanism of Pore Formation. The Journal of Biological Chemistry.2001,276:5643～5649
[76]柴晓杰,王丕武.抗真菌肽的研究进展及应用前景.中国饲料.2005,4：15～17
[77]HV Westerhoff, M Zasloff, JL Rosner, et al. Functional Synergism of the Magainins PGLa and Magainin-2 in Escherichia coli, Tumor Cells and Liposomes. European Journal of Biochemistry.1995,228(2):257～264
[78]K Matsuzaki, Y Mitani, K Akada, et al. Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa. Biochemistry.1998,37(43):15144～15153
[79]REW Hancock, MG Scott. The Role of Antimicrobial Peptides in Animal Defenses. Proceedings of the National Academy of Sciences of United States of America.2000, 97(16):8856～8861
[80]REW Hancock, G Diamond. The Role of Cationic Antimicrobial Peptides in Innate Host Defences. Trens in Microbiology.2000,8:402～410
[81]L Silvestro. Antibacterial and Antimembrane Activities of Cecropin A in E. coli. Antimicrobial Agents and Chemotherapy.2000,44:602～607
[82]JL Arques, E Rodriguez, M Nunez, M Medinal. Antimicrobial Activity of Nisin, Reuterin, and the Lactoperoxidase System on Listeria monocytogenes and Staphylococcus aureus in Cuajada, a Semisolid Dairy Product Manufactured in Spain. Dairy Science.2008, 91:70～75
[83]Y Park, SN Park, SC Park, et al. Synergism of Leu-Lys Rich Antimicrobial Peptides and Chloramphenicol against Bacterial Cells. Biochimica et Biophysica Acta-Proteins & Proteomics.2006,1764(1):24～32
[84]柳爱华,宝福凯.近年来固有免疫研究中的一些重要进展.自然杂志.2009,31(4)：218～222
[85]REW Hancock. Cationic Peptides:Effectors in Innate Immunity and Novel Antimicrobials. Lancet Infectious Diseases.2001,1:156～164
[86]SM Travis, N Anderson, WR Forsyth, et al. Bactericidal Activity of Mammalian Cathelicidin Peptides. Infect Immun.2000,68:2748～2755
[87]DC Woodhams, J Voyles, KR Lips, et al. In a Panamanian Amphibian Assemblage Based on Skin Peptide Defenses. Journal of Wildlife Diseases.2006,42(2):207～218
[88]金钺,刘慧敏,耿娟等.粗提牛白细胞抗菌肽对小鼠免疫功能的影响.河南农业大学学报.2009,43(6)：630～633
[89]麻延峰,周文仙,应志锐等.抗菌肽防治金华猪呼吸道疾病综合症的疗效研究.家畜生态学报.2010,31(1)：90～92
[90]邱晓燕.抗菌肽的免疫功能和应用前景.生物学通报.2002,37(1)：24～25
[91]JD Heilborn, MF Nilsson, CI Jimenez, et al. Antimicrobial Protein hCAP18/LL237 is Highly Expressed in Breast Cancer and is a Putative Growth Factor for Epithelial Cells. Int J Cancer.2005,114(5):713～719
[92]C Ye, L Fei, S Hu. Rare and Economic Amphibians of China. Sichuan House of Science and Technology. Chengdu, China,1993
[93]JB Kim, MS Min, SY Yang, et al. Genetic Relationships among Korean Brown Frog Species (Anura, Ranidae), with Special Reference to Evolutionary Divergences between Two Allied Species Rana dybowskii and Rana huanrenensis. Zool Sci.2002,19:369～382
[94]JM Conlon, J Kolodziejek, N Nowotny, et al. Cytolytic Peptides belonging to the Brevinin-1 and Brevinin-2 Families Isolated from the Skin of the Japanese Brown Frog, Rana dybowskii. Toxicon.2007,50:746～756
[95]韩俊友,赵瑞利,韩文瑜等.4种蛙皮肤活性肽的提取及生物活性比较.中国兽医学报.2009,29(5)：576～579
[96]C Lacombe, C Cifuentes-Diaz, I Dunia, et al. Peptide Secretion in the Cutaneous Glands of South American tree frog Phyllomedusa bicolor: an Ultrastructural Study. European Journal of Cell Biology.2000,79(9):631～641
[97]SS Kim, MS Shim, J Chung, et al. Purification and Characterization of Antimicrobial Peptides from the Skin Secretion of Rana dybowskii. Peptides.2007,28:1532～1539
[98]L Jin, Q Li, S Song, et al. Characterization of Antimicrobial Peptides Isolated from the Skin of the Chinese Frog, Rana dybowskii. Comparative Biochemistry and Physiology, Part B.2009,154:174～178
[99]金莉莉.东北林蛙抗菌肽及其生物学特性.中国医科大学博士学位论文.2009：34～36
[100]T Halverson, YJ Basir, FC Knoop, et al. Purification and Characterization of Antimicrobiald Peptide from the Skin of the North American Green Frog Rana clamitans. Peptides.2000,21(4):496～497
[101]A Giacometti, O Cirioni, R Ghiselli, et al. Polycationic Peptides as Prophylacticagents agsinst Methicillin-susceptible or Methicillin-resistant Staphylococcus Epidermidis Vascular Graft Infection. Antimicrob Agents Chemothe.2000,44(12):3306～3309
[102]申屠德君.特定细菌刺激下东北林蛙皮肤抗菌肽基因克隆及其表达.东北林业大学硕士学位论文.2009：23～28
[103]F Xie, MW Lau, SN Stuart, et al. Conservation Needs of Amphibians in China:a review. Sci China C Life Sci.2007,50(2):265～276
[104]A Ohnuma, JM Conlon, H Kawasaki, et al. Developmental and Triiodothyronine-induced Expression of Genes Encoding Preprotemporins in the Skin of Tago's Brown Frog Rana tagoi. Gen Comp Endocrinol.2006,146:242～250
[105]ML Mangoni, R Miele, TG Renda, et al. The Synthesis of Antimicrobial Peptides in the Skin of Rana esculenta is Stimulated by Microorganisms. FASEB Journal.2006, 15:1431～1432
[106]M Simmaco, ML Mangoni, A Boman. Experimental Infections of Rana esculenta with Aeromonas hydrophila:A Molecular Mechanism for the Control of the Normal Flora. Scandinavian Journal of Immunology.1998,48:357～363
[107]L Berger, R Speare, P Daszak, et al. Chytridiomycosis Causes Amphibian Mortality Associated with Population Declines in the Rain Forests of Australia and Central America. Proc Natl Acad Sci USA.1998,95:9031～9036
[108]JE Longcore, AP Pessier, DK Nichols. Batrachochytrium Dendrobatidis gen. et sp. nov., a Chytrid Pathogenic to Amphibians. Mycologia.1999,91:219～227
[109]AP Pessier, DK Nichols, JE Longcore, et al. Cutaneous Chytridiomycosis in Poison Dart Frogs (Dendrobates spp.) and White's Tree Frogs (Litoria caerulea). J Vet Diagn Invest. 1999,11:194～199
[110]M Simmaco, A Boman, ML Mangoni, et al. Effect of Glucocorticoids of the Synthesis of Antimicrobial Peptides in Amphibian Skin. Federation of European Biochemical Societies Letters.1997,416:273～275
[111]LA Rollins-Smith, LK Reinert, CJ O'Leary, et al. Antimicrobial Peptide Defenses in Amphibian Skin. Integrative and Comparative Biology.2005,45:137～142
[112]B Matutte, KB Storey, FC Knoop, et al. Induction of Synthesis of an Antimicrobial Peptide in the skin of the Freeze-tolerant frog, Rana sylvatica, in Response to Environmental Stimuli. FEBS Letters.2000,483:135～138
[113]C Cellura, M Toubiana, N Parrinello, et al. Specific Expression of Antimicrobial Peptide and HSP70 Genes in Response to Heat-shock and Several Bacterial Challenges in Mussels. Fish & Shellfish Immunology.2007,22:340～350
[114]G Mitta, F Hubert, EA Dyrynda, et al. Mytilin B and MGD2, Two Antimicrobial Peptides of Marine Mussels:Gene Structure and Expression Analysis. Developmental and Comparative Immunology.2000,24(4):381～393
[115]M Wachinger, A Kleinschmidt, D Winder, et al. Antimicrobial Peptides Melttin and Cecropin Inhibit Replication of Human Immunodeficiency Virus I by Suppressing Viral Gene Expression. Gen Virol.1998,79(4):731～740
[116]C Gestal, MM Costa, A Figueras, et al. Analysis of Differentially Expressed Genes in Response to Bacterial Stimulation in Hemocytes of the Carpet-shell Clam Ruditapes Decussatus:Identification of New Antimicrobial Peptides. Gene.2007,406(1-2):134～143
[117]李伟,孙文秀.黄鳝Ⅰ型hepcidin抗菌肽基因的诱导表达分析.长江大学学报(自然科学版).2009,6：52～56
[118]柳峰松,王丽娜,唐婷等.家蝇抗菌肽Diptericin基因的克隆与分析.昆虫学报.2009,52(10)：1078～1082
[119]EA Levashina, S Ohresser, B Lemaitre, et al. Two Distinct Pathways can Control Expression of the Gene Encoding the Drosophila Antimicrobial Peptide Metchnikowin. Journal of Molecular Biology.1998,278(3):515～527
[120]JA Tennessen. Molecular Evolution of Animal Antimicrobial Peptides:Widespread Moderate Positive Selection. Journal of Evolutionary Biology.2005,18:1387～1394
[121]KT Miyasaki, R Lofel, RI Lehrer. Sensitivity of Periodontal Pathongens to the Bacterial Activity of Synthetic Protegrins, Antibiotic Peptides Derived from Procine Leukocytes. J Dent Res.1997,76:1453～1459
[122]刘进,奚涛.人β防御素23的研究进展.药物生物技术.2005,12(5)：337～341
[123]LA Haverson, GI Engber, L Baltzer, et al. Human Lactoferrin and Peptides Derived from a Surface Exposed Helical Region Reduce Experimental Escherichia coli Urinary Tract Infection in Mice. Infect Immun.2000,68(10):5816～5823
[124]JM Guthmiller, KG Vargas, R Srikantha, et al. Susceptibilities of Oral Bacteria and Yeast to Mammalian Cathelicidins. Antimicrob Agents Chemother.2001,45(11):3216～3219
[125]W Paquetted, DM Simpson, P Friden, et al. Safety and Clinical Effects of Topical Histatin Gels in Humans with Experimental Gingivitis J Clin Periodontol.2002, 29(12):1051～1058
[126]TJ Fella, REW Hancock. Improved Activity of a Synthetic Indolicidin Analog. Antimicrob Agents Chemother.1997,41(4):771～775
[127]A Giacometti, O Cirioni, R Ghiselli, et al. Effects of Pexiganan Alone and Combined with Betalactams in Experimental Endotoxic Shock. Peptides.2005,26(2):207～216
[128]KA Daher, ME Selsted, RI Lehrer. Direct Inactivation of Viruses by Human Granulocyte Defensins. J Virol.1986,60(3):1068～1074
[129]H Tamamura, T Ishihara, A Otaka, et al. Analysis of the Interaction of an anti-HIV Peptide T-2 ([Tyr5,12,Lys7]-polyphemusin2Ⅱ),with gp120 and CD4 by Surface Plasmon Resonance. Biochim Biophys Acta.1996,1298(1):37～44
[130]M Siep rawska-Lupa, P Mydel, K Krawczyk, et al. Degradation of Human Antimicrobial Peptide LL-37 by Staphylococcus Aureus-derived Proteinases. Antimicrob Agents Chemother.2004,48(12):4673～4679
[131]陈福,罗玉萍,抗菌肽耐药性研究进展.微生物学通报.2008,35(11)：1786～1790
[132]GG Perron, M Zasloff, G Bell. Experimental Evolution of Resistance to an Antimicrobial Peptide. Proc Biol Sci.2006,273(1583):251～256
[133]M Pietiainen, P Francois, HL Hyyrylainen, et al. Transcriptome Analysis of the Responses of Staphylococcus Aureus to Antimicrobial Peptides and Characterization of the Roles of vraDE and vraSR in Antimicrobial Resistance. BMC Genomics.2009, 10(1):429～431
[134]布冠好,李宏基,杨国宇,汪国和等.抗菌肽的作用特点及应用前景.动物医学进展.2005,26(3)：26～28
[135]D Barra, M Simmaco. Amphibian Skin:A Promising Resource for Antimicrobial Peptides. Trends Biotechnol.1995,13(6):205～209
[136]LA Rollins-Smith. The Role of Amphibian Antimicrobial peptides in Protection of Amphibians from Pathogens Linked to Global Amphibian Declines. Biochim Biophys Acta. 2009,1788(8):1593～1599
[137]孙芳.东北林蛙皮肤活性肽HPLC分离纯化及体外抗病毒活性研究.东北林业大学硕士学位论文.2007：26～42
[138]张晶钰.东北林蛙皮肤活性肽对MCF7和平滑肌细胞膜离子通道的影响.东北林业大学硕士学位论文.2007：20～29
[139]乔军,孟庆铃.运用OD值法进行细菌计数的研究.中国家禽.1996,4：26～27
[140]M Simmaco, ML Mangoni, A Boman, et al. Experimental Infections of Rana esculenta with Aeromonas hydrophila:A Molecular Mechanism for the Control of the Normal Flora. Scand J Immunol.1998,48(4):357～363
[141]M Murakami, T Ohtake, RA Dorschner, et al. Cathelicidin Antimicrobial Peptides are Expressed in Salivary Glands and Saliva. Journal of Dental Research.2002,81:845～850
[142]F Barchiesi, AL Colombo, DA Mcgough, et al. Comparative Study of Broth Microdilution and Macrodilution Techniques for in vitro Antifungal Susceptibility Testing of Yeasts by Using the National Committee for Clinical Laboratory Standards' Proposed Standards. Journal of Clinical Microbiology.1994,32:2494～2500
[143]JM Conlon, A al-Dhaheri, E al-Mutawa, et al. Peptide Defenses of the Cascades Frog Rana cascadae:Implications for the Evolutionary History of Frogs of the Amerana Species Group. Peptide.2007,28:1268～1274
[144]Lu Yi, Ma Yufang, Wang Xu, et al. The First Antimicrobial Peptide from Sea Amphibian. Molecular Immunology.2008,45:678～681
[145]苏立强,郑永杰.色谱分析方法.北京：清华大学出版社,2009：41～49
[146]RJ Simpson. Protein and Proteomics:A Laboratory Manual. New York:Cold Spring Harbor Laboratory,2004:219～230
[147]REW Hancock, R Lehrer. Cationic Peptides:a New Source of Antibiotics. Trends in Biotechnology.1998,16:82～88
[148]O Chertov, DF Michiel, L Xu, et al. Identification of Defensin-1, Defensin-2, and CAP37/azurocidin as T cell Chemoattractant Proteins Released from Interleukin28-stimulated Neutrophils. J Biol Chem.1996,271(6):2935～2940
[149]DJ Davidson, AJ Currie, GS Reid, et al. The Cationic Antimicrobial Peptide LL-37 Modulates Dendritic Cell Differentiation and Dendritic Cell-induced T cell Polarization. J Immunol.2004,172(4):1146～1156
[150]HJ Huang, CR Ross, F Blecha. Chemoattractant Properties of PR-39, a Neutrophil Antibacterial Peptide. J Leukoc Biol.1997,61(5):624～629
[151]B Agerberth, J Charo, J Werr, et al. The Human Antimicrobial and Chemotactic Peptides LL-37 and Alpha-defensins are Expressed by Specific Lymphocyte and Monocyte Populations. Blood.2000,96(9):3086～3093
[152]N Mookherjee, REW Hancock. Cationic Host Defence Peptides:Innate Immune Regulatory Peptides as a Novel Approach for Treating Infections. Cell Mol Life Sci,2007. 64(728):922～933
[153]DM Bowdish, DJ Davidson, DP Speert, et al. The Human Cationic Peptide LL-37 Induces Activation of the Extracellular Signal Regulated Kinase and p38 Kinase Pathways in Primary Human Monocytes. J Immunol.2004,172(6):3758～3765
[154]X Chen, F Niyonsaba, H Ushio, et al. Human Cathelicidin LL-37 Increases Vascular Permeability in the Skin via Mast Cell Activation and Phosphorylates MAP Kinases p38 and ERK in Mast Cells. J Dermatol Sci.2006,43(1):63～66
[2]GL Dockray, CR Hopkins. Caerulein Secretion by Dermal Glands in Xenopus laevis. J Cell Biol.1975,64:724～733
[3]D Hultmark, H Steiner, GH Boman. Insect Immunity Purification and Properties of Three Inducible Bactericidal Proteins from Hemolymph of Immunized Pupae of Hyalophora cecropia. Eur Biochem.1980,106:7～16
[4]S Seebah, A Suresh, S Zhuo, et al. Defensins Knowledge Base:A Manually Curated Database and Information Source Focused on the Defensins Family of Antimicrobial Peptides. Nucleic Acids Res.2007,35:D262～D268
[5]W Kamysz. Are Antimicrobial Peptides an Alternative for Conventional Antibiotics? Nucl Med Rev Cent East Eur.2005,8(1):78～86
[6]Z Wang, G Wang. APD:the Antimicrobial Peptide Database. Nucleic Acids Res.2004, 32:D590～D592
[7]BT Clarke. The Natural History of Amphibian Skin Secretions, their Normal Function and Potential Medical Applications. Biol Rev.1997,72(3):365～379
[8]K Tsuruda, O Arakawa, K Kawatsu, et al. Secretary Glands of Tetrodotoxin in the Skin of the Japanese Newt Cynops Pyrrhogadter. Toxicon.2002,40(2):131～136
[9]H Greven, K Zanger, G Schwinger. Mechanical Properties of the Skin of Xenopus laevis (Anura, Amphibia). J Morphol.1995,224:15～22
[10]C Lacombe, C Cifuentes-Diaz, I Dunia, et al. Peptide Secretion in the Cutaneous Glands of South American Tree Frog Phyllomedusa bicolor: an Ultrastructural Study. European Journal of Cell Biology.2000,79:631～641
[11]AE Levashina, S Ohresser, B Lemaitre, et al. Two Distinct Pathways can Control Expression of the Gene Encoding the Drosophila Antimicrobial Peptide Metchnikowin. J Mol Biol.1998,278:515～527
[12]M Zasloff. Antimicrobial Peptides of Multicellular Organisms. Nature.2002,415:389～395
[13]R Miele, G Bjorklund, D Barra, et al. Involvement of Rel Factors in the Expression of Antimicrobial Peptide Genes in Amphibia. Eur J Biochem.2001,268:443～449
[14]O Froy. Regulation of Mammalian Defensin Expression by Toll-like receptor Dependent and Independent Signaling Pathways. Cell Microbiol.2005,7(10):1387～1397
[15]M Zasloff. Fighting Infections with Vitamin D. Nat Med.2006,12(4):388～390
[16]宋珊珊,胡国斌,董先智.抗菌肽基因异源表达的研究进展.生物医学工程学杂志. 2009,26(6):1372～1375
[17]UniProtKB/SWISS-PROT at EBI. http://www.expasy.org/sprot/relnotes/relstat.html
[18]岳昌武,莫宁萍,刘坤祥等.抗菌肽的结构特点作用机理及应用前景.安徽农业科学.2008,36(5)：1736～1739
[19]SY Choi, YM Choo, KS Lee, et al. Cloning and Expression Profiling of Four Antibacterial Peptide Genes from the Bumblebee Bombus ignites. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology.2008,150(2):141～146
[20]RI Lehrer, T Ganz. Defensins of Vertebrate Animals. Curr Opin Immunol.2002, 14:96～102
[21]KVR Reddy, RD Yedery, C Aranha. Antimicrobial peptides:Premises and Promises. Int J Antimicrob Agents.2004,24 (6):536～573
[22]CB Park, KS Yi, K Matsuzaki, et al. Structure-Activity Analysis of Buforin Ⅱ, a Histone H2 Aderived Antimicrobial Peptide:The Proline Hinge is Responsible for the cell-Penetrating Aability of Buforin Ⅱ. Proc Natl Acad Sci.2000,97:8245～8250
[23]CV Kalfa. Congeners of SMAP29 kill Ovine Pathogens and Induce Ultrastructural Damage in Bacterial Cells. Antimicrob Agents Chemother.2001,45:3256～3261
[24]SA Ladokhin, EM Selsted, HS White. Sizing Membrane Pores in Lipid Vesicles by Leakage of Coencapsulated Markers:Pore Formation by Melittin Biophys J.1997, 72:1762～1766
[25]K Matsuzaki, S Yoneyama, K Miyajima. Pore Formation and Translocation of Melittin. Biophys J.1997,73:831～838
[26]HJ Kang, YS Shin, YS Jang, et al. Release of Aqueous Contents from Phospholipid Vesicles Induced by Cecropin A (1-8) Magainin 2 (1-12) Hybrid and its Analogues. J Peptide Res.1998,52:45～50
[27]K Hristova, EM Selsted, HS White. Critical Role of Lipid Composition in Membrane Permeabilization by Rabbit Neutrophil Defensins. J Biol Chem.1997,272:24224～24233
[28]H Zhao, PJ Mattila, MJ Holopainen, et al. Comparison of the Membrane Association of Two Antimicrobial Peptides, Magainin 2 and Indolicidin. Biophys J.2001,81:2979～2991
[29]B Christensen, J Fink, BR Merrifield, et al. Channel-Forming Properties of Cecropins and Related Model Compounds Incorporated into Planar Lipid Membranes. Proc Natl Acad Sci USA.1988,85:5072～5076
[30]TM Lee, YF Chen, WH Huang. Energetics of Pore Formation Induced by Membrane Active Peptides. Biochemistry.2004,43:3590～3599
[31]Y Wu, WH Huang, AG Olah. Method of Oriented Circular Dichroism. Biophys J.1990, 57:797～806
[32]B Bechinger. The Structure, Dynamics and Orientation of Antimicrobial Peptides in Membranes by Multidimensional Solid-State NMR Spectroscopy. Biochim Biophys Acta. 1999,1462:157～183
[33]S Yamaguchi, T Hong, A Waring, et al. Solid-State NMR Investigations of Peptide-lipid Interaction and Orientation of a β-sheet Antimicrobial Peptide, Protegrin. Biochemistry. 2002,41:9852～9862
[34]B Bechinger, M Zasloff, SJ Opella. Structure and Orientation of the Antibiotic Peptide Magainin in Membranes by Solid-State Nuclear Magnetic Resonance Spectroscopy. Protein Sci.1993,2:2077～2084
[35]S Yamaguchi, et al. Orientation and Dynamics of an Antimicrobial Peptide in the Lipid Bilayer by Solid-State NMR Spectroscopy. Biophys J.2001,81:2203～2214
[36]A Spaar, C Munster, T Salditt. Conformation of Peptides in Lipid Membranes Studied by X-ray Grazing Incidence Scattering. Biophys J.2004,87:396～407
[37]K He, SJ Ludtke, HW Huang, et al. Antimicrobial Peptide Pores in Membranes Detected by Neutron in-plane Scattering. Biochemistry.1995,34:15614～15618
[38]SJ Ludtke, K He, WT Heller, et al. Membrane Pores Induced by Magainin. Biochemistry. 1996,35:13723～13728
[39]L Yang, TA Harroun, WT Heller, et al. Neutron Off-plane Scattering of Aligned Membranes. I. Method of Measurement. Biophys J.1998,75:641～645
[40]L Yang, TM Weiss, TA Harroun, et al. Supramolecular Structures of Peptide Assemblies in Membranes by Neutron Off-plane Scattering:Method of Analysis. Biophys J.1999, 77:2648～2656
[41]H Zhao, JP Mattila, JM Holopainen, et al. Comparison of the Membrane Association of Two Antimicrobial Peptides, Magainin 2 and Indolicidin. Biophys J.2001,81:2979～2991
[42]L Silvestro, K Gupta, JN Weiser, et al. The Concentration-dependent Membrane Activity of Cecropin A. Biochemistry.1997,36:11452～11460
[43]A Shizuo, U Satoshi, T Osamu. Pathogen Recognition and Innate Immunity. Cell.2006, 124:783～801
[44]KA Brogden. Antimicrobial Peptides:Pore Formers or Metabolic Inhibitors in Bacteria? Nature Reviews.2005,3:238～250
[45]HW Huang. Action of Antimicrobial Peptides:Two-state Model. Biochemistry.2000, 39:8347～8352
[46]FY Chen, MT Lee, HW Huang. Evidence for Membrane Thinning Effect as the Mechanism for Peptide Induced Pore Formation. Biophys J.2003,84:3751～3758
[47]L Yang, TA Harroun, TM Weiss, et al. Barrel-stave Model or Toroidal Model? A Case Study on Melittin Pores. Biophys J.2001,81:1475～1485
[48]G Ehrenstein, H Lecar. Electrically Gated Ionic Channels in Lipid Bilayers. Rev Biophys. 1977,10:1～34
[49]Y Pouny, D Rapaport, A Mor, et al. Interaction of Antimicrobial Dermaseptin and its Fluorescently Labeled Analogues with Phospholipid Membranes. Biochemistry.1992, 31:12416～12423
[50]A Spaar, C Munster, T Salditt. Conformation of Peptides in Lipid Membranes Studied by X-ray Grazing Incidence Scattering. Biophys J.2004,87:396～407
[51]Y Shai. Mechanism of the Binding, Insertion and Destabilization of Phospholipid Bilayer Membranes by Alpha-helical Antimicrobial and Cell Non-selective Membrane-lytic peptides. Biochim Biophys Acta.1999,1462:55～70
[52]AS Ladokhin, SH White.'Detergent-like' Permeabilization of Anionic Lipid Vesicles by Melittin. Biochim Biophys Acta.2001,1514:253～260
[53]K Matsuzaki, O Murase, N Fujii, et al. An Antimicrobial Peptide, Magainin 2, Induced Rapid Flip-flop of Phospholipids Coupled with Pore Formation and Peptide Translocation. Biochemistry.1996,35:11361～11368
[54]KJ Hallock, DK Lee, A Ramamoorthy. MSI-78, an Analogue of the Magainin Antimicrobial Peptides, Disrupts Lipid Bilayer Structure via Positive Curvature Strain. Biophys J.2003,84:3052～3060
[55]K Matsuzaki, K Sugishita, N Ishibe, et al. Relationship of Membrane Curvature to the Formation of Pores by Magainin 2. Biochemistry.1998,37(34):11856～11863
[56]K Matsuzaki, K Sugishita, M Harada, et al. Interactions of an Antimicrobial Peptide, Magainin 2, with Outer and Inner Membranes of Gram-negative Bacteria. Biochim Biophys Acta.1997,1327:119～130
[57]S Yamaguchi, T Hong, A Waring, et al. Solid-state NMR Investigations of Peptide-lipid Interaction and Orientation of a β-sheet Antimicrobial Peptide, Protegrin. Biochemistry. 2002,41:9852～9862
[58]R Gennaro, M Zanetti. Structural Features and Biological Activities of the Cathelicidin-derived Antimicrobial Peptides. Biopolymers.2000,55:31～49
[59]J Shi, CR Ross, MM Chengappa, et al. Antibacterial Activity of a Synthetic Peptide (PR-26) Derived from PR-39, a Proline-arginine-rich Neutrophil Antimicrobial Peptide. Antimicrob Agents Chemother.1996,40(1):115～121
[60]C Subbalakshmi, N Sitaram. Mechanism of Antimicrobial Action of Indolicidin. FEMS Microbiol Lett.1998,160:91～96
[61]CB Park, KS Yi, K Matsuzaki, et al. Structure-activity Analysis of Buforin Ⅱ, a Histone H Aderived Antimicrobial Peptide:the Proline Hinge is Responsible for the Cell-penetrating Ability of Buforin Ⅱ. Proc Natl Acad Sci USA.2000,97:8245～8250
[62]WS Home, CM Wiethoff, C Cui, et al. Antiviral Cyclic D,L-a-peptides:Targeting a General Biochemical Pathway in Virus Infections. Bioorganic and Medicinal Chemistry. 2005,13:5145～5153
[63]GJ Kotwal, JN Kaczmarek, S Leivers, et al. Anti-HIV, Anti-poxvirus, and Anti-SARS Activity of a Nontoxic, Acidic Plant Extract from the Trifollium Species Secomet-V/anti-Vac Suggests that it Contains a Novel Broad-spectrum Antiviral. Annals New York Academy of Sciences.2005,1056:293～302
[64]Y Yu, X Huang, Q Wang, et al. A Novel Peptide that Inhibits HIV-1 Entry. Chinese Science Bulletin.2004,49 (19):2110～2112
[65]C Lorin, H Saidi, A Belaid, et al. The Antimicrobial Peptide Dermaseptin S4 Inhibits HIV-1 Infectivity in Vitro.Virology.2005,334:264～275
[66]M Wachinger, A Kleinschmidt, D Winder. Antimicrobial Peptides Melttin and Cecropin Inhibit Replication of Human Immunodeficiency Virus I by Suppressing Viral Gene Expression. Journal of General Virology.1998,79:731～740
[67]MA Clark, TM Conway, RGL Shorr.Identification and Isolation of a Mammalian Protein which is Antigenically and Functionally Related to the Phospholipase A2 Stimulatory Peptide Melittin. Journal of Biological Chemistry.1987,262:4402～4406
[68]牛国琴.空间稳定免疫脂质体体内过程及其对策的研究进展.国外医学药学分册.2002,29(5)：301～306
[69]SV Sharma. Melittin-induced Hyperactivation of Phospholipase A2 Activity and Calcium Influx in Rastransformed Cells. Oncogene.1993,8:939～947
[70]PJ Fisher, FG Prendergast, MR Ehrhardt. Calmodulin Interacts with Amphiphilic Peptides Composed of all Amino Acids. Nature.1994,368:651～653
[71]KR Gravitt, NE Ward, CA Brian. Inhibition of Protein Kinase C by Melittin:Antagonism of Binding Interactions between Melittin and the Catalytic Domain by Active Site Binding of Mg ATP. Biochemical Pharmacology.1994,47:425～427
[72]D Andreu, L Rivas. Animal Antimicrobial Peptides:an Overview. Biopolymers.1999, 47:413～433
[73]Y Aboudy, E Mendelson, I Shalit. Activity of Two Synthetic Amphiphilic Peptides and Magainin-2 against Herpes Simplex Virus Types 1 and 2. International Journal of Peptide and Protein Research.1994,43(6):573～582
[74]EJ Helmeerhorst, P Breeuwer, WV Hof, et al. The Cellular Target of Histatin5 on Candida Albicans is the Energized Mitochondrion. J oil Chem.1999,274(11):7286～7291
[75]EJ Helmeerhorst, W van't Hof, P Breeuwer, et al. Characterization of Histatin 5 with Respect to Amphipathicity and effects on Cell and Mitochondrial Membrane Integrity Excludes a Candidacidal Mechanism of Pore Formation. The Journal of Biological Chemistry.2001,276:5643～5649
[76]柴晓杰,王丕武.抗真菌肽的研究进展及应用前景.中国饲料.2005,4：15～17
[77]HV Westerhoff, M Zasloff, JL Rosner, et al. Functional Synergism of the Magainins PGLa and Magainin-2 in Escherichia coli, Tumor Cells and Liposomes. European Journal of Biochemistry.1995,228(2):257～264
[78]K Matsuzaki, Y Mitani, K Akada, et al. Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa. Biochemistry.1998,37(43):15144～15153
[79]REW Hancock, MG Scott. The Role of Antimicrobial Peptides in Animal Defenses. Proceedings of the National Academy of Sciences of United States of America.2000, 97(16):8856～8861
[80]REW Hancock, G Diamond. The Role of Cationic Antimicrobial Peptides in Innate Host Defences. Trens in Microbiology.2000,8:402～410
[81]L Silvestro. Antibacterial and Antimembrane Activities of Cecropin A in E. coli. Antimicrobial Agents and Chemotherapy.2000,44:602～607
[82]JL Arques, E Rodriguez, M Nunez, M Medinal. Antimicrobial Activity of Nisin, Reuterin, and the Lactoperoxidase System on Listeria monocytogenes and Staphylococcus aureus in Cuajada, a Semisolid Dairy Product Manufactured in Spain. Dairy Science.2008, 91:70～75
[83]Y Park, SN Park, SC Park, et al. Synergism of Leu-Lys Rich Antimicrobial Peptides and Chloramphenicol against Bacterial Cells. Biochimica et Biophysica Acta-Proteins & Proteomics.2006,1764(1):24～32
[84]柳爱华,宝福凯.近年来固有免疫研究中的一些重要进展.自然杂志.2009,31(4)：218～222
[85]REW Hancock. Cationic Peptides:Effectors in Innate Immunity and Novel Antimicrobials. Lancet Infectious Diseases.2001,1:156～164
[86]SM Travis, N Anderson, WR Forsyth, et al. Bactericidal Activity of Mammalian Cathelicidin Peptides. Infect Immun.2000,68:2748～2755
[87]DC Woodhams, J Voyles, KR Lips, et al. In a Panamanian Amphibian Assemblage Based on Skin Peptide Defenses. Journal of Wildlife Diseases.2006,42(2):207～218
[88]金钺,刘慧敏,耿娟等.粗提牛白细胞抗菌肽对小鼠免疫功能的影响.河南农业大学学报.2009,43(6)：630～633
[89]麻延峰,周文仙,应志锐等.抗菌肽防治金华猪呼吸道疾病综合症的疗效研究.家畜生态学报.2010,31(1)：90～92
[90]邱晓燕.抗菌肽的免疫功能和应用前景.生物学通报.2002,37(1)：24～25
[91]JD Heilborn, MF Nilsson, CI Jimenez, et al. Antimicrobial Protein hCAP18/LL237 is Highly Expressed in Breast Cancer and is a Putative Growth Factor for Epithelial Cells. Int J Cancer.2005,114(5):713～719
[92]C Ye, L Fei, S Hu. Rare and Economic Amphibians of China. Sichuan House of Science and Technology. Chengdu, China,1993
[93]JB Kim, MS Min, SY Yang, et al. Genetic Relationships among Korean Brown Frog Species (Anura, Ranidae), with Special Reference to Evolutionary Divergences between Two Allied Species Rana dybowskii and Rana huanrenensis. Zool Sci.2002,19:369～382
[94]JM Conlon, J Kolodziejek, N Nowotny, et al. Cytolytic Peptides belonging to the Brevinin-1 and Brevinin-2 Families Isolated from the Skin of the Japanese Brown Frog, Rana dybowskii. Toxicon.2007,50:746～756
[95]韩俊友,赵瑞利,韩文瑜等.4种蛙皮肤活性肽的提取及生物活性比较.中国兽医学报.2009,29(5)：576～579
[96]C Lacombe, C Cifuentes-Diaz, I Dunia, et al. Peptide Secretion in the Cutaneous Glands of South American tree frog Phyllomedusa bicolor: an Ultrastructural Study. European Journal of Cell Biology.2000,79(9):631～641
[97]SS Kim, MS Shim, J Chung, et al. Purification and Characterization of Antimicrobial Peptides from the Skin Secretion of Rana dybowskii. Peptides.2007,28:1532～1539
[98]L Jin, Q Li, S Song, et al. Characterization of Antimicrobial Peptides Isolated from the Skin of the Chinese Frog, Rana dybowskii. Comparative Biochemistry and Physiology, Part B.2009,154:174～178
[99]金莉莉.东北林蛙抗菌肽及其生物学特性.中国医科大学博士学位论文.2009：34～36
[100]T Halverson, YJ Basir, FC Knoop, et al. Purification and Characterization of Antimicrobiald Peptide from the Skin of the North American Green Frog Rana clamitans. Peptides.2000,21(4):496～497
[101]A Giacometti, O Cirioni, R Ghiselli, et al. Polycationic Peptides as Prophylacticagents agsinst Methicillin-susceptible or Methicillin-resistant Staphylococcus Epidermidis Vascular Graft Infection. Antimicrob Agents Chemothe.2000,44(12):3306～3309
[102]申屠德君.特定细菌刺激下东北林蛙皮肤抗菌肽基因克隆及其表达.东北林业大学硕士学位论文.2009：23～28
[103]F Xie, MW Lau, SN Stuart, et al. Conservation Needs of Amphibians in China:a review. Sci China C Life Sci.2007,50(2):265～276
[104]A Ohnuma, JM Conlon, H Kawasaki, et al. Developmental and Triiodothyronine-induced Expression of Genes Encoding Preprotemporins in the Skin of Tago's Brown Frog Rana tagoi. Gen Comp Endocrinol.2006,146:242～250
[105]ML Mangoni, R Miele, TG Renda, et al. The Synthesis of Antimicrobial Peptides in the Skin of Rana esculenta is Stimulated by Microorganisms. FASEB Journal.2006, 15:1431～1432
[106]M Simmaco, ML Mangoni, A Boman. Experimental Infections of Rana esculenta with Aeromonas hydrophila:A Molecular Mechanism for the Control of the Normal Flora. Scandinavian Journal of Immunology.1998,48:357～363
[107]L Berger, R Speare, P Daszak, et al. Chytridiomycosis Causes Amphibian Mortality Associated with Population Declines in the Rain Forests of Australia and Central America. Proc Natl Acad Sci USA.1998,95:9031～9036
[108]JE Longcore, AP Pessier, DK Nichols. Batrachochytrium Dendrobatidis gen. et sp. nov., a Chytrid Pathogenic to Amphibians. Mycologia.1999,91:219～227
[109]AP Pessier, DK Nichols, JE Longcore, et al. Cutaneous Chytridiomycosis in Poison Dart Frogs (Dendrobates spp.) and White's Tree Frogs (Litoria caerulea). J Vet Diagn Invest. 1999,11:194～199
[110]M Simmaco, A Boman, ML Mangoni, et al. Effect of Glucocorticoids of the Synthesis of Antimicrobial Peptides in Amphibian Skin. Federation of European Biochemical Societies Letters.1997,416:273～275
[111]LA Rollins-Smith, LK Reinert, CJ O'Leary, et al. Antimicrobial Peptide Defenses in Amphibian Skin. Integrative and Comparative Biology.2005,45:137～142
[112]B Matutte, KB Storey, FC Knoop, et al. Induction of Synthesis of an Antimicrobial Peptide in the skin of the Freeze-tolerant frog, Rana sylvatica, in Response to Environmental Stimuli. FEBS Letters.2000,483:135～138
[113]C Cellura, M Toubiana, N Parrinello, et al. Specific Expression of Antimicrobial Peptide and HSP70 Genes in Response to Heat-shock and Several Bacterial Challenges in Mussels. Fish & Shellfish Immunology.2007,22:340～350
[114]G Mitta, F Hubert, EA Dyrynda, et al. Mytilin B and MGD2, Two Antimicrobial Peptides of Marine Mussels:Gene Structure and Expression Analysis. Developmental and Comparative Immunology.2000,24(4):381～393
[115]M Wachinger, A Kleinschmidt, D Winder, et al. Antimicrobial Peptides Melttin and Cecropin Inhibit Replication of Human Immunodeficiency Virus I by Suppressing Viral Gene Expression. Gen Virol.1998,79(4):731～740
[116]C Gestal, MM Costa, A Figueras, et al. Analysis of Differentially Expressed Genes in Response to Bacterial Stimulation in Hemocytes of the Carpet-shell Clam Ruditapes Decussatus:Identification of New Antimicrobial Peptides. Gene.2007,406(1-2):134～143
[117]李伟,孙文秀.黄鳝Ⅰ型hepcidin抗菌肽基因的诱导表达分析.长江大学学报(自然科学版).2009,6：52～56
[118]柳峰松,王丽娜,唐婷等.家蝇抗菌肽Diptericin基因的克隆与分析.昆虫学报.2009,52(10)：1078～1082
[119]EA Levashina, S Ohresser, B Lemaitre, et al. Two Distinct Pathways can Control Expression of the Gene Encoding the Drosophila Antimicrobial Peptide Metchnikowin. Journal of Molecular Biology.1998,278(3):515～527
[120]JA Tennessen. Molecular Evolution of Animal Antimicrobial Peptides:Widespread Moderate Positive Selection. Journal of Evolutionary Biology.2005,18:1387～1394
[121]KT Miyasaki, R Lofel, RI Lehrer. Sensitivity of Periodontal Pathongens to the Bacterial Activity of Synthetic Protegrins, Antibiotic Peptides Derived from Procine Leukocytes. J Dent Res.1997,76:1453～1459
[122]刘进,奚涛.人β防御素23的研究进展.药物生物技术.2005,12(5)：337～341
[123]LA Haverson, GI Engber, L Baltzer, et al. Human Lactoferrin and Peptides Derived from a Surface Exposed Helical Region Reduce Experimental Escherichia coli Urinary Tract Infection in Mice. Infect Immun.2000,68(10):5816～5823
[124]JM Guthmiller, KG Vargas, R Srikantha, et al. Susceptibilities of Oral Bacteria and Yeast to Mammalian Cathelicidins. Antimicrob Agents Chemother.2001,45(11):3216～3219
[125]W Paquetted, DM Simpson, P Friden, et al. Safety and Clinical Effects of Topical Histatin Gels in Humans with Experimental Gingivitis J Clin Periodontol.2002, 29(12):1051～1058
[126]TJ Fella, REW Hancock. Improved Activity of a Synthetic Indolicidin Analog. Antimicrob Agents Chemother.1997,41(4):771～775
[127]A Giacometti, O Cirioni, R Ghiselli, et al. Effects of Pexiganan Alone and Combined with Betalactams in Experimental Endotoxic Shock. Peptides.2005,26(2):207～216
[128]KA Daher, ME Selsted, RI Lehrer. Direct Inactivation of Viruses by Human Granulocyte Defensins. J Virol.1986,60(3):1068～1074
[129]H Tamamura, T Ishihara, A Otaka, et al. Analysis of the Interaction of an anti-HIV Peptide T-2 ([Tyr5,12,Lys7]-polyphemusin2Ⅱ),with gp120 and CD4 by Surface Plasmon Resonance. Biochim Biophys Acta.1996,1298(1):37～44
[130]M Siep rawska-Lupa, P Mydel, K Krawczyk, et al. Degradation of Human Antimicrobial Peptide LL-37 by Staphylococcus Aureus-derived Proteinases. Antimicrob Agents Chemother.2004,48(12):4673～4679
[131]陈福,罗玉萍,抗菌肽耐药性研究进展.微生物学通报.2008,35(11)：1786～1790
[132]GG Perron, M Zasloff, G Bell. Experimental Evolution of Resistance to an Antimicrobial Peptide. Proc Biol Sci.2006,273(1583):251～256
[133]M Pietiainen, P Francois, HL Hyyrylainen, et al. Transcriptome Analysis of the Responses of Staphylococcus Aureus to Antimicrobial Peptides and Characterization of the Roles of vraDE and vraSR in Antimicrobial Resistance. BMC Genomics.2009, 10(1):429～431
[134]布冠好,李宏基,杨国宇,汪国和等.抗菌肽的作用特点及应用前景.动物医学进展.2005,26(3)：26～28
[135]D Barra, M Simmaco. Amphibian Skin:A Promising Resource for Antimicrobial Peptides. Trends Biotechnol.1995,13(6):205～209
[136]LA Rollins-Smith. The Role of Amphibian Antimicrobial peptides in Protection of Amphibians from Pathogens Linked to Global Amphibian Declines. Biochim Biophys Acta. 2009,1788(8):1593～1599
[137]孙芳.东北林蛙皮肤活性肽HPLC分离纯化及体外抗病毒活性研究.东北林业大学硕士学位论文.2007：26～42
[138]张晶钰.东北林蛙皮肤活性肽对MCF7和平滑肌细胞膜离子通道的影响.东北林业大学硕士学位论文.2007：20～29
[139]乔军,孟庆铃.运用OD值法进行细菌计数的研究.中国家禽.1996,4：26～27
[140]M Simmaco, ML Mangoni, A Boman, et al. Experimental Infections of Rana esculenta with Aeromonas hydrophila:A Molecular Mechanism for the Control of the Normal Flora. Scand J Immunol.1998,48(4):357～363
[141]M Murakami, T Ohtake, RA Dorschner, et al. Cathelicidin Antimicrobial Peptides are Expressed in Salivary Glands and Saliva. Journal of Dental Research.2002,81:845～850
[142]F Barchiesi, AL Colombo, DA Mcgough, et al. Comparative Study of Broth Microdilution and Macrodilution Techniques for in vitro Antifungal Susceptibility Testing of Yeasts by Using the National Committee for Clinical Laboratory Standards' Proposed Standards. Journal of Clinical Microbiology.1994,32:2494～2500
[143]JM Conlon, A al-Dhaheri, E al-Mutawa, et al. Peptide Defenses of the Cascades Frog Rana cascadae:Implications for the Evolutionary History of Frogs of the Amerana Species Group. Peptide.2007,28:1268～1274
[144]Lu Yi, Ma Yufang, Wang Xu, et al. The First Antimicrobial Peptide from Sea Amphibian. Molecular Immunology.2008,45:678～681
[145]苏立强,郑永杰.色谱分析方法.北京：清华大学出版社,2009：41～49
[146]RJ Simpson. Protein and Proteomics:A Laboratory Manual. New York:Cold Spring Harbor Laboratory,2004:219～230
[147]REW Hancock, R Lehrer. Cationic Peptides:a New Source of Antibiotics. Trends in Biotechnology.1998,16:82～88
[148]O Chertov, DF Michiel, L Xu, et al. Identification of Defensin-1, Defensin-2, and CAP37/azurocidin as T cell Chemoattractant Proteins Released from Interleukin28-stimulated Neutrophils. J Biol Chem.1996,271(6):2935～2940
[149]DJ Davidson, AJ Currie, GS Reid, et al. The Cationic Antimicrobial Peptide LL-37 Modulates Dendritic Cell Differentiation and Dendritic Cell-induced T cell Polarization. J Immunol.2004,172(4):1146～1156
[150]HJ Huang, CR Ross, F Blecha. Chemoattractant Properties of PR-39, a Neutrophil Antibacterial Peptide. J Leukoc Biol.1997,61(5):624～629
[151]B Agerberth, J Charo, J Werr, et al. The Human Antimicrobial and Chemotactic Peptides LL-37 and Alpha-defensins are Expressed by Specific Lymphocyte and Monocyte Populations. Blood.2000,96(9):3086～3093
[152]N Mookherjee, REW Hancock. Cationic Host Defence Peptides:Innate Immune Regulatory Peptides as a Novel Approach for Treating Infections. Cell Mol Life Sci,2007. 64(728):922～933
[153]DM Bowdish, DJ Davidson, DP Speert, et al. The Human Cationic Peptide LL-37 Induces Activation of the Extracellular Signal Regulated Kinase and p38 Kinase Pathways in Primary Human Monocytes. J Immunol.2004,172(6):3758～3765
[154]X Chen, F Niyonsaba, H Ushio, et al. Human Cathelicidin LL-37 Increases Vascular Permeability in the Skin via Mast Cell Activation and Phosphorylates MAP Kinases p38 and ERK in Mast Cells. J Dermatol Sci.2006,43(1):63～66