大鲵皮肤分泌液中抗菌肽对铜绿假单胞菌感染小鼠创面的抗菌作用

观察大鲵皮肤分泌液中抗菌肽对铜绿假单胞菌感染小鼠创面的抗菌作用.方法用5％醋酸浸提和Sephadex G -50、G-25凝胶过滤层析等方法分离抗菌肽;采用Tricine - SDS - PAGE电泳鉴定,抑菌圈法检测抗菌活性;切除30只ICR小鼠背部1 cm×1 cm全层皮肤,涂抹铜绿假单胞菌菌液制成感染模型,随机均...     

两栖类皮肤抗菌肽的分离纯化与抗菌活性检测技术

两栖类皮肤抗菌肽由于具有广谱、高效的抗菌活性,且不引起微生物耐药性等特点,已成为目前开发新型抗菌药物的理想材料。文章概述了两栖类抗菌肽的主要特点及其分离纯化和抗菌活性检测的主要方法,并对这些技术的原理、方法及其近年来在抗菌肽研究中的进展进行了介绍。同时本文还比较了各种方法的优缺点,对当前研究中所存在的一些问题进行了初步的探讨,以期为两栖类抗菌肽的应用提供理论依据。     

羊小肠抗菌肽分离及其生物活性研究

目的:从羊小肠提取抗菌肽并对其抑菌作用进行研究,寻找廉价易得的广谱高效天然抗菌肽。方法:葡聚糖凝胶分离及反相高效液相色谱法提纯羊小肠活性抗菌肽;采用琼脂平板打孔法进行抑菌效果的测定;SDS-PAGE电泳分析相对分子质量。结果:反相高效液相色谱法分离出现3个主峰,其中之一对大肠杆菌和金葡球菌有较高抑菌活性(蛋白浓度分别为38,19,10 m.gmL-1);相对分子质量约为40 Ku。结论:实验得到的抗菌肽材料易得,对G-菌和G+菌均有较强的抑制作用。     

人内源性抗菌肽的多样性研究

目的　从人单个核白细胞和子宫颈及子宫内膜粘液酸溶性提取物中分离纯化新的抗菌肽。方法　电泳凝胶琼脂糖弥散法分析 r IL- 2刺激培养的人单个核白细胞和子宫颈及子宫内膜粘液酸溶性提取物抗菌组分 ,用制备性酸性尿素聚丙烯酰胺凝胶电泳初步分离并制备电泳纯的各抗菌组分 ,反相高压液相色谱技术进一步纯化 ,琼脂糖弥散法分析其抗菌活性 ,Tricine- SDS- PAGE鉴定其纯度和分子量 ,用 Edman降解法进行氨基酸序列测定。结果　从刺激培养的人单个核白细胞分别纯化出 HL P- 2 / 2 3、HL P- 3/ 2 1两个抗菌肽分子 ,分子量分别是 7.84和 16 .86 ku;从子宫颈粘液及子宫内膜粘液分别分离纯化出 HCP- 1/ 2 1、HUP1/ 38两个抗菌肽分子 ,分子量分别为 13.89和 5 .95 ku。测定 HL P- 3/ 2 1分子的前 10位 N端氨基酸序列为PKRKAEGDAK。结论　人体抗菌肽分子存在多样性 ,HL P- 2 / 2 3、HL P- 3/ 2 1、HCP- 1/ 2 1和 HU P1/ 38可能为新的抗菌肽分子     

一种高效制备重组抗菌肽S-thanatin的方法

S-thanatin是一个含有21个氨基酸的抗菌肽,以融合形式在大肠杆菌体内表达,载体构建时人工设计了一个凝血酶位点。为降低重组抗菌肽S-thanatin的生产成本以利于工业化生产,探索了一个处理速度快且成本较低的分离纯化策略。通过大肠杆菌pET-32a/BL21(DE3)系统高效可溶表达融合蛋白后,首先分析了融合蛋白的表达部位,然后利用融合蛋白热稳定性高的性质通过加热去除不耐热的大分子质量蛋白,同时降低蛋白酶对融合蛋白的影响。离心后溶液通过中空纤维超滤来浓缩融合蛋白,之后进行固定化凝血酶酶切。酶切后的TrxA片段和目的多肽利用其分子质量的差异,使用超滤膜包进行分离,最后使用制备反相高效液相对S-thanatin进行精制,并检测其对大肠杆菌ATCC 25922的抗菌活性。结果表明,通过这一简单的纯化工艺,可制备出纯度95%以上的S-thanatin,并具有很好的抗菌活性。     

蛙类皮肤及其分泌物中生物活性肽的研究进展

蛙类的皮肤及其分泌物中包含了种类繁多、功能复杂的生理活性物质。此文综述了蛙类皮肤及其分泌物中生物活性多肽的最新研究进展,重点介绍了多肽的种类以及它们的结构与功能,并展望了蛙类活性肽在食品保鲜与防腐和临床上的应用前景。     

板栗中蛋白质的分离鉴定及活性研究

目的探讨板栗中蛋白质的分离纯化及其促肾细胞生长作用和抗肿瘤作用及机制。方法从板栗中提取出的水溶性蛋白质,经鉴定是欧栗球蛋白(CAS)。利用MTT法检测该蛋白对正常肾细胞和肝癌细胞HepS,小鼠肉瘤S180腹水型肿瘤细胞增殖的影响;检验其对小鼠T细胞和B细胞增殖作用的影响;用DNA电泳方法检测该蛋白诱导HepS细胞凋亡的作用。结果 CAS在体外对正常肾细胞的增殖有促进作用,对试验的肿瘤细胞的增殖均有抑制作用,对T细胞和B细胞的增殖有促进作用,对HepS细胞具有诱导凋亡的作用。结论 CAS具有促肾细胞生长和抗肿瘤作用,其抗肿瘤作用机制可能通过提高机体免疫力和诱导细胞凋亡来实现。     

人源性抗菌肽的研究现况

抗菌肽又称为肽抗生素是约于20～40个氨基酸组成的一类具有生物活性多肽，它具有分子量小、热稳定、水溶性好、抗菌谱广等优点。除有非特异性的抗细菌、病毒、真菌、原虫、及抗肿瘤作用外，还是生物体天然免疫的重要组成。因此近年来人源性抗菌肽成为了国际国内研究的热点之一。目前世界已发现的抗菌肽类物质已超过1700多种，广泛存在于动植物界，而人源性抗菌肽发现甚少，主要由三类组成，分别是防御素、cathecidins，histatins。但人源性抗菌肽同其它生物抗菌肽相比人源性抗菌肽具有难以比拟的优越性，且应用前景巨大。本文就人源性抗菌肽上述三类以及它们作用机制、应用前景及意义进行综述。     

杂合抗菌肽HMCM的原核表达及其活性鉴定

将哺乳动物抗菌肽(Hexapeptide)、两栖类抗菌肽(Magainin)、昆虫抗菌肽(Cecropin A和Melittin)的有效作用部分拼接在一起,以获得一种新型的杂合抗菌肽HMCM。根据HMCM氨基酸序列和大肠杆菌B细胞的密码子偏好性设计了HMCM的基因序列;将目的基因克隆到原核表达载体pET-32a-c(+),构建了重组表达载体pET-32a-HMCM;再转化原核表达菌株E.coli BL21(DE3)实现融合表达,通过摸索诱导时间、IPTG浓度、OD600和温度等条件确定了表达最佳条件,并确定了融合蛋白以可溶形式存在;通过亲和层析纯化了融合蛋白,并进行了Western-blotting鉴定;最后用EK酶酶切融合蛋白释放出HMCM,对HMCM的抗菌性进行了初步研究。结果表明HMCM对枯草芽孢杆菌有良好的抑菌作用。     

硫酸皮肤素的研究概况

介绍了硫酸皮肤素的基本性质、生理功能、应用及国内外对硫酸皮肤素的提取、纯化研究的概况。     

Characterization of antimicrobial peptides from the skin secretions of the Malaysian frogs, Odorrana hosii and Hylarana picturata (Anura:Ranidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions from Hose's rock frog Odorrana hosii (Boulenger, 1891) led to the isolation of 8 peptides with differential antibacterial activities. Structural characterization demonstrated that the peptides belonged to the esculentin-1 (1 peptide), esculentin-2 (1 peptide), brevinin-1 (2 peptides), brevinin-2 (2 peptides), and nigrocin-2 (2 peptides) families of antimicrobial peptides. Similar analysis of skin secretions from the Malaysian fire frog Hylarana picturata (Boulenger, 1920) led to the isolation and characterization of peptides belonging to the brevinin-1 (2 peptides), brevinin-2 (5 peptides), and temporin (1 peptide) families. The differences in antimicrobial activities of paralogous peptides provide insight into structure-activity relationships, emphasizing the importance of cationicity in determining potency. The substitution Lys(11)-->Gln in brevinin-1HSa (FLPAVLRVAAKIVPTVFCAISKKC) from O. hosii abolishes growth inhibitory activity against Escherichia coli but has no effect on the high potency (MIC=8mug/ml) against Staphylococcus aureus. In contrast, the substitution (Gly(4)-->Asp) in brevinin-2PTb (GFKGAFKNVMFGIAKSAGKSALNALACKIDKSC) from H. picturata reduces activity against both E. coli and S. aureus. Cladistic analysis based upon the amino acid sequences of the brevinin-2 peptides from Asian frogs provides evidence for sister taxon relationships between O. hosii and O. livida and between H. picturata and H. güntheri.     

Two antimicrobial peptides from skin secretions of Rana grahami.

Two antimicrobial peptides manifested a broad spectrum of antimicrobial activity against various microorganisms have been isolated from skin secretions of Rana grahami. These antimicrobial peptides were named grahamin 1 and grahamin 2. Their primary structures are GLLSGILGAGKNIVCGLSGLC and GLLSGILGAGKHIVCGLSGLC, respectively, determined by Edman degradation and mass spectrometry. They are structurally related to nigrocins identified from skin secretions of the dark-spotted frog, Rana nigromaculata. The cDNA clones encoding the precursor of grahamins were screened and sequenced from the skin cDNA library of R. grahami. The amino sequences deduced from the cDNA sequences match well with the results from Edman degradation. As other antimicrobial peptides from Rana species, grahamins contain a C-terminal loop region delineated by an intra-disulfide bridge named Rana box. Based on structural comparison of grahamin with other known antimicrobial peptides, grahamins could be classified into the family of antimicrobial peptides containing a single intra-disulfide bridge.     

Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog,Rana sphenocephala

Abstract: Three peptides with growth-inhibitory activity towards the Gram-negative bacterium Eschericia coli were isolated from electrically stimulated secretions from the skin of the southern leopard frog, Rana sphenocephala. Structural characterization demonstrated that the peptides [brevinin-1Sa, minimum inhibitory concentration (MIC) = 55µm ; brevinin-1Sb, MIC = 17 µm ; brevinin-1Sc, MIC = 14 µm ] represent new members of the brevinin-1 family of antimicrobial peptides, previously isolated from several other species of frogs of the genus Rana. Their high concentration in skin secretions and extreme variability in amino acid sequence suggest that the brevinin family of peptides may be of value as molecular markers for the identification and taxonomic classification of Ranid frogs.     

抗菌肽的抗肿瘤作用研究新进展

抗菌肽是一类广泛存在于生物体内的天然小分子多肽,它是机体免疫系统的天然组分,具有抗菌、抗炎及抗病毒等作用.许多抗菌肽具有抗肿瘤活性,而且在细胞选择性和耐药性方面也拥有独特的优势.本文综述抗菌肽的抗肿瘤机制及应用.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic α-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no α-helicity in buffer or liposomes, exhibits a modest degree of α-helical structure in the presence of the a-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an α-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog,Rana ornativentris

Abstract: Six peptides with antimicrobial activity were isolated from an extract of freeze‐dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin‐20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin‐20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin‐2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin‐1Oa FLPLLASLFSRLL.NH₂, 13 nmol/g; temporin‐1Ob FLPLIGKILGTI L.NH₂, 350 nmol/g; temporin‐1Oc FLPLLASLFSRLF.NH₂, 14 nmol/g; and temporin‐1Od FLPLLASLFSGLF.NH₂, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin‐2 peptides showed broad‐spectrum activity against the gram‐positive bacterium, Staphylococcus aureus, the gram‐negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic α‐helical conformation but it is significant that temporin‐1Od, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 µm compared with 2 µm for temporin‐1Oa). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Hydrophobic interactions in complexes of antimicrobial peptides with bacterial polysaccharides

Biofilms of Pseudomonas aeruginosa are responsible for chronic lung infections in cystic fibrosis patients, where they are characterized by overproduction of the exopolysaccharide alginate and are recalcitrant to treatment with conventional antibiotics. Cationic antimicrobial peptides (CAPs) are potential alternatives for the treatment of multi-drug-resistant P. aeruginosa. However, alginate in P. aeruginosa biofilms has been proposed to bind these peptides through hydrophobic interactions, consequently reducing their activity [Chan et al., J Biol Chem 2004; 279: 38749-38754]. Here we perform biophysical analyses of the interactions of alginate with a series of novel peptide antibiotics (alpha-CAPs) of prototypic sequence KK-AAAXAAAAAXAAWAAXAAA-KKKK (where X = Phe, Trp or Leu). The hydrophobic interaction interface in alginate was investigated by examining (i) the effects of polysaccharide composition with respect to D-mannuronate and L-guluronate content; (ii) glycan chain length; (iii) alpha-CAP Trp fluorescence; and (iv) 1-anilinonaphthalene-8-sulfonate fluorescence. The results show that, while M and G residues produce equivalent effects, hydrophobic interactions between alginate and alpha-CAPs require a minimal glycan chain length. Peptide interactions with alginate are deduced to be mediated by hydrophobic microdomains comprised of pyranosyl C-H groups that are inducible upon formation of alpha-CAP-alginate complexes due to charge neutralization between the two species.     

Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides

Abstract: Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram‐negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.