Ocellatins: New Antimicrobial Peptides from the Skin Secretion of the South American Frog <Emphasis Type="Italic">Leptodactylus ocellatus</Emphasis> (Anura: Leptodactylidae)

The emergence, in recent years, of microbial resistance to commonly used antibiotics has aroused a search for new naturally occurring bactericidal and fungicidal agents that may have clinical utility. In the present study, three new antimicrobial peptides were purified from the electrical-stimulated skin secretion of the South American frog Leptodactylus ocellatus by reversed-phase chromatographic procedures. Ocellatin 1 (¹GVVDILKGAGKDLLAHLVG ISEKV²⁵-CONH²), ocellatin 2 (¹GVVDILKGAGKDLLAHLVGKISEKV²⁵-CONH₂) and ocellatin 3 (¹GVLDILKNAAKNILAHAAEQI²¹-CONH₂) are structurally related peptides. These peptides present hemolytic activity against human erythrocytes and are also active against Escherichia coli. Ocellatins exhibit significant sequence similarity to other amphibian antimicrobial peptides, mainly to brevinin 2ED from Rana esculenta.     

Molecular cloning of skin peptide precursor-encoding cDNAs from tibial gland secretion of the Giant Monkey Frog, Phyllomedusa bicolor (Hylidae,Anura)

The skins of phyllomedusine frogs have long been considered as being tremendously rich sources of bioactive peptides. Previous studies of both peptides and cloning of their precursor encoding cDNAs have relied upon methanolic skin extracts or the dissected skins of recently deceased specimens and have not considered the different glands in isolation. We therefore focused our attention on the tibial gland of the Giant Monkey Frog, Phyllomedusa bicolor and constructed a cDNA library from the skin secretion that was obtained via mechanical stimulation of this macrogland. Using shotgun cloning, four precursors encoding host-defense peptides were identified: two archetypal dermaseptins, a phyllokinin and a phylloseptin that is new for this species but has been recently described from the Waxy Monkey Leaf Frog, Phyllomedusa sauvagii. Our study is the first to report defensive peptides specifically isolated from anuran tibial glands, confirming the hypothesis that these glands also contribute to chemical defense. Moreover, the discovery of novel compounds for this otherwise very well characterized species suggests that this largely neglected gland might possess a different cocktail of secretions from glands elsewhere in the same animal. We will also discuss some evolutionary implications of our findings with respect to the adaptive plasticity of secretory glands.     

Purification and cDNA Cloning of Antimicrobial Peptides from the Skin Secretion of the Chinese Frog Rana chensinensis

The skin secretions of amphibians are a rich source of bioactive peptides. We isolated chensirin-1 and chensirin-2 from the skin secretion of the Chinese frog Rana chensinensis. Sephadex-G-50 and RP-HPLC were employed to purify these peptides. The amino acid sequences of these peptides were VLPLVGNLLNDLLGE and IIPLPLGYFAKKT, respectively, as determined by Edman degradation. The molecular weights were 1578.7 and 1460.8 Da, respectively, as analyzed by HPLC-ESI-MS. The chensirin cDNA was cloned by 5′ and 3′ amplification of cDNA ends, synthesized and purified. The antibacterial activities of the chensirins were tested using minimum inhibitory concentration, the results indicated that chensirins inhibit the growth of gram-negative and gram-positive bacteria. Among them, chensirin-1 is a novel peptide with a higher antibacterial activity compared to other similar antimicrobial peptides. These low molecular weight peptides with good antimicrobial efficacy are considered potential sources for developing new antimicrobial agents to improve traditional drug resistance.

     

蛙皮抗菌肽促胰岛素分泌功能研究进展

随着对蛙皮抗菌肽功能研究的不断深入,陆续发现部分肽具有促胰岛素分泌活性,该活性对于2型糖尿病治疗具有较好的应用前景。蛙皮抗菌肽即可以通过克服注射胰岛素产生的低血糖反应,又能改善2型糖尿病胰岛素抵抗的问题,这使其有希望成为安全、高效治疗2型糖尿病药物的新药物。本文综述了具有促胰岛素分泌功能的蛙皮抗菌肽的序列特征和工作机制的研究进展,为进一步开展相关研究提供参考。     

多疣狭口蛙皮肤分泌物中FSH和LH的分离及在不同繁殖期的表达

运用聚丙烯酰胺凝胶电泳、葡聚糖凝胶层析、酶联免疫等实验方法,对多疣狭口蛙Kaloula verrucosa繁殖前期、繁殖期、繁殖后期的皮肤分泌物进行初步的成分分离,并对其不同时期皮肤分泌物成分中卵泡刺激素(Follicle stimulating hormone,FSH)及黄体生成素(Luteinizing hormone, LH)进行了免疫检测和定量比较.结果 表明:多疣狭口蛙皮肤分泌物中FSH的含量在其繁殖前期处于三个时期中的最高,繁殖期次之,繁殖后期最低;而LH则是在繁殖期含量最高,繁殖前期含量略偏低,到繁殖后期含量最低.     

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH₂ inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH₂ is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH₂ induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH₂ was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH₂ was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH₂ has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH₂ is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.     

The Maculatin peptides from the skin glands of the tree frog Litoria genimaculata: a comparison of the structures and antibacterial activities of Maculatin 1.1 and Caerin 1.1

Six peptides have been isolated and characterized from the dorsal glands of the tree frog Litoria genimaculata. One of these is the known hypotensive peptide caerulein; the others have been named maculatins. The amino acid sequences of the maculatin peptides have been determined using a combination of fast atom bombardment mass spectrometry and automated Edman sequencing. Four of the maculatin peptides show antibiotic activity, with maculatin 1.1 [GLFGVLAKVAAHVVPAIAEHF(NH₂;)] showing the most pronounced activity, particularly against Gram-positive organisms. Maculatin 1.1 resembles the known caerin 1 antibiotic peptides, except that four of the central amino acid residues (of the caerin 1 system) are missing in maculatin 1.1. A comparison of the antibiotic activity of maculatin 1.1 with those of caerin 1.1 is reported. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

黄粉虫防御性分泌物抑菌活性的研究

目的:研究黄粉虫防御性分泌物的化学成分及其抑菌活性。方法:用二氯甲烷萃取分泌物并经GC/MS分析,后用牛津杯法和平板连续稀释法分别就该分泌物对大肠杆菌、金黄色葡萄球菌、白色假丝酵母、黑曲霉、桔青霉的抑菌作用及2-甲基对苯醌和对甲酚标准品对上述供试菌的最低抑菌浓度进行测定和比较。结果:分泌物含2-甲基对苯醌、对甲酚和正二十三烷等7种成分,抑菌强度:桔青霉>大肠杆菌和金黄色葡萄球菌>白色假丝酵母>黑曲霉。结论:该分泌物对这5种供试菌的抑制效果优于2-甲基对苯醌和对甲酚标准品。     

Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, <Emphasis Type="Italic">Pelophylax kl. Esculentus</Emphasis>

The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts’ innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a ‘shotgun’ cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.     

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μm) than against E. coli (64 μm) and an intermediate cytotoxicity against hRBC (30 μm). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.     

Vasorelaxin: A Novel Arterial Smooth Muscle-Relaxing Eicosapeptide from the Skin Secretion of the Chinese Piebald Odorous Frog (Odorrana schmackeri)

The defensive skin secretions of amphibians are a rich resource for the discovery of novel, bioactive peptides. Here we report the identification of a novel vascular smooth muscle-relaxing peptide, named vasorelaxin, from the skin secretion of the Chinese piebald odorous frog, Odorrana schmackeri. Vasorelaxin consists of 20 amino acid residues, SRVVKCSGFRPGSPDSREFC, with a disulfide-bridge between Cys-6 and Cys-20. The structure of its biosynthetic precursor was deduced from cloned skin cDNA and consists of 67 amino acid residues encoding a single copy of vasorelaxin (vasorelaxin, accession number: {"type":"entrez-nucleotide","attrs":{"text":"HE860494","term_id":"429534177","term_text":"HE860494"}}HE860494). Synthetic vasorelaxin caused a profound relaxation of rat arterial smooth muscle with an EC₅₀ of 6.76 nM.     

肺内调节肽对兔支气管上皮细胞分泌白介素的影响

为探讨肺内调节肽对气管上皮细胞（brochial epithelial cells,BECs）分泌功能的影响,实验观察了兔BECs在未受应激与臭氧应激两种条件下白细胞介素（interleukin,ILs）的分泌。结果发现：血管活性肠肽（vasoac-tive intestinal peptide,VIP）以未受应激BECs存在抑制作用,并使用臭氧应激BECs分泌ILs下降;表皮生长因子（epi-dermal growth factor,EGF）使未受应激BECs IL-1、IL-8分泌增加,使臭氧应激的BECs ILs分泌降低;内皮素-1（endothelin-1,ET-1）、降钙素基因相关肽(calctionin gene-related peptide,CGRP）可使未受应激的BECs分泌ILs增加,CGRP还可使臭氧应激的BECs ILs分泌增加。结果提示：肺内调节肽可调控BECs ILs的分泌,在调控气道炎症损伤信号传递方面具有一定的作用。     

Carboxypeptidase Activity of the Zinc Metalloprotease in the Cement Precursor Secretion of the Barnacle, Chthamalus fragilis Darwin

The liquid cement precursor secretion (CPS) of the adult barnacle, Chthamalus fragilis, exhibits Zn-metalloprotease activity. To assess the bond specificity of the Zn-metalloprotease, samples of liquid CPS were collected by glass micropipette from the exposed bases of adult barnacles and incubated in 50 mM Tris buffer, pH 8.0, containing 10 mM Ca⁺⁺, at 27°C or 20°C for 6 or 24 hr. in the presence of fluorescent dye-labeled synthetic C-1 and A-1 PepTag peptides. These peptides have a net positive charge and migrate toward the anode during agarose gel electrophoresis. When incubated with the C-1 PepTag peptide, CPS samples generated F1 hydrolysis fragments that failed to migrate during electrophoresis in 0.8% agarose gels, indicating the presence of proteolytic activity in the CPS. Proteolysis of the C-1 peptide was inhibited by 2.0 mM orthophenanthroline in the presence of 10 mM Ca⁺⁺ ions. No hydrolysis products were generated when samples of CPS were incubated in the presence of the A-1 PepTag peptide. This suggested that the CPS contained a Zn-metalloprotease that exhibited a preference for the carboxy-terminal lysine of the C-1 PepTag peptide. There were no indications of aminopeptidase or endopeptidase activities in the barnacle CPS. When incubated with hippuryl-lysine and hippuryl-arginine substrates, samples of CPS gave activities ranging from 111 to 319 μmol hippuric acid formed/min/mg of CPS protein. These observations indicate that the CPS of C. fragilis contains a Zn-metallo-exoprotease with a preference for carboxy-terminal basic amino acids.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

Factors contributing to the potency of antimicrobial cationic peptides from the N-terminal region of human lactoferrin

This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.