Antimicrobial peptide defenses of the Tarahumara frog,Rana tarahumarae

Populations of the Tarahumara frog Rana tarahumarae have decreased markedly in recent years in the northern part of their range. Infection by the chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines. To determine whether antimicrobial peptides in the skin provide protection against this pathogen, norepinephrine-stimulated skin secretions were tested for their ability to inhibit growth of B. dendrobatidis in vitro. After concentration, crude mixtures of skin peptides inhibited the growth of the chytrid in a concentration-dependent manner. Proteomic analysis led to the identification and characterization of three peptides belonging to the brevinin-1 family of antimicrobial peptides and three belonging to the ranatuerin-2 family. The two most abundant peptides, ranatuerin-2TRa (GIMDSIKGAAKEIAGHLLDNLKCKITGC) and brevinin-1TRa (FLPVIAGIAANVLPKLFCKLTKRC), were active against B. dendrobatidis (MIC of 50 microM for ranatuerin-2TRa and 12.5 microM for brevinin-1TRa against zoospores). These data clearly show that antimicrobial peptides in the skin secretions of the Tarahumara frog are active against B. dendrobatidis and should provide some protection against infection. Therefore, the observed susceptibility of these frogs to this pathogen in the wild may be due to the effects of additional environmental factors that impair this innate defense mechanism, leading to the observed population declines.     

Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex

Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC<5 microM)), were identified. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships.     

Evidence from peptidomic analysis of skin secretions that the red-legged frogs, Rana aurora draytonii and Rana aurora aurora, are distinct species

The northern red-legged frog Rana aurora aurora and the California red-legged frog Rana aurora draytonii are traditionally classified together in the same species group. Ten peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of R. aurora draytonii and purified to near homogeneity. The peptides were identified as belonging to the ranatuerin-2 family (two peptides), brevinin-1 family (four peptides), temporin family (three peptides), and a novel peptide, RV-23 (RIGVLLARLPKLFSLFKLMGKKV) that has limited structural similarity to the bee venom peptide, melittin. This distribution of peptides contrasts with that found previously in skin secretions from R. aurora aurora collected under the same conditions and at the same time of year (one ranatuerin-2 peptide, two brevinin-1 peptides, and one temporin peptide). The variation in amino acid sequences between corresponding R. aurora draytonii and R. aurora aurora peptides is comparable with the variation in sequences of orthologs from other members of the Amerana group of New World ranid frogs (Rana boylii, Rana muscosa, and Rana luteiventris). It is proposed, therefore, that the red-legged frogs should be regarded as separate species (R. aurora and R. draytonii) within the Amerana group rather than conspecific subspecies. The data emphasize that amino acid sequences of antimicrobial peptides in skin secretions may be used to infer taxonomic and phylogenetic relationships between species of ranid frogs.     

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.     

Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami

Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 microM against Escherichia coli, 12.5 microM against Staphylococcus aureus and 50 microM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50 = 40 microM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.     

Isolation of peptides of the brevinin-1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii.

The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions from the foothill yellow-legged frog Rana boylii. Brevinin-1BYa (FLPILASLAA10KFGPKLF CLV20TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 microm] and also active against Escherichia coli (MIC = 17 microm) and Staphylococcus aureus (MIC = 2 microm), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC50 = 4 microm). The single amino acid substitution (Phe12 --> Leu) in brevinin-1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys11 --> Thr, Phe17 --> Leu and Val20 --> Ile) in brevinin-1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin-2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.     

An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina

A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1 Da, against the Gram-positive bacterium Staphylococcus aureus was 7 microM and against the Gram-negative bacterium Escherichia coli was 30 microM.     

A family of brevinin-2 peptides with potent activity against Pseudomonas aeruginosa from the skin of the Hokkaido frog, Rana pirica

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.     

Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis)

Skin secretions were obtained from male, female, and juvenile specimens of the mink frog (Rana septentrionalis) by electric stimulation and shown to contain 10 peptides that differentially inhibited the growth of microorganisms. The elution profiles of secretions from the three groups following reverse-phase HPLC were almost identical indicating that there were no major sexual or developmental differences in chemical composition. Four peptides of the brevinin-1 family, with potent antimicrobial activity and strong hemolytic activity, two members of ranatuerin-2 family and three members of the temporin family, were purified and characterized structurally. A 21-amino-acid C-terminally alpha-amidated peptide (GIWDTIKSMGKVFAGKILQNL.NH(2)) with broad-spectrum antimicrobial activity was also isolated from the skin secretions. This peptide shows limited structural similarity with the N-terminal region of brevinin-2 peptides previously isolated from R. temporaria skin but lacks the C-terminal cyclic heptapeptide domain associated with this family. Molecular and morphological data support the placement of R. septentrionalis in the R. catesbeiana species group, but analysis based upon the distribution of the molecular forms of the antimicrobial peptides is indicative of a closer phylogenetic relationship between R. septentrionalis and the frogs of the R. pipiens and R. boylii groups.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

A family of acyclic brevinin-1 peptides from the skin of the Ryukyu brown frog Rana okinavana

The 24 amino-acid residue antimicrobial peptide, brevinin-1 is synthesized in the skins of a wide range of species of Eurasian and North American frogs belonging to the genus Rana. All previously characterized brevinin-1 peptides contain the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule. Four structurally related peptides were isolated from an extract of the skin of the Ryukyu brown frog Rana okinavana. The amino acid sequences of the peptides [Phe-(Xaa)4-Ile-(Xaa)2-Leu-Ala-Lys-Gly-Leu-Pro-Ser-Leu-Ile-Xaa-Leu-Xaa-Lys-Lys.NH2] identified them as members of the brevinin-1 family that lacked the COOH-terminal cyclic domain but contained a C-terminally alpha-amidated residue. It is suggested, as one possibility, that the Cys18 in the brevinin-1 consensus sequence has been deleted and the Cys24 residue has mutated to a glycine that acts as substrate for peptidyl-glycine alpha-amidating monooxygenase. The peptides potently inhibited the growth of Escherichia coli and Staphylococcus aureus confirming that a cyclic domain is not necessary for antimicrobial activity. A fifth peptide (SFLNFFKGAA10KNLLAAGLDK20LKCKISGTQC30), that also displayed broad-spectrum antimicrobial activity, was isolated from the skin extract and showed structural similarity with members of the ranatuerin-2 family previously isolated from the skin of North American ranid frogs.     

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.     

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

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-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d 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 alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). 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.     

Antimicrobial peptides from the skin of the Tsushima brown frog Rana tsushimensis

The Tsushima brown frog Rana tsushimensis Stejneger, 1907 exists in reproductive isolation on the island of Tsushima, Japan. Six peptides with antimicrobial activity were isolated in pure form from an extract of the skin of this species and their amino acid sequences identified them as members of the brevinin-1 (one peptide), brevinin-2 (one peptide) and temporin (four peptides) families. The C-terminally alpha-amidated brevinin-1 peptide (FLGSIVGALASALPSLISKIRN.NH2) lacks the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule that characterizes other members of that family. A structurally similar brevinin-1 peptide, also lacking the cyclic domain, was previously isolated from the skin of the Ryukyu brown frog Rana okinavana, indicative of a close phylogenetic relationship between the species. Brevinin-2TSa (GIMSLFKGVLKTAGKHVAGSLVDQLKCKITGGC) showed broad-spectrum growth inhibitory activity against a range of Gram-negative and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) (minimum inhibitory concentrations< or =25 microM) and relatively low hemolytic activity against human erythrocytes (LD50=100 microM). The peptide therefore represents a candidate for drug development.     

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.     

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1-3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.     

Antimicrobial properties of two purified skin peptides from the mink frog (Rana septentrionalis) against bacteria isolated from the natural habitat

Numerous peptides exhibiting antimicrobial properties have been isolated from the skins of many amphibian species. These peptides offer an innate chemical defense system against various microbial agents that exist in the amphibian's environment. Amphibian skin peptides are typically tested for antimicrobial activity against microbial strains that are pathogenic to humans, but not on potential pathogenic or opportunistic bacteria that exist in the organism's habitat. Two peptides, a brevinin-2-related peptide and temporin-1SPb previously isolated from secretions of the mink frog, Rana septentrionalis, were tested for antimicrobial activity on bacterial isolates endemic to the frog's habitat. Ten isolates were identified, using 16S rRNA gene sequencing techniques, in the genera Pseudomonas, Serratia, Bacillus, Aeromonas, Burkholderia, Microbacterium, and Delftia. Bacterial isolates were tested with peptides at concentrations ranging from 0.8 microM to 1000 microM to determine the minimum inhibitory concentration (MIC) to inhibit growth. Growth of four of the isolates was inhibited by temporin-1SPb at the concentrations used, but all of the isolates were inhibited by the brevinin-2-related within the range of peptide concentrations used. This demonstrates the efficacy of both peptides as a component of the frog's innate chemical defense system.     

Purification and characterization of antimicrobial peptides from the skin of the North American green frog Rana clamitans

Ten 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 a North American frog, the green frog Rana clamitans. Ranatuerin-1C (SMLSVLKNLGKVGLGLVACKINKQC), ranalexin-1Ca (FLGGLMKAFPALICAVTKKC), ranalexin-1Cb (FLGGLMKAFPAIICAVTKKC), ranatuerin-2Ca (GLFLDTLKGAAKDVAGKLLEGLKCKIAGC KP), and ranatuerin-2Cb (GLFLDTLKGLAGKLLQGLKCIKAGCKP), are members of three previously characterized families of antimicrobial peptides, first identified in the North American bullfrog Rana catesbeiana. In addition, five structurally related peptides (temporin-1Ca, -1Cb, -1Cc, -1Cd, and -1Ce), comprising 13 amino acid residues and containing a C-terminally alpha-amidated residue, belong to 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 Asian and European Ranid frogs, were not identified in the extract. The data support the hypothesis that the distribution and amino acid sequences of the skin antimicrobial peptides are valuable tools in the identification and classification of Ranid frogs.     

Purification and characterization of antimicrobial peptides from the skin secretion of Rana dybowskii

Six antimicrobial peptides designated dybowskins were isolated from the skin secretion of Rana dybowskii, an edible frog in Korea. Dybowskin-1 (FLIGMTHGLICLISRKC) and dybowskin-2 (FLIGMTQGLICLITRKC) were isoforms differing in only two amino acid residues at the 7th and 14th positions from the N-terminus, and they showed amino acid sequence similarities with ranalexin peptides. Dybowskin-3 (GLFDVVKGVLKGVGKNVAGSLLEQLKCKLSGGC), dybowskin-4 (VWPLGLVICKALKIC), dybowskin-5 (GLFSVVTGVLKAVGKNVAKNVGGSLLEQLKCKISGGC), and dybowskin-6 (FLPLLLAGLPLKLCFLFKKC) differed in both size and sequence, and they were, in terms of amino acid sequence similarities, related to brevinin-2, japonicin-2, esculentin-2, and brevinin-1 peptides, respectively. All the peptides presented in this paper contained Rana-box, the cyclic heptapeptide domain, which is conserved in other antimicrobial peptides derived from the genus Rana. All the dybowskin peptides showed a broad spectrum of antimicrobial activity against the Gram-positive and Gram-negative bacteria (minimum inhibition concentrations (MIC), 12.5 to >100 microg/ml) and against Candida albicans (MIC, 25 to >100 microg/ml). Especially, dybowskin-4 with valine at its N-terminus was the most abundant and showed the strongest antimicrobial activity among all the dybowskin peptides. This result indicates that the dybowskin peptides from R. dybowskii, whose main habitats are mountains or forests, have evolved differently from antimicrobial peptides isolated from other Korean frogs, whose habitats are plain fields.     

A potent, non-toxic insulin-releasing peptide isolated from an extract of the skin of the Asian frog, Hylarana guntheri (Anura:Ranidae)

Peptides in extract of the skin of the Asian frog Hylarana guntheri Boulenger,1882 were purified by reversed-phase HPLC and individual components analysed for their ability to release insulin from the rat BRIN-BD11 clonal beta cell line. The most potent peptide identified in the extract belonged to the brevinin-2 family (brevinin-2GUb; GVIIDTLKGAAKTVAAELLRKAHCKLTNSC). Other peptides with weaker insulin-releasing activity belonged to the brevinin-1 (2 peptides), brevinin-2 (2 peptides) and temporin (3 peptides) families. Only the brevinin-1 peptides showed cytolytic activity against the BRIN-BD11 cells, as demonstrated by an increased rate of release of the cytosolic enzyme, lactate dehydrogenase. A synthetic replicate of brevinin-2GUb produced a significant stimulation of insulin release (139% of basal rate; P<0.05) at a concentration of 100 nM with a maximum response of 373% of basal rate at a concentration of 3 microM) by a mechanism that did not involve mobilization of intracellular calcium. Brevinin-2GUb also inhibited the growth of microorganisms (MIC against Escherichia coli=32 microM, Staphylococcus aureus=64 microM, and Candida albicans=64 microM) but had only weak hemolytic activity against human erythrocytes (LC(50)=700 microM). Administration of brevinin-2GUb (75 nmol/kg body weight) into mice significantly (P<0.05) improved glucose tolerance following a intraperitoneal injection of glucose, thereby demonstrating that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.