Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Host-defense peptides in skin secretions of African clawed frogs (Xenopodinae, Pipidae).

African clawed frogs of the Xenopodinae (Xenopus+Silurana) constitute a well-defined system in which to study the evolutionary trajectory of duplicated genes and are a source of antimicrobial peptides with therapeutic potential. Allopolyploidization events within the Xenopodinae have given rise to tetraploid, octoploid, and dodecaploid species. The primary structures and distributions of host-defense peptides from the tetraploid frogs Xenopus borealis, Xenopus clivii, Xenopus laevis, Xenopus muelleri, "X. muelleri West", and Xenopus petersii may be compared with those from the octoploid frogs Xenopus amieti and X. andrei. Similarly, components in skin secretions from the diploid frog Silurana tropicalis may be compared with those from the tetraploid frog Silurana paratropicalis. All Xenopus antimicrobial peptides may be classified in the magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) families. However, the numbers of paralogs from the octoploid frogs were not significantly greater than the corresponding numbers from the tetraploid frogs. Magainins were not identified in skin secretions of Silurana frogs and the multiplicity of the PGLa, CPF, and XPF peptides from S. paratropicalis was not greater than that of S. tropicalis. The data indicate, therefore, that nonfunctionalization (gene silencing) has been the most common fate of antimicrobial peptide genes following polyploidization. While some duplicated gene products retain high antimicrobial potency (subfunctionalization), the very low activity of others suggests that they may be evolving towards a new biological role (neofunctionalization). CPF-AM1 and PGLa-AM1 from X. amieti show potential for development into anti-infective agents for use against antibiotic-resistant gram-negative bacteria.

Radiographic diagnosis of metabolic bone disease in captive bred mountain chicken frogs (Leptodactylus fallax).

Asymptomatic captive bred and wild-caught mountain chicken frogs (Leptodactylus fallax) were radiographed for evidence of metabolic bone disease (MBD). All 22 captive bred frogs had multiple folding fractures of long bones, decreased bone density, and cortical thinning, whereas none of the 11 wild-caught frogs had any radiographic evidence of MBD. These findings suggest that the nutritional requirements of L. fallax need to be examined for captive management purposes.

Evaluation of three immobilization combinations in the capybara (Hydrochoerus hydrochaeris).

Capybaras (Hydrochoerus hydrochaeris) are the world's largest rodent. Owing to its uniqueness, 50 AZA institutions in North America display this species. As shown by a survey, no standard anesthetic protocol has been developed for this species. As a part of an ongoing behavioral study in Venezuela, capybaras were surgically implanted with radio transmitters. Animals were randomly assigned to one of the three immobilization protocols: (1) Tiletamine HCl/Zolazepam HCl, (2) Tiletamine HCl/Zolazepam HCl/Medetomidine HCl, and (3) Tiletamine HCl/Zolazepam HCl/Medetomidine HCl/Butorphanol tartrate. The protocol recommended for minimally invasive procedures when inhalant anesthetics are unavailable is a combination of Tiletamine HCl/Zolazepam HCl/Medetomidine HCl/Butorphanol tartrate. This is based on ease of administration, volume, onset of action, depth of anesthetic achieved, reversibility, safety, and costs.

Purification and characterization of antimicrobial peptides from the Caribbean frog, Leptodactylus validus (Anura: Leptodactylidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Caribbean frog Leptodactylus validus Garman, 1888 led to the identification of three peptides with previously undescribed sequences that were structurally similar to those of antimicrobial peptides isolated from other species of leptodactylid frogs. These paralogs have been termed ocellatin-V1 (GVVDILKGAGKDLLAHALSKLSEKV.NH(2)), ocellatin-V2 (GVLDILKGAGKDLLAHALSKISEKV.NH(2)), and ocellatin-V3 (GVLDILTGAGKDLLAHALSKLSEKV.NH(2)). The very low antimicrobial potency (MIC>200microM) against Escherichia coli and Staphylococcus aureus associated with the peptides is probably a consequence of their lack of amphipathicity and reduced cationicity compared with active members of the ocellatin family from related species.

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 Lys11-->Gln in brevinin-1HSa (FLPAVLRVAAKIVPTVFCAISKKC) from O. hosii abolishes growth inhibitory activity against Escherichia coli but has no effect on the high potency (MIC = 8 microg/ml) against Staphylococcus aureus. In contrast, the substitution (Gly4-->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.

Peptidomic analysis of skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae): Insight into the origin of host-defense peptides within the Pipidae and characterization of a proline-arginine-rich peptide.

The Mexican burrowing toad Rhinophrynus dorsalis is the sole extant representative of the Rhinophrynidae. United in the superfamily Pipoidea, the Rhinophrynidae is considered to be the sister-group to the extant Pipidae which comprises Hymenochirus, Pipa, Pseudhymenochirus and Xenopus. Cationic, α-helical host-defense peptides of the type found in Hymenochirus, Pseudhymenochirus, and Xenopus species (hymenochirins, pseudhymenochirins, magainins, and peptides related to PGLa, XPF, and CPF) were not detected in norepinephrine-stimulated skin secretions of R. dorsalis. Skin secretions of representatives of the genus Pipa also do not contain cationic α-helical host-defense peptides which suggest, as the most parsimonious hypothesis, that the ability to produce such peptides by frogs within the Pipidae family arose in the common ancestor of (Hymenochirus+Pseudhymenochirus)+Xenopus after divergence from the line of evolution leading to extant Pipa species. Peptidomic analysis of the R. dorsalis secretions led to the isolation of rhinophrynin-27, a proline-arginine-rich peptide with the primary structure ELRLPEIARPVPEVLPARLPLPALPRN, together with rhinophrynin-33 containing the C-terminal extension KMAKNQ. Rhinophrynin-27 shows limited structural similarity to the porcine multifunctional peptide PR-39 but it lacks antimicrobial and cytotoxic activities. Like PR-39, the peptide adopts a poly-l-proline helix but some changes in the circular dichroism spectrum were observed in the presence of anionic sodium dodecylsulfate micelles consistent with the stabilization of turn structures.

Purification and properties of laticeptin, an antimicrobial peptide from skin secretions of the South American frog Leptodactylus laticeps.

Norepinephrine-stimulated skin secretions from the Sante Fe frog Leptodactylus laticeps contained high concentrations of a peptide, termed laticeptin, with the primary structure Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Leu-Ala-Gly-His-Leu-Ala-Thr-Lys-Val-Met-Asn-Lys-Leu.NH(2). Laticeptin inhibited the growth of selected Gram-negative bacteria but the lack of activity against Gram-positive bacteria and the very low hemolytic activity is probably a consequence of the weak amphipathicity of the peptide in its alpha-helical conformation.

Peptidomic analysis of the extensive array of host-defense peptides in skin secretions of the dodecaploid frog Xenopus ruwenzoriensis (Pipidae).

The Uganda clawed frog Xenopus ruwenzoriensis with a karyotype of 2n=108 is one of the very few vertebrates with dodecaploid status. Peptidomic analysis of norepinephrine-stimulated skin secretions from this species led to the isolation and structural characterization of 23 host-defense peptides belonging to the following families: magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 6 peptides), xenopsin precursor fragment (XPF; 3 peptides), caerulein precursor fragment (CPF; 8 peptides), and caerulein precursor fragment-related peptide (CPF-RP; 3 peptides). In addition, the secretions contained caerulein, identical to the peptide from Xenopus laevis, and two peptides that were identified as members of the trefoil factor family (TFF). The data indicate that silencing of the host-defense peptide genes following polyploidization has been appreciable and non-uniform. Consistent with data derived from comparison of nucleotide sequences of mitochrondrial and nuclear genes, cladistic analyses based upon the primary structures of the host-defense peptides provide support for an evolutionary scenario in which X. ruwenzoriensis arose from an allopolyploidization event involving an octoploid ancestor of the present-day frogs belonging to the Xenopus amieti species group and a tetraploid ancestor of Xenopus pygmaeus.

Characterization of a peptide from skin secretions of male specimens of the frog, Leptodactylus fallax that stimulates aggression in male frogs.

During the breeding season of the mountain chicken frog Leptodactylus fallax, fighting between males results in the emergence of dominant animals that subsequently attract females to nesting sites. A peptide, termed Leptodactylus aggression-stimulating peptide (LASP), was isolated from norepinephrine-stimulated skin secretions from male specimens of L. fallax that was not present in skin secretions obtained from females. The primary structure of the peptide was established as: Gly-Leu-Trp-Asp-Asp-Leu-Lys-Ala-Ala-Ala-Lys-Lys-Val-Val-Ser-Ser-Leu-Ala-Ser-Ala-Ala-Ile-Glu-Lys-Leu NH2. LASP had no pheromone-like action on females but had a chemoattractive effect on males and stimulated aggressive behaviors, such as rearing and leaping. It is suggested that this peptide may play an important role in initiating the competitive male-male interactions that are associated with the onset of reproductive behavior in L. fallax.

An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae).

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.

Host-defense and trefoil factor family peptides in skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from the octoploid Mawa clawed frog Xenopus boumbaensis Loumont, 1983 led to the identification and characterization of 15 host-defense peptides belonging to the magainin (two peptides), peptide glycine-leucine-amide (PGLa; three peptides), xenopsin precursor fragment (XPF; three peptides), caerulein precursor fragment (CPF; two peptides), and caerulein precursor fragment-related peptide (CPF-RP; five peptides) families. In addition, caerulein and three peptides with structural similarity to the trefoil factor family (TFF) peptides, xP2 and xP4 from Xenopus laevis were also present in the secretions. Consistent with data from comparisons of the nucleotides sequence of mitochondrial and nuclear genes, the primary structures of the peptides suggest a close phylogenetic relationship between X. boumbaensis and the octoploid frogs Xenopus amieti and Xenopus andrei. As the three species occupy disjunct ranges within Cameroon, it is suggested that they diverged from a common ancestor by allopatric speciation.

A family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae, Hylinae) revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2 from Litoria infrafrenata (Hylidae, Pelodryadinae) and frenatin 2D from Discoglossus sardus (Alytidae). Frenatin 2.1S (GLVGTLLGHIGKAILG.NH2) and frenatin 2.2S (GLVGTLLGHIGKAILS.NH2) are C-terminally α-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG) is not. Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MIC ≤16µM) but are less active against a range of Gram-negative bacteria. Frenatin 2.1S (LC50=80±6 µM) and 2.2S (LC50=75±5 µM) are cytotoxic against non-small cell lung adenocarcinoma A549 cells but are less hemolytic against human erythrocytes (LC50=167±8 µM for frenatin 2.1S and 169±7 µM for 2.2S). Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal α-amidation for activity. Frenatin 2.1S and 2.2S significantly (P<0.05) increased production of proinflammatory cytokines IL-1ß and IL-23 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-α. Effects on IL-6 production were not significant. Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10 by LPS-stimulated cells. The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms. They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Host defense peptides from Lithobates forreri, Hylarana luctuosa, and Hylarana signata (Ranidae): phylogenetic relationships inferred from primary structures of ranatuerin-2 and brevinin-2 peptides.

The primary structures of host-defense peptides present in frog skin secretions constitute useful molecular markers for establishing taxonomic classifications and investigating phylogenetic relationships between species within a particular genus. Peptidomic analysis has led to the characterization of multiple host-defense peptides in norepinephrine-stimulated skin secretions of three species of frogs from the family Ranidae: Lithobates forreri (Boulenger, 1883), Hylarana luctuosa (Peters, 1871), and Hylarana signata (Günther, 1872). The L. forreri secretions contain ranatuerin-2 (2 peptides), brevinin-1 (4 peptides), and temporin (1 peptide). The H. luctuosa secretions contain brevinin-2 (4 peptides), esculentin-1 (1 peptide), esculentin-2 (1 peptide), palustrin-2 (2 peptides), and temporin (2 peptides). The H. signata secretions contain brevinin-2 (4 peptides), brevinin-1 (5 peptides), palustrin-2 (1 peptide), and temporin (2 peptides). Cladistic analysis based upon the primary structures of 44 ranatuerin-2 peptides from 20 Lithobates species indicates a close phylogenetic relationship between L. forreri, Lithobates onca, and Lithobates yavapaiensis. A similar cladistic analysis based upon the primary structures of 27 brevinin-2 peptides from 8 Hylarana species provides support for a close phylogenetic relationship between H. signata and Hylarana picturata, while showing that the species are not conspecific, with H. luctuosa more distantly related.

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae).

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianusAhl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensisPerret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.

Characterization of the host-defense peptides from skin secretions of Merlin's clawed frog Pseudhymenochirus merlini: insights into phylogenetic relationships among the Pipidae.

The family Pipidae comprises the genera Hymenochirus, Pipa, Pseudhymenochirus, Silurana, and Xenopus but phylogenetic relationships within the family are unclear. Peptidomic analysis of norepinephrine-stimulated skin secretions from Pseudhymenochirus merlini Chabanaud, 1920, the single species within the genus Pseudhymenochirus, led to identification of 13 host-defense peptides with antimicrobial activity. Two peptides (hymenochirin-1Pa and -1Pb) show structural similarity to hymenochirin-1B from Hymenochirus boettgeri and eight peptides (hymenochirin-5Pa, -5Pb, -5Pc, -5Pd, -5Pe, -5Pf, 5Pg and -5Ph) are structurally similar to each other and to hymenochirin-5B from H. boettgeri. Two peptides differing by a single amino acid (IKIPSFFRNILKKVGKEAVSLM/I AGALKQS), termed pseudhymenochirin-1Pa and -1Pb, and pseudhymenochirin-2Pa (GIFPIFAKLLGKVIKVASSLISKGRTE) do not resemble host-defense peptides previously isolated from pipid frogs. Hymenochirin-5Pe was the most abundant peptide in the secretions and hymenochirin-1Pa the most potent against Staphylococcus aureus (MIC=2.5µM) and Escherichia coli (MIC=10µM). The data support a close phylogenetic relationship between Hymenochirus and Pseudhymenochirus that is distinct from the Xenopodinae (Xenopus+Silurana) clade with Pipa sister-group to all other extant pipids.

Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of host-defense peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families from the tetraploid frogs, Xenopus petersii (Peters' clawed frog) and Xenopus pygmaeus (Bouchia clawed frog), and the octoploid frog Xenopus lenduensis (Lendu Plateau clawed frog). Xenopsin-precursor fragment (XPF) peptides were not detected. The primary structures of the antimicrobial peptides from X. petersii demonstrate a close, but not conspecific relationship, with Xenopus laevis whereas the X. pygmaeus peptides show appreciable variation from previously characterized orthologs from other Xenopus species. Polyploidization events within the Xenopodinae (Silurana+Xenopus) are associated with extensive gene silencing (nonfunctionization) but unexpectedly the full complement of four PGLa paralogs were isolated from X. lenduendis secretions. Consistent with previous data, the CPF peptides showed the highest growth-inhibitory activity against bacteria with CPF-PG1 (GFGSLLGKALKIGTNLL.NH(2)) from X. pygmaeus combining high antimicrobial potency against Staphylococcus aureus (MIC=6 µM) with relatively low hemolytic activity (LC(50)=145 µM).

The hymenochirins: a family of host-defense peptides from the Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae).

Skin secretions of frogs from the subfamily Xenopodinae (Xenopus+Silurana) within the family Pipidae are a rich source of antimicrobial peptides with therapeutic potential but species from the sister taxon Hymenochirus in the subfamily Pipinae (Hymenochirus+Pseudhymenochirus+Pipa) have not been investigated. Peptidomic analysis of norepinephrine-stimulated skin secretions from two distinct populations of the Congo dwarf clawed frog Hymenochirus boettgeri (Tornier, 1896) has led to identification of five structurally related peptides with broad-spectrum antimicrobial activity. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH(2)) is C-terminally α-amidated whereas hymenochirins-2B-5B have the general structure XKIPX(2)VKDTLKKVAKGX(2)SX(2)AGAX(3).COOH. Hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ) was the most abundant peptide in the secretions. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of Silurana tropicalis and Xenopus laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. Synthetic replicates of hymenochirin-1B-4B inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus (MIC in the range 10-40 µM) and Candida albicans (MIC=80 µM). The peptides display relatively weak hemolytic activity against human erythrocytes (LC(50) in the range 160 to >300 µM).

Host-defense peptides in skin secretions of the tetraploid frog Silurana epitropicalis with potent activity against methicillin-resistant Staphylococcus aureus (MRSA).

A putative genome duplication event within the Silurana lineage has given rise to the tetraploid Cameroon clawed frog Silurana epitropicalis (Fischberg, Colombelli, and Picard, 1982). Peptidomic analysis of norepinephrine-stimulated skin secretions of S. epitropicalis led to identification of 10 peptides with varying degrees of growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization identified the peptides as belonging to the magainin family (magainin-SE1), the caerulein-precursor fragment family (CPF-SE1, -SE2 and -SE3), the xenopsin-precursor fragment family (XPF-SE1, SE-2, SE-3 and -SE4), and the peptide glycine-leucine-amide family (PGLa-SE1 and -SE2). In addition, peptide phenylalanine-glutamine-amide (FLGALLGPLMNLLQ·NH(2)) was isolated from the secretions that lacked antimicrobial activity. Comparison of the multiplicity of orthologous peptides in S. epitropicalis and the diploid Silurana tropicalis indicates that extensive nonfunctionalization (deletion or silencing) of antimicrobial peptide genes has occurred after polyploidization in the Silurana lineage, as in the Xenopus lineage. CPF-SE2 (GFLGPLLKLGLKGAAKLLPQLLPSRQQ; MIC=2.5µM) and CPF-SE3 (GFLGSLLKTGLKVGSNLL·NH(2); MIC=5µM) showed potent growth-inhibitory activity against a range of clinical isolates of methicillin-resistant S. aureus (MRSA). Their utility as systemic anti-infective drugs is limited by significant hemolytic activity against human erythrocytes (LC(50)=50µM for CPF-SE2 and 220µM for CPF-SE3) but the peptides may find application as topical agents in treatment of MRSA skin infections and decolonization of MRSA carriers.

Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific.

Mueller's clawed frog Xenopus muelleri (Peters 1844) occupies two non-contiguous ranges in east and west Africa. The phylogenetic relationship between the two populations is unclear and it has been proposed that the western population represents a separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions from X. muelleri from the eastern range resulted in the identification of five antimicrobial peptides structurally related to the magainins (magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and caerulein-precursor fragments (CPF-M1 and -M2) previously found in skin secretions of other Xenopus species. A cyclic peptide (WCPPMIPLCSRF.NH2) containing the RFamide motif was also isolated that shows limited structural similarity to the tigerinins, previously identified only in frogs of the Dicroglossidae family. The components identified in skin secretions from X. muelleri from the western range comprised one magainin (magainin-MW1), one XPF peptide (XPF-MW1), two peptides glycine-leucine amide (PGLa-MW1 and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the primary structures of these peptides suggest that western population of X. muelleri is more closely related to X. borealis than to X. muelleri consistent with its proposed designation as a separate species. The CPF peptides showed potent, broad-spectrum activity against reference strains of bacteria (MIC 3-25 µM), but were hemolytic against human erythrocytes.