Molecular Cloning of a Novel Tryptophyllin Peptide from the Skin of the Orange-Legged Monkey Frog, Phyllomedusa hypochondrialis

Tryptophyllins are a group of small (4–14 amino acids), heterogenous peptides, mostly from the skins of hylid frogs from the genera, Phyllomedusa and Litoria. To date, more than forty TPHs have been discovered in species from these two genera. Here, we describe the identification of a novel tryptophyllin type 3 peptide, PhT-3, from the extracts of skin of the orange-legged monkey frog, Phyllomedusa hypochondrialis, and molecular cloning of its precursor-encoding cDNA from a cDNA library constructed from the same skin sample. Full primary structural characterization was achieved using a combination of direct Edman degradation, mass spectrometry and deduction from cloned skin-derived cDNA. The open-reading frame of the precursor cDNA was found to consist of 63 amino acid residues. The mature peptide arising from this precursor contains a post-translationally modified N-terminal pyroglutamate (pGlu) residue, formed from acid-mediated cyclization of an N-terminal Gln (Q) residue, and with the structure: pGlu-Asp-Lys-Pro-Phe-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met. Pharmacological assessment of a synthetic replicate of this peptide on phenylephrine preconstricted rat tail artery segments, revealed a reduction in relaxation induced by bradykinin. PhT-3 was also found to mediate antiproliferative effects on human prostate cancer cell lines.

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: a novel CRF-like peptide with smooth muscle contraction activity

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.

Ecologia trófica e reprodutiva de Phyllomedusa hypochondrialis (Daudin, 1802) (Hylidae, Phyllomedusinae) na Amazônia Oriental, Pará, Brasil

Este estudo está dividido em dois capítulos formatados de acordo com as normas da revista Amphibia-Reptilia. No capítulo 1, intitulado “Ecologia trófica de Phyllomedusa hypochondrialis (Daudin, 1802) (Hylidae, Phyllomedusinae) na Amazônia Oriental, Pará, Brasil”, verificamos a contribuição das presas na dieta da espécie assim como diferenças na dieta em relação ao sexo, tamanho corporal e estações pluviométrica e seca. No capítulo 2 intitulado “Biologia reprodutiva e uso de hábitat por Phyllomedusa hypochondrialis (Daudin, 1802) (Hylidae, Phyllomedusinae) na Amazônia Oriental, Pará, Brasil”, descrevemos a biologia reprodutiva e o uso de recursos espaciais da espécie. período sazonal, já fêmeas tendem a procurar itens que lhes garantam maior retorno energético.

Medusins: a new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog, (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group were named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles

While bradykinin has been identified in the skin secretions from several species of amphibian, bradykinin-related peptides (BRPs) are more common constituents. These peptides display a plethora of primary structural variations from the type peptide which include single or multiple amino acid substitutions, N- and/or C-terminal extensions and post-translational modifications such as proline hydroxylation and tyrosine sulfation. Such modified peptides have been reported in species from many families, including Bombinatoridae, Hylidae and Ranidae. The spectrum of these peptides in a given species is thought to be reflective of its predator profile from different vertebrate taxa. Here we report the isolation of BRPs and parallel molecular cloning of their respective biosynthetic precursor-encoding cDNAs from the skin secretions of the Mexican leaf frog (Pachymedusa dacnicolor), the Central American red-eyed leaf frog (Agalychnis callidryas) and the South American orange-legged leaf frog (Phyllomedusa hypochondrialis). Additionally, the eight different BRPs identified were chemically synthesized and screened for bioactivity using four different mammalian smooth muscle preparations and their effects and rank potencies were found to be radically different in these with some acting preferentially through bradykinin B1-type receptors and others through B2-type receptors.

Leishmanicidal Activity and Immobilization of dermaseptin 01 antimicrobial peptides in ultrathin films for nanomedicine applications

Antimicrobial peptides (AMPs) are essential for the innate immune system of eukaryotes, imparting protection against pathogens and their proliferation in host organisms. The recent interest in AMPs as active materials in bionanostructures is due to the properties shown by these biological molecules, such as the presence of an alpha-helix structure and distribution of positive charges along the chain. In this study the antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines. The leishmanicidal activity of DS 01 was confirmed using kinetic essays, in which DS 01 promoted death of all metacyclic promastigote cells in 45 minutes. Surprisingly, the immobilized DS 01 molecules displayed electroactivity, as revealed by electrochemical experiments, in which an oxidation peak at about 0.61 V was observed for a DS 01 monolayer deposited on top of a conductive electrode. Such electroactivity was used to investigate the sensing abilities of the nanostructured films toward Leishmania. We observed an increase in the oxidation current as a function of number of Leishmania cells in the electrolytic solution at concentrations down to 10(3) cells/mL. The latter is indicative that the use of AMPs immobilized in electroactive nanostructured films may be of interest for applications in the pharmaceutical industry and diagnosis.

Ecologia de comunidades e comportamento reprodutivo de anfíbios anuros em savana amazônica

The Amazon savannas occur as isolated patches throughout extensive areas of forest in the states of Amapá, Amazonas, Pará, and Roraima. There is a considerable variation in the composition of anuran assemblages in the localities and phytophysiognomies of Amazon savannas and given the absence of studies on reproductive behavior, a systematic and geographically wide sampling has been carried out in the Amapá savanna, located in the Eastern Amazon. The study was conducted in a savanna area in the state of Amapá to examine the composition, ecology, and reproductive behavior of anuran amphibians. We carried out 24 field trips in each phytophysiognomy (gramineous-woody savana, gramineous-herbaceous-woody savana, park savana, and arboreal savanna); for analysis of reproductive behavior observations were made during the period January to December 2013, lasting four consecutive days. Samples were collected by active and acoustic search along 20 plots of 100x50 meters. Twenty-one anuran species were recorded, of which four are new records for the state of Amapá: Dendropsophus walfordi, Scinax fuscomarginatus, Pseudopaludicola boliviana e Elachistocleis helianneae. The KruskalWallis ANOVA revealed significant differences between richness and species diversity in the phytophysiognomies (p < 0.05). The Bray-Curtis similarity coefficient divided the phytophysiognomies into three groups: arboreal savana, gramineous-woody savanna and gramineous-herbaceous-woody savanna, and park savanna. According to the non-metric multidimensional scaling, the structure of the anuran community resulted in a separation into three phytophysiognomies, with significant differences in the structure of communities (ANOSIM, R = 0.823; p < 0.001). In the study of community ecology, the results obtained for spatial, temporal, and trophic niche breadth suggest that the assemblage of anurans of the Amapá savanna is not composed of predominantly generalist species. Also, the presence of other specialist anurans may explain the processes of speciation associated with the isolation of habitats, resulting in heterogeneity and spatial discontinuity in the phytophysiognomies with open formations. The null model analysis revealed that the community is structured based on temporal and trophic niche, indicating a significant influence of contemporary ecological factors on the assemblage. The absence of structure based on spatial niche might be explained by the spatial segregation in the distribution and occupation of anurans in the different phytophysiognomies of the Amapá savanna. Regarding the reproductive behavior of anurans, 11 species were classified as having a long breeding season, intrinsically associated with the rainy season and the reproductive mode of most species that lay egg clutches in lentic water bodies. Six reproductive modes were recorded and parental care was observed in Leptodactylus macrosternum and L. podicipinus, whose reproductive mode is characterized by foam nests. Regarding behavioral reproductive strategies, calling males were observed in all species of anurans, satellite males were recorded only for D. walfordi, Hypsiboas multifasciatus, S. nebulosus and S. fuscomarginatus; active search for females was observed for Phyllomedusa hypochondrialis and L. fuscus, and male displacement was recorded only for Rhinella major and R. margaritifera. Of the reproductive behaviors observed, throat and vocal sac display is associated with courtship and territorial behavior exhibited by males. In addition to courtship behavior, visual signals associated with courtship strategies were recorded for the anurans of the Amapá savanna.

Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

The defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel dermaseptin-related peptides (dermaseptins sVI–sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian skin antimicrobial peptides may in part be explained by individual variation—a factor essential for selective functional molecular evolution and perhaps, ultimately in speciation.