Pseudis Paradoxa Research Articles

Insulin-releasing properties of the frog skin peptide pseudin-2 and its [Lys18]-substituted analogue

Abstract Pseudin-2 is a cationic alpha-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10(-9)-10(-6) m, pseudin-2, and its [Lys18], [Phe8], and [d-Lys3,d-Lys10,d-Lys14] derivatives, stimulated insulin release from the BRIN-BD11 clonal beta-cell line without increasing release of lactate dehydrogenase. The [Lys18] analogue was the most potent (46% increase in insulin release at 10(-9) m) and the most effective (215% increase in insulin release at 10(-6) m). The more cationic [Lys3,Lys10,Lys14] and [Lys3,Lys10,Lys14,Lys21] analogues lacked insulinotropic action and the more hydrophobic [Phe16] analogue was cytotoxic at concentrations > or =10(-7) m. Pseudin-2 and [Lys18]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys18]-pseudin-2 (10(-8) m) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca2+-independent pathways and identify [Lys18]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.

Cytogenetic analysis of four species of Pseudis (Anura, Hylidae), with the description of ZZ/ZW sex chromosomes in P. tocantins

Pseudis paradoxa paradoxa, P. p. platensis, P. bolbodactyla, P. fusca and P. tocantins were analyzed cytogenetically by conventional chromosomal staining, C-banding, silver staining and fluorescent in situ hybridization with an rDNA probe. Pseudis tocantins chromosomes were also stained with distamycin A/DAPI. All of the species had a diploid number of 2n = 24 chromosomes and the nucleolar organizer region (NOR) was located on pair 7. However, the karyotypes could be differentiated based on the morphology of chromosomal pairs 2 and 8, the region that the NORs occupied on the long arms of the homologous of pair 7, and the pattern of heterochromatin distribution. The subspecies P. p. paradoxa and P. p. platensis had identical karyotypes. Heteromorphism in NOR size was seen in P. p. paradoxa, P. p. platensis, P. bolbodactyla and P. fusca. Heteromorphic sex chromosomes (ZZ/ZW) were identified in P. tocantins. The W chromosome was subtelocentric and larger than the metacentric Z chromosomes. The differences observed in the C-banding pattern and in the position of the NOR on the sex chromosomes suggested that inversions and heterochromatinization were responsible for the morphological differentiation of these chromosomes.

Phylogenetic relationships of Pseudis and Lysapsus (Anura, Hylidae, Hylinae) inferred from mitochondrial and nuclear gene sequences

AbstractThe previous uncertain placement of Lysapsus and Pseudis within the neobatrachians was recently resolved by molecular and morphological studies, which supported them as members of the Hylinae subfamily. Their inter‐ and intrageneric relationships, however, have long been under debate and no studies shed light on these questions. Aiming to elucidate such questions, this paper used 3.2 kb comprising the mitochondrial genes 12S, tRNA valine, 16S and cytochrome b, and the nuclear exon 1 coding for rhodopsin, to all representatives of both genera (except to two subspecies of Pseudis paradoxa). The results identified three major clades: the clade 1 was composed by Lysapsus species and subspecies; clade 2 included the subspecies of the Pseudis paradoxa (Pseudis paradoxa paradoxa, P. paradoxa platensis and P. paradoxa occidentalis), P. fusca, P. bolbodactyla and P. tocantins, and clade 3 was composed by Pseudis southern Brazil species (Pseudis cardosoi and P. minuta). As closely related taxa we found Pseudis minuta + P. cardosoi; P. tocantins + P. fusca, and the subspecies within each genus. Evidence that Pseudis is not monophyletic with respect to Lysapsus was found and we suggest Lysapsus to be a junior synonym of Pseudis. Based on pair‐wise comparison among gene sequences, we also suggest that the subspecies of Pseudis paradoxa and Lysapsus limellum must be considered as full species.© The Willi Hennig Society 2007.

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic α-helical conformation. Analogs of the peptide with increased cationicity and α-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the α-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [ d-Lys 3, d-Lys 10, d-Lys 14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 μM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC 50>500 μM) and cytolytic activity against L929 fibroblasts (LC 50=215 μM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys 3, Lys 10, Lys 14, Lys 21]pseudin-2 at a concentration of 1×MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the α-helix by l-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.

Spermatozoa of Pseudinae (Amphibia, Anura, Hylidae), with a test of the hypothesis that sperm ultrastructure correlates with reproductive modes in anurans.

We describe, for the first time, the sperm ultrastructure of the two genera of Pseudinae. Based on sperm ultrastructure, the five species herein examined can be separated into three groups: one containing Pseudis paradoxa, P. bolbodactyla, and P. tocantins, the second containing P. minuta, and the third containing Lysapsus laevis. The midpiece is similar in all species and auxiliary fibers and the undulating membrane are absent. In Pseudis a subacrosomal cone and a multilaminar structure (P. minuta) or a granular material (P. paradoxa group) are seen above the nucleus. Lysapsus laevis has only remnants of the subacrosomal cone. All species have peripheral fibers associated with the outer doublets of the axoneme. We tested the hypothesis of correlation between the presence of an undulating membrane and fertilization environments in anurans using a concentrated changes test (CCT) based on the Hay et al. (Mol Biol Evol 1995;12:928-937) hypothesis of phylogenetic relationships among anuran families. Only a subset of the resolved topologies derived from the Hay et al. (1995) cladogram, where Ranoidea is the sister-group of Sooglossidae, produced significant probabilities of the CCT. Therefore, support for the correlation between sperm ultrastructure and fertilization environments in anurans is, at best, equivocal.

Diet of the giant tadpole Pseudis paradoxa platensis (Anura, Pseudidae) from Argentina

97 Pseudis paradoxa platensis is restricted to areas along the Paraguay and Parana Rivers, from Paraguay, Bolivia and Mato Grosso (Brazil) to northeast of Argentina (Cei 1980). In Argentina, this subspecies has been found in Formosa, Chaco, Corrientes, Misiones and Santa Fe provinces (Frost 1985, Gallardo and Varela de Olmedo 1992). The adults are very aquatic, occurring in semipermanent and permanent ponds (Dixon et al. 1995, Dure and Kehr 2001). In Argentina breeding occurs between November and March. Tadpoles of P. paradoxa platensis reach lengths up to 168 mm (Bokermann 1967, Emerson 1988). Studies on the biology of this giant tadpole in nature are extremely scarce. This study describes the tadpole diet of P. paradoxa platensis and presents some behavior data. Tadpoles were collected in a semipermanent pond located 30 km from the city of Corrientes (27o25’S 58o44’W, Province of Corrientes, Argentina) during the summer season (December, January and February). The area corresponds to the Oriental Chaquena phytogeographical region (Carnevali 1994, Burkart et al. 1999). Climatically, this area has a mean annual rainfall of 1100 mm and a mean annual temperature of 20o C. The pond area was Diet of the giant tadpole Pseudis paradoxa platensis (Anura, Pseudidae) from Argentina

Cutaneous serous glands in South‐American anurans: An ultrastructural comparison between hylid and pseudid species

An ultrastructural analysis of the secretory processes in serous cutaneous glands of some hylid species (Scinax acuminata, S. fuscovaria, S. nasica and Hyla nana) and one pseudid species (Pseudis paradoxa) revealed similar features, from the early steps to advanced maturational phases. The most consistent and peculiar trait was the occurrence of granules with repeating substructures, resulting from multi‐focal condensation of the dispersed material contained in the Golgian vesicles, which represent the early feature of the secretory activity. Since the ultimate granule features derive from comparable pathways of sequential processes ‐ including early secretory activity and maturation ‐ the similar repetitive substructure is proposed as a trait informative on the taxonomic relationships between the species considered. Comparable granule features in serous glands of different species confirm the close systematic framing of Hyla and Scinax among Hyli‐nae, while stressing phylogenetic relationships between the hylid and pseudid families.

Pseudin-2: An Antimicrobial Peptide with Low Hemolytic Activity from the Skin of the Paradoxical Frog

Four structurally related peptides (pseudins 1–4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC = 2.5 μM against Escherichia coli, 80 μM against Staphylococcus aureus, and 130 μM against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 μM. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic α-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis.

Development of blood pressure and cardiac reflexes in the frog Pseudis paradoxsus.

Systemic arterial blood pressure and heart rate (fH) were measured in unanesthetized, unrestrained larvae and adults of the paradoxical frog, Pseudis paradoxus from São Paulo State in Brazil. Four developmental groups were used, representing the complete transition from aquatic larvae to primarily air-breathing adults. fH (49-66 beats/min) was not significantly affected by development, whereas mean arterial blood pressure was strongly affected, being lowest in the stage 37-39 larvae (10 mmHg), intermediate in the stage 44-45 larvae (18 mmHg), and highest in the juveniles and adults (31 and 30 mmHg, respectively). Blood pressure was not significantly correlated with body mass, which was greatest in the youngest larvae and smallest in the juveniles. In the youngest larvae studied (stages 37-39), lung ventilation was infrequent, causing a slight decrease in arterial blood pressure but no change in heart rate. Lung ventilation was more frequent in stages 44-45 larvae and nearly continuous in juveniles and adults floating at the surface. Bradycardia during both forced and voluntary diving was observed in almost every advanced larva, juvenile, and adult but in only one of four young larvae. Developmentally related changes in blood pressure were not complete until metamorphosis, whereas diving bradycardia was present at an earlier stage.

The giant tadpole of Pseudis paradoxa

Pseudis paradoxa has an extremely large tadpole with a long, deep tail- These features are also found in overwintering tadpoles of temperate species where low temperatures delay development and prolong prolactin exposure. Pseudis paradoxa does not occur in localities with marked seasonal temperature fluctuations. Low temperature cannot be implicated in the development of the tadpole. However, the parallels in shape between Pseudis tadpoles and those of temperate overwintering species suggest that Pseudis tadpoles may have a prolonged exposure to prolactin, higher levels of prolactin during development or an increased sensitivity to prolactin.