Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the viruss glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.(AU)

Antileishmanial and antitrypanosomal activity of the cutaneous secretion of Siphonops annulatus

Background Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity.Methods Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantumpromastigotes and intracellular amastigotes, trypomastigotes ofTrypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania.Results The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 μg/mL against promastigotes and 2.75 μg/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms ofLeishmania. Ultrastructural studies withLeishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasite's death within a few hours. Nevertheless, it caused no alteration in the plasma membrane permeability as detected by the fluorescent probe and TEM.Conclusions The present study demonstrated for the first time the antiparasitic activity of the skin secretion of the caecilian S. annulatus againstLeishmania and T. cruzi, confirming that skin secretions of these amphibians, similarly to those of anurans and salamanders, are also potential tools for the development of new drug candidates against neglected diseases.(AU)

Antileishmanial and antitrypanosomal activity of the cutaneous secretion of Siphonops annulatus

Background Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity.Methods Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantumpromastigotes and intracellular amastigotes, trypomastigotes ofTrypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania.Results The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 g/mL against promastigotes and 2.75 g/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms ofLeishmania. Ultrastructural studies withLeishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasites death within a few hours...

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...

Contribuição ao estudo das anfisbenias Reptilia, Squamata, Amphisbaenia): morfologia de glândulas cutâneas e do tegumento, e alguns aspectos da biologia reprodutiva / Contribuition to the knowledge of amphisbaenians(Reptilia, Squamata, Amphisbaenia): morphology of cutaneous glands and of the integument, and some aspects of reprodutive biology

Esta tese é baseada em cinco trabalhos publicados e um submetido à publicaçao, que tratam de aspectos morfógicos ligados à biologia de três espécies de Amphisbaenia (Squamata), Amphisbaena alba, Leposternon microcephalum e Leposternon infraorbitale. A maior ênfase foi dada à histologia, à ultra-estrutura e à histoquímica das glândulas pré-cloacais e de sua secreçao em Amphisbaena alba. Essas glândulas secretam uma substância que parece ser um feromônio, provavelmente utilizado na reproduçao e/ou na marcaçao territorial desses animais. A secreçao sólida, constituída principalmente de polissacarídeos e proteínas, obstrui os poros glandulares na forma de um "plug" e, à medida que o animal se locomove, é espalhada no ambiente subterrâneo formando uma trilha, cujo aspecto microscópico foi aqui descrito através de microscopia eletrônica de varredura. Um dos trabalhos relata, ainda, o nascimento em cativeiro de uma ninhada de Leposternon infraorbitale. Trata-se da primeira descriçao na literatura de nascimento de uma anfisbênia. Por fim, foi realizado um trabalho sobre a histologia e a morfometria da pele de Amphisbaena alba, analisando os dados obtidos em funçao do "display" de defesa apresentado por esta espécie, e comparando-os com aqueles encontrados em Leposternon microcephalum, anfisbenídeo que nao apresenta esse comportamento defensivo. Devido ao fato de as anfisbênias serem animais bastante inacessíveis, já que possuem hábito fossório, trabalhos desse tipo podem contribuir sobremaneira para o conhecimento da biologia e da sistemática desse grupo dos Squamata

Estudo histológico das glândulas lombares de Pleurodema thaul (Amphibia, Anura, Leptodactylidae) / Histologic study of lumbar glands of Pleurodema thaul (Amphibia, Anura, Leptodactylidae)

As räs da espécie Pleurodema thaul possuem duas protuberâncias cutâneas dorsolaterais, uma de cada lado do corpo, em posiçäo imediatamente posterior ao sacro. Essas protuberâncias, denominadas glândulas lombares, foram estudadas histologicamente verificando-se que seus cromatóforos apresentam-se, sobretudo, dispostos organizadamente logo abaixo da epiderme, constituindo uma unidade cromatofórica dérmica. Como outras macroglândulas de anuros, as protuberâncias lombares säo constituídas principalmente pelo acúmulo de alvéolos granulosos repletos de secreçäo. A prsença de alvéolos granulosos e a organizaçäo cracterística dos cromatófaros dérmicos permitem sugerir um papel defensivo para as glândulas lombares. Na maioria dos anfíbios, os alvéolos granulosos acumulam uma secreçäo tóxica para diversos vertebrados. Os cromatóforos, nesta espécie, talves desempenhem uma funçäo apossemática pois sua disposiçäo na pele faz com que as glândulas lombares se assemelhem a olhos, dando, possivelmente, a um eventual predador da espécie em questäo, a impressäo de que se trata de animal de maiores dimensöes

Consideraçoes sobre o veneno dos anfíbios / Considerations on amphibian venoms

Este artigo é uma revisäo a respeito dos principais componentes da secreçäo cutânea dos anfíbios, enfatizando, sobretudo, alguns de seus aspectos bioquímicos e farmacológicos