Osmindo Rodrigues Pires Júnior

Composition of indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications

The composition of indolealkylamines of Bufo rubescens cutaneous secretions was compared to those from six other Brazilian bufonids. Skin, parotoid and tibial gland secretions were obtained for analysis by thin-layer chromatography. A triple-quadrupole mass spectrometer was used to confirm the indolealkylamines standards (serotonin, 5-HT; bufotenin, BTN; dehydrobufotenin, DHB and bufotenidin, BTD). We observed clear variation in the composition of indolealkylamines of the cutaneous secretions studied and also between those found in the skin and parotoid gland secretions of the same species. We discuss the utility of indolealkylamines to the phylogeny of this group of toads.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Histopathological effects of [D-Leu1]Microcystin-LR variants on liver, skeletal muscle and intestinal tract of Hypophthalmichthys molitrix (Valenciennes, 1844)

This study evaluated the effects of [D-Leu(1)]Microcystin-LR variants, by the exposure of Hypophthalmichthys molitrix to Microcystis aeruginosa NPLJ4. Fish was placed in aquariums and exposed to 10(5) cells mL(-1). For 15 days, 05 individuals were removed every 05 days, and tissue samples of liver, skeletal muscle and intestinal tract were collected for histopathologic analyses. Following exposure, those surviving were placed in clean water for 15 days to evaluate their recovery. A control without toxins was maintained in the same conditions and exhibited normal histology and no tissue damage. In exposed fish, samples were characterized by serious damages that similarly affected the different organs, such as dissociation of cells, necrosis and haemorrhage. Samples showed signs of recovery but severe damages were still observed. The results should be valuable to analyze the potency of microcystin toxicity and to help in the diagnosis of fish deaths.

Toxicity and genotoxicity in Astyanax bimaculatus (Characidae) induced by microcystins from a bloom of Microcystis spp

Studies of genotoxicity in fish caused by cyanobacterial microcystins can be useful both in determining the sensitivity of native species, as well as comparing exposure routes. The genotoxicity caused by the microcystins LR and LA from a bloom collected in a eutrophic lake, was revealed in the fish Astyanaxbimaculatus, a native species from South America. LC50 (72 h) was determined as 242.81 μg L -1 and LD50 (72 h) as 49.19 μg kg -1 bw. There was a significant increase of DNA damage in peripheral erythrocytes, following intraperitoneal injection (ip) with tested concentrations of 24.58 μg kg -1 bw and 36.88 μg kg -1 bw, as well as through body exposure to a concentration of 103.72 μg L -1 . Micronucleus (MN) induction was observed after ip injections of 24.58 μg kg -1 bw and 36.88 μg kg -1 bw for 72 h, as well as following body exposure for 72 at 103.72 μg L -1 . Thus, both exposure routes resulted in MN induction and DNA damage. Apoptosis-necrosis testing was carried out only by ip injection with concentrations of 24.58 μg kg -1 bw and 36.88 μg kg- 1 bw. Exposure to microcystins at lower concentrations induced more apoptosis than necrosis in peripheral erythrocytes, whereas exposure at higher concentrations gave rise to both conditions. Thus, Astyanax bimaculatus can be considered as a species sensitive to the genotoxic effects caused by microcystins.

The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases.

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

A Staging Table of Post-Ovipositional Development for the South American Collared LizardTropidurus torquatus(Squamata: Tropiduridae): IN OVO STAGING TABLE OFTROPIDURUS TORQUATUS

The mouse, chicken, African clawed frog, and zebrafish are considered ¨model organisms¨ due to their extensive embryological and genetic characterization. However, they are far from representative of known diversity, impairing phylogenetic analyses of developmental patterns. Since squamates have historically received limited attention in developmental studies, we here describe the developmental sequence for Tropidurus torquatus, and provide the first post‐ovipositional developmental series for the lizard family Tropiduridae. Fifteen developmental stages are described based on morphological traits such as the eye and accessory visual structures, pharyngeal arches, fusion of facial primordia, limb development, pigmentation, and scales. Organogenesis is already in progression at oviposition (Stage 28), with embryos continuing to develop at the incubation temperature of 30°C ± 1°C, and hatching after 75 ± 5 days, at Stage 42. Comparisons with other lizards show a conserved embryonic sequence, however developmental timing differences were found in features such as the pharyngeal arches, endolymphatic sacs, pigmentation and scales. The development of the phallic and cranial lip of the cloaca anlages are compared with that of other lizards. The order of T. torquatus fore‐ and hindlimb formation differs from that most commonly observed in lizards. The abundance and close association of this species with urban environments, as well as the ease of capturing and managing females, makes T. torquatus an attractive source of developmental data for future experimental and ontogenetic studies. Anat Rec, 300:277–290, 2017.

Marine Depsipeptides as Promising Pharmacotherapeutic Agents

Depsipeptides are a group of biologically active peptides that have at least one of the amide bonds replaced by an ester bond. These peptides sometimes present additional chemical modifications, including unusual amino acid residues in their structures. Depsipeptides are known to exhibit a large array of bioactivities, such as anticancer, antiproliferative, antimicrobial, antiviral and antiplasmodial properties. They are commonly found in marine organisms: bacteria, tunicates, mollusks, sponges, and others. Herein, we summarize the latest insights about marine depsipeptides, their mechanisms of action and potential as therapeutic agents.

Improving carotenoids and amino-acids in Cassava

More than 800 million people in tropics and sub tropics use cassava as food. However, its roots are poor in protein content (0.7-2%). Amino acids such as lysine and methionine are also low, and some research reports indicate the absence of methionine in cassava edible roots. By inter-specific hybridization it was possible to increase true protein in cassava roots measured by amino acid contents. The amino acid profiles of a common cassava cultivar and an inter-specific hybrid, namely ICB 300, were determined using the computerized amino acid analyzer Hitachi L-8500. The inter-specific hybrid has 10-fold lysine and 3-fold methionine than common cassava cultivar: lysine content was 0.010 g per 100 g in the common cassava cultivar while it reached 0.098 in the inter-specific hybrid. Methionine in the common cassava cultivar was 0.014 g per 100 g whereas it reached 0.041 g per 100 g in the inter-specific hybrid. Total amino acid content in the common cassava cultivar was 0.254 g per 100 g viz. a viz. 1.664 g per 100 g in the inter-specific hybrid. The genetic variability of the profile and quantity of amino acids indicate the feasibility of selecting inter-specific hybrids that are rich in both crude protein and amino acids. Carotenoid content could be improved in cassava edible roots by selecting cultivars rich in carotenoids. In Brazil, the center of cassava origin, cassava landraces have acquired through their domestication a large diversity in relation to many economic traits such as high content of carotenoids and excellent palatability among other characters. One of these clones, which has been grown by indigenous farmers in Brazil and available at the University of Brasília genebank, showed a high level of lycopene content (5 mg/kg viz. a viz. zero in common cultivars, and 12-20 mg/kg in tomato-a lycopene-rich vegetable). The cassava landrace UnB 400 had a high content of beta-carotene (up to 4 mg/kg). This article also discusses relevant patents to the main subject of this research.

The Amazing World of Peptide Engineering: the Example of Antimicrobial Peptides from Frogs and Their Analogues.

This review discusses the importance and properties of antimicrobial peptides from frogs and their synthetic analogues as potential therapeutic alternatives in fighting not only bacterial infections, but also protozoans involved with the major neglected diseases, which afflict human populations (e.g., Chagas disease, African sleeping sickness, Leishmaniasis and malaria). Here, we emphasize their multifunctional properties such as promising broad-spectrum drugs that target protozoan parasites too.

HPLC-FLD method for itraconazole quantification in poly lactic-co-glycolic acid nanoparticles, plasma and tissue

Itraconazole, a broad-spectrum anti-fungal, has many side effects, and nanosystems for drug delivery have been proposed as a method to optimize the drugs pharmacokinetics and reduce side effects. An high performance liquid chromatography (HPLC) procedure using fluorometric detection was developed for determination of itraconazole in polymeric poly(lactic-co-glycolic acid) nanoparticles, plasma and tissue. Linearity, limits of detection and quantification, recovery, precision, selectivity and stability were established. The developed method was tested in itraconazole detection and quantification of biodistribution of nanoparticles administered intraperitoneally to Balb/C female mice. This study developed an analytical method for HPLC with fluorometric detection for quantification of itraconazole in polymeric nanoparticles, tissue and plasma, which is sensitive, low cost, viable for routine usage and with potential for application in itraconazole biodistribution and pharmacokinetics studies.