Eder Alves Barbosa

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram-negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60-240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy 1H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.

Characterization of a novel peptide toxin from Acanthoscurria paulensis spider venom: a distinct cysteine assignment to the HWTX-II family.

Spider venom toxins have raised interest in prospecting new drugs and pesticides. Nevertheless, few studies are conducted with tarantula toxins, especially with species found in Brazil. This study aims to characterize chemically and biologically the first toxin isolated from Acanthoscurria paulensis venom. Ap1a consists of 48 amino acid residues and has a molecular mass of 5457.79 Da. The cloned gene encodes a putative sequence of 23 amino acid residues for the signal peptide and 27 for the pro-peptide. The sequence of the mature peptide is 60-84% identical with those of toxins of the HWTX-II family. Different from the structural pattern proposed for these toxins, the disulfide pairing of Ap1a is of the ICK type motif, which is also shared by the U1-TRTX-Bs1a toxin. Ap1a induced a dose-dependent and reversible paralytic effect in Spodoptera frugiperda caterpillars, with an ED50 of 13.0 ± 4.2 μg/g 8 h after injections. In the Drosophila melanogaster Giant Fiber circuit, Ap1a (1.14-22.82 μg/g) reduces both the amplitude and frequency of responses from GF-TTM and GF-DLM pathways, suggesting an action at the neuromuscular junction, which is mediated by glutamatergic receptors. It is also lethal to mice (1.67 μg/g, intracranial route), inducing effects similar to those reported with intracerebroventricular administration of NMDA. Ap1a (1 μM) does not alter the response induced by acetylcholine on the rhabdomyosarcoma cell preparation and shows no significant effects on hNav1.2, hNav1.4, hNav1.5, and hNav1.6 channels. Because of its unique sequence and cysteine assignment to the HWTX-II family, Ap1a is a significant contribution to the structure-function study of this family of toxins.

Lycopene-rich extract from red guava (Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M]+• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC50]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC50=1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC50≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway.

Skin secretion peptides: the molecular facet of the deimatic behavior of the four‐eyed frog, Physalaemus nattereri (Anura, Leptodactylidae)

RATIONALE Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri. METHODS Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data. RESULTS Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions. CONCLUSIONS The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frogs deimatic behavior.

Structure and function of a novel antioxidant peptide from the skin of tropical frogs

ABSTRACT The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin‐I, has a molecular weight [M+H]+ = 1543.69 Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin‐I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin‐I has a novel primary sequence with low similarity compared with previously described amphibians AOPs. Antioxidin‐I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin‐I presented a low cytotoxicity and suppressed menadione‐induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia‐induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide. Graphical abstract Figure. No caption available. HighlightsAntioxidin‐I is a new antioxidant peptide isolated from the skin tropical frogs.The bioactive peptide presented very low cytotoxicity against mammalian cells.It was able to avoid redox imbalance in oxidative challenged cells.Antioxidin‐I had the capacity to suppress ROS levels in hypoxia‐exposed microglia.Results support the application of the peptide for neuroprotection.

To4, the first Tityus obscurus β-toxin fully electrophysiologically characterized on human sodium channel isoforms

&NA; Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named &agr;‐ or &bgr;‐types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked &bgr;‐type effect on different sodium channel subtypes (hNav1.1–hNav1.7) at low concentrations, and has more pronounced activity on hNav1.1, hNav1.2 and hNav1.4. By comparing To4 primary structure with other Tityus &bgr;‐toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms. HighlightsTo4 is the first toxin from Tityus obscurus venom characterized on hNav isoforms.To4 shows a &bgr;‐toxin effect when tested on human sodium channels isoforms.To4 showed promiscuous activity on human sodium channels.