Lycopene from Red Guava (Psidium guajava L.): From Hepatoprotective Effect to Its Use as Promising Self-Emulsifying Drug Delivery System for Anti-Inflammatory and Antioxidant Applications.

Lycopene is a carotenoid with potential use in the treatment of chronic illnesses. Here, different formulations of lycopene were studied: lycopene-rich extract from red guava (LEG), purified lycopene from red guava (LPG) and a self-emulsifying drug delivery system loaded with LPG (nanoLPG). The effects of administering orally various doses of LEG to hypercholesterolemic hamsters were evaluated regarding the liver function of the animals. The cytotoxicity of LPG in Vero cells was analyzed by a crystal violet assay and by fluorescence microscopy. In addition, nanoLPG was employed in stability tests. LPG and nanoLPG were tested for their cytotoxic effect on human keratinocytes and antioxidant capacity on cells in an endothelial dysfunction model in an isolated rat aorta. Finally, the effect of different nanoLPG concentrations on the expression of immune-related genes (IL-10, TNF-α, COX-2 and IFN-γ) from peripheral blood mononuclear cells (PBMC) using real-time PCR was also analyzed. Results suggest that LEG, despite not being able to improve blood markers indicative of liver function in hypercholesterolemic hamsters, reduced hepatic degenerative changes. Additionally, LPG did not show cytotoxicity in Vero cells. In relation to nanoLPG, the effects produced by heat stress evaluated by Dynamics Light Scattering (DLS) and visually were loss of color, texture change and phase separation after 15 days without interfering with the droplet size, so the formulation proved to be efficient in stabilizing the encapsulated lycopene. Although LPG and nanoLPG showed moderate toxicity to keratinocytes, which may be related to cell lineage characteristics, both revealed potent antioxidant activity. LPG and nanoLPG showed vasoprotective effects in aortic preparations. The gene expression assay indicates that, although no significant differences were observed in the expression of IL-10 and TNF-α, the PBMCs treated with nanoLPG showed a reduction in transcriptional levels of IFN-γ and an increased expression of COX-2. Thus, the work adds evidence to the safety of the use of lycopene by humans and shows that tested formulations, mainly nanoLPG due to its stability, stand out as promising and biosafe products for the treatment of diseases that have oxidative stress and inflammation in their etiopathology.

Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

Structure and function of cationic hylin bioactive peptides from the tree frog Boana pulchella in interaction with lipid membranes.

Amphibians have a great diversity of bioactive peptides in their skin. The cDNA prepro-peptide sequencing allowed the identification of five novel mature peptides expressed in the skin of Boana pulchella, four with similar sequences to hylin peptides having a cationic amphipathic-helical structure. Whole mature peptides and some of their fragments were chemically-synthesized and tested against Gram-positive and Gram-negative bacterial strains. The mature peptide hylin-Pul3 was the most active, with a MIC= 14 µM against Staphylococcus aureus. Circular dichroism assays indicated that peptides are mostly unstructured in buffer solutions. Still, adding large unilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoylphosphatidylglycerol increased the α-helix content of novel hylins. These results demonstrate the strong influence of the environment on peptide conformation and highlight its significance while addressing the pharmacology of peptides and their biological function in frogs.

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (Bothrops moojeni).

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated.

Mass Spectrometric Identification of Licania rigida Benth Leaf Extracts and Evaluation of Their Therapeutic Effects on Lipopolysaccharide-Induced Inflammatory Response.

Licania rigida Benth has been evaluated as an alternative drug to treat diseases associated with inflammatory processes. This study evaluated the anti-inflammatory effects of aqueous and hydroalcoholic leaf extracts of L. rigida with inflammation induced by lipopolysaccharides in in vitro and in vivo inflammation models. The phytochemical profile of the extracts, analyzed by ultra-fast liquid chromatography coupled with tandem mass spectrometry, revealed the presence of gallic and ellagic acids in both extracts, whereas isovitexin, ferulate, bulky amino acids (e.g., phenylalanine), pheophorbide, lactic acid, and pyridoxine were detected in the hydroalcoholic extract. The extracts displayed the ability to modulate in vitro and in vivo inflammatory responses, reducing approximately 50% of pro-inflammatory cytokine secretion (TNF-α, IL-1ß, and IL-6), and inhibiting both NO production and leukocyte migration by approximately 30 and 40% at 100 and 500 µg/mL, respectively. Overall, the results highlight and identify, for the first time, the ability of L. rigida leaf extract to modulate inflammatory processes. These data suggest that the leaf extracts of this plant have potential in the development of herbal formulations for the treatment of inflammation.

BR-bombesin: a novel bombesin-related peptide from the skin secretion of the Chaco tree frog (Boana raniceps) with physiological gastric effects.

Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.

A first look at the N- and O-glycosylation landscape in anuran skin secretions.

Amphibians secrete a complex array of molecules that shape their interactions with coinhabiting microorganisms and macroscopic predators. Glycans are a rapidly evolving and complex class of biomolecules implicated in intrinsic and extrinsic recognition events. Despite the numerous studies aiming at the biochemical characterization of anuran skin secretions, little is known about protein-linked oligosaccharides, their synthesis pathways, and their homing secreted glycoproteins. In the present report, LC-MS/MS was used to investigate the diversity of N- and O-linked oligosaccharides in the skin secretion of two South American frogs, Pithecopus azureus and Boana raniceps. Additionally, the enzymes responsible for glycan synthesis pathways were evaluated based on their skin tissue transcriptome. Our analyses allowed the annotation of various N- and O-glycan structures commonly found in vertebrate proteins. Paucimannosidic glycans were abundant in the skin secretion of both amphibians; however, hybrid and complex N-glycan structures were detected only in B. raniceps. A good correlation between the structures discovered in glycomic analyses and transcripts encoding enzymes necessary for their synthesis was obtained. Some transcripts such as those of MAN1A2, FUT8, and ST6GALNAC were found solely in B. raniceps. Finally, secreted N- and O- linked glycoproteins were predicted from the transcriptomic data, indicating that proteases and protease inhibitors are putative sources of the glycans described herein. Overall, our results show the presence of oligosaccharides in amphibians skin secretions and suggest that their diversity is species-specific, paving the way for novel perspectives involving amphibian evolution and ecology.

Characterization of the first two toxins isolated from the venom of the ancient scorpion Tityus (Archaeotityus) mattogrossensis (Borelli, 1901).

BACKGROUND: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. METHODS: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. RESULTS: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are α-NaScTx. CONCLUSION: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are α-NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

Characterization of the first two toxins isolated from the venom of the ancient scorpion Tityus (Archaeotityus) mattogrossensis (Borelli, 1901)

Abstract Background: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. Methods: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. Results: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are -NaScTx. Conclusion: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are -NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.

Isolation and Sequencing of Cu-, Fe-, and Zn-Binding Whey Peptides for Potential Neuroprotective Applications as Multitargeted Compounds.

This study aims to isolate metal-binding peptides and synthesize promising amino acid sequences to potentially act as neuroprotective compounds in the future, targeting different mechanisms. Fractions of whey metal-binding peptides (Cu, Fe, and Zn) isolated by immobilized metal affinity chromatography showed different amino acid profiles according to the metal. The Cu-binding peptides presented roughly twofold increase in the in vitro antioxidant, as assessed by oxygen radical absorbance capacity and anticholinesterase activities over the hydrolysate. This is probably because of the higher concentration of aromatic and basic residues, the latter being crucial for binding to the anionic sites of acetylcholinesterase. Six peptide sequences were synthesized based on the metal-binding sites, molecular mass, hydrophobicity, and bioactivity probability. Among the synthetic peptides, the VF dipeptide stood out both for its in vitro antioxidant and anticholinesterase activities. This peptide, as well as the fraction of Cu-binding peptides, should be further studied because it may act through different mechanisms related to neurodegenerative diseases, in addition to the chelation of the excess of metals in the central nervous system.

Novel Ocellatin Peptides Mitigate LPS-induced ROS Formation and NF-kB Activation in Microglia and Hippocampal Neurons.

Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.

Imaging Mass Spectrometry of Endogenous Polypeptides and Secondary Metabolites from Galls Induced by Root-Knot Nematodes in Tomato Roots.

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation; mainly, the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared with nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI time-of-flight tandem mass spectrometry and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.

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.

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

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]+ = 1543.69Da 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 amphibian's 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.

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

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 α- or ß-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 ß-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 ß-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.

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 frog's deimatic behavior.

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.