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.

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes.

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.

Antimicrobial peptide from the skin secretion of the frog Leptodactylus syphax.

Antimicrobial peptides are considered part of the innate immune system of the majority of living organisms. Most of these molecules are small, cationic and show amphiphilic nature. The skin secretions of Leptodactylus syphax were extracted by mild electrical stimulation and its semipreparative reverse-phase chromatography was resolved in more than 40 fractions. Among these fractions, an antimicrobial peptide was isolated and its amino acid sequence determined by de novo sequencing. Six other truncated forms were characterized in skin secretion. The longest one (25 amino acid residues), named syphaxin (SPX), is amidated at the C-terminal, and shares strong sequence similarity with antimicrobial peptides found in the skin secretion of leptodactylid frogs. Two of the truncated peptides (SPX(1-22) and SPX(1-16)) were tested against Escherichia coli and Staphylococcus aureus, showing low minimal inhibitory concentration (MIC) and no significant toxicity towards blood cells, including both leukocytes and erythrocytes, based on their direct incubation in whole blood at the highest MIC concentration (64 microg/mL).