Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia) Guilherme D. Brand a,b , Jose ´ Roberto S.A. Leite a , Saulo Martins de Sa ´ Mandel a , Darlan A. Mesquita a , Luciano P. Silva a , Maura V. Prates a , Eder A. Barbosa c , Felipe Vinecky c , Graciella R. Martins d , Joa ˜o Henrique Galasso d , Selma A.S. Kuckelhaus e , Raimunda N.R. Sampaio f , Jose ´ R. Furtado Jr. g , Alan C. Andrade c , Carlos Bloch Jr. a, * a Laborato ´ rio de Espectrometria de Massa, EMBRAPA-Recursos Gene ´ticos e Biotecnologia, Brası ´lia, DF, Brazil b Programa de po ´ s-graduac ¸a ˜o em Biologia Animal, Universidade de Brası ´lia, Brazil c Laborato ´ rio de Gene ´tica Molecular, EMBRAPA-Recursos Gene ´ticos e Biotecnologia, Brazil d Laborato ´ rio SABIN de Ana ´ lises Clı ´ nicas, Brası ´lia, DF, Brazil e Faculdade de Medicina, A ´ rea de Morfologia, Universidade de Brası ´lia, Brazil f Laborato ´ rio de Dermatomicologia, Faculdade de Cie ˆncias da Sau ´ de, Universidade de Brası ´lia, Brazil g Universidade Paulista-UNIP, Brası ´lia, DF, Brazil Received 16 June 2006 Available online 10 July 2006 Abstract Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphib- ian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were deter- mined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging tech- niques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, asso- ciated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Frog skin; Phyllomedusa; Imaging mass spectrometry; Leishmania; Surface plasmon resonance Antimicrobial peptides represent an essential part of the innate immunity of eukaryotes by preventing the invasion of pathogens and their proliferation in host organisms [1–3]. The expression and accumulation of antimicrobial peptides in frog skin secretions is one of the most conserved features and is shared by anurans from all continents. It is postulated that these molecules have endured a diversifying selection proportional to the variety of microorganisms in the frogs’ environments, resulting in the peptide families known nowadays [4,5]. The great majority of these peptides are rather distinct in their primary structures apart from attributes such as high amphiphilicity and net positive charge. Although their precise mechanism of action is not fully understood, those two elements seem to play impor- tant roles in the functionality, selectiveness, and their abil- ity to destabilize cell membranes [6–9]. The dermaseptins (DSs) are a family of peptides isolated from the skin secretion of frogs from the Phyllomedusa genus [10–12]. They are 24–34 amino acids long cationic molecules that fold into amphiphilic helices when in con- tact to a hydrophobic media. The DS polypeptide chains are gene encoded as part of larger precursor molecules comprising a signal peptide of 22 residues, followed by an acidic propeptide, a typical prohormone processing 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.168 * Corresponding author. Fax: +55 6133403658. E-mail address: cbloch@cenargen.embrapa.br (C. Bloch Jr.). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 347 (2006) 739–746 BBRC