RESUMO
Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.
RESUMO
Antimicrobial peptides (AMPs) are considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and alternative mechanisms of action compared to conventional antibiotics. Although AMPs present considerable advantages over conventional antibiotics, their clinical and commercial development still have some limitations, because of their potential toxicity, susceptibility to proteases, and high cost of production. To overcome these drawbacks, the use of peptides mimics is anticipated to avoid the proteolysis, while the identification of minimalist peptide sequences retaining antimicrobial activities could bring a solution for the cost issue. We describe here new polycationic ß-amino acids combining these two properties, that we used to design small dipeptides that appeared to be active against Gram-positive and Gram-negative bacteria, selective against prokaryotic versus mammalian cells, and highly stable in human plasma. Moreover, the in vivo data activity obtained in septic mice reveals that the bacterial killing effect allows the control of the infection and increases the survival rate of cecal ligature and puncture (CLP)-treated mice.
Assuntos
Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/síntese química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/síntese química , Sepse/tratamento farmacológico , Aminoácidos/química , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Mimetismo Molecular , Proteólise , Sepse/etiologia , Sepse/microbiologiaRESUMO
The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.
Assuntos
Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros/metabolismo , Enterococcus faecium/efeitos dos fármacos , Membranas/efeitos dos fármacos , Peptidoglicano/farmacologia , Pele/química , Staphylococcus aureus/efeitos dos fármacos , Células A549/efeitos dos fármacos , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/isolamento & purificação , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Dicroísmo Circular , Sinergismo Farmacológico , Humanos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade MicrobianaRESUMO
CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.
Assuntos
Movimento Celular/fisiologia , Quimiocina CCL2/metabolismo , Oligopeptídeos/farmacologia , Receptores CCR2/metabolismo , Animais , Células CHO , Quimiocina CCL2/genética , Cricetulus , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Oligopeptídeos/química , Ligação Proteica , Conformação Proteica , Receptores CCR2/genéticaRESUMO
Recruitment of leukocytes is essential to fight infections or to heal injuries; however, excessive and/or prolonged responses favor the development of major inflammatory pathologies, such as cardiovascular or neurodegenerative diseases. Thus, it is of great interest to seek novel compounds that can regulate leukocyte recruitment depending on the degree of inflammation. We have isolated and characterized, by different chromatographic techniques, mass spectrometry, and Edman sequencing, a new hexapeptide (SSLSKL) from the Mexican frog Pachymedusa dacnicolor, which we named pachymodulin. In vitro, pachymodulin promotes the migration of leukocytes through the binding and activation of the human and mouse N-formyl peptide receptor 2 (huFPR2). In vivo, it exhibits opposite biological activities: under homeostatic conditions, pachymodulin induces the recruitment of leukocytes, whereas under inflammatory conditions, it inhibits this process. Therefore, pachymodulin represents an interesting template in the quest to design new immunomodulatory drugs in the therapy of immune-related diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Fatores Imunológicos/farmacologia , Oligopeptídeos/farmacologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Lipoxinas/antagonistas & inibidores , Pele/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anuros , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Inflamação/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Ligantes , Camundongos , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Plasticins (PTC) are dermaseptin-related antimicrobial peptides characterized by a large number of leucine and glycine residues arranged in GXXXG motifs that are often described to promote helix association within biological membranes. We report the structure and interaction properties of two plasticins, PTC-B1 from Phyllomedusa bicolor and a cationic analog of PTC-DA1 from Pachymedusa dacnicolor, which exhibit membrane-lytic activities on a broad range of microorganisms. Despite a high number of glycine, CD and NMR spectroscopy show that the two plasticins adopt mainly alpha-helical conformations in a wide variety of environments such as trifluoroethanol, detergent micelles and lipid vesicles. In DPC and SDS, plasticins adopt well-defined helices that lie parallel to the micelle surface, all glycine residues being located on the solvent-exposed face. Spectroscopic data and cross-linking experiments indicate that the GXXXG repeats in these amphipathic helices do not provide a strong oligomerization interface, suggesting a different role from GXXXG motifs found in transmembrane helices.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas do Olho/química , Proteínas do Tecido Nervoso/química , Motivos de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Calorimetria , Dicroísmo Circular , Difusão , Proteínas do Olho/síntese química , Proteínas do Olho/metabolismo , Glicina/química , Leucina/química , Lipossomos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/síntese química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Cell penetrating peptides (CPPs) are peptides displaying the ability to cross cell membranes and transport cargo molecules inside cells. Several uptake mechanisms (endocytic or direct translocation through the membrane) are being considered, but the interaction between the CPP and the cell membrane is certainly a preliminary key point to the entry of the peptide into the cell. In this study, we used three basic peptides: RL9 (RRLLRRLRR-NH(2)), RW9 (RRWWRRWRR-NH(2)) and R9 (RRRRRRRRR-NH(2)). While RW9 and R9 were internalised into wild type Chinese Hamster Ovary cells (CHO) and glycosaminoglycan-deficient CHO cells, at 4°C and 37°C, RL9 was not internalised into CHO cells. To better understand the differences between RW9, R9 and RL9 in terms of uptake, we studied the interaction of these peptides with model lipid membranes. The effect of the three peptides on the thermotropic phase behaviour of a zwitterionic lipid (DMPC) and an anionic lipid (DMPG) was investigated with differential scanning calorimetry (DSC). The presence of negative charges on the lipid headgroups appeared to be essential to trigger the peptide/lipid interaction. RW9 and R9 disturbed the main phase transition of DMPG, whereas RL9 did not induce significant effects. Isothermal titration calorimetry (ITC) allowed us to study the binding of these peptides to large unilamellar vesicles (LUVs). RW9 and R9 proved to have about ten fold more affinity for DSPG LUVs than RL9. With circular dichroism (CD) and NMR spectroscopy, the secondary structure of RL9, RW9 and R9 in aqueous buffer or lipid/detergent conditions was investigated. Additionally, we tested the antimicrobial activity of these peptides against Escherichia coli and Staphylococcus aureus, as CPPs and antimicrobial peptides are known to share several common characteristics. Only RW9 was found to be mildly bacteriostatic against E. coli. These studies helped us to get a better understanding as to why R9 and RW9 are able to cross the cell membrane while RL9 remains bound to the surface without entering the cell.
Assuntos
Arginina/química , Membrana Celular/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Células CHO , Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular , Cricetinae , Cricetulus , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Staphylococcus aureus/metabolismoRESUMO
Antimicrobial peptides participate in innate host defense by directly eliminating pathogens as a result of their ability to damage the microbial membrane and by providing danger signals that will recruit innate immune cells to the site of infection. Dermaseptin DA4 (DRS-DA4), a new antimicrobial peptide of the dermaseptin superfamily, was identified based on its chemotactic properties, contrasting with the currently used microbicidal properties assessment. The peptide was isolated and purified by size exclusion HPLC and RP-HPLC from the skin of the Mexican frog, Pachymedusa dacnicolor. MS and amino acid sequence analyses were consistent with the structure GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM. CD experiments showed that, unlike most antimicrobial peptides of the dermaseptin superfamily, DRS-DA4 is not structured in the presence of zwitterionic lipids. DRS-DA4 is a potent chemoattractant for human leukocytes and is devoid of hemolytic activity; in addition, bactericidal tests and membrane perturbation assays on model membranes and on Escherichia coli and Staphylococcus aureus strains have shown that the antibacterial effects of DRS-DA4 and permeabilization of the inner membrane are exclusively selective for Gram-negative bacteria. Interestingly, despite high sequence homology with dermaseptin S4, dermaseptin B2 was not able to induce directional migration of leukocytes, and displayed a broader bactericidal spectrum. A detailed structure-function analysis of closely related peptides with different capabilities, such as DRS-DA4 and dermaseptin B2, is critical for the design of new molecules with specific attributes to modulate immunity and/or act as microbicidal agents.
Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fatores Quimiotáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Anuros/metabolismo , Dicroísmo Circular , Humanos , Leucócitos/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Homologia de Sequência de Aminoácidos , Pele/metabolismoRESUMO
A comparative study was designed to evaluate the staphylococcidal efficiency of two sequence-related plasticins from the dermaseptin superfamily we screened previously. Their bactericidal activities against Staphylococcus aureus as well as their chemotactic potential were investigated. The impact of the GraS/GraR two-component system involved in regulating resistance to cationic antimicrobial peptides (CAMPs) was evaluated. Membrane disturbing activity was quantified by membrane depolarization assays using the diS-C3 probe and by membrane integrity assays measuring beta-galactosidase activity with recombinant strain ST1065 reflecting compromised membranes and cytoplasmic leakage. Interactions of plasticins with membrane models composed of either zwitterionic lipids mimicking the S. aureus membrane of CAMP-resistant strains or anionic lipids mimicking the negative charge-depleted membrane of CAMP-sensitive strains were analyzed by jointed Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and differential scanning calorimetry (DSC) to yield detailed information about the macroscopic interfacial organization, in situ conformation, orientation of the peptides at the lipid-solvent interface, and lipid-phase disturbance. We clearly found evidence of distinct interfacial behaviors of plasticins we linked to the distribution of charges along the peptides and structural interconversion properties at the membrane interface. Our results also suggest that amidation might play a key role in GraS/GraR-mediated CAMP sensing at the bacterial surface.
Assuntos
Antibacterianos/química , Antibacterianos/toxicidade , Proteínas do Olho/química , Proteínas do Olho/toxicidade , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Adulto , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Farmacorresistência Bacteriana , Proteínas do Olho/antagonistas & inibidores , Inibidores do Crescimento/antagonistas & inibidores , Inibidores do Crescimento/química , Inibidores do Crescimento/toxicidade , Humanos , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Conformação Proteica , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/crescimento & desenvolvimentoRESUMO
Dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM, isolated from frog skin, does not resemble any of the cationic and amphipathic antimicrobial peptides identified to date, having a highly hydrophobic core sequence flanked at either side by cationic termini. Previous studies [Lequin O, Ladram A, Chabbert A, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P & Amiche M (2006) Biochemistry45, 468-480] demonstrated that this peptide adopted a non-amphipathic alpha-helical conformation in trifluoroethanol/water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles. Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered beta-sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles. These structures slowly assembled into amyloid-like fibrils in aqueous environments via spherical intermediates, as revealed by electron microscopy and Congo red staining. Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes. Interestingly, the antimicrobial and chemotactic activities of Drs S9 are modulated by its amyloid-like properties. Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self-associated forms of Drs S9 act on human leukocytes to promote chemotaxis and/or immunological response activation in the same range of concentration as amyloidogenic peptides Abeta(1-42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106-126).
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Fatores Quimiotáticos/química , Sequência de Aminoácidos , Amiloide/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Bicamadas Lipídicas/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The present study investigates the relationships between structural polymorphism, adsorption onto membrane mimetic support, lipid disturbance, and biological activity of bactericidal 23-residue, glycine-leucine-rich dermaseptin orthologues from the Phyllomedusinae frog skin, the "plasticins". Biological activities were evaluated using the membrane models DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) for prokaryotic membranes and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) for eukaryotic membranes. We performed a conformational analysis of plasticins by molecular simulations and spectroscopic methods and analyzed phospholipid perturbations by infrared spectroscopy. Adsorption onto synthetic model membranes was quantified by surface plasmon resonance. Biological assays including antimicrobial and membrane potential-dissipating activities, together with hemolytic tests and imaging analysis of cytotoxicity, were carried out to clarify the peptide-membrane interactions. Two major groups were distinguished: (i) Neutral plasticins revealed the presence of strong beta-structures with the zwitterionic or anionic phospholipid vesicles. They were weakly adsorbed in the range of antibacterial activity concentrations (micromolar). Nevertheless, for millimolar concentrations, they caused perturbations at the interface peptide-DMPG vesicles and in the bilayer alkyl chains, suggesting insertion into bacterial membranes. (ii) Cationic plasticins revealed multiple conformational transitions, including destabilized helix states, beta-structures, and disordered states. Peptide-lipid complex densities depended on hydrophobic bond strengths. The most soluble cationic plasticins were strongly adsorbed, with stable peptide-lipid interactions inducing noticeable perturbations of bilayer alkyl chains, pointing out possible insertion into bacterial membranes. In contrast, cytotoxic plasticins were less adsorbed, with less stable peptide-lipid interactions causing membrane dehydration, formation of peptide-membrane hydrogen bonds, and little disturbances of lipid alkyl chains. These characteristics could be compatible with their putative action on intracellular targets leading to apoptosis.
Assuntos
Proteínas do Olho/química , Proteínas do Olho/farmacologia , Lipídeos/química , Membranas Artificiais , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Adsorção , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Dicroísmo Circular , Simulação por Computador , Escherichia coli/efeitos dos fármacos , Proteínas do Olho/metabolismo , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Potenciais da Membrana , Conformação Molecular , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Ressonância de Plasmônio de SuperfícieRESUMO
The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.