A cDNA,library from the skin of Kuna temporaria has been screened using a cDNA fragment probe that encodes the signal peptide of the precursor of esculentin from the skin secretion of Rana esculentu. With this approach, the cDNAs encoding the precursors of three peptides were isolated. Subsequently, the peptides predicted from the sequence of the cloned cDNAs as well as several structurally related peptides could be isolated from the skin secretion of R. temporuria. These peptides, which were named temporins, have a length of 10-13 residues and show some sequence similarity to hemolytic peptides Natural and synthetic temporins have antibacterial activity against gram-positive bacteria, but they are not hemolytic. Temporins are the smallest antibacterial peptides hitherto found in nature.

Epigenetic mechanisms are involved in the pathophysiology of depressive disorders and are unique potential targets for therapeutic intervention. The acetylating agent L-acetylcarnitine (LAC), a welltolerated drug, behaves as an antidepressant by the epigenetic regulation of type 2 metabotropic glutamate (mGlu2) receptors. It caused a rapid and long-lasting antidepressant effect in Flinders Sensitive Line rats and in mice exposed to chronic unpredictable stress, which, respectively, model genetic and environmentally induced depression. In both models, LAC increased levels of acetylated H3K27 bound to the Grm2 promoter and also increased acetylation of NF-ĸB-p65 subunit, thereby enhancing the transcription of Grm2 gene encoding for the mGlu2 receptor in hippocampus and prefrontal cortex. Importantly, LAC reduced the immobility time in the forced swim test and increased sucrose preference as early as 3 d of treatment, whereas 14 d of treatment were needed for the antidepressant effect of chlorimipramine. Moreover, there was no tolerance to the action of LAC, and the antidepressant effect was still seen 2 wk after drug withdrawal. Conversely, NF-ĸB inhibition prevented the increase in mGlu2 expression induced by LAC, whereas the use of a histone deacetylase inhibitor supported the epigenetic control of mGlu2 expression. Finally, LAC had no effect on mGlu2 knockout mice exposed to chronic unpredictable stress, and a single injection of the mGlu2/3 receptor antagonist LY341495 partially blocked LAC action. The rapid and long-lasting antidepressant action of LAC strongly suggests a unique approach to examine the epigenetic hypothesis of depressive disorders in humans, paving the way for more efficient antidepressants with faster onset of action.BDNF | histone acetylation | MDD | glutamatergic neurotransmission | chromatin

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus PhyUomedusa, that have a higher affinity and selectivity for 6 opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of PhyUomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, deltorphins I and II, show an even higher affinity for 6 receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phylomedusa skin, dermorphin, which is highly selective for ,u-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both pA and 6 opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.The endogenous opioid ligands isolated from vertebrate brain show little selectivity toward the different types of opioid receptors. Peptides isolated from amphibian skin appear to be more selective. In 1981 Montecucchi et al. (1) extracted from the skin of the Argentinian frog Phyllomedusa sauvagei a heptapeptide named dermorphin, which preferentially binds to A-type opioid receptors (2). By recombinant DNA techniques, it was demonstrated that dermorphin, like numerous other peptides, is derived in multiple copies from larger precursors. In addition, from inspection of the sequence of one of the cloned cDNAs for these precursors, the existence of another heptapeptide with an N-terminal region similar to that of dermorphin was predicted (3). We recently succeeded in isolating small quantities of this peptide from the skin ofP. sauvagei and named it deltorphin, because of its high affinity and selectivity for the 8 opioid binding site (4). Both dermorphin and deltorphin contain a D amino acid (D-alanine and D-methionine, respectively) as the second amino acid. In the cloned cDNAs, codons for the corresponding L amino acids-i.e., GCG for alanine and ATG for methionine-were found at these positions. This characteristic suggested that the processing of these peptides includes a reaction whereby an L amino acid residue is converted to its D isomer within peptide linkage (3). Here we describe the isolation of two other heptapeptides from the skin of Phyllomedusa bicolor, which show an affinity and selectivity for 8 opioid receptors several times higher than that of deltorphin and the cyclic enkephalin derivative enkephalin (DPDPE, where D-Pen is D-penicillamine) (5). Once again, these peptides contain a D-alanine residue in the second position and share with dermorphin and deltorphin the N-terminal sequence Tyr-D-Xaa-Phe. We refer to these peptides, which differ by the presence of an aspartic or glutamic residue in position 4,...

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The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.

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Temporins are short and homologous antimicrobial peptides (AMPs) isolated from the frog skin of Rana genus. To date, very little is known about the biological significance of the presence of closely related AMPs in single living organisms. Here we addressed this question using temporins A, B, and L isolated from Rana temporaria. We found that temporins A and B are only weakly active toward Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L. To shed light on the underlying mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the effect of the peptides' interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction with lipopolysaccharides (LPS) and the effect of LPS on the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution and when bound to LPS, by using circular dichroism and ATR-FTIR spectroscopies. Our data reveal that temporin L synergizes with A and B by preventing their oligomerization in LPS. This should promote their translocation across the outer membrane into the cytoplasmic membrane. To the best of our knowledge, this is the first study that explains how a combination of native AMPs from the same species can overcome bacterial resistance imposed by the LPS leaflet.

Temporins, antimicrobial peptides of 10±13 residues, were isolated from secretions of Rana temporaria [Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem. 242, 788±792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13 residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the membrane composition; temporins A and B allow the leakage of large-size molecules from the bacterial cells; temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes. The results allow important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity of temporins.