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Frogs and toads appear at first sight to be easy prey for a host of predators wherever they live. In the temperate European countryside or the deep, humid forests of the tropics, there would seem to be plenty of adversaries for them to watch out for. Even in my own back garden in middle England, a heron visits regularly to plunder my pond for frogs and newts. But many of these small amphibians have a secret weapon that keeps them safe, even snatching them from the jaws of their killers. Dogs give us a clue to the nature of this weapon. Often when a dog succeeds in grabbing a toad it will drop it almost immediately, even before it has had a chance to dig in its teeth or swallow. Toads rapidly secrete an irritant liquid from glands in their skin that is downright unpleasant and literally saves their skins. Even the common toad (Bufo bufo) issues these secretions but, unfortunately for them, there are a few predators, such as hedgehogs and grass snakes, that are not deterred (although I would expect a toad to outrun a hedgehog). The amphibian skin secretions act as a defence mechanism but scientific examination has revealed that their components possess a range of medicinal properties. In Japan and China, toad venom has been used as an expectorant, anti-haemorrhagic, diuretic and cardiac stimulant. Some peptides in the secretions of South American frogs and toads have antibiotic and anticancer properties. Others accelerate the healing of wounds and discarded frog skin from butchers' shops is used to dress burns. In South America, some toxic amphibian secretions are used to coat the tips of blowpipe darts and others are used by the natives as a hunting magic. Psychoactive components heighten the senses and give a feeling of increased strength and bravery. The compositions of toad and frog venoms are complex and varied and have been proposed as the best method for taxonomic classification. This biodiversity has led scientists to collect species and isolate the bioactive components in the secretions in the search for new drug candidates. One of the most common methods of venom analysis is proteomics. It requires samples of the skin secretions, so the animals are not killed. This method is often supported by transcriptome analysis, which used to require dissection of the skin for cDNA library construction, so the animals had to be sacrificed. However, this lethal route was circumvented in 2003 when scientists from the University of Ulster founds that the skin secretions contain polyadenylated mRNAs that encode dermal gland peptides. So, both proteome and transcriptome analysis could be carried out humanely, while preserving the species. Now, that team has extended its combined methodology to the skin secretions of the South American tiger leg leaf frog (Phyllomedusa hypochondrialis azurea), so named for the characteristic patterns on the legs. Stephen McLean and colleagues from Ulster, with Chris Shaw from Queen's University Belfast, realised that most of the peptides in this secretion were relatively short, containing up to 15 amino acid residues. So they were able to use LC/MS/MS to sequence the peptides individually, since the fragmentation profiles were not too complicated. Secretions were gathered by manual massaging or by gentle electrical stimulation and subjected to transcriptome analysis to create a cDNA library. The secretions were also fractionated by simple reversed-phase HPLC and each fraction was subjected to high resolution LC/MS/MS with electrospray ionisation on a hybrid quadrupole-time-of-flight mass spectrometer for peptide sequencing. A total of 22 peptides were identified in the tiger leg leaf frog skin secretions and 19 of them were novel, finding no sequence matches in online databases. A series of tryptophan-containing peptides, termed tryptophyllins, was found with 11 of the 13 exhibiting previously unreported post-translational modifications such as C-terminal amidation or hydroxyproline formation. This group had strong sequence similarity, but their bioactivities remain to be determined. Five peptides were assigned to a new group and called hyposins. They too have sequence similarities, with four residues near the N-terminus conserved in all of them, as well as a high proportion of basic residues near the C-terminus. Four of them were also C-terminally amidated. No peptides with significant sequence matching were found by database searching. Various other peptides were also identified, including [Hyp-6]dermorphin, a defence peptide known to have cataleptic and sedative properties and with an analgesic power 100-fold stronger than that of morphine. All of these peptides were identified from a single skin secretion, and the method could be extended to secretion collection in the field, say the researchers. This will help to preserve the species and will provide secretions collected in their natural environment. Combined with recent work, the team have now used cDNA/mass spectrometry to identify a total of more than 50 novel peptides from this frog, some with antimicrobial properties. Their short sequences suggest that they could be synthesised with relative ease by solid-phase synthesis for bioassay testing, rather than killing multiple species to gather sufficient samples. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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 The tiger leg leaf frog
Shaw and McClean - frog fanciers |
