Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

Helokinestatin: a new bradykinin B2 receptor antagonist decapeptide from lizard venom.

Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Mr 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.

Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion

Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained non-invasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n = 14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at - 20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykinin-related peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology.

Parasitic peptides! The structure and function of neuropeptides in parasitic worms

Parasitic worms come from two very different phyla-Platyhelminthes (flatworms) and Nematoda (roundworms). Although both phyla possess nervous systems with highly developed peptidergic components. there are key differences in the structure and action of native neuropeptides in the two groups. For example, the most abundant neuropeptide known in platyhelminths is the pancreatic polypeptide-like neuropeptide F, whereas the most prevalent neuropeptides in nematodes an FMRFamide-related peptides (FaRPs), which are also present in platyhelminths. With respect to neuropeptide diversity, platyhelminth species possess only one or two distinct FaRPs, whereas nematodes have upwards of 50 unique FaRPs. FaRP bioactivity in platyhelminths appears to be restricted to myoexcitation, whereas both excitatory and inhibitory effects have been reported in nematodes. Recently interest has focused on the peptidergic signaling systems of both phyla because elucidation of these systems will do much to clarify the basic biology of the worms and because the peptidergic systems hold the promise of yielding novel targets for a new generation of antiparasitic drugs. (C) 1999 Elsevier Science Inc. All rights reserved.

APEASPFIRFamide, a novel FMRFamide-related decapeptide from Caenorhabditis elegans: Structure and myoactivity

To date, 9 FMRF amide-related peptides (FaRPs) have been identified in Caenorhabditis elegans. Eight of these peptides are encoded on the flp-1 gene. However, AF2 (KHEYLRF amide) which was not co-encoded was the most abundant FaRP identified in ethanolic extracts. Further radioimmunometrical screening of acidified ethanol extracts of C. elegans has revealed the presence of other novel FaRPs, which are not encoded on the flp-l gene. One of these peptides has been isolated by sequential rpHPLC and subjected to Edman degradation analysis and gas-phase sequencing and the unequivocal primary structure of the decapeptide Ala-Pro-Glu-Ala-Ser-Pro-Phe-Ile-Arg-Phe-NH2 was determined following a single gas-phase sequencing run. The molecular mass of the peptide was found to be 1133.7 Ha, determined using a time-of-flight mass spectrometer. Synthetic replicates of this peptide were found to induce a profound relaxation of both dorsal and ventral somatic muscle-strip preparations of Ascaris suum with a threshold for activity of 10 nM. The inhibitory response was not dependent on the presence of nerve cords, indicating a post-synaptic site-of-action. The relaxation was Ca++- and Cl--independent but was abolished in high-KI medium and could be distinguished from those of other inhibitory nematode FaRPs, including PF1 (SDPNFLRFamide)and PF1 (KPNFIRF amide). (C) 1997 Academic Press.

Physiological effects of platyhelminth RFamide peptides on muscle-strip preparations of Fasciola hepatica (Trematoda: Digenea)

The effects of each of the known platyhelminth neuropeptides were determined on muscle-strip preparations from the liver fluke, Fasciola hepatica. The activity of synthetic replicates of the C-terminal nonapeptide of neuropeptide F (NPF9, Moniezia expansa), and the FMRFamide-related peptides (FaRPs), GNFFRFamide, RYIRFamide, GYIRFamide and YIRFamide, were examined. Muscle-strip activity was recorded from 1 mm segments of muscle prepared from 28 to 32-day-old worms, using a photo-optic transducer system. None of the peptides (less than or equal to 10 mu M) altered baseline tension significantly; however, each of the peptides increased the amplitude and frequency of muscle contraction. The threshold for activity of each of the peptides examined was, respectively, 1 nM (RYIRFamide), 0.3 mu M (GYIRFamide and YIRFamide), and 10 mu M (GNFFRFamide and NPF9). All of the effects were reversible and repeatable, following wash-out. Muscle-strip integrity was tested following experimentation, using arecoline (10 mu M) and high-K+ bathing medium (90 mM K+).

Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: Evidence for the presence of more than one FMRFamide related peptide-encoding gene

Numerous FMRF amide-related peptides (FaRPs) have been isolated and sequenced from extracts of free-living and parasitic nematodes. The most abundant FaRP identified in ethanolic/methanolic extracts of the parasitic forms, Ascaris suum and Haemonchus contortus and from the free-living nematode, Panagrellus redivivus, was KHEYLRF amide (AF2). Analysis of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris identified a series of related FaRPs which did not include AF2. An acid-ethanol extract of Caenorhabditis elegans was screened radioimmunometrically for the presence of FaRPs using a C-terminally directed FaRP antiserum. Approximately 300 pmols of the most abundant immunoreactive peptide was purified to homogeneity and 30 pmols was subjected to Edman degradation analysis and gas-phase sequencing. The unequivocal primary structure of the heptapeptide, Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a time-of-flight mass spectrometer and was found to be 920 (MH(+))(-), which was consistent with the theoretical mass of C-terminally amidated AF2. These results indicate that C. elegans possesses more than one FaRP gene. (C) 1995 Academic Press, Inc.

KSAYMRFAMIDE - A NOVEL FMRFAMIDE-RELATED HEPTAPEPTIDE FROM THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS, WHICH IS MYOACTIVE IN THE PARASITIC NEMATODE, ASCARIS-SUUM

In nematodes, FMRFamide-related peptides (FaRPs) have been structurally characterised from the parasite, Ascaris suum, and from two free-living species, Panagrellus redivivus and Caenorhabditis elegans. While both FaRPs isolated from P. redivivus (PF1 and PF2) have been identified in C. elegans the two heptapeptides isolated from A. suum (AF1 and AF2) have until recently been considered unique to this parasitic species. We have recently isolated AF2 from P. redivivus and, during this study, an additional novel heptapeptide amide, Lys-Ser-Ala-Tyr-Met-Arg-Phe amide (KSAYMRFamide), was structurally characterised. A synthetic replicate of this peptide induced a rapid concentration-dependent muscle tension increase in an isolated A. suum somatic muscle preparation, with a threshold of approximately 0.1 mu M. These data suggest that the complement of FaRPs in parasitic and free-living nematodes may not be as radically different as preliminary studies would suggest, and that the absence of AF1, AF2 and KSAYMRFamide on the C. elegans FMRFamide-related peptide gene (flp-1) may imply the presence of at least two different FaRP genes in nematodes. (C) 1994 Academic Press, Inc.

RYIRFAMIDE - A TURBELLARIAN FMRFAMIDE-RELATED PEPTIDE (FARP)

FMRFamide was isolated originally from neural-tissue extracts of a bivalve mollusc, since when either authentic FMRFamide or a series of structurally-related peptides have been isolated from representative arthropods, annelids and many additional molluscs. However, to date no information exists as to the definitive presence and primary structure of a FaRP in a free-living flatworm. Here, we report the isolation and primary structure of a FaRP from the free-living turbellarian, Artioposthia triangulata, a species from which NPF has been previously structurally-characterised. Unlike molluscs and insects, in which several FaRP a are expressed, only a single member of this family was detected in this turbellarian. The primary structure of this turbellarian FaRP was established as Arg-Tyr-Ile-Arg-Phe-NH2 (RYIRFamide) and the molecular mass as 752.7 Da. These data have established unequivocally that FaRPs occur in the nervous systems of the most phylogenetically-ancient invertebrates which display bilaterally-symmetrical neuronal plans and that authentic FMRFamide is probably not the original member of the family in molecular evolutionary terms.

Antimicrobial peptides and alytesin are co-secreted from the venom of the Midwife toad, Alytes maurus (Alytidae, Anura): Implications for the evolution of frog skin defensive secretions

The skin secretions of frogs and toads (Anura) have long been a known source of a vast abundance of bioactive substances. In the past decade, transcriptome data of the granular glands of anuran skin has given new impetus to investigations of the putative constituent peptides. Alytes obstetricans was recently investigated and novel peptides with antimicrobial activity were isolated and functionally characterised. However, genetic data for the evolutionarily ancient lineage to which Alytes belongs (midwife toads; Alytidae) remains unavailable.

Here we present the first such genetic data for Alytidae, derived via the granular gland transcriptome of a closely-related species of midwife toad, Alytes maurus. First, we present nucleotide sequences of the entire peptide precursors for four novel antimicrobial peptides (AMPs). The two precursors resemble those from Bombinatoridae in both their structural architecture and amino acid sequence. Each precursor comprises two AMPs as tandem repeats, with a member of the alyteserin-1 family (alyteserin-1Ma: GFKEVLKADLGSLVKGIAAHVAN-NH2 or alyteserin-1Mb: GFKEVLKAGLGSLVKGIPAHVAN-NH2) followed by its corresponding member from the alyteserin-2 family (alyteserin-2Ma: FIGKLISAASGLLSHL-NH2 or alyteserin-2Mb: ILGAIIPLVSGLLSHL-NH2). Synthetic replicates of the four AMPs possessed minimal inhibitory concentrations (MICs) ranging from 9.5 to 300 µM, with the most potent being alyteserin-2Ma. Second, we also cloned the cDNA encoding an alytesin precursor, with the active alytesin exhibiting high sequence identity to bombesin-related peptides from other frogs. All putative mature peptide sequences were confirmed to be present in the skin secretion via LC/MS.

The close structural resemblance of the alyteserin genes that we isolated for A. maurus with those of Bombina provide independent molecular evidence for a close evolutionary relationship between these genera as well as more support for the convergent evolution of the AMP system within anurans. In contrast to the more evolutionarily conserved nature of neuropeptides (including alytesin, which we also isolated), the more variable nature of the AMP system together with the sporadic distribution of AMPs among anuran amphibians fuels in part our hypothesis that the latter system was co-opted secondarily to fulfil a function in the innate immune system, having originally evolved for defence against potential macropredators.

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

Synucleins are small proteins that are highly expressed in brain tissue and are localised at presynaptic terminals in neurons. alpha-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms of alpha-synuclein have been associated with autosomal-dominant Parkinson's Disease. A fragment of alpha-synuclein has also been identified as the non-Abeta component of Alzheimer's Disease amyloid. In this review we describe some structural properties of alpha-synuclein and the two mutant forms, as well as alpha-synuclein fragments, with particular emphasis on their ability to form beta-sheet on ageing and aggregate to form amyloid-like fibrils. Differences in the rates of aggregation and morphologies of the fibrils formed by alpha-synuclein and the two mutant proteins are highlighted. Interactions between alpha-synuclein and other proteins, especially those that are components of amyloid or Lewy bodies, are considered. The toxicity of alpha-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases.

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

The defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel dermaseptin-related peptides (dermaseptins sVI–sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian skin antimicrobial peptides may in part be explained by individual variation—a factor essential for selective functional molecular evolution and perhaps, ultimately in speciation.

The musculature and associated innervation of adult and intramolluscan stages of Echinostoma caproni (Trematoda) visualised by confocal microscopy

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.

Neuroactive substances and associated major muscle systems in Bucephaloides gracilescens (Trematoda : Digenea) metacercaria and adult

Cholinergic, serotoninergic and neuropeptidergic components of the nervous system were examined and compared in the progenetic metacercaria and adult gasterostome trematode, Bucephaloides gracilescens in order to provide baseline information on neuronal control of the musculature involved in egg-assembly. Enzyme cytochemistry and indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy demonstrated all three classes of neuroactive substance throughout the central and peripheral nervous systems. A comparable orthogonal arrangement of the central nervous system (CNS) and peripheral array of nerve plexuses was observed in both metacercaria and adult. Staining patterns for cholinergic and peptidergic substances showed significant overlap, while the serotoninergic system was confined to a separate set of neurons. Immunostaining for FMRFamide-related peptides (FaRPs) was strong in the CNS and peripheral innervation to the attachment apparatus of metacercaria and adult but was only found in the innervation of the ootype in ovigerous adults, implicating FaRPs in neuronal control of the muscle of the female reproductive tract during egg-assembly.

Isolation and preliminary biological assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans

To date, 9 FMRFamide-related peptides (FaRPs) have been structurally characterised from Caenorhabditis elegans. Radioimmunometrical screening of an ethanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradation analysis and gas-phase sequencing. Unequivocal primary structures for the two FaRPs were determined as Ala-Ala-Asp-Gly-Ala-Pro-Leu-Ile-Arg-Phe-NH2 and Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH2. Using MALDI-TOF mass. spectrometry, the molecular masses of the peptides were found to be 1032 Da (MH) and 875 Da (MH)(+), respectively. Two copies of AADGAPLIRFamide are predicted to be encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFamide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had inhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 muM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 muM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dorsal and ventral muscle strips as well as innervated and denervated preparations, suggesting that these physiological effects are not nerve cord dependent. Addition of ADDGAPLIRFamide (10 muM) to muscle strips preincubated in high-K+ and -Ca2+-free medium resulted in a normal inhibitory response. Peptide addition to muscle strips preincubated in Cl--free medium showed no inhibitory response, suggesting that the inhibitory response of the peptide may be chloride mediated. A normal excitatory response was noted following the addition of 10 muM SVPGVLRFamide to muscle strips preincubated in high-K+, Ca2+- and Cl--free media. (C) 2001 Academic Press.