NEUROPEPTIDE-F - A NOVEL PARASITIC FLATWORM REGULATORY PEPTIDE FROM MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

Using a C-terminally directed pancreatic polypeptide (PP) antiserum and immunocytochemical methods, PP-immunoreactivity (IR) was localized throughout the central (CNS) and peripheral nervous systems (PNS) of the cestode, Moniezia expansa. In the CNS, immunostaining was evident in the paired cerebral ganglia (primitive brain), connecting commissure, and the paired longitudinal nerve cords that are cross-linked by numerous regular transverse connectives. The PNS was seen to consist of a fine anastomosing nerve-net of immunoreactive fibres, many of which were closely associated with reproductive structures. Radioimmunoassay of this peptide IR in acid-alcohol extracts of the worm measured 192.8 ng/g of PP-IR. HPLC analyses of the M. expansa PP-IR identified a single molecular form which was purified to homogeneity. Plasma desorption mass spectrometry (PDMS) of purified parasite peptide resolved a single peptide with a molecular mass of 4599 +/- 10 Da. Automated gas-phase Edman degradation identified a 39-amino acid peptide with a C-terminal phenylalaninamide. Examination of its primary structure shows that it displays significant sequence homology with the vertebrate neuropeptide Y superfamily, suggesting that this platyhelminth-derived peptide is the phylogenetic precursor. Neuropeptide F (M. expansa) is the first regulatory peptide to be fully sequenced from the phylum Platyhelminthes and may represent a member of an important new class of invertebrate neuropeptide.

Pharmacological effects of nematode FMRFamide-related peptides (FaRPs) on muscle contractility of the trematode, Fasciola hepatica

The physiological effects of synthetic replicates of the nematode FaRPs, AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), PF1 (SDPNFLRFamide), PF2 (SADPNFLRFamide), AF8/PF3 (KSAYMRFamide) and PF4 (KPNFIRFamide) were examined on muscle preparations of the liver fluke, Fasciola hepatica. Changes in contractility following the addition of the test compound were recorded using a photo-optic transducer system. Unlike the varied effects these peptides have on nematode somatic musculature, all were found to induce excitatory responses in the muscle activity of F. hepatica. While qualitative effects of the nematode peptides were similar in that they induced increases in both the amplitude and frequency of F. hepatica muscle contractions, they varied considerably in the potency of their excitatory effects. The threshold activity for each peptide was as follows: 10 mu M, PF1 and PF2; 3 mu M, AF1 and PF3; 1 mu M, AF2; and 30 nM, PF4. The results demonstrate, for the first time, the cross-phyla activity of nematode neuropeptides on the neuromuscular activity of a trematode.

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.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),Leu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for Ile(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier Science Inc.

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.

Influence of alpha-helicity, amphipathicity and D-amino acid incorporation on the peptide-induced mast cell activation

Mast cell activation by polycationic substances is believed to result from a direct activation of G protein alpha subunits and it was suggested that the adaption of amphipathic, alpha-helical conformations would allow the peptide to reach the cytosolic compartment to interact with G proteins (Mousli et al., 1994, Immunopharmacology 27, 1, for review). We investigated the histamine-releasing activity of model peptides as well as analogues of magainin 2 amide and neuropeptide Y with different amphipathicities and alpha-helix content on rat peritoneal mast cells. Amphipathic helicity is not a prerequisite for mast cell activation. Moreover, non-helical magainin peptides with high histamine-releasing activity were less active in the liberation of carboxyfluoresceine from negatively charged liposomes, indicating that peptide-induced mast cell activation and peptide-induced membrane perturbation do not correlate. In contrast to the negligible influence of the secondary structure, amino acid configuration may exert a striking influence on peptide-induced mast cell activation. Thus histamine-release by substance P was markedly impaired when the L-amino acids in the positively charged N-terminal region were replaced by D-amino acids, with [D-Arg(1)]substance P being the most inactive substance P diastereoisomer.

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.

ELECTRON IMMUNOGOLD LABELING OF REGULATORY PEPTIDE IMMUNOREACTIVITY IN THE NERVOUS-SYSTEM OF MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

An electron immunogold-labeling technique was used in conjunction with a post-embedding procedure to demonstrate for the first time the ultrastructural distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF), in the nervous system of the cestode Moniezia expansa. Two axon types, distinguished by their populations of different-sized electron-dense vesicles, were identified. Immunogold labeling demonstrated an apparent homogeneity of PP, FMRFamide and NPF (M. expansa) antigenic sites throughout the larger dense-cored vesicles within the central nervous system. Triple labeling clearly demonstrated the co-localisation of immunoreactivities (IR) for NPF, PP and FMRFamide within the same dense-cored vesicles. The presence of NPF-IR within the vesicles occupying the perikaryon of the neuronal cell body indicated that the peptides had undergone post-translational C-terminal amidation prior to entering the axon. Antigen pre-absorption experiments using NPF prevented labeling with either PP or FMRFamide antisera, and the failure of these antisera to block NPF-IR supports the view that some, if not all, of the PP/FMRFamide-IR is due to NPF-like peptides.

NEUROPEPTIDE-Y AND NEUROPEPTIDE-Y 3-36 - ISOLATION FROM HUMAN PANCREATIC ENDOCRINE TUMORS

Using an antiserum raised to the C-terminal region of neuropeptide Y (NPY) which does not cross-react with pancreatic polypeptide (PP), immunoreactivity has been detected in two different endocrine tumours of the human pancreas in concentrations permitting isolation and structural analysis. In a clinically-typical gastrinoma, resected from the head of pancreas, the concentration of NPY immunoreactivity was 3.4 nmol/g. Reverse phase HPLC analysis of extracts of this tumour resolved a single immunoreactive peptide coeluting with synthetic human NPY. The molecular mass of the isolated peptide, determined by mass spectroscopy, was 4270 Da, which was in close agreement with that derived from the deduced primary structure of human tumour NPY (4271.7 Da), obtained by gas-phase sequencing. A somatostatinoma, resected from the region of the ampulla of Vater, contained 3.8 nmol/g of NPY immunoreactivity and isolation of this immunoreactive peptide followed by structural analyses, indicated a molecular structure consistent with NPY 3-36. These data suggest that NPY immunoreactivity detected in human pancreatic endocrine tumours is molecularly heterogenous, a finding which may be of relevance in the symptomatology of such tumours as attenuation of the N-terminus of this peptide generates receptor selectivity.