Biomedical subjects

N J Marks

Publications and source records attributed to N J Marks.

At least 19 recordsLinked to original sources

PubMed2005

Inter-phyla studies on neuropeptides: the potential for broad-spectrum anthelmintic and/or endectocide discovery.

Flatworm, nematode and arthropod parasites have proven their ability to develop resistance to currently available chemotherapeutics. The heavy reliance on chemotherapy and the ability of target species to develop resistance has prompted the search for novel drug targets. In view of its importance to parasite/pest survival, the neuromusculature of parasitic helminths and pest arthropod species remains an attractive target for the discovery of novel endectocide targets. Exploitation of the neuropeptidergic system in helminths and arthropods has been hampered by a limited understanding of the functional roles of individual peptides and the structure of endogenous targets, such as receptors. Basic research into these systems has the potential to facilitate target characterization and its offshoots (screen development and drug identification). Of particular interest to parasitologists is the fact that selected neuropeptide families are common to metazoan pest species (nematodes, platyhelminths and arthropods) and fulfil specific roles in the modulation of muscle function in each of the three phyla. This article reviews the inter-phyla activity of two peptide families, the FMRFamide-like peptides and allatostatins, on motor function in helminths and arthropods and discusses the potential of neuropeptide signalling as a target system that could uncover novel endectocidal agents.

Animals↗

PubMed2003

The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamide-related peptides.

Caenorhabditis elegans possesses 22 FMRFamide-like peptide (flp) genes predicted to encode 60 different FMRFamide-related peptides with a range of C-terminal signatures. Peptides from five flp genes (1, 6, 8, 9 and 14) are known to modulate the ovijector of Ascaris suum in vitro. This study examines the physiological effects of peptides from the remaining 17 flp genes such that the variety of FMRFamide-related peptide-induced ovijector response types can be delineated. Five categories of response were identified according to the pattern of changes in contractile behaviour and baseline tension. Peptides encoded on 16 flp genes (1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 20) had qualitatively similar inhibitory (response type 1) actions, with the lowest activity thresholds (1 nM) recorded for peptides with FIRFamide or FLRFamide C-terminal signatures. Peptides encoded on four flp genes (2, 18, 19 and 21), and on the A. suum afp-1 gene, had excitatory actions on the ovijector (response type 2), with PGVLRFamides having the lowest activity threshold (1 nM). An flp-2 peptide (LRGEPIRFamide) induced a transient contraction of the ovijector (activity threshold, 10nM) that was designated response type 3. Response type 4 comprised a transient contraction followed by an extended period of inactivity and was observed with peptides encoded on flp-5 (AGAKFIRFamide, APKPKFIRFamide), flp-8 (KNEFIRFamide) and flp-22 (SPSAKWMRFamide). SPSAKWMRFamide was the most potent peptide tested with an activity threshold of 0.1 nM. A single peptide (AMRNALVRFamide; activity threshold 0.1 microM), encoded on flp-11, induced response type 5, a shortening of the ovijector coupled with an increase in contraction frequency. Although most flp genes encode structurally related peptides that trigger one of the five ovijector response types, flp-2 and flp-11 co-encode FMRFamide-related peptides that induce distinct responses. Within the ovijector of A. suum FaRPs play a complex role involving at least five receptor subtypes or signalling pathways.

Animals↗

PubMed2003

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle.

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations </=1 microM. AF2-induced stimulation of cAMP was unaffected by removal of the dorsal or ventral nerve cord, even though this form of denervation abolished the excitatory phase of the tension response. The effects of 0.1 and 1 microM AF2 on cAMP were also unaffected by 10 microM SDPNFLRF-NH(2) (PF1, an inhibitory FaRP) and 10 microM PF1022A (an inhibitory cyclodepsipeptide), even though each of these peptides abolished the excitatory phase of the tension response induced by AF2. Within an alanine-scan series of AF2 analogues, only KHAYLRF-NH(2) stimulated cAMP production with equipotency to AF2; the effects of this peptide on muscle tension also mimicked AF2. Another excitatory FaRP present in nematodes, KNEFIRF-NH(2) (AF1), also stimulated cAMP production, but was 100-fold less potent than AF2. The stimulatory effects of AF1 on tension and cAMP levels were blocked by an alanine-substituted analogue of this peptide (Ala(6)-AF1, KNEFIAF-NH(2)), while the stimulatory effects of AF2 on tension and cAMP were not affected by this analogue. AF2 and AF1 increase A. suum somatic muscle cAMP by targeting different receptors. Increases in cAMP stimulated by AF2 can be decoupled from the excitatory response caused by this peptide, and it is not possible to establish a causal linkage between the contractile response elicited by this peptide and its effects on cAMP accumulation.

Animals↗

PubMed2003

Gross anatomy of the muscle systems and associated innervation of Apatemon cobitidis proterorhini metacercaria (Trematoda: Strigeidea), as visualized by confocal microscopy.

The major muscle systems of the metacercaria of the strigeid trematode, Apatemon cobitidis proterorhini have been examined using phalloidin as a site-specific probe for filamentous actin. Regional differences were evident in the organization of the body wall musculature of the forebody and hindbody, the former comprising outer circular, intermediate longitudinal and inner diagonal fibres, the latter having the inner diagonal fibres replaced with an extra layer of more widely spaced circular muscle. Three orientations of muscle fibres (equatorial, meridional, radial) were discernible in the oral sucker, acetabulum and paired lappets. Large longitudinal extensor and flexor muscles project into the hindbody where they connect to the body wall or end blindly. Innervation to the muscle systems of Apatemon was examined by immunocytochemistry, using antibodies to known myoactive substances: the flatworm FMRFamide-related neuropeptide (FaRP), GYIRFamide, and the biogenic amine, 5-hydroxytryptamine (5-HT). Strong immunostaining for both peptidergic and serotoninergic components was found in the central nervous system and confocal microscopic mapping of the distribution of these neuroactive substances revealed they occupied separate neuronal pathways. In the peripheral nervous system, GYIRFamide-immunoreactivity was extensive and, in particular, associated with the innervation of all attachment structures; serotoninergic fibres, on the other hand, were localized to the oral sucker and pharynx and to regions along the anterior margins of the forebody.

Animals↗

PubMed2001

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-NH(2) and Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH(2). 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 microM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 microM. 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 microM) to muscle strips preincubated in high-K(+) and -Ca(2+)-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 microM SVPGVLRFamide to muscle strips preincubated in high-K(+), Ca(2+)- and Cl(-)-free media.

Animals↗

PubMed2000

Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4.

Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at > or = 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 microM) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.

Animals↗

PubMed2000

Classical neurotransmitters in the ovijector of Ascaris suum: localization and modulation of muscle activity.

Ascaris suum possesses a well-developed nervous system which is regulated by a number of classical neurotransmitters including acetylcholine (ACh), gamma-aminobutyric acid (GABA), glutamate and serotonin. The vagina vera, the distal part of the ovijector, displays intrinsic, rhythmic activity which has been shown to be modulated by FMRFamide-related peptides (FaRPs) in vitro. Confocal scanning laser microscopy coupled with immunocytochemistry, and histochemical studies, revealed that the nerve plexus of the ovijector contains GABAergic and glutamatergic innervation. Although no distinctive cholinergic or serotoninergic innervation was apparent, cholinesterase activity was localized to discrete areas of the musculature of the vagina vera. The effects of classical transmitters on the activity of the vagina vera in vitro were examined. ACh was excitatory, stimulating a brief but powerful contraction of the vagina vera with a threshold for activity of 1 microM. Both GABA and glutamate were inhibitory, causing a cessation of contractile activity at high concentrations (> 10 microM). Although less potent than glutamate, GABA had more profound effects and induced longer-lasting paralysis of the tissue. The threshold concentrations for activity were 5 microM for glutamate and 10 microM for GABA. Serotonin had no consistent effect on the vagina vera. This study demonstrates that classical transmitters modulate the activity of the ovijector of A. suum.

Acetylcholine↗

PubMed1999

Neuromusculature of the ovijector of ascaris suum (Ascaroidea, nematoda): an ultrastructural and immunocytochemical study.

This study used electron microscopy and confocal scanning laser microscopy interfaced with cytochemistry to study neuromuscular interrelationships in the ovijector of Ascaris suum. An extensive nerve plexus with both FaRPergic and non-FaRPergic components extends over the outer surface of the ovijector. The non-FaRPergic component is derived from nerve branches of the ventral nerve cord, whereas the FaRPergic component emanates from two large FMRFamide-immunoreactive neurons. In the vagina vera, most myofibrils are circular in orientation and a number of them divide and run for short distances in longitudinal and diagonal directions, their myofilaments are also orientated in a variety of directions. Parallel nerve fibres run in tracts along the length of the vagina vera with branches that penetrate the muscle layers. The vagina uteri possesses a thicker hypodermis than that of the vagina vera. It appears rich in secretory and phagocytic vesicles and the luminal side is invested with an electron-dense substance. The musculature of the vagina uteri is less well developed than that of the vagina vera, being restricted to circular myofibrils, with an apparent diagonal arrangement of myofilaments. Also, the innervation is less extensive in the vagina uteri with many fibres returning to the vagina vera to rejoin the nerve net and others continuing into the uteri.

Animals↗

PubMed1999

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus.

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 microM)-induced contractions, with a threshold for activity of 1 microM. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus.

Amino Acid Sequence↗

PubMed1999

Isolation, pharmacology and gene organization of KPSFVRFamide: a neuropeptide from Caenorhabditis elegans.

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH]+) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, >/=0.1 microM) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, >/=10 nM).

Amino Acid Sequence↗

PubMed1999

Pharmacology of FMRFamide-related peptides in helminths.

Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide-related peptides (FaRPs), with more yet to be identified. To date, few non-FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free-living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand-receptor recognition features among the Nematoda. Structure-activity studies on nematode FaRPs have revealed that structure-activity relationship (SAR) "rules" differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross-phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross-phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non-neuropeptidergic systems, also remains to be elucidated.

Amino Acid Sequence↗

PubMed1998

KSAYMRFamide (PF3/AF8) is present in the free-living nematode, Caenorhabditis elegans.

To date, seven FMRFamide-related peptides (FaRPs) have been structurally characterized from C. elegans, of which one is structurally identical to the parasitic nematode peptide AF2 (KHEYLRFamide). The other six FaRPs have so far been identified in free-living forms only. In the present study an additional FaRP was isolated and structurally characterized from an ethanolic extract of C. elegans. The extract was screened using a C-terminally directed FaRP antiserum, and the FMRFamide-immunoreactive peptide purified to homogeneity using HPLC. Approximately 80 pmol of the peptide was subjected to Edman degradation and the unequivocal primary structure of the K7-amide, KSAYMRFamide (PF3/AF8) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a MALDI-TOF mass spectrometer and was found to be 919 (MH+), which is in agreement with the theoretical mass of C-terminally amidated PF3. A new flp-gene, designated flp-6, has recently been identified which encodes six copies of KSAYMRFamide (PF3/AF8).

Amino Acids↗

PubMed1998

Modulation of the motility of the vagina vera of Ascaris suum in vitro by FMRF amide-related peptides.

Ascaris suum contains a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8, also called PF3) have been extensively studied and are known to exert actions on somatic muscle strips of the worm. In the present study, the effects of AF1, AF2 and AF8 on the activity of the vagina vera of female A. suum have been examined in vitro. The vagina vera is a muscular tube connecting the uterus and vagina uteri to the gonopore and is probably involved in regulating egg output. The tissue exhibited spontaneous, rhythmic contractions in vitro, which were modulated by each of the FaRPs tested. The effects of each of the peptides were qualitatively and quantitatively different, and in each case were reversible. AF1 (1 microM) caused a biphasic response in the form of a transient lengthening of the preparation, followed by a shortening; contractions were initially inhibited but resumed 5 min post-addition of the peptide. Lower concentrations (< or = 0.1 microM) induced a less marked effect, with rhythmic contractions returning 5 min post-addition. AF2 and AF8 reduced contraction frequency at concentrations > or = 0.1 microM. Both peptides also caused the tissue to shorten, although the effects of AF8 on baseline tension were inconsistent. The apparent potencies of AF1 and AF8 on contraction frequency of the vagina vera were 10-fold greater than AF2 and, unlike their actions on A. suum somatic body wall muscles, the actions of AF1 and AF2 were qualitatively different. Indeed, the effects of each of these FaRPs on the vagina vera were markedly different from those observed on the somatic muscle.

Animals↗

PubMed1997

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

To date, 9 FMRFamide-related peptides (FaRPs) have been identified in Caenorhabditis elegans. Eight of these peptides are encoded on the flp-1 gene. However, AF2 (KHEYLRFamide) 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-1 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 Da, 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(+2)- and Cl(-)-independent but was abolished in high-K+ medium and could be distinguished from those of other inhibitory nematode FaRPs, including PF1 (SDPNFLRFamide) and PF4 (KPNFIRFamide).

Amino Acid Sequence↗

PubMed1997

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 microM, PF1 and PF2; 3 microM, AF1 and PF3; 1 microM, 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.

Animals↗

PubMed1996

Cholinergic, serotoninergic and peptidergic components of the nervous system of Discocotyle sagittata (Monogenea:Polyopisthocotylea).

Cholinergic, serotoninergic (5-HT) and peptidergic neuronal pathways have been demonstrated in both central and peripheral nervous systems of adult Discocotyle sagittata, using enzyme histochemistry and indirect immunocytochemistry in conjunction with confocal scanning laser microscopy. Antisera to 2 native flatworm neuropeptides, neuropeptide F and the FMRFamide-related peptide (FaRP), GNFFRFamide, were employed to detect peptide immunoreactivity. The CNS is composed of paired cerebral ganglia and connecting dorsal commissure, together with several paired longitudinal nerve cords. The main longitudinal nerve cords (lateral, ventral and dorsal) are interconnected at intervals by a series of annular cross-connectives, producing a ladder-like arrangement typical of the platyhelminth nervous system. At the level of the haptor, the ventral cords provide nerve roots which innervate each of the 9 clamps. Cholinergic and peptidergic neuronal organisation was similar, but distinct from that of the serotoninergic components. The PNS and reproductive system are predominantly innervated by peptidergic neurones.

Animals↗

PubMed1996

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 [Leu5]PF4,[Ala2]PF4, [Gly2]PF4, [Ala2,Leu5]PF4, and [Gly2,Leu5]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 = [Leu5]PF4 > > [Ala2]PF4 = [Ala2,Leu5]PF4 > > [Gly2]PF4 = [Gly2,Leu5]PF4, Leu5 for Ile5 substitutions in PF4 did not alter the activity of this peptide: however, Gly2/Ala2 for Pro2 substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly2]PF4(2-7) and -(3-7) and [Ala2]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 Pro2 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.

Amino Acid Sequence↗

PubMed1996

FMRFamide-related peptides (FaRPs) in nematodes: occurrence and neuromuscular physiology.

The occurrence of classical neurotransmitter molecules and numerous peptidic messenger molecules in nematode nervous systems indicate that although structurally simple, nematode nervous systems are chemically complex. Thus far, studies on one nematode neuropeptide family, namely the FMRFamide-related peptides (FaRPs), have revealed an unexpected variety of neuropeptide structures in both free-living and parasitic species. To date 23 nematode FaRPs have been structurally characterized including 12 from Ascaris suum, 8 from Caenorhabditis elegans, 5 from Panagrellus redivivus and 1 from Haemonchus contortus. Ten FaRP-encoding genes have been identified in Caenorhabditis elegans. However, the full complement of nematode neuronal messengers has yet to be described and unidentified nematode FaRPs await detection. Preliminary characterization of the actions of nematode neuropeptides on the somatic musculature and neurones of A, suum has revealed that these peptidic messengers have potent and complex effects. Identified complexities include the biphasic effects of KNEFIRFamide/KHEYLRFamide (AF1/2; relaxation of tone followed by oscillatory contractile activity) and KPNFIRFamide (PF4; rapid relaxation of tone followed by an increase in tone), the diverse actions of KSAYMRFamide (AF8 or PF3; relaxes dorsal muscles and contracts ventral muscles) and the apparent coupling of the relaxatory effects of SDPNFLRFamide/SADPNFLRFamide (PF1/PF2) to nitric oxide release. Indeed, all of the nematode FaRPs which have been tested on somatic muscle strips of A. suum have actions which are clearly physiologically distinguishable. Although we are a very long way from understanding how the actions of these peptides are co-ordinated, not only with those of each other but also with those of the classical transmitter molecules, to control nematode behaviour, their abundance coupled with their diversity of structure and function indicates a hitherto unidentified sophistication to nematode neuromuscular intergration.

Animals↗