Schistosome I/Lamides - A new family of bioactive helminth neuropeptides

Here we report the identification of a new family of helminth neuropeptides with members in both nematodes and flatworms, and include preliminary cell biological and functional characterisation of one of the peptides from the trematode parasite of humans, Schistosoma mansoni. Bioinformatics and Rapid Amplification of cDNA Ends (RACE)-PCR were used to identify the completes. mansoni neuropeptide precursor gene Sm-npp-1, which encodes three pentapeptides bearing the motif (A/G)FVR(I/L).NH2. Similar peptides were identified in three other flatworm species and in 15 nematode species. Quantitative PCR (qPCR) and immunocytochemical (ICC) analyses showed that Sm-npp-1 is constitutively expressed in larval and adult worms. ICC and confocal microscopy were employed to localise one of the schistosome NPP-1 peptides (GFVRIamide) in adult worms and schistosomules; antibodies labelled a pair of neurones in the cerebral ganglia that extend posteriorly along the main nerve cords. GFVRIamide displayed no detectable co-localisation with FMRFamide-like peptides (FLPs), nor was it detectable in muscle innervation. Exogenously applied peptide had a significant inhibitory effect on the mobility of whole adult worm pairs at 10(-5) M (n = 9). Finally, we explored Sm-npp-1 function in schistosomules using RNA interference (RNAi); we successfully achieved specific knockdown of the Sm-npp-1 transcript (54.46 +/- 10.41% knockdown, n = 3), but did not detect any clear, aberrant mobility or morphological phenotypes. NPP-1-like peptides are a new family of helminth peptides with a cell-specific expression pattern distinct from FLPs and a modulatory effect on schistosome muscular activity. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Electrophysiological, pharmacological and molecular profile of the transient outward rectifying conductance in neonatal rat symapthetic preganglionic neurons in vitro.

Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K + channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1-10 µM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Ka (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Ka and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 µM). Single-cell RT-PCR revealed mRNA expression for the a-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory ß-subunits was detected for Kvß2 in all SPN with differential expression of mRNA for KChIP1, Kvß1 and Kvß3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the ß-subunit Kvß2. Differential expression of the accessory ß subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons. © 2011 IBRO.

Immunocytochemical localization of glutamate-like immunoreactivity within the nervous system of the cestode Mesocestoides corti and the trematode Fasciola hepatica

The localization and distribution of glutamate-like immunoreactivity (IR) in the nervous system of both the cestode Mesocestoides corti and the trematode Fasciola hepatica has been determined by an indirect immunofluorescent technique, in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the central (CNS) and peripheral (PNS) nervous systems of both species examined. In the CNS, IR was evident in nerve cells and fibres in the cerebral ganglia, the cerebral commissure and the dorsal, ventral and longitudinal nerve cords. In the peripheral nervous system (PNS) of M. corti, IR was apparent in nerve plexuses associated with the subtegmental musculature and the musculature associated with the anteriorly positioned suckers. In F. hepatica, IR was evident in the innervation of both the oral and the ventral suckers, In the reproductive system of F. hepatica, glutamate-IR was observed around the ootype/Mehlis' gland complex.

NPY and cardiac diseases

Hypertension-induced left ventricular hypertrophy (LVH), along with ischemic heart disease, result in LV remodeling as part of a continuum that often leads to congestive heart failure. The neurohormonal model has been used to underpin many treatment strategies, but optimal outcomes have not been achieved. Neuropeptide Y (NPY) has emerged as an additional therapeutic target, ever since it was recognised as an important mediator released from sympathetic nerves in the heart, affecting coronary artery constriction and myocardial contraction. More recent interest has focused on the mitogenic and hypertrophic effects that are observed in endothelial and vascular smooth muscle cells, and cardiac myocytes. Of the six identified NPY receptor subtypes, Y-1, Y-2, and Y-5 appear to mediate the main functional responses in the heart. Plasma levels of NPY become elevated due to the increased sympathetic activation present in stress-related cardiac conditions. Also, NPY and Y receptor polymorphisms have been identified that may predispose individuals to increased risk of hypertension and cardiac complications. This review examines what understanding exists regarding the likely contribution of NPY to cardiac pathology. It appears that NPY may play a part in compensatory or detrimental remodeling of myocardial tissue subsequent to hemodynamic overload or myocardial infarction, and in angiogenic processes to regenerate myocardium after ischemic injury. However, greater mechanistic information is required in order to truly assess the potential for treatment of cardiac diseases using NPY-based drugs.

The nervous system of Amphilina foliacea (Platyhelminthes, Amphilinidea). An immunocytochemical, ultrastructural and spectrofluorometrical study

The nervous system of young and adult Amphilina foliacea was studied with immunocytochemical, electron microscopical and spectrofluorometrical methods. The general neuroanatomy is described in detail. New data on the structure and development of the brain were obtained. The 5-HT and GYIRFamide-immunoreactivities occur in separate sets of neurones. The innervation of the reproductive organs is described. The fine structure of 2 types of neurones in the CNS, a sensory neurone, a 'glial' cell type, the neuropile and the synapses are described. The level of 5-HT varies between 0.074 and 0.461 mug/g wet weight. This is the first detailed study of the nervous system of A. foliacea. Earlier data on the structure of the nervous system in A. foliacea published in Russian are introduced into the discussion. The study provides data that can be used when considering the phylogenetic position of Amphilinidea.

Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

THE GULL-TAPEWORM, DIPHYLLOBOTHRIUM-DENDRITICUM AND NEUROPEPTIDE-F - AN IMMUNOCYTOCHEMICAL STUDY

Neuropeptide F (Moniezia expansa) immunoreactivity (NPF-IR) has been detected in the nervous system of plerocercoid and adult stages of the gull-tapeworm Diphyllobothrium dendriticum, using immunocytochemical methodology. The application of the antiserum for this authentic flatworm neuropeptide to whole-mounts and frozen sections of the worm has resulted in new information about its neuroanatomy. Thus, at regular intervals, transverse nerves extend from the main nerve cords laterally, joining the longitudinal lateral minor cords in the cortical parenchyma. In the adult worm, the transverse nerves are located at the posterior border of each proglottis. The medullary parenchyma lacks NPF-IR. The NPF-immunoreactive cell bodies are bi- to multipolar and preferentially located in the peripheral nervous system, in close association with the holdfast musculature of the scolex and the extensive body musculature. NPF-IR was observed in the innervation to the muscular ducts of the reproductive system. The pattern of NPF-IR was compared with that recorded for RFamide- and 5-HT-IR and double-immunostaining has revealed separate populations of serotoninergic and peptidergic neurones.

GRILLOTIA ERINACEUS (CESTODA, TRYPANORHYNCHA) - LOCALIZATION OF NEUROACTIVE SUBSTANCES IN THE PLEROCERCOID, USING CONFOCAL AND ELECTRON-MICROSCOPIC IMMUNOCYTOCHEMISTRY

Indirect immunocytochemistry, in conjunction with confocal scanning laser microscopy and electron-microscopic immunogold labeling, has been used to localize neuropeptide and 5-hydroxytryptamine (5-HT) immunereactivities (IRs) in the plerocercoid (scolex and surrounding blastocyst) of the trypanorhynch tapeworm, Grillotia erinaceus. Antisera directed to two native cestode neuropeptides, neuropeptide F and the FMRFamide-related peptide, GNFFRFamide, were used to demonstrate the presence of a well-developed and extensive peptide-immunoreactive nervous system of central and peripheral elements in the juvenile scolex. Neuronal connectivity exists between the scolex and the surrounding blastocyst, in which there is a rich innervation of varicose fibers displaying peptide IR. Ultrastructurally, gold labeling of the peptide IR was found exclusively over the contents of dense secretory vesicles in the axons and somatic cytoplasm of neurons. Double-labeling experiments demonstrated an apparent colocalization of peptide IR, although the results of antigen preadsorption procedures indicated substantial cross-reactivity of the two antisera. A separate and well-differentiated 5-HT-immunoreactive nervous system, with a similar anatomical arrangement as the peptide-immunoreactive nervous system, is present in both the scolex and blastocyst (C) 1994 Academic Press, Inc.

5-HYDROXYTRYPTAMINE (SEROTONIN)-IMMUNOREACTIVITY IN THE NERVOUS-SYSTEM OF MESOCESTOIDES-CORTI TETRATHYRIDIA (CESTODA, CYCLOPHYLLIDEA)

An indirect immunocytochemical technique has been interfaced with confocal scanning laser microscopy to investigate the occurrence and distribution of serotoninergic (5-HT) nerve elements in Mesocestoides corti tetrathyridia. Cell bodies and nerve fibers immunoreactive to 5-HT were found concentrated in the innervation around the 4 suckers and associated commissures and in the 5 pairs of longitudinal nerve cords and their cross-connectives. Immunoreactivity was evident also in the extensive, peripheral network of fine fibers of the subtegumental region and in the plexus of varicose fibers that innervate the muscle in each of the suckers. In dividing stages of the tetrathyridium, the immunoreactive lateral nerve cords of adjoining progeny were in continuity around the base of the division cleft.

AN IMMUNOCYTOCHEMICAL STUDY OF PUTATIVE NEUROTRANSMITTERS IN THE METACERCARIAE OF 2 STRIGEOID TREMATODES FROM RAINBOW-TROUT (ONCORHYNCHUS-MYKISS)

Whole mounts of the metacercariae of Diplostomum sp. and Cotylurus erraticus from rainbow trout have been treated cytochemically for the demonstration of cholinergic, serotoninergic (5-hydroxytryptamine) and peptidergic elements in the nervous system. Antisera directed against four vertebrate (pancreatic polypeptide, peptide YY, substance P and peptide histidine isoleucine) and two invertebrate peptides (neuropeptide F and FMRFamide) were used in an indirect immunofluorescence procedure in conjunction with confocal scanning laser microscopy (CSLM). Of the seven antisera tested, all except peptide histidine isoleucine showed significant immunoreactivity. Cholinergic and serotoninergic staining was found primarily in the central nervous system (CNS) and in cell bodies associated with the ventral and dorsal nerve cords in both trematodes. Peptidergic immunoreactivity was localised in the CNS and PNS of both genera, revealing an extensive innervation within the holdfast organ and in and around the oral and ventral suckers.

THE CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC COMPONENTS OF THE NERVOUS-SYSTEM OF MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

The central (CNS) and peripheral (PNS) nervous systems of the cyclophyllidean tapeworm, Moniezia expansa, were examined for the presence of cholinergic, serotoninergic and peptidergic elements using enzyme cytochemical and immunocytochemical techniques in conjunction with light and confocal scanning laser microscopy. Cholinesterase activity and 5-hydroxytryptamine- and regulatory peptide-immunoreactivities (IRs) were localized to the nerve fibres and cell bodies of all of the major neuronal components in the CNS of the worm, including the cerebral ganglia and connecting commissure, the 10 longitudinal nerve cords and associated transverse ring commissures. Although each of the 3 systems appeared well developed and comprised a significant portion of the nervous system, the serotoninergic constituent was the most highly developed, consisting of a vast array of nerve fibres and cell bodies distributed throughout the strobila of the worm. A close association of cholinesterase reactivity and peptide-IRs was evident throughout the CNS, indicating the possible co-localization of acetylcholine and neuropeptides. Within the PNS, cholinergic activity and serotoninergic- and peptidergic-IRs occurred in the subtegumental network of nerve fibres and somatic musculature. Although all 3 neurochemical elements were present in the acetabula, they were found in different nerve fibres; only cholinergic and peptidergic cell bodies were found. The common genital opening, vagina and ootype regions of the reproductive system displayed a rich innervation of all 3 types of neuronal populations. Within the peptidergic system, immunostaining with antisera raised to the C-terminus of the neuropeptide Y superfamily of peptides and the invertebrate peptides, neuropeptide F (M. expansa) and FMRFamide was the most prevalent. Limited positive-IR for substance P and neurokinin A were also recorded in the CNS of the worm.

NEUROPEPTIDE F-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE DICLIDOPHORA-MERLANGI

The localisation and distribution of neuropeptide F (NPF)-immunoreactivity (IR) in the monogenean fish-gill parasite, Diclidophora merlangi, have been investigated by whole-mount immunocytochemistry interfaced with confocal scanning laser microscopy and, at the ultrastructural level, by indirect immunogold labeling. Using antisera directed to intact synthetic NPF (Moniezia expansa, residues 1-39) or to the C-terminal decapeptide (residues 30-39) of synthetic NPF (M. expansa), immunostaining was found throughout the central (CNS) and peripheral nervous systems (PNS), including the innervation of the reproductive system. Immunoreactivity was found to be more intense using the antiserum to the C-terminal decapeptide fragment of NPF. At the subcellular level, gold labeling of NPF-IR was found exclusively over the contents of dense-cored vesicles that occupied nerve axons of both the CNS and the PNS. The distribution pattern of immunostaining for NPF mirrored exactly that previously documented for the vertebrate pancreatic polypeptide (PP) family of peptides and for FMRFamide. This finding and the results of preabsorption experiments strongly suggest that NPF is the predominant native neuropeptide in D. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.

5-HYDROXYTRYPTAMINE-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE, ENTOBDELLA-SOLEAE

5-HT-immunoreactivity in Entobdella soleae was found to be extensive throughout both the central and peripheral nervous systems, with the strongest staining occurring in the innervation of the forebody, most notably in the paired cerebral ganglia, pharynx and adhesive pads. In the reproductive system, staining was evident throughout the numerous cell bodies and fibres innervating the musculature of the egg-assembly apparatus. The haptor contained an extensive array of serotoninergic fibres derived from the main longitudinal cords; this array was associated with the haptoral muscles and sclerites, and possibly with the ventral sensory papillae.

A CYTOCHEMICAL STUDY OF THE NERVOUS-SYSTEM OF THE PROTEOCEPHALIDEAN CESTODE, PROTEOCEPHALUS-POLLANICOLA

The localization and distribution of cholinergic, serotoninergic and peptidergic nerve elements in the proteocephalidean tapeworm, Proteocephalus pollanicola, have been investigated by enzyme histochemistry, and by an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy. Cholinesterase (ChE) activity was localized in the major components of the central nervous system (CNS) and the peripheral nervous system (PNS), including the innervation of the reproductive structures of the worm. Serotoninergic (5-HT) nerves were found in the paired cerebral ganglia, transverse commissure and in the 10 longitudinal nerve cords. Antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide have been used to explore the peptidergic nervous system of the worm. The most extensive immunostaining occurred with antisera raised to members of the neuropeptide Y superfamily, namely neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP). In all cases, intense immunoreactivity was found in numerous cell bodies and fibres of both the CNS and PNS, including the innervation of the reproductive apparatus. FMRFamide antisera stained the same structures to a comparable degree as those raised to the NPY superfamily. Cholinergic and peptidergic elements were much more prevalent within the CNS, while the serotoninergic nerve fibres tended to dominate in the PNS. The overlap obtained in staining patterns for the peptidergic and cholinergic components suggests that there may be a certain amount of co-localization of peptides with small-molecule transmitter substances in the same neurone. Weak staining for the tachykinin, substance P and for calcitonin gene-related peptide(CGRP) was confined to the major longitudinal nerve cords.