Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24 h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4 h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4 h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24 h soaks. However, a 10-fold increase in dsRNA to 1 mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24 h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

Antimicrobial activity of neuropeptides against a range of micro-organisms from skin, oral, respiratory and gastrointestinal tract sites

Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. This study aimed to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), displayed antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. SP, NPY, VIP and CGRP displayed variable degrees of antimicrobial activity against all the pathogens tested with the exception of S. aureus. These antimicrobial activities add a further dimension to the immunomodulatory roles for neuropeptides in the inflammatory and immune responses. (c) 2008 Elsevier B.V. All rights reserved.

Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts

Abstract
INTRODUCTION:
Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro
METHODS:
An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts.
RESULTS:
Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro.
CONCLUSIONS:
The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair.

SRIF receptor subtype expression and involvement in positive and negative contractile effects of somatostatin-14 (SRIF-14) in ventricular cardiomyocytes.

Background/Aims: Somatostatin-14 (SRIF-14), a neuropeptide co-stored with acetylcholine in the cardiac parasympathetic innervation, exerts both positive and negative influences directly on contraction of ventricular cardiomyocytes, indicative of involvement of more than one of five known SRIF (SSTR) receptor subtypes. The aim was to characterize receptor subtype expression in adult rat ventricular cardiomyocytes and to investigate the influence of a series of SRIF (SSTR) subtype-selective agonists on contractile parameters. Methods: mRNA and protein expression of each receptor subtype were quantified by RT-PCR and immunoblotting respectively; for contraction studies, cells were stimulated at 0.5 Hz under basal conditions and in the presence of isoprenaline (ISO, 10-8M). Results: all five SRIF (SSTR) receptor subtypes were expressed in cardiomyocytes although SRIF1A (SSTR2) and SRIF2A (SSTR1) were less abundant than the other subtypes. L803087 (10-8M), a SRIF2B (SSTR4) agonist, attenuated ISO-stimulated peak contractile amplitude and prolonged relaxation time (T50). L796778 (10-7M), a SRIF1C (SSTR3) agonist, augmented basal and ISO-stimulated peak contractile amplitude; L779976 (10-8M) and L817818 (10-9M), agonists at SRIF1A (SSTR2) and SRIF1B (SSTR5) receptors, respectively, also augmented ISO-stimulated peak amplitude. Conclusion: these data support involvement of SRIF2B (SSTR4) receptors in the negative contractile effects of SRIF-14, while one or more of the three SRIF1 receptor subtypes (SSTR2, 3 or 5) may contribute to the positive contractile effects of SRIF-14.

Substance P induces histamine release from human pulmonary mast cells.

Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non-preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 μM) and A23187 caused histamine release (median 26.7%, range 6.2–62.8% and 32.1%, 7.7–56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1–33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen.

RNA interference in a cestode reveals specific silencing of selected highly expressed gene transcripts

Evolving RNA interference (RNAi) platforms are providing opportunities to probe gene function in parasitic helminths using reverse genetics. Although relatively robust methods for the application of RNAi in parasitic flatworms have been established, reports of successful RNAi are confined to three genera and there are no known reports of the application of RNAi to the class Cestoda. Here we report the successful application of RNAi to a cestode. Our target species was the common ruminant tapeworm, Moniezia expansa which can significantly impact the health/productivity of cattle, sheep and goats. Initial efforts aimed to silence the neuronally expressed neuropeptide F gene (Me-npf-1), which encodes one of the most abundant neuropeptides in flatworms and a homologue of vertebrate neuropeptide Y (NPY). Double stranded (ds)RNAs, delivered by electroporation and soaking (4-8 h), failed to trigger consistent Me-npf-1 transcript knock-down in adult worms; small interfering RNAs (siRNAs) were also ineffective. Identical approaches resulted in significant and consistent transcript knock-down of actin transcript (71 +/- 4%) following soaking in Me-act-1 dsRNA. Similar successes were seen with hydrophobic lipid-binding protein (Me-lbp-1), with a dsRNA inducing significant target transcript reduction (72 +/- 5%). To confirm the validity of the observed transcript knock-downs we further investigated Me-act-1 RNAi worms for associated changes in protein levels, morphology and phenotype. Me-act-1 RNAi worms displayed significant reductions in both filamentous actin immunostaining (62 +/- 3%) and the amount of actin detected in Western blots (54 +/- 13%). Morphologically, Me-act-1 RNAi worms displayed profound tegumental disruption/blebbing. Further, muscle tension recordings from Me-act-1 RNAi worms revealed a significant reduction in both the number of worms contracting in response to praziquantel (20 +/- 12%) and in their contractile ability. These data demonstrate, to our knowledge for the first time, a functional RNAi pathway in a cestode and show that the robust knock-down of abundant gene transcripts is achievable using long dsRNAs following short exposure times. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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.

SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN THE PROTOSCOLEX OF ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and neuropeptides in the nervous system of the protoscolex of the hydatid organism Echinococcus granulosus were determined by an indirect immunofluorescence technique. Nerve-cell bodies immunoreactive for 5-HT occurred in the lateral ganglia and in association with the lateral longitudinal nerve cords. 5-HT immunostaining was also evident in the central nerve ring, in the rostellar nerves and in the nerve plexus innervating the suckers. Of the antisera used to screen the protoscolex for neuropeptide immunoreactivity (IR), immunostaining was obtained with those raised against pancreatic polypeptide (PP), peptide YY (PYY), substance P (SP), peptide histidine isoleucine (PI-II) and vasoactive intestinal peptide (VIP). The most extensive pattern of IR occurred with antisera to PP and PYY. Immunoreactive nerve elements were evident in the lateral ganglia, central nerve ring, rostellar nerves, rostellar ganglia, sucker plexus and longitudinal nerve cords. The distribution of SP-, PHI- and VIP-IRs was more restricted: SP-IR occurred in the lateral ganglia and sucker nerves, whilst PHI- and VIP-immunoreactive nerve elements were associated with the lateral longitudinal nerve cords. Protoscoleces cultured in vitro for 29 days were also examined and neuroanatomical changes noted. A greater development of the longitudinal nerve cords and their cross-connectives in the body of the worm was evident, and a group of nerve cells were seen to develop at the posterior end of the main lateral nerve cords.

IMMUNOELECTRON MICROSCOPIC STUDIES OF REGULATORY PEPTIDES IN THE NERVOUS-SYSTEM OF THE MONOGENEAN PARASITE, DICLIDOPHORA-MERLANGI

Specific antisera, directed against the highly conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) and the invertebrate peptide FMRFamide, have been used in conjunction with post-embedding, IgG-conjugated colloidal gold immunostaining to demonstrate peptide immunoreactivity at subcellular level in the nervous system of adult Diclidophora merlangi. Gold labelling revealed that immunoreactivity for PP and FMRFamide was localized exclusively in dense-cored vesicles occupying the majority of axons in the central nervous system. Double-labelling demonstrated an apparent co-localization of PP and FMRFamide in the same dense-cored vesicles. Antigen preabsorption experiments indicated cross-reactivity of the two antisera as unlikely, and that some if not all of the PP/FMRFamide immunostaining in the parasite was due to a neuropeptide F-like peptide.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE CENTRAL-NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

CYTOCHEMICAL DEMONSTRATION OF CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN GORGODERINA-VITELLILOBA (TREMATODA, DIGENEA)

Standard enzyme cytochemical and indirect immunocytochemical techniques have been used in conjunction with light and confocal scanning laser microscopy (CSLM) to visualize cholinergic, serotoninergic and peptidergic nerve elements in whole-mount preparations of the amphibian urinary-bladder fluke, Gorgoderina vitelliloba. Cholinesterase (ChE) activity was localized in paired anterior ganglia, a connecting dorsal commissure and in the origins of the ventral nerve cords. Cholinergic ganglia were also evident in shelled embryos in the uterus. Serotonin-immunoreactivity (IR) was more extensive than ChE activity and was identified in both the central and peripheral nervous systems. Serotoninergic nerve fibres were associated with the somatic musculature and female reproductive ducts. Antisera to nine mammalian peptides and one invertebrate (FMRFamide) peptide have been used to investigate the peptidergic nervous system in the parasite. Immunoreactivity was obtained to five peptides, namely pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP) and FMRFamide. Peptidergic nerve fibres were found to be more abundant than demonstrable cholinergic or serotoninergic nerve fibres. NPY-IR was identified only in the main components of the central nervous system. However, PP- and PYY-IR occurred in the anterior ganglia, dorsal commissure, main nerve cords and in numerous small varicose fibres that ramified throughout the worm. Additionally, PP-immunoreactive nerve fibres were found to innervate the musculature of the female reproductive tracts. Six sites of IR were found in the acetabulum, using antisera directed towards the C-terminal end of PP and PYY, and these matched with the distribution of six non-ciliated rosette-like papillae observed by scanning electron microscopy. SP- and FMRFamide-IR were identified in the CNS, and FMRFamide-immunopositive nerve fibres were also evident in association with the gonopore/cirrus region and with the terminal excretory pore. Results are discussed with respect to possible roles for each of the neurochemical types.

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.

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.

The nervous system of Procerodes littoralis (Maricola, Tricladida). An ultrastructural and immunoelectron microscopical study

The ultrastructure of the nervous system of a planarian, Procerodes littoralis, belonging to the taxon Maricola is described for the first time. The study has revealed the presence of two neuronal cell types and a glia-like cell. Immunogold labelling with antibodies to two native flatworm neuropeptides-neuropeptide F and GNFFRFamide-has been localised to one neuronal cell type and associated processes and synapses, thus indicating its peptidergic nature. The ultrastructural features are compared to those of other investigated turbellarian species. The number of features shared by species from the Proseriata, Lecitoepitheliata and Tricladida show that in respect of the nervous system these taxa form a closely related group. (C) 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.