RESUMEN
Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 µM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 µM; fmoc-1, IC50, 10.00 ± 1.38 µM; fmoc-2, IC50, 16.67 ± 1.95 µM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.
Asunto(s)
Ascaris suum/anatomía & histología , Ascaris suum/efectos de los fármacos , Ascaris suum/metabolismo , Descubrimiento de Drogas/métodos , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Regulación Alostérica , Sitio Alostérico/genética , Animales , Ascaris suum/genética , Sitios de Unión/genética , Butaclamol/farmacología , Simulación por Computador , Sistemas de Liberación de Medicamentos , Fluorenos/metabolismo , Fluorenos/farmacología , Humanos , Concentración 50 Inhibidora , Ácidos Isonipecóticos/metabolismo , Ácidos Isonipecóticos/farmacología , Ligandos , Modelos Moleculares , Agonistas Nicotínicos/química , Nitrilos/farmacología , Oocitos , Técnicas de Placa-Clamp , Tetrahidroisoquinolinas/farmacología , Torpedo/genética , Torpedo/fisiología , Xenopus/genéticaRESUMEN
The authors describe an individual of the female Ascaris suum Goeze with a unique genital system. A female with such an anomaly was found during laboratory classes of Invertebrate Zoology at the Faculty of Biology and Environmental Protection, NCU. The specimen was about 200 mm long, and the total length of the reproductive system was 1970 mm. Further comparative analysis between unchanged and changed individuals revealed differences in the length of individual sections of the studied system, as well as in the diameter of the uterus. The described case is extremely interesting because of the phenomenon of eutely occurring in nematodes. The exact cause and mechanism of abnormalities described in Ascaris suum are not known and difficult to explain experimentally because of the extremely small number of these anomalies. Moreover, the interpretation of the anomaly is difficult because of the specific behavior and complex morphogenesis of this endoparasite.
Asunto(s)
Ascaris suum/anatomía & histología , Animales , Femenino , Genitales Femeninos/anomalías , PoloniaRESUMEN
SUMMARY: The genotypes of both host and parasite may influence the outcome of parasitic infections, but few attempts have been made to quantify the effect of parasite genotype on macroparasite infections of socio-economic importance. We examined variation in particular traits during the infection in pigs with the parasitic nematode Ascaris suum. We infected 26 pigs with mixtures of equal proportions of embryonated eggs from 4 single female worms each with a unique mtDNA haplotype--the eggs from each female worm were a mixture of siblings and half-siblings. Pigs were necropsied on days 14, 17 and 28 following inoculation, which corresponded to time-points before, during and after the main immune responses against the nematode. A total of approximately 11,000 worms were recovered at necropsy. The location in the small intestine was recorded for all worms and the length and mtDNA haplotype were determined for about 4200 individual worms. There were significant differences in the distribution and abundance of the 4 individual haplotypes among individual pigs demonstrating strong interactions between parasite and host. We found significant differences in the abundance and position in the small intestine as well as the size of worms among haplotypes. We conclude that both parasite and host effects as well as the interplay between them play important roles in determining the characteristics and outcome of infection.
Asunto(s)
Ascariasis/parasitología , Ascaris suum/genética , Ascaris suum/patogenicidad , Parasitosis Intestinales/parasitología , Análisis de Varianza , Animales , Ascariasis/patología , Ascaris suum/anatomía & histología , Ascaris suum/aislamiento & purificación , ADN Mitocondrial , Femenino , Haplotipos , Interacciones Huésped-Parásitos/genética , Parasitosis Intestinales/patología , Intestino Delgado/parasitología , Intestino Delgado/patología , Larva/genética , Fenotipo , PorcinosRESUMEN
In the present study, larvae of Ascaris suum and Trichuris muris were investigated by light and electron microscopy after incubation in a hatching medium containing 89% phosphate-buffered saline (pH 7.4), 10% RPMI-1640 and 1% sodiumhypochlorite at 40 and 37 degrees C, respectively. The larvae were obtained from fertilised eggs of the worms during defined phases of development (A. suum, 36th-50th day of development; T. muris, once a week from week 16 to 20). Light and electron micrographs of the larvae gave evidence that the third larval stage of A. suum is probably the infectious stage. The first moult of the larvae had already taken place before the 36th day of incubation starting at day 1. After 36 days of incubation, only the second larval stage was found within eggs. Some of these larvae were coated by a separated sheath so that a second moult of the larvae is reasonable. On the other hand, no sheathed larvae of T. muris were found in the eggs incubated for 20 weeks in distilled water. No signs of moult were seen for 20 weeks neither on light nor on the electron micrographs. Therefore, in T. muris, the first larval stage is the infectious stage, which was proven by means of re-infections of mice 16, 18 or 20 weeks after incubation of the eggs.
Asunto(s)
Ascaris suum/anatomía & histología , Ascaris suum/ultraestructura , Larva/anatomía & histología , Larva/ultraestructura , Trichuris/anatomía & histología , Trichuris/ultraestructura , Animales , Ascariasis/parasitología , Medios de Cultivo/química , Ratones , Microscopía , Microscopía Electrónica de Transmisión , Muda , Tricuriasis/parasitologíaRESUMEN
Monoclonal antibody G15-6A was generated by immunizing mice with Ascaris head extracts. It recognizes an antigen present in a single neuron, with a cell body in the dorsal rectal ganglion, that projects along the ventral cord to the nerve ring. Ascaris extracts were fractionated by HPLC and ammonium sulfate precipitation, and fractions assayed by dot-blotting with antibody G15-6A. A single immunoreactive polypeptide was purified; mass spectrometry showed a molecular weight of 11,542 Da. Partial N-terminal sequencing, followed by cloning of the transcript encoding the peptide, revealed a predicted peptide product comprising 109 amino acids, and a molecular mass of 11,863 Da. The N-terminus of the predicted peptide includes four more amino acids than are found in the isolated product.
Asunto(s)
Antígenos Helmínticos/genética , Ascaris suum/química , Neuronas/química , Neuropéptidos/genética , Secuencia de Aminoácidos , Sulfato de Amonio/química , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos Helmínticos/análisis , Antígenos Helmínticos/aislamiento & purificación , Ascaris suum/anatomía & histología , Ascaris suum/fisiología , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Bases de Datos de Proteínas , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Immunoblotting , Inmunohistoquímica , Métodos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Neuronas/inmunología , Neuropéptidos/química , Neuropéptidos/aislamiento & purificación , Pronasa/metabolismo , Inhibidores de Proteasas/química , ARN de Helminto/genética , ARN de Helminto/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Previous electrophysiological and anatomical studies of Ascaris suum motor neurons demonstrated a strong correlation between functional interactions and the presence of anatomically defined synapses. However, one example of a physiologically robust synaptic connection was encountered for which no anatomical evidence of direct chemical synapses was found. This involved synaptic transmission from an identified excitatory motor neuron to its inhibitory partner. In this study, pressure injection of horseradish peroxidase or nickel lysine into inhibitory motor neurons revealed numerous spines projecting from the main neuronal process toward the neuromuscular surface that then branched and extended fine, longitudinal processes up to 130 microm in length. Subsequent examination of nickel lysine-injected spines by electron microscopy revealed numerous chemical synapses, including for the first time direct inputs from the excitatory neuron. However, the numbers of synapses from this motor neuron were very small relative to inputs from other identified cells. Thus, direct synapses are unlikely to explain the robust nature of this physiological interaction.
Asunto(s)
Ascaris suum/anatomía & histología , Ascaris suum/fisiología , Dendritas/ultraestructura , Neuronas Motoras/ultraestructura , Sinapsis/ultraestructura , Animales , Estructuras de la Membrana Celular/ultraestructura , Antagonistas Colinérgicos/farmacología , Cobalto/farmacología , Dendritas/fisiología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Técnicas In Vitro , Mecamilamina/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Vías Nerviosas/citología , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
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.
Asunto(s)
Ascaris suum/anatomía & histología , Ascaris suum/química , Genitales Femeninos/química , Genitales Femeninos/ultraestructura , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestructura , Neuronas/química , Neuronas/ultraestructura , Animales , Ascaris suum/fisiología , FMRFamida/análisis , Femenino , Ganglios de Invertebrados/química , Ganglios de Invertebrados/fisiología , Ganglios de Invertebrados/ultraestructura , Genitales Femeninos/fisiología , Fibras Musculares Esqueléticas/fisiología , Neuronas/fisiología , Faloidina/análisis , Serotonina/análisis , Tubulina (Proteína)/análisisRESUMEN
Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.
Asunto(s)
Ascaris suum/crecimiento & desarrollo , Ascaris suum/patogenicidad , Animales , Ascariasis/veterinaria , Ascaris suum/anatomía & histología , Heces/parasitología , Intestinos/parasitología , Larva/anatomía & histología , Larva/crecimiento & desarrollo , Hígado/parasitología , Ratones , Muda , Morfogénesis , Óvulo , Enfermedades de los Roedores/parasitologíaRESUMEN
The pharyngeal muscle of nematodes consists of a syncytium of radial muscle whereby feeding occurs by the process of pharyngeal pumping. It is believed that the pumping behaviour of the pharynx may be partly controlled by the enteric nervous system (ENS), a component of the nematode nervous system which is associated predominantly with the pharynx. The distribution of serotoninergic and peptidergic (especially SALMF-amide-like) immunostaining is widespread in the ENS of Ascaris, being localized within the lateral and dorsal pharyngeal nerve tracts, the pharyngeal commissures, the nerve plexuses and associated nerve cells and fibres. Immunostaining for serotonin (5-HT) was only localized within the ENS. This paper also describes a method to enable in vitro pharmacological studies on the Ascaris pharynx. The effects of "classical" neurotransmitters and native nematode peptides on pharyngeal pumping behaviour in Ascaris have been investigated. The function of the pharynx in Ascaris is discussed.
Asunto(s)
Ascaris suum/anatomía & histología , Ascaris suum/fisiología , Faringe/anatomía & histología , Faringe/fisiología , Secuencia de Aminoácidos , Animales , Sistema Nervioso Entérico/anatomía & histología , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/fisiología , Inmunohistoquímica , Hormonas de Invertebrados/química , Hormonas de Invertebrados/farmacología , Neuropéptidos/química , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Músculos Faríngeos/anatomía & histología , Músculos Faríngeos/efectos de los fármacos , Músculos Faríngeos/fisiología , Faringe/inervación , Serotonina/metabolismo , Serotonina/farmacologíaRESUMEN
The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction.