RESUMO
Crustaceans are major constituents of aquatic ecosystems and, as such, changes in their behavior and the structure and function of their bodies can serve as indicators of alterations in their immediate environment, such as those associated with climate change and anthropogenic contamination. We have used bioinformatics and a de novo transcriptome assembly approach to identify potential targets for developing specific antibodies to serve as nervous system function markers for freshwater prawns of the Macrobrachium spp. Total RNA was extracted from brain ganglia of Macrobrachium carcinus freshwater prawns and Illumina Next Generation Sequencing was performed using an Eel Pond mRNA Seq Protocol to construct a de novo transcriptome. Sequencing yielded 97,202,662 sequences: 47,630,546 paired and 1,941,570 singletons. Assembly with Trinity resulted in 197,898 assembled contigs from which 30,576 were annotated: 9,600 by orthology, 17,197 by homology, and 3,779 by transcript families. We looked for glutamate receptors contigs, due to their main role in crustacean excitatory neurotransmission, and found 138 contigs related to ionotropic receptors, 32 related to metabotropic receptors, and 18 to unidentified receptors. After performing multiple sequence alignments within different biological organisms and antigenicity analysis, we were able to develop antibodies for prawn AMPA ionotropic glutamate receptor 1, metabotropic glutamate receptor 1 and 4, and ionotropic NMDA glutamate receptor subunit 2B, with the expectation that the availability of these antibodies will help broaden knowledge regarding the underlying structural and functional mechanisms involved in prawn behavioral responses to environmental impacts. The Macrobrachium carcinus brain transcriptome can be an important tool for examining changes in many other nervous system molecules as a function of developmental stages, or in response to particular conditions or treatments.
Assuntos
Anticorpos/imunologia , Encéfalo/metabolismo , Ecossistema , Anotação de Sequência Molecular/métodos , Palaemonidae/genética , Receptores de Glutamato/genética , Animais , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Palaemonidae/metabolismo , Receptores de Glutamato/imunologia , Receptores de Glutamato/metabolismo , TranscriptomaRESUMO
Glutamate is the main neurotransmitter of photoreceptors, bipolar cells, and ganglion cells of the vertebrate retina. Three main classes of ionotropic glutamate receptors comprising different subunits can be distinguished: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionate), KA (kainate), and NMDA (N-methyl-D-aspartate). This study was undertaken to characterize the AMPA (GluR1, GluR2/3, and GluR4), KA (GluR5/6/7), and NMDA (NR1) ionotropic glutamate receptor subunits and to determine their distribution during the development of the chick retina by Western blotting and immunohistochemistry. Western blotting analysis at 1 day after hatching indicated that the antibodies against GluR1, 2/3, 4, and 5/6/7 and NR1 recognized specifically a single band of 100-110 kDa. In turn, immunohistochemistry at P1 showed that all subunits were expressed in cells of the inner nuclear and ganglion cell layers of the chick retina, mostly amacrine and ganglion cells, and their processes in the inner plexiform layer. In addition, stained processes in the outer plexiform layer were observed with the antibodies against GluR2/3, GluR4, and GluR5/6/7. Although all subunits appeared around E5-E6 in the prospective ganglion cell layer, and later in the prospective inner nuclear layer, the distribution of cells containing these glutamate receptor subunits revealed distinct ontogenetic patterns. This multiplicity of glutamate receptors may contribute to different processes that occur in the chick retina during development.
Assuntos
Embrião de Galinha/embriologia , Receptores de Glutamato/análise , Retina/química , Retina/embriologia , Animais , Especificidade de Anticorpos , Imuno-Histoquímica , Receptores de AMPA/análise , Receptores de AMPA/imunologia , Receptores de Glutamato/imunologia , Receptores de Ácido Caínico/análise , Receptores de Ácido Caínico/imunologia , Receptores de N-Metil-D-Aspartato/análise , Receptores de N-Metil-D-Aspartato/imunologiaRESUMO
INTRODUCTION: Rasmussen's encephalitis is a neurological disorder probably of immunological origin, characterized by intractable epilepsy, neurological deterioration and the presence of antibodies against R3 glutamate receptors. OBJECTIVE: To present the experience of an Argentinian Paediatric Neurology Service during the past 7 years. MATERIAL AND METHODS: We analyzed the clinical histories of 12 patients with RS as defined by the diagnostic criteria proposed by Hart et al. We evaluated the following parameters: neurological examination, neuropsychological assessment, types of crises, age of onset, frequency, distribution, electroencephalogram, video EEG, evoked potentials, neuro-radiological studies; CT and cerebral MR using gadolinium, SPECT, lumbar puncture and cerebral biopsy. RESULTS: The crises were of simple partial motor type in 12 cases, secondarily generalized partial crises in 7 cases, complex partial crises in 4 cases and partial and generalized motor states in 8 cases. RESULTS. The ictal EEGs during the continuous partial crises showed contralateral focal slow activity of the affected side of the body in 30% of cases. The neuro-radiological studies (CT and MR) showed signs of cerebral hemiatrophy in all 12 cases, hyper-intense images in the T2 sequences of the fronto-temporo-parietal regions in 5 cases, an asymmetric megacisterna magna in 1 case and hypoplasia of the cerebellar vermis in another case. CONCLUSIONS: The continuous partial crises showed little correlation with the electro-encephalography. Following administration of i.v. gammaglobulin there was a transient halt in progression of the neurological deterioration and control of the crises in 55% of the cases. Surgery continued to be the treatment recommended for control of the epileptic crises.