RESUMEN
The California sea hare (Aplysia californica) is a model for age associated cognitive decline. Recent researched identified a novel nidovirus, Aplysia Abyssovirus 1, with broad tropism enriched in the Aplysia nervous system. This virus is ubiquitous in wild and maricultured, young and old animals without obvious pathology. Here we re-evaluated gene expression data from several previous studies to investigate differential expression in the nervous system and gill in response to virus and aging as well as the mutational spectrum observed in the viral sequences obtained from these datasets. Viral load and age were highly correlated, indicating persistent infection. Upregulated genes in response to virus were enriched for immune genes and signatures of ER and proteostatic stress, while downregulated genes were enriched for mitochondrial metabolism. Differential expression with respect to age suggested increased iron accumulation and decreased glycolysis, fatty acid metabolism, and proteasome function. Interaction of gene expression trends associated with viral infection and aging suggest that viral infection likely plays a role in aging in the Aplysia nervous system. Mutation analysis of viral RNA identified signatures suggesting ADAR and AID/APOBEC like deaminase act as part of Aplysia anti-viral defense.
Asunto(s)
Aplysia , Nodaviridae , Animales , Envejecimiento/inmunología , Aplysia/inmunología , Branquias/virología , Branquias/inmunología , Interacciones Huésped-Patógeno/inmunología , Sistema Nervioso/virología , Sistema Nervioso/inmunología , Nodaviridae/fisiología , ARN Viral/genética , Carga ViralRESUMEN
All haemolymph lectins with uniquely juxtaposed N-terminal domain similar to the immunoglobulin superfamily (IgSF) and C-terminal fibrinogen (FBG) termed FBG-related proteins (FREP) are documented till now only in the pulmonate mollusc Biomphalaria glabrata. Using genomic WGS database we have found two FREP genes from marine opistobranch Aplysia californica named AcFREP1 and AcFREP2. The AcFREP1 and AcFREP2 mRNA molecules have been subsequently isolated from cDNA of sea hare larvae as well as adult mollusc tissues. These genes encode proteins (504 and 510aa respectively) with domain architecture typical for FREPs with two N-terminal IgSF domains and C-terminal FBG domain. Although cDNA sequences of AcFREP1 and AcFREP2 are 81% identical, their genomic structure is entirely different: AcFREP1 is intronless and AcFREP2 is encoded in four exons. These genes are paralogous pair in which AcFREP2 is a parental gene and AcFREP1 is the new transposed copy that has lost the introns (retrogene). Using RT-PCR analysis, expression of AcFREP1 and AcFREP2 was shown to be developmentally and tissue-specific and no constitutive expression in haemocytes was found. The overall frequency of nucleotide substitutions in genomic DNA trace sequences of coding region of the AcFREP1 and AcFREP2 is not higher than in the sequences of control conserved genes (actin, FMRFamide). Thus, previously reported high diversification of Biomphalaria FREP gene, BgFREP3, is not detected in Aplysia FREPs. A search for FREP homologs in other available complete genome of mollusc, Lottia gigantea (Patellogastropoda), a representative of the evolutionary earliest gastropod clade, did not reveal any DNA sequences coding for similar lectins. We suggest that unique domain architecture of FREPs is an evolutionary novelty that appeared and evolved only within one branch of Protostomata species, exclusively in heterobranch molluscs (Pulmonata and Opistobranchia).
Asunto(s)
Aplysia/inmunología , Fibrinógeno/metabolismo , Inmunoglobulinas/metabolismo , Secuencia de Aminoácidos , Animales , Biomphalaria/inmunología , Clonación Molecular , Fibrinógeno/inmunología , Frecuencia de los Genes , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Filogenia , Polimorfismo Genético , Alineación de SecuenciaRESUMEN
CD38 is a novel multifunctional protein that serves not only as an antigen but also as an enzyme. It catalyzes the metabolism of cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, two structurally and functionally distinct Ca(2+) messengers targeting, respectively, the endoplasmic reticulum and lysosomal Ca(2+) stores. The protein has recently been crystallized and its three-dimensional structure solved to a resolution of 1.9 A. The crystal structure of a binary complex reveals critical interactions between residues at the active site and a bound substrate, providing mechanistic insights to its novel multi-functional catalysis. This article reviews the current advances in the understanding of the structural determinants that control the multiple enzymatic reactions catalyzed by CD38.
Asunto(s)
ADP-Ribosil Ciclasa 1/química , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD/química , Antígenos CD/metabolismo , Aplysia/enzimología , Aplysia/inmunología , ADP-Ribosa Cíclica/metabolismo , Humanos , NADP/metabolismoRESUMEN
This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.
Asunto(s)
ADP-Ribosil Ciclasa 1/inmunología , ADP-Ribosil Ciclasa/inmunología , Antígenos CD/inmunología , Inmunidad Innata , Animales , Aplysia/inmunología , Membrana Celular/inmunología , Proteínas Ligadas a GPI , HumanosRESUMEN
Members of the actin-depolymerizing factor (ADF)/cofilin family of proteins are expressed in all eukaryotic cells. In higher vertebrates, cells often express as many as three different ADF/cofilin genes and each of these proteins may be phosphorylated on serine 3, giving rise to up to six different species. Also, many avian, amphibian, and invertebrate systems have been useful in studying different aspects of ADF/cofilin function. Antibodies have been prepared against different members of the ADF/cofilin family, but no systematic examination of their cross-reactivity has been reported. Although ADF and cofilins within a single vertebrate species have about a 70% sequence homology, antibodies often differentiate between these proteins. Here, Western blotting was used with chemiluminescence substrates of different sensitivities to determine the relative immunoreactivities of different polyclonal rabbit antibodies and a mouse monoclonal antibody to purified ADF/cofilins from plants, protists, nematodes, insects, echinoderms, birds, and mammals. From immunocross-reactivities and sequence alignments, the principal epitope in mammalian ADF and cofilin-1 recognized by an antibody raised against avian ADF was identified. The specificity of an antibody to the phosphopeptide epitope of metazoan ADF/cofilins was confirmed by two-dimensional (2-D) immunoblot analysis. Futhermore, this bank of antibodies was used to identify by Western blotting a putative member of the ADF/cofilin family in the sea slug, Aplysia californica.
Asunto(s)
Anticuerpos/inmunología , Aplysia/inmunología , Proteínas de Microfilamentos/inmunología , Serina/metabolismo , Factores Despolimerizantes de la Actina , Secuencia de Aminoácidos , Animales , Western Blotting , Encéfalo/metabolismo , Células Cultivadas , Embrión de Pollo , Destrina , Drosophila/inmunología , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia , Pez Cebra/inmunologíaRESUMEN
Axon growth during development and after injury has processes in common, but also differs in that regeneration requires the participation of cells of the immune system. To investigate how neuron-immunocyte interactions might influence regeneration, we developed an in vitro model whereby neurons and hemocytes from Aplysia californica were cocultured. The hemocytes, which behave like vertebrate macrophages, migrated randomly throughout the dish. When a neuron was encountered, some hemocytes exhibited an avoidance response, whereas others formed stable contacts. Hemocytes did not distinguish between neurons from different animals. Stable contacts occurred on neurites and growth cones, but not the cell soma, and were benign in that the hemocytes did not impede neurite growth. When hemocytes attached to the cell body, it presaged the destruction of the neuron. Destruction was a dynamic process that was initiated when groups of one to three hemocytes adhered to various regions of the cell soma. Each group was then joined by other hemocytes. They did not contact the neuron, but interconnected the initial groups, forming a network around the neuron. The network then contracted to dismember the cell. Once a neuron was destroyed, hemocytes removed the debris by phagocytosis. Both damaged neurons and those without apparent damage were targets for destruction. Severing neurites with a needle resulted in the destruction of only one of six cells. Our studies suggest that hemocytes, and by extrapolation, vertebrate macrophages, exhibit highly complex interactions with neurons that can exert a variety of influences on the course of nerve regeneration.
Asunto(s)
Aplysia/inmunología , Comunicación Celular/inmunología , Hemocitos/inmunología , Macrófagos/inmunología , Regeneración Nerviosa/inmunología , Neuronas/inmunología , Animales , Aplysia/citología , Axotomía , Muerte Celular/inmunología , Movimiento Celular/inmunología , Tamaño de la Célula/inmunología , Células Cultivadas/citología , Células Cultivadas/inmunología , Conos de Crecimiento/inmunología , Conos de Crecimiento/ultraestructura , Hemocitos/citología , Macrófagos/citología , Neuronas/citología , Fagocitosis/inmunologíaRESUMEN
VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum-resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Aplysia/inmunología , Proteínas Portadoras/inmunología , Células Cultivadas , Potenciales Postsinápticos Excitadores , Técnica del Anticuerpo Fluorescente Indirecta , Hipocampo/metabolismo , Hipocampo/ultraestructura , Humanos , Inmunohistoquímica , Sustancias Macromoleculares , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/inmunología , Neuroglía/metabolismo , Neuronas/metabolismo , Especificidad de Órganos , Proteínas R-SNARE , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Vesículas Sinápticas/ultraestructuraRESUMEN
Nerve injury in Aplysia californica is accompanied by a profound long-lasting enhancement of the excitability of nociceptive sensory neurons that have axons in injured nerves. It is likely that a variety of signals are involved in triggering this injury-induced sensory plasticity. The objective of the present study was to determine whether cells of the cellular defense system (hemocytes) play a role in the modulation of sensory excitability following injury. In support of such an idea, we have shown previously that the induction of a cellular defense reaction close to sensory axons is accompanied by an increase in the excitability of sensory neurons with axons close to responding hemocytes. Furthermore, in the present study, we verified that, following axonal crush, numerous hemocytes accumulate at the injured site on the nerve. Using a hemocyte/nervous system co-culture preparation, we found that there were no significant differences in the expression of injury-induced sensory plasticity between sensory neurons incubated in the presence or absence of hemocytes. To overcome some potential limitations of our co-culture preparation, we used the endotoxin lipopolysaccharide (LPS) as a tool to activate the hemocytes. Sensory cells incubated in the presence of LPS and hemocytes were significantly more excitable than sensory cells incubated in the presence of LPS alone. We speculate that the addition of LPS to the incubation medium containing hemocytes enhanced the release of hemocyte-derived cytokine-like factors such as interleukin-1 and tumor necrosis factor. These cytokine-like factors may act as signals to modulate the expression of injury-induced sensory hyperexcitability.
Asunto(s)
Aplysia/fisiología , Neuronas Aferentes/fisiología , Nociceptores/fisiología , Animales , Aplysia/inmunología , Axones/fisiología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Hemocitos/inmunología , Hemocitos/fisiología , Inmunidad , Lipopolisacáridos/farmacología , Compresión Nerviosa , Plasticidad Neuronal , Neuronas Aferentes/ultraestructuraRESUMEN
Efforts to understand how the immune system can influence nervous system function are hampered by the complexity of mammalian nervous and immune systems. The marine mollusc Aplysia californica has recently emerged as a useful model system to investigate cellular mechanisms underlying neural-immune interactions. Aplysia has a relatively simple, well-characterized nervous system that is accessible for intracellular recording. Moreover, it shares with mammals basic cellular defensive responses to non-self or wounded-self, i.e. the accumulation of numerous defense cells (hemocytes) around foreign objects or at injured sites. We have shown that the excitability of a population of nociceptive sensory neurons in Aplysia can be influenced by the presence of hemocytes close to their axons. These sensory neurons also show profound, long-lasting increases in their excitability following axonal injury. Hemocytes are attracted to injured sites on peripheral nerves, and we have developed an in vitro nervous system-hemocyte coculture system to demonstrate that hemocytes can also influence the expression of this injury-induced sensory hyperexcitability. Immunoreactive interleukin-1 (IL-1) and tumor necrosis factor have been identified in Aplysia. Preliminary in vitro studies showing that IL-1 can modulate the expression of injury-induced sensory hyperexcitability raise the interesting possibility that hemocyte-derived cytokine-like factors can modulate sensory neuron functioning. The relevance of this work to more phylogenetically advanced organisms is also discussed.
Asunto(s)
Aplysia/inmunología , Evolución Biológica , Neuroinmunomodulación , Neuronas Aferentes/inmunología , AnimalesRESUMEN
Male copulatory behavior in the snail Lymnaea stagnalis is controlled by several types of peptidergic neurons, including a cluster of neurons in the ventral lobe of the right cerebral ganglion that show immunoreactivity to myomodulin-A of Aplysia and innervate the penis complex. We identified structurally myomodulin-A and three related peptides from Lymnaea and showed that they are present in a characteristic ratio in both the penis nerve and penis complex, suggesting that they are processed from a single precursor and transported from the ventral lobe to the penis complex. All four peptides decreased the relaxation time of electrically evoked contractions of the penis retractor muscle. However, their effects on the amplitude of contraction were different, ranging from no effect to an increase or a decrease in the amplitude. A mixture of the peptides in a ratio as determined by direct mass spectrometry of the penis nerve decreased the contraction time, the relaxation time, and the amplitude. These effects resemble those of one particular peptide in the mixture. The direct mass spectrometry determinations of the peptide profile in the penis nerve suggest that many more, as yet unidentified, neuropeptides are involved in modulation of muscle activities of the penis complex.
Asunto(s)
Lymnaea/química , Músculo Liso/efectos de los fármacos , Neuropéptidos/fisiología , Pene/química , Secuencia de Aminoácidos , Animales , Aplysia/química , Aplysia/inmunología , Relación Dosis-Respuesta a Droga , Masculino , Datos de Secuencia Molecular , Moluscos/química , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/fisiología , Neuropéptidos/clasificación , Neuropéptidos/aislamiento & purificación , Pene/inervación , Pene/fisiología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Alineación de Secuencia , Conducta Sexual Animal/fisiología , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Synapsins are a well-characterized class of phosphoproteins found at synapses in the mammalian nervous system. One member of this family, synapsin I, has been extensively studied and shown to associate in a phosphorylation-dependent manner with both small synaptic vesicles and cytoskeletal elements. Though the characteristics of synapsin I suggest an important function in synaptic transmission, its definitive role is still in question. In an effort to find a model system in which to test directly the function of synapsin I, we have looked in the nervous system of the marine mollusc Aplysia californica for synapsin I-like antigens (SILA). Light microscope immunocytochemical studies using polyclonal and monoclonal antibodies to bovine brain synapsin I demonstrate Aplysia SILA in neuronal somata, in the neuropil, and at some identified synapses. Though SILA were exclusively associated with neuronal structures in Aplysia, the pattern of staining suggested that they are not present at all synaptic terminals. This interpretation was corroborated by ultrastructural studies in which SILA were present at some synaptic terminals but absent, or in low abundance, in adjacent terminals. In axons, SILA were associated with vesicles of 120-150 nm diameter, as well as with filamentous structures. Biochemical studies identified small amounts of SILA of 40 and 50 kD molecular weight that are recognized by several antibodies to mammalian synapsin I, and are acid extractable, collagenase-sensitive phosphoproteins; these are criteria used to define synapsin I homologues in other species. Our studies indicate that SILA are present in neurons in Aplysia californica but suggested that they represent only a small percentage of the total protein within the nervous system.
Asunto(s)
Antígenos/química , Aplysia/inmunología , Sistema Nervioso/ultraestructura , Sinapsinas/inmunología , Absorción , Animales , Anticuerpos Monoclonales/inmunología , Citratos , Ácido Cítrico , Inmunohistoquímica , Microscopía Electrónica , Sistema Nervioso/inmunología , Fosforilación , Sinapsis/inmunología , Sinapsis/ultraestructuraRESUMEN
A bacteriostatic factor, aplysianin P, was purified from the purple fluid of a sea hare, Aplysia kurodai, as a glycoprotein of 60 K daltons. A cytolytic glycoprotein purified from the purple fluid of Dolabella auricularia did not show antibacterial activity. Aplysianin P was half-maximally active for gram-positive and -negative bacteria at 0.2-5.8 micrograms/mL and its action was not bactericidal but bacteriostatic. Aplysianin P completely inhibited the syntheses of DNA and RNA by E. coli, but it did not induce the release of ATP from bacteria. These results suggest that aplysianin P, found in an invertebrate, the sea hare, is a new bacteriostatic protein and that it exerts its action by inhibiting nucleic acid syntheses, as a DNA-inhibiting, chemotherapeutic drug does.
Asunto(s)
Antibacterianos , Glicopéptidos , Glicoproteínas/inmunología , Moluscos/inmunología , Animales , Aplysia/inmunología , Bacterias/efectos de los fármacos , Líquidos Corporales/inmunología , Citotoxinas/aislamiento & purificación , Citotoxinas/farmacología , Glicoproteínas/aislamiento & purificación , Glicoproteínas/farmacologíaRESUMEN
A phosphonoglycosphingolipid, designated as FGL-IIb, was first identified in nerve fibers of Aplysia kurodai by two-dimensional TLC (Abe, S. et al. (1986) Biomed. Res. 7, 47-51), and its chemical structure has been determined to be 3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNac alpha 1----3(Fuc alpha 1----2)(2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide (Araki, S. et al., submitted). Cryostat and paraffin sections of the nervous tissue and skin of Aplysia were examined immunohistochemically with antiserum against FGL-IIb. With this antiserum, only nerve bundles were stained distinctly: nerve cells in ganglia and in subcutaneous and muscular tissues and other cell elements were not stained. From histochemical findings in cryostat sections pretreated with chloroform-methanol (2 : 1, v/v) and from the results of Western blot analysis of the nervous tissue, the staining was concluded to be due to glycolipid antigens. The antiserum reacted with FGL-IIb and other phosphonoglycosphingolipids named FGL-I, FGL-IIa, FGL-V, and F-9 on TLC plates. This reactivity of FGL-IIb was abolished by mild acid-methanol treatment, and the lost reactivity was recovered by alkaline hydrolysis. These findings suggest that the free carboxyl group of the pyruvic acid of FGL-IIb is essential for the immunological reaction and that all the glycolipids listed above have the same epitope as that of FGL-IIb. Immunohistochemical findings indicated that these glycolipids including FGL-IIb are localized specifically in nerve bundles of Aplysia.