RESUMEN
Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions.
Asunto(s)
Actinas/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Xenopus , Citoesqueleto de Actina/metabolismo , Animales , Dominio Catalítico/fisiología , Línea Celular , Clonación Molecular , Células Epiteliales/citología , Regulación Enzimológica de la Expresión Génica , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes , Técnicas de Dilución del Indicador , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Polímeros/metabolismo , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transfección , Tirosina/metabolismo , Xenopus laevis , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Penaeidins are members of a new family of antimicrobial peptides isolated from a crustacean, which present both Gram-positive antibacterial and antifungal activities. We have studied the localization of synthesis and storage of penaeidins in the shrimp Penaeus vannamei. The distribution of penaeidin transcripts and peptides in various tissues reveals that penaeidins are constitutively synthesized and stored in the shrimp haemocytes. It was shown by immunocytochemistry, at both optical and ultrastructural levels, that the peptides are localized in granulocyte cytoplasmic granules. The expression and localization of penaeidins were further analysed in shrimp subjected to microbial challenge. We found that (1) penaeidin mRNA levels decrease in circulating haemocytes in the first 3 hours following stimulation and (2) an increase in plasma penaeidin concentration occurs after microbial challenge, together with (3) a penaeidin immunoreactivity in cuticular tissue, which can be related to the chitin-binding activity we demonstrate here for penaeidins.
Asunto(s)
Decápodos/metabolismo , Granulocitos/metabolismo , Hemocitos/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Fusarium/inmunología , Datos de Secuencia Molecular , Péptidos , Streptococcaceae/inmunología , Vibrio/inmunologíaRESUMEN
In this study, Dicentrarchus labrax encephalitis virus (DlEV), which causes sea bass encephalitis, was propagated in cell culture, thus allowing study of its lytic cycle. DlEV infection of mammalian and fish cells induced different patterns of expression of capsid proteins, which were assembled as virus-like particles, accumulating in the cytoplasm either as diffuse masses or in vesicles, as shown by electron microscopy. These particles correspond to virions, as shown by their ability to induce secondary infection. Fish cells proved to be more permissive for DlEV than mammalian cells, although virus yield remained low. RNA analysis of infected sea bass cells revealed DlEV RNA3, in addition to genomic RNA1 and RNA2, and the presence of the RNA2 minus strand, thus demonstrating the replication of the DlEV genome. In addition, DlEV RNA-dependent RNA polymerase was associated with mature virions even after purification by a CsCl gradient, but it was dissociated when capsids were destabilized. In addition to providing more information about the relatedness of DlEV to the members of the family Nodaviridae, this study shows that fish nodaviruses may not be able to infect as wide a variety of cells as insect nodaviruses can.
Asunto(s)
Enfermedades de los Peces/virología , Peces/virología , Virus ARN/fisiología , Animales , Células COS , Cápside/genética , Cápside/metabolismo , Citoplasma/virología , Células HeLa , Humanos , Mamíferos , Oncorhynchus mykiss , Virus ARN/genética , Virus ARN/metabolismo , ARN Viral/biosíntesis , ViriónRESUMEN
Tachyplesin 1 is an antimicrobial peptide extracted from hemocytes of the Japanese horseshoe crab Tachypleus tridentatus. We studied the in vitro activity of tachyplesin I against bivalve pathogens: the oyster parasites Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis and Perkinsus marinus, the histozoic parasite of the Eastern oyster Crassostrea virginica, and the bacterium Vibrio P1, pathogenic for the clam Tapes philippinarum. Viability of the protozoans was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide. Following exposure to tachyplesin I, B. ostreae and P. marinus viabilities were reduced in a dose-dependent manner, up to, respectively, 94 and 62% within a 500 µg/ml peptide concentration. The fine structure of P. marinus was highly altered by the peptide. Tachyplesin I also displayed a potent activity against marine vibrios, with a MIC of 0.4-0.8 µg/ml against Vibrio P1. We examined the morphology of oyster hemocytes treated by tachyplesin I, together with the cell functional capabilities to produce chemiluminescence. No effect of the peptide was found on bivalve host cells. As transgenic technology is currently being applied to marine invertebrates, these results indicate that tachyplesin I may provide effective gene sequences to be manipulated in order to produce disease-resistant bivalves.
RESUMEN
RNA2, the short segment of the genome of Dicenthrarchus labrax encephalitis virus (DIEV), a fish nodavirus causing seabass encephalitis, was cloned. Sequence analysis revealed that DIEV RNA2 contains a single open reading frame (ORF), which carries the catalytic D-75 residue but lacks the site for autocatalytic proteolysis, the process yielding the two capsid proteins of insect nodaviruses. Nevertheless, SDS-PAGE analysis of mature virions revealed a 43-45 kDa protein doublet. In order to determine the mechanism of synthesis of the two capsid proteins in DIEV, wild type and mutagenized forms of RNA2 were expressed in cell-free translation extracts and in transfected cells. Results showed that, despite the presence of the catalytic D-75 residue, the DIEV capsid protein doublet did not result from the assembly-dependent autocatalytic cleavage of a protein precursor. Moreover, our data show that, although suggested by sequence analysis, the DIEV capsid protein doublet results from neither an alternative initiation codon usage nor from a--1 ribosomal frameshift. Results of cell-free translation experiments demonstrate that the capsid protein doublet neither results of the proteolytic cleavage of a precursor nor of a degradation process. Kinetics of capsid protein synthesis in cell-free translation programmed with RNA2 revealed, instead, that the two capsid proteins are cosynthesized. Together these data strongly suggest that the DIEV capsid protein doublet results from cotranslational modification(s) of the ORF-encoded protein.
Asunto(s)
Lubina/virología , Proteínas de la Cápside , Cápside/metabolismo , Virus de la Encefalitis/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Cápside/genética , Clonación Molecular , Virus de la Encefalitis/genética , Datos de Secuencia Molecular , Virus ARN/genética , Virus ARN/metabolismo , ARN Viral , Análisis de Secuencia de ADNRESUMEN
A cytoplasmic dsRNA virus, rotifer birnavirus (RBV), has recently been isolated from the rotifer Brachionus plicatilis and is associated with a high mortality rate. Histologically, the viral lesions consist of characteristic inclusions, particularly amorphous dense bodies containing occluded particles. Purified virions are about 59 nm in diameter, single-shelled and display four capsomers per edge. The purified virions have a buoyant density of 1.290 (full particles) and 1.250 (empty particles) in CsCl gradients. Four major structural polypeptides of MrS 60K, 52K, 33K and 27K were detected by SDS-PAGE. The genome is composed of two linear segments of dsRNA with MrS of 2.45 x 10(6) and 2.31 x 10(6); additionally, small circular ssRNA molecules were detected by electrophoresis in overloaded agarose gels, but their significance is currently unknown. Except for this last feature and the structural instability of purified virions under freeze storage, all the other biochemical and biophysical characters indicate that RBV is a member of the Birnaviridae family with, for the moment, a unique position in this group.
Asunto(s)
Genes Virales , Rotíferos/microbiología , Virión/aislamiento & purificación , Virus/aislamiento & purificación , Animales , Línea Celular , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Microscopía Electrónica , Peso Molecular , ARN Bicatenario/genética , ARN Bicatenario/aislamiento & purificación , ARN Bicatenario/ultraestructura , ARN Viral/genética , ARN Viral/aislamiento & purificación , ARN Viral/ultraestructura , Proteínas Estructurales Virales/aislamiento & purificación , Virión/análisis , Virión/genética , Virión/ultraestructura , Virus/análisis , Virus/genética , Virus/ultraestructuraRESUMEN
Virus particles and their morphogenesis have been observed in the cytoplasm of connective cells in the Japanese Oyster Crassostrea gigas. This virus seems to be similar to those described in the Portuguese Oyster Crassostrea angulata when high rates of mortality were recorded in 1970-1973 and those found in gill disease of this species.