Juan Ramón Oreja Rodríguez

Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

ABSTRACT Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements.

Protection in dogs against visceral leishmaniasis caused by Leishmania infantum is achieved by immunization with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK.

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rVV) vectors expressing relevant antigens has been shown to enhance specific cellular immune responses and to elicit protection against a variety of pathogens in animal models. In this paper, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a plasmid carrying the gene for the LACK antigen from Leishmania infantum (DNA-LACK) followed by a booster with a rVV containing the same gene (rVV-LACK). Thereafter, animals were challenged with L. infantum to induce visceral leishmaniasis (VL). In the vaccinated dogs as compared with the controls, the outcome of the infection after challenge with a high inoculum (10(8)) of L. infantum stationary promastigotes was assessed by tissue parasite load, specific anti-Leishmania antibody production, cytokine level and development of clinical signs of leishmaniasis. We observed a 60% protection against infection in dogs immunized by DNA-LACK prime/rVV/-LACK boost while two doses of DNA-LACK did not elicit protection against the disease. The interleukin 4 (IL-4), interferon gamma (IFNgamma) and IL-12 (p40 subunit) cytokine mRNA expression profiles in PBMC as well as lymphocyte proliferative response and the IgG2/IgG1 ratios specific for LACK suggest that in vaccinated animals there is triggering of cellular immune responses. This type of DNA/rVV prime/boost immunization approach may have utility against visceral leishmaniasis in dogs.

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

ABSTRACT The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of theEscherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Discurso argumentativo en episodios de conflictos entre padres e hijos adolescentes

Resumen Analizamos el discurso oral en conflictos entre padres e hijos adolescentes desde una perspectiva transactional y evolutiva. Ocho familias fueron grabadas mientras discutían un conflicto. Siguiendo la metodología observational se calculó la frecuencia de ocurrencia y duración de categorías de regulación, de argumentación y emocionales en el discurso del padre, la madre y del hijo y las diferencias según la edad de los adolescentes. Se realizaron análisis secuenciales para detectar la secuencia de las intervenciones de los participantes en la discusión. Según los resultados, padres e hijos elaboran un discurso fundamentalmente argumentativo. La madre incluye elementos de negociación en la adolescencia tardía mientras que el padre elabora un discurso de confrontación. Con la edad, el discurso de los hijos aumenta en complejidad y confrontación, incluyendo argumentos y contraargumentos. La secuencia de interacción en la triada es jerárquica tornándose más democrática hacia el final de la adolescencia.

¿Peleamos o negociamos? La argumentación de los padres y las madres con sus hijos e hijas adolescentes durante los conflictos familiares

The polarization of postures in conflicts among parents and adolescents involve a threat for family harmony. Skills for argumentation and perspective taking are required to enable the achievement of satisfactory agreements during the processes of negotiation. The purpose of this paper has been to analyze the skills of parents and adolescents to adopt other perspectives when discussing a conflict by means of observational methodology. The study was carried out with 29 families (constituted by father, mother and an adolescent son or daughter) who were recorded while they argued about a real conflict. Frequency and duration of five levels of perspective-taking were registered (Myself; Reasoned myself; the other like an obstacle; the other adding to me; and us) in the discourse of the family, and also in the different members of the triad, considering their interrelations. The results were showing a moderated level of perspective-taking in family discussions, predominating the levels Reasoned myself, focused on explaining own perspective, and The other like an obstacle, focused on reasons to contest the perspective of the other. In general parents showed more perspective-taking. Particularly the mothers exhibited more sensitivity with her adolescent son or daughter, providing higher levels of perspective-taking and argumentation.

Multiperspectivistic management of discourse in oral argumentation in academic settings / Gestión multiperspectivista del discurso en la argumentación oral en contextos académicos

Abstract The relationship between perspectivism and oral argumentation is explored by observing eight triads of high school and university students discussing an environmental dilemma, with the aim of convincing their peers. No differences are appreciated between high school and university students regarding the frequency and duration of perspectivistic levels of discourse. When analysing such levels in the whole sample, significant differences are revealed in favour of the me (defence and explanation of own perspective) and other as an obstacle (demolition of others’ positions) levels, compared to the other strategy (legitimate persuasion) and us (search for agreement) levels. Therefore, argumentative discourse is anchored in a reasoned defense of one’s own perspective as well as in the attack on others, displaying a limited perspectivistic development. In relation to the interaction sequences, symmetrical responses are observed towards the me and other as obstacle levels, both in high school and university students, and also towards us in university students. The potential of perspectivism to improve expert argumentation is discussed.