Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.     

Late Hauterivian coralline algae (Rhodophyta,Corallinales) from the Iberian Chain (E Spain). Taxonomy and the evolution of multisporangial reproductive structures

Upper Hauterivian reefal carbonates of the Llàcova Formation (Maestrat Basin, Iberian Chain, E Spain) contain Sporolithon phylloideum (Bucur and Dragastan) Tomás, Aguirre, Braga and Martín-Closas comb. nov. and Sporolithon rude (Lemoine) Ghosh and Maithy (1996). Moussavian et al. (1993) identified them as Parakymalithon phylloideum (Bucur and Dragastan) Moussavian 1987 and Archaeolithothamnium rude Lemoine 1925. The re-assessment of the type of P. phylloideum and additional material indicate that the diagnostic characters of the genus do not warrant separation from Sporolithon and the new combination Sporolithon phylloideum is proposed. The lectotype of Sporolithon rude presents sporangial cavities grouped in sori that can be merged originating a structure that resembles the multiporate tetrasporangial conceptacles of the Hapalidiaceae. We hypothesize that multiporate tetrasporangial conceptacles could have originated from the fusion of several sporangial cavities, suggesting a phylogenetic linkage between Sporolithaceae and Hapalidiaceae supported by other anatomical features, molecular phylogeny and the fossil record.     

Detection of different mRnas expressed in the thyro-parathyroid complex of the rat by in situ hybridization using digoxigenin-labelled oligonucleotide probes

The effects have been examined of different methods and regimens for tissue fixation, preservation, permeabilization and immunostaining of different mRNAs detected by in situ hybridization in paraffin-embedded samples. The three main hormone mRNAs expressed in the thyro–parathyroid glands, namely thyroglobulin, calcitonin and parathyroid hormone mRNAs, were chosen as the target nucleic acid sequences to be detected using digoxigenin-labelled probes. Our results suggest that chemical fixation and permeabilization of tissue samples are restrictive steps. Thus, paraformaldehyde fixation provides excellent signal intensities and non-detectable background levels whereas routine formalin and Bouin's solution give unsatisfactory results. A clear linear correlation was also found between signal intensity and proteinase K permeabilization. Moreover, the optimization of immunohistochemical steps, such as anti-digoxigenin antibody concentration and colour development times, enhance the intensity and specificity of hybrid signals. Furthermore, our results show that, in contrast to some data in the literature, paraffin-embedded tissue is suitable for detection of mRNAs by in situ hybridization. It gives equivalent intensities of specific signal and superior histological and cellular resolutions when compared to cryopreserved tissue.     

Mitochondrial DNA and the peopling of South America

The initial peopling of South America is largely unresolved, in part because of the unique distribution of genetic diversity in native South Americans. On average, genetic diversity estimated within Andean populations is higher than that estimated within Amazonian populations. Yet there is less genetic differentiation estimated among Andean populations than estimated among Amazonian populations. One hypothesis is that this pattern is a product of independent migrations of genetically differentiated people into South America. A competing hypothesis is that there was a single migration followed by regional isolation. In this study we address these hypotheses using mtDNA hypervariable region 1 sequences representing 21 South American groups and include new data sets for four native Peruvian communities from Tupe, Yungay, and Puno. An analysis of variance that compared the combined data from western South America to the combined data from eastern South America determined that these two regional data sets are not significantly different. As a result, a migration from a single source population into South America serves as the simplest explanation of the data.     

Synthesis and structural characterization of two new polynuclear metal thiosaccharinates: Hexakis(thiosaccharinato)hexasilver(I) and tetrakis(thiosaccharinato)tetracopper(I)

The title compounds, for short Ag₆(tsac)₆ (1) and [Cu₄(tsac)₄(MeCN)₂] · 2MeCN (2), were prepared by the reaction of thiosaccharin with Ag(I) or Cu(II) salts in different solvents. The new complexes were characterized by FT-IR, Raman, UV-Vis and NMR spectroscopy. Their crystal and molecular structures were determined by X-ray diffraction methods. The structures were solved from 1621 (1) and 7080 (2) reflections with I > 2σ(I) and refined to agreement R1-factors of 0.0261 (1) and 0.0456 (2). Ag₆(tsac)₆ molecule derives from the clustering of six Ag(tsac) moieties related to each other through the crystallographic 3-bar (S₆) symmetry operations of the space group. This results in a highly regular molecular structure where the silver atoms are at the corners of an octahedral core slightly compressed along one of its three-fold axis [inter-metallic Ag?Ag contacts of 3.1723(4) and 3.1556(4) Å]. The six thiosaccharinate ligands bridge neighboring Ag atoms along the C₃-axis through Ag-N bonds [d(Ag-N) = 2.285(2) Å] at one end and bifurcated Ag-S(thione)-Ag bonds [Ag-S distances of 2.4861(7) and 2.5014(8) Å] at the other end. In contrast, the 2 compound is arranged in the lattice as an irregular tetrameric copper complex [Cu₄(tsac)₄(MeCN)₂] where the metals show different environments. Two copper ions are four-fold coordinated to three tsac ions through the N-atom of one tsac [Cu-N distances of 2.112(3) and 2.064(3) Å] and the thione sulfur atom of the other two [Cu-S distances in the range from 2.284(1) to 2.358(1) Å] and to a MeCN solvent molecule [Cu-N distances of 1.983(4) and 2.052(3) Å]. The other two copper ions are in three-fold environment, one trans-coordinated to two tsac ions [Cu-N distances of 1.912(3) and 1.920(3) Å] and to the thione S-atom of a third ligand [d(Cu-S) = 2.531(1) Å], the other one to the thione sulfur atom of three tsac ligands [Cu-S distances in the range from 2.229(1) to 2.334(1) Å]. The clustering renders the metals to short distances from each other, the shorter Cu?Cu distance being 2.6033(7) Å, as to presume the existence of weak inter-metallic interaction within the cluster.     

Opposite regulation of endothelial NO synthase by HSP90 and caveolin in liver and lungs of rats with hepatopulmonary syndrome

The hepatopulmonary syndrome is a complication of cirrhosis that associates an overproduction of nitric oxide (NO) in lungs and a NO defect in the liver. Because endothelial NO synthase (eNOS) is regulated by caveolin that decreases and heat shock protein 90 (HSP90) that increases NO production, we hypothesized that an opposite regulation of eNOS by caveolin and HSP90 might explain the opposite NO production in both organs. Cirrhosis was induced by a chronic bile duct ligation (CBDL) performed 15, 30, and 60 days before sample collection and pharmacological tests. eNOS, caveolin, and HSP90 expression were measured in hepatic and lung tissues. Pharmacological tests to assess NO released by shear stress and by acetylcholine were performed in livers (n = 28) and lungs (n = 28) isolated from normal and CBDL rats. In lungs from CBDL rats, indirect evidence of high NO production induced by shear stress was associated with a high binding of HSP90 and a low binding of caveolin to eNOS. Opposite results were observed in livers from CBDL rats. Our study shows an opposite posttranslational regulation of eNOS by HSP90 and caveolin in lungs and liver from rats with CBDL. Such opposite posttranslational regulation of eNOS by regulatory proteins may explain in part the pulmonary overproduction of NO and the hepatic NO defect in rats with hepatopulmonary syndrome.