Protein kinase CK2 phosphorylation of EB2 regulates its function in the production of Epstein-Barr virus infectious viral particles

The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2alpha and -beta subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2beta regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293(BMLF1-KO) cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293(BMLF1-KO) cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.     

Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans

His6-tagged xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotope effect when [8-2H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, alphaKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/alphaKG dioxygenase.     

Delayed erythropoietin therapy reduces post-MI cardiac remodeling only at a dose that mobilizes endothelial progenitor cells

We examined the cardiac effects of chronic erythropoietin (EPO) therapy initiated 7 days after myocardial infarction (MI) in rats. A single high dose of EPO has been shown to reduce infarct size by preventing apoptosis when injected immediately after myocardial ischemia. The proangiogenic potential of EPO has also been reported, but the effects of chronic treatment with standard doses after MI are unknown. In this study, rats underwent coronary occlusion followed by reperfusion or a sham procedure. Infarcted rats were assigned to one of three treatment groups: 1) 0.75 microg/kg darbepoetin (MI+darb 0.75, n = 12); 2) 1.5 microg/kg darbepoetin (MI+darb 1.5, n = 12); 3) vehicle (MI+PBS, n = 16), once a week from day 7 postsurgery. Sham rats received the vehicle alone (n = 10). After 8 wk of treatment, the animals underwent echocardiography, left ventricular pressure-volume measurements, and peripheral blood endothelial progenitor cell (EPC) counting. MI size and capillary density in the border zone and the area at risk (AAR) were measured postmortem. The AAR was similar in the three MI groups. Compared with MI+PBS, the MI+darb 1.5 group showed a reduction in the MI-to-AAR ratio (20.8% vs. 38.7%; P < 0.05), as well as significantly reduced left ventricle dilatation and improved cardiac function. This reduction in post-MI remodeling was accompanied by increased capillary density (P < 0.05) and by a higher number of EPC (P < 0.05). Both darbepoetin doses increased the hematocrit, whereas MI+darb 0.75 did not increase EPC numbers or capillary density and had no functional effect. We found that chronic EPO treatment reduces MI size and improves cardiac function only at a dose that induces EPC mobilization in blood and that increases capillary density in the infarct border zone.     

Phylogeography of the piranha genera Serrasalmus and Pygocentrus: implications for the diversification of the Neotropical ichthyofauna

The phylogenetic relationships within the piranhas were assessed using mitochondrial sequences with the aim of testing several hypotheses proposed to explain the origin of Neotropical diversity (palaeogeography, hydrogeology and museum hypotheses). Sequences of the ribosomal 16S gene (510 bp) and control region (980 bp) were obtained from 15 localities throughout the main South American rivers for 21 of the 28 extant piranha species. The results indicate that the genus Serrasalmus is monophyletic and comprises three major clades. The phylogeographical analyses of these clades allowed the identification of five vicariant events, extensive dispersal and four lineage duplications suggesting the occurrence of sympatric speciation. Biogeographical patterns are consistent with the prediction made by the museum hypothesis that lineages from the Precambrian shields are older than those from the lowlands of the Amazon. The vicariant events inferred here match the distribution of the palaeoarches and several postdispersal speciation events are identified, thereby matching the predictions of the palaeogeography and hydrogeology hypotheses, respectively. Molecular clock calibration of the control region sequences indicates that the main lineages differentiated from their most recent common ancestor at 9 million years ago in the proto Amazon-Orinoco and the present rate of diversification is the highest reported to date for large carnivorous Characiformes. The present results emphasize that an interaction among geology, sea-level changes, and hydrography created opportunities for cladogenesis in the piranhas at different temporal and geographical scales.     

Functional annotation strategy for protein structures

Whole-genome sequencing projects are a major source of unknown function proteins. However, as predicting protein function from sequence remains a difficult task, research groups recently started to use 3D protein structures and structural models to bypass it. MED-SuMo compares protein surfaces analyzing the composition and spatial distribution of specific chemical groups (hydrogen bond donor, acceptor, positive, negative, aromatic, hydrophobic, guanidinium, hydroxyl, acyl and glycine). It is able to recognize proteins that have similar binding sites and thus, may perform similar functions. We present here a fine example which points out the interest of MED-SuMo approach for functional structural annotation.     

Emergence of symmetry breaking in fucoid zygotes

Fucoid zygotes are model cells for the study of symmetry breaking in plants. After fertilization, their initial spherical symmetry reduces to an axial symmetry, even in the absence of any external cue. This indicates that zygotes have an intrinsic ability to break symmetry in a way that is solely dependent on their internal biochemical and/or biophysical state. In our opinion, symmetry breaking is a self-organized process. It arises around the fucoid zygotes from the ion dynamics through channels (voltage-dependent calcium channels and a potassium leak) and outside the membrane (electrodiffusion owing to slower calcium diffusion compared with potassium). The robustness of this self-organized process and its lability ensure its relevance in plants where symmetry breaking is correlated with transcellular ion currents.     

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca²⁺ releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy. ryanodine receptor; smooth muscle; alternative splicing     

Vertical distribution of picoeukaryotic diversity in the Sargasso Sea

Eukaryotic molecular diversity within the picoplanktonic size-fraction has primarily been studied in marine surface waters. Here, the vertical distribution of picoeukaryotic diversity was investigated in the Sargasso Sea from euphotic to abyssal waters, using size-fractionated samples (< 2 microm). 18S rRNA gene clone libraries were used to generate sequences from euphotic zone samples (deep chlorophyll maximum to the surface); the permanent thermocline (500 m); and the pelagic deep-sea (3000 m). Euphotic zone and deep-sea data contrasted strongly, the former displaying greater diversity at the first-rank taxon level, based on 232 nearly full-length sequences. Deep-sea sequences belonged almost exclusively to the Alveolata and Radiolaria, while surface samples also contained known and putative photosynthetic groups, such as unique Chlorarachniophyta and Chrysophyceae sequences. Phylogenetic analyses placed most Alveolata and Stramenopile sequences within previously reported 'environmental' clades, i.e. clades within the Novel Alveolate groups I and II (NAI and NAII), or the novel Marine Stramenopiles (MAST). However, some deep-sea NAII formed distinct, bootstrap supported clades. Stramenopiles were recovered from the euphotic zone only, although many MAST are reportedly heterotrophic, making the observed distribution a point for further investigation. An unexpectedly high proportion of radiolarian sequences were recovered. From these, five environmental radiolarian clades, RAD-I to RAD-V, were identified. RAD-IV and RAD-V were composed of Taxopodida-like sequences, with the former solely containing Sargasso Sea sequences, although from all depth zones sampled. Our findings highlight the vast diversity of these protists, most of which remain uncultured and of unknown ecological function.     

A new look towards BAC-based array CGH through a comprehensive comparison with oligo-based array CGH

Background

Natural populations of the teleost fish Fundulus heteroclitus tolerate a broad range of environmental conditions including temperature, salinity, hypoxia and chemical pollutants. Strikingly, populations of Fundulus inhabit and have adapted to highly polluted Superfund sites that are contaminated with persistent toxic chemicals. These natural populations provide a foundation to discover critical gene pathways that have evolved in a complex natural environment in response to environmental stressors.

Results

We used Fundulus cDNA arrays to compare metabolic gene expression patterns in the brains of individuals among nine populations: three independent, polluted Superfund populations and two genetically similar, reference populations for each Superfund population. We found that up to 17% of metabolic genes have evolved adaptive changes in gene expression in these Superfund populations. Among these genes, two (1.2%) show a conserved response among three polluted populations, suggesting common, independently evolved mechanisms for adaptation to environmental pollution in these natural populations.

Conclusion

Significant differences among individuals between polluted and reference populations, statistical analyses indicating shared adaptive changes among the Superfund populations, and lack of reduction in gene expression variation suggest that common mechanisms of adaptive resistance to anthropogenic pollutants have evolved independently in multiple Fundulus populations. Among three independent, Superfund populations, two genes have a common response indicating that high selective pressures may favor specific responses.