The coxsackievirus 2B protein increases efflux of ions from the endoplasmic reticulum and Golgi, thereby inhibiting protein trafficking through the Golgi

Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca2+ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca2+ into the stores. Measurement of intra-organelle-free Ca2+ in permeabilized cells revealed that the ability of 2B to reduce the Ca2+ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cell-free system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. To investigate whether 2B specifically affects Ca2+ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H+ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.     

The Pole3 bidirectional unit is regulated by MYC and E2Fs

Pole3 (DPB4/YBL1/CHRAC17) is one of the subunits of the DNA polymerase e. It contains a histone-like domain required for the hererodimerization with its Pole4 (DPB3) partner. In another interaction, Pole3 heterodimerizes with YCL1/CHRAC15 and associates with the ACF1/SNF2H remodelling complex. We find that the Pol3 gene is regulated in starved NIH3T3 fibroblasts upon induction with serum, with a peak at the entry in the S phase. We characterized the Pole3 promoter, which is linked bidirectionally to C9Orf46, a gene of unknown function: it has no CCAAT nor TATA-boxes, and contains an E box and two potential E2F sites. Mutagenesis analysis points to a minimal promoter region as sufficient for activation; the E box and a neighbouring direct repeat are important for regulation. Cell-cycle regulation was reproduced in stable clones and an additional E2F site was found to be important. Chromatin immunoprecipitation analysis indicates that E2F1/4, as well as MYC, are associated with the Pole3 promoter in a phase-specific way. These data highlight coregulation of a histone-like gene with core histones upon DNA synthesis, and represent a first dissection of the interplay between two essential cell-cycle regulators on a bidirectional promoter.     

Glyphosate Resistance by Engineering the Flavoenzyme Glycine Oxidase

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the k_cat/K_m ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the α2-α3 loop (comprising residues 50–60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg⁵⁴ could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide.     

Reweighting of molecular simulations with explicit-solvent SAXS restraints elucidates ion-dependent RNA ensembles

Small-angle X-ray scattering (SAXS) experiments are increasingly used to probe RNA structure. A number of forward models that relate measured SAXS intensities and structural features, and that are suitable to model either explicit-solvent effects or solute dynamics, have been proposed in the past years. Here, we introduce an approach that integrates atomistic molecular dynamics simulations and SAXS experiments to reconstruct RNA structural ensembles while simultaneously accounting for both RNA conformational dynamics and explicit-solvent effects. Our protocol exploits SAXS pure-solute forward models and enhanced sampling methods to sample an heterogenous ensemble of structures, with no information towards the experiments provided on-the-fly. The generated structural ensemble is then reweighted through the maximum entropy principle so as to match reference SAXS experimental data at multiple ionic conditions. Importantly, accurate explicit-solvent forward models are used at this reweighting stage. We apply this framework to the GTPase-associated center, a relevant RNA molecule involved in protein translation, in order to elucidate its ion-dependent conformational ensembles. We show that (a) both solvent and dynamics are crucial to reproduce experimental SAXS data and (b) the resulting dynamical ensembles contain an ion-dependent fraction of extended structures.     

What are we dealing with? An explicit test reveals different levels of taxonomical diagnosability in the Sylvia cantillans species complex

Diagnosability is the ability to discriminate between similar taxa, including sibling or cryptic taxa. We have developed an explicit test of diagnosability, using the Sylvia cantillans species complex as a model, which compares an identification based on phenotype with that based on genotype. Individual warblers sampled during their migration in central Italy were first identified to the (sub)species level using putatively diagnostic plumage traits. Nucleotide sequences of a (598-bp) fragment of the mitochondrial (mt)DNA cytochrome b were then used to assign each individual to distinct phylogenetic clades, as determined by reference haplotypes that had been sequenced in breeding individuals. This resulted in the construction of clearly distinct clades corresponding to known taxa of the complex. The new haplotypes were assigned to one of the previously identified groups (corresponding to three different taxa); no sample was assigned outside of them. In contrast, when plumage traits were used to assign the birds into distinct phylogenetic clades, 11 of 58 birds were classified as ‘uncertain/intermediate’ among two taxa, while five were classified differently with the two methods. A perfect agreement between the two methods was found for only for one taxon (Sylvia subalpina, syn. S. moltonii). For the other two taxa of the complex, diagnosability is therefore not guaranteed, and their field identification by hand should be carefully addressed. We provide here an example of an explicit test for establishing the diagnosability of taxa in which two or more ‘markers’ can be used for determining discordant identification and/or unambiguous diagnosability. Our results outline the importance of considering different features for taxa diagnosis and illustrate the weakness of visual appearance-based identification (currently widely used for taxa determination) in our study complex.     

Defecting or not defecting: how to "read" human behavior during cooperative games by EEG measurements

Understanding the neural mechanisms responsible for human social interactions is difficult, since the brain activities of two or more individuals have to be examined simultaneously and correlated with the observed social patterns. We introduce the concept of hyper-brain network, a connectivity pattern representing at once the information flow among the cortical regions of a single brain as well as the relations among the areas of two distinct brains. Graph analysis of hyper-brain networks constructed from the EEG scanning of 26 couples of individuals playing the Iterated Prisoner's Dilemma reveals the possibility to predict non-cooperative interactions during the decision-making phase. The hyper-brain networks of two-defector couples have significantly less inter-brain links and overall higher modularity--i.e., the tendency to form two separate subgraphs--than couples playing cooperative or tit-for-tat strategies. The decision to defect can be "read" in advance by evaluating the changes of connectivity pattern in the hyper-brain network.     

Immunological characterization and fine localization of a lizard beta-keratin

Scales of lizards contain beta-keratin of poorly known composition. In the present study, a rat polyclonal serum against a lizard beta-keratin of 14-15 kDa has been produced and the relative protein has been immunolocalized in the epidermis. The observations for the first time show that the isolated protein band derives from the extraction of a protein component of the beta-keratin filaments of lizard epidermis. In immunoblots and immunocytochemistry, the antiserum recognizes most lizard beta-keratins, but produces a variable cross-reactivity with snake beta-keratins, and weak or no reactivity with beta-keratins isolated from tuatara, turtles, alligator and birds. In bidimensional immunoblots of lizard epidermis, three main spots at 15-16 kDa with isoelectric point at 7.0, 7.6 and 8.0, and an unresolved large spot at 29-30 kDa and with pI at 7.5-8.0, are obtained, may be derived from the aggregation of smaller beta-keratin proteins. The ultrastructural immunolocalization with the antibody against lizard beta-keratin shows that only small and large beta-keratin filaments of beta-cells of lizard epidermis are labeled. Keratin bundles in oberhautchen cells are less immunolabeled. Beta-keratin is rapidly polymerized into beta-packets that merge into larger beta-keratin filaments. No labeling is present over other cell organelles or cell layers of lizard epidermis, and is absent in non-epidermal cells. The antiserum recognizes epitope(s) characteristics for lizard beta-keratins, partially recognized in snakes and absent in non-lepidosaurian species. This result indicates that beta-keratins among different reptilian groups posses different immunoreactive regions.     

Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.