Confocal images of Neuro2A cells transfected with the wild-type or the mutant construct.

<p>To investigate intracellular distribution of CRH, cell were fixed in PFA and probed with mouse polyclonal anti-GM130 (red) for Golgi visualization and rabbit polyclonal anti-CRH (green) antibodies.</p

Ability to express CRH in Neuro2A cells transiently transfected with wild-type or mutant preproCRH construct.

<p>A) CRH levels of expression detected by realtime quantitative PCR in not transfected (NT) or transfected cells (wt or p.Pro30Arg) at three different times: 24 h, 48 h and 72 h. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) of mRNA levels normalized to the basal CRH expression in Neuro2A cells (NT values) and to a housekeeping control gene (b-Actin). * t = −3.676 and p = 0.020 compared with wt at 24 h; ** t = 5.274 and p = 0.002 compared with wt at 24 h. B) Densitometric analysis of CRH immunoreactive proteins in subcellular fractions of the Neuro2A cells. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) and protein content is expressed in arbitrary units. C) Levels of secreted CRH protein measured by ELISA. The ability of cells to secrete the CRH hormone was evaluated by measuring the protein level in cultured media of cells transfected either with the wild-type or the mutant construct at 24 h or 48 h after the transfection. Each bar represents the mean ± S.E.M. (<i>n</i> = 2) and protein content is expressed as % in respect to the mean value of wt 24 h (assumed equal to 100%).* t = −7.403 and p = 0.005 compared with wt at 24 h; ** t = 7.796 and p = 0.004 compared with wt at 24 h.</p

Complement_gene_alignments

Primates Multispecies alignments used in the evolutionary analyse

Diverse selective regimes shape genetic diversity at <i>ADAR</i> genes and at their coding targets

<div><p>A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 <i>ADAR</i> family genes and of their coding targets. Evolutionary analysis indicated that <i>ADAR</i> evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both <i>ADAR</i> genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for <i>ADAR</i> genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis.</p></div

omegaMap_alignments

complement bacterial-encoded interactors alignment file

Looking at Human Cytosolic Sialidase NEU2 Structural Features with an Interdisciplinary Approach

Circular dichroism (CD) spectra at variable temperatures have been recorded for human cytosolic sialidase NEU2 in buffered water solutions and in the presence of divalent cations. The results show the prevalence of β-strands together with a considerable amount of α-helical structure, while in the solid state, from both previous X-ray diffraction analysis and our CD data on film samples, the content of β-strands is higher. In solution, a significant change in CD spectra occurs with an increase in temperature, related to a decrease in α-helix content and a slight increase in β-strand content. In the same range of temperatures, the enzymatic activity decreases. Although the overall structure of the protein appears to be particularly stable, molecular dynamics simulations performed at various temperatures evidence local conformational changes possibly relevant for explaining the relative lability of enzymatic activity