Glucocorticoids reduce inflammation in cystic fibrosis bronchial epithelial cells

Reduction of lung inflammation is one of the goals of cystic fibrosis (CF) therapy. Among anti-inflammatory molecules, glucocorticoids (GC) are one of the most prescribed. However, CF patients seem to be resistant to glucocorticoid treatment. Several molecular mechanisms that contribute to decrease anti-inflammatory effects of glucocorticoids have been identified in pulmonary diseases, but the molecular actions of glucocorticoids have never been studied in CF. In the cytoplasm, glucocorticoids bind to glucocorticoid receptor (GR) and then, control NF-κB and MAPK pathways through direct interaction with AP-1 and NF-κB in the nucleus. Conversely, MAPK can regulate glucocorticoid activation by targeting GR phosphorylation. Together these pathways regulate IL-8 release in the lung. Using bronchial epithelial cell lines derived from non CF and CF patients, we analyzed GR-based effects of glucocorticoids on NF-κB and MAPK pathways, after stimulation with TNF-α. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-κB activity in CF cells in a pro-inflammatory context. Moreover, we show that p38 MAPK controls IL-8 release by determining GR activation through specific phosphorylation on serine 211. Finally, we demonstrate a synergistic effect of dexamethasone treatment and inhibition of p38 MAPK inducing more than 90% inhibition of IL-8 production in CF cells. All together, these results demonstrate the good responsiveness to glucocorticoids of CF bronchial epithelial cells and the reciprocal link between glucocorticoids and p38 MAPK in the control of CF lung inflammation.     

A H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates

Background

The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).

Methods

Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.

Results

The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.

Conclusions and general significance

The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.     

Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors

Serine/arginine-rich (SR) proteins are splicing regulators that share a modular structure consisting of one or two N-terminal RNA recognition motif domains and a C-terminal RS-rich domain. We investigated the dynamic localization of the Arabidopsis thaliana SR protein RSZp22, which, as we showed previously, distributes in predominant speckle-like structures and in the nucleolus. To determine the role of RSZp22 diverse domains in its nucleolar distribution, we investigated the subnuclear localization of domain-deleted mutant proteins. Our results suggest that the nucleolar localization of RSZp22 does not depend on a single targeting signal but likely involves different domains/motifs. Photobleaching experiments demonstrated the unrestricted dynamics of RSZp22 between nuclear compartments. Selective inhibitor experiments of ongoing cellular phosphorylation influenced the rates of exchange of RSZp22 between the different nuclear territories, indicating that SR protein mobility is dependent on the phosphorylation state of the cell. Furthermore, based on a leptomycin B- and fluorescence loss in photobleaching-based sensitive assay, we suggest that RSZp22 is a nucleocytoplasmic shuttling protein. Finally, with electron microscopy, we confirmed that RSp31, a plant-specific SR protein, is dynamically distributed in nucleolar cap-like structures upon phosphorylation inhibition. Our findings emphasize the high mobility of Arabidopsis SR splicing factors and provide insights into the dynamic relationships between the different nuclear compartments.     

Geophagy in Wild Yellow-Tailed Woolly Monkeys (Lagothrix flavicauda) Is Brief and Rare

International Journal of Primatology - Many primates intentionally consume earth. This geophagy has probable health benefits, such as mineral supplementation or gastrointestinal tract protection,...     

Identification of a crucial amino acid implicated in the hydroxylation/desaturation ratio of CpFAH12 bifunctional hydroxylase

Claviceps purpurea bifunctional Δ12-hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. To identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine-box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing the only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di-iron center geometry rather than changes in the substrate binding mode.     

Diene-modified nucleotides for the Diels–Alder-mediated functional tagging of DNA

We explore the potential of the Diels–Alder cycloaddition for the functional tagging of DNA strands. A deoxyuridine triphosphate derivative carrying a diene at position 5 of the pyrimidine base was synthesized using a two-step procedure. The derivative was efficiently accepted as substrate in enzymatic polymerization assays. Diene carrying strands underwent successful cycloaddition with maleimide-terminated fluorescence dyes and a polymeric reagent. Furthermore, a nucleotide carrying a peptide via a Diels–Alder cyclohexene linkage was prepared and sequence-specifically incorporated into DNA. The Diels–Alder reaction presents a number of positive attributes such as good chemoselectivity, water compatibility, high-yield under mild conditions and no additional reagents apart from a diene and a dienophile. Furthermore, suitable dienophiles are commercially available in the form of maleimide-derivatives of fluorescent dyes and bioaffinity tags. Based on these advantages, diene- and cyclohexene-based nucleotide triphosphates are expected to find wider use in the area of nucleic acid chemistry.