Molecular mechanisms of O-GlcNAcylation

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of serines/threonines on metazoan proteins and occurring with similar time scales, dynamics and stoichiometry as protein phosphorylation. Levels of this modification are regulated by two enzymes-O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Although the biochemistry of these enzymes and functional implications of O-GlcNAc have been studied extensively, until recently the structures and molecular mechanisms of OGT/OGA were not understood. This review covers a body of recent work that has led to an understanding of the structure of OGA, its catalytic mechanism and the development of a plethora of different inhibitors that are finding their use in cell biological studies towards the functional implications of O-GlcNAc. Furthermore, the very recent structure determination of a bacterial OGT orthologue has given the first insights into the contribution of the tetratricopeptide repeats (TPRs) to the active site and the role of some residues in catalysis and substrate binding.     

The chemokine interleukin-8 acutely reduces Ca(2+) currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs

The chemokine IL-8 is known to be synthesized by glial cells in the brain. It has traditionally been shown to have an important role in neuroinflammation but recent evidence indicates that it may also be involved in rapid signaling in neurons. We investigated how IL-8 participates in rapid neuronal signaling by using a combination of whole-cell recording and single-cell RT-PCR on dissociated rat septal neurons. We show that IL-8 can acutely reduce Ca(2+) currents in septal neurons, an effect that was concentration-dependent, involved the closure of L- and N-type Ca(2+) channels, and the activation of G(ialpha1) and/or G(ialpha2) subtype(s) of G-proteins. Analysis of the mRNAs from the recorded neurons revealed that the latter were all cholinergic in nature. Moreover, we found that all cholinergic neurons that responded to IL-8, expressed mRNAs for either one or both IL-8 receptors CXCR1 and CXCR2. This is the first report of a chemokine that modulates ion channels in neurons via G-proteins, and the first demonstration that mRNAs for CXCR1 are expressed in the brain. Our results suggest that IL-8 release by glial cells in vivo may activate CXCR1 and CXCR2 receptors on cholinergic septal neurons and acutely modulate their excitability by closing calcium channels.     

Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) inhibits myogenic differentiation via the activation of nuclear factor-kappaB (NF-kappaB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C(2)C(12) cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha-treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha-induced activation of NF-kappaB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappaB. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.     

Resveratrol metabolites in urine as a biomarker of wine intake in free-living subjects: The PREDIMED Study

Several clinical and epidemiological studies have shown that moderate wine consumption may exert a protective effect against oxidative stress involved in several diseases, such as cardiovascular and neurodegenerative disorders. However, the epidemiological assessment of wine consumption has usually been obtained using self-reported questionnaires containing less reliable information for assessing total intake than nutritional biomarkers. A reliable biomarker for wine consumption is, therefore, needed. To validate urinary resveratrol metabolites (RMs) as a biomarker of wine consumption in a large cohort of free-living subjects, 1000 consecutive subjects entering a substudy of the PREDIMED trial (Prevención con Dieta Mediterránea) were evaluated. Data were collected in a validated semiquantitative food frequency questionnaire. RMs were measured in morning urine by LC-MS/MS. Urinary RM values correlated directly with reported daily amounts of wine consumed (r = 0.895; p < 0.001). One drink of wine per week can be detected. Using a cut-off of 411.4 nmol/g creatinine, the measurement of urinary RMs could discriminate wine consumers from non-wine consumers with a sensitivity of 93.3% (95% confidence interval (CI) 91.5–94.7%) and a specificity of 92.1% (CI 90.2–93.7%). Urinary RMs fulfill the criteria to be considered as a nutritional biomarker of wine consumption in a large sample of free-living subjects. This biomarker would provide an additional tool for investigating more precisely the relationship between wine consumption and health benefits.     

The E3 ubiquitin ligase adaptor Ndfip1 regulates Th17 differentiation by limiting the production of proinflammatory cytokines

Ndfip1 is an adaptor for the E3 ubiquitin ligase Itch. Both Ndfip1- and Itch-deficient T cells are biased toward Th2 cytokine production. In this study, we demonstrate that lungs from Ndfip1(-/-) mice showed increased numbers of neutrophils and Th17 cells. This was not because Ndfip1(-/-) T cells are biased toward Th17 differentiation. In fact, fewer Ndfip1(-/-) T cells differentiated into Th17 cells in vitro due to high IL-4 production. Rather, Th17 differentiation was increased in Ndfip1(-/-) mice due to increased numbers of IL-6-producing eosinophils. IL-6 levels in mice that lacked both Ndfip1 and IL-4 were similar to wild-type controls, and these mice had fewer Th17 cells in their lungs. These results indicate that Th2 inflammation, such as that observed in Ndfip1(-/-) mice, can increase Th17 differentiation by recruiting IL-6-producing eosinophils into secondary lymphoid organs and tissues. This may explain why Th17 cells develop within an ongoing Th2 inflammatory response.     

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Añana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.     

A Recombinant Saccharomyces cerevisiae Strain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

Stabilization against protein haze was one of the first positive properties attributed to yeast mannoproteins in winemaking. In previous work we demonstrated that deletion of KNR4 leads to increased mannoprotein release in laboratory Saccharomyces cerevisiae strains. We have now constructed strains with KNR4 deleted in two different industrial wine yeast backgrounds. This required replacement of two and three alleles of KNR4 for the EC1118 and T73-4 backgrounds, respectively, and the use of three different selection markers for yeast genetic transformation. The actual effect of the genetic modification was dependent on both the genetic background and the culture conditions. The fermentation performance of T73-4 derivatives was clearly impaired, and these derivatives did not contribute to the protein stability of the wine, even though they showed increased mannoprotein release in vitro. In contrast, the EC1118 derivative with both alleles of KNR4 deleted released increased amounts of mannoproteins both in vitro and during wine fermentation assays, and the resulting wines were consistently less susceptible to protein haze. The fermentation performance of this strain was slightly impaired, but only with must with a very high sugar content. These results pave the way for the development of new commercial strains with the potential to improve several mannoprotein-related quality and technological parameters of wine.