Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc–modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs

Anyone for non-scalarity?

This paper examines the status of scalarity in the analysis of the meaning of the English determiner any. The latter’s position as a prime exemplar of the category of polarity-sensitive items has led it to be generally assumed to have scalar meaning. Scalar effects are absent however from a number of common uses of this word. This suggests that any does not involve scales as part of its core meaning, but produces them as a derived interpretative property. The role of three factors in the derivation of the expressive effect of scalarity is explored: grammatical number, stress and the presence of gradable concepts in the NP. The general conclusions point to the importance of developing a causal semantic analysis in which the contributions of each of the various meaningful components of an utterance to the overall message expressed are carefully distinguished

Chemoenzymatic Probes for Detecting and Imaging Fucose-α(1-2)-galactose Glycan Biomarkers

The disaccharide motif fucose-α(1-2)-galactose (Fucα(1-2)Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα(1-2)Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα(1-2)Gal glycans. We demonstrate that the approach is highly selective for the Fucα(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease

Caracterización fisiológica de tres razas de maíz peruano cultivadas con altos contenidos de boro

El boro (B) es un nutriente esencial para un adecuado desarrollo de las plantas. En regiones áridas o semiáridas es frecuente encontrar suelos con elevado contenido de B. Este exceso de B desencadena problemas importantes de toxicidad en las plantas, que conducen a una reducción de su crecimiento y a una disminución de la fotosíntesis, entre otros efectos. El maíz es uno de los cultivos de mayor importancia en Perú. Muchas de las tierras de cultivo del sur del Perú presentan contenidos elevados de B. La selección de razas que sean tolerantes a estas condiciones es fundamental para obtener mejores rendimientos en las cosechas. El objetivo de este trabajo fue caracterizar la tolerancia al exceso de B de las razas de maíz Pachía, Lipe-Coruca y Sama, originarias de la región de Tacna (Perú). Para ello, se cultivaron estas razas de maíz en macetas con tierras con alto contenido de B (214 ppm), y fueron regadas con aguas del río Sama cuya concentración de B fue de 14.63 mgL-1. Las razas Sama y Lipe-Coruca mostraron las mayores alturas con respecto al vástago. Además, la raza Sama presentó los más bajos contenidos foliares de B soluble e insoluble, y los mayores contenidos de clorofilas y carotenoides. En resumen, estos resultados sugieren que Sama es la raza más tolerante al exceso de B. Así, los menores contenidos de B y el mayor contenido de estos pigmentos de Sama permitirían una mejor adaptación al estrés generado por la acumulación de B en las tierras de cultivo

Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics

The addition of the monosaccharide beta-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons

On-line estimation of O2 production, CO2 uptake, and growth kinetics of microalgal cultures in a gas-tight photobioreactor

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO2, H2, and N2 were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO2 uptake was estimated from the addition of CO2 as acidic titrant and O2 evolution was estimated from titration by H2, which was used to reduce O2 over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O2 evolution and CO2 up-take rates. NH4+, NO2−, or NO3− was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH4+ as the nitrogen source and 1.3 when NO3− was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3–4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO2 and H2 into the reactor headspace to estimate the up-take of CO2, the production of O2, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified

Role of ABA in the adaptive response of Arabidopsis plants to long-term boron toxicity treatment

Boron (B) toxicity causes impairments in several plant metabolic and physiological processes. Under conditions of excessive B availability, this micronutrient is passively transported through the transpiration stream and accumulates in leaves, causing the development of necrotic regions in leaf tips. Some plants have developed adaptive mechanisms to minimize the toxic effects of excessive B accumulation in their tissues. Thus, for instance, in Arabidopsis it has been described an ABA-dependent decrease in the transpiration rate that would restrict B accumulation in aerial plant tissues in response to short-term B toxicity, this effect being mediated by AtNCED3 (which encodes a key enzyme for ABA biosynthesis). The present work aimed to study the possible involvement of ABA in the adjustment of plant water balance and B homeostasis during the adaptive response of Arabidopsis to prolonged B toxicity. For this purpose, Arabidopsis wild-type and the ABA-deficient nced3-2 mutant plants were subjected to B toxicity for 7 days. We show that ABA-dependent stomatal closure is determinant for the adjustment of plant water relations under conditions of prolonged B toxicity. Results suggest that, in addition to the AtNCED3 gene, the AtNCED5 gene could also be involved in this ABA-dependent stomatal closure. Finally, our results also indicate the possible role of endogenous root ABA content in the mechanism of active efflux of B via BOR4 (efflux-type B transporter) from the root to the external environment under excess B conditions.Departamento de Fisiología, Anatomía y Biología Celular, Universidad Pablo de OlavideDepartamento de Biología Animal, Biología Vegetal y Ecología, Facultad de Ciencias Experimentales, Campus de Las Lagunillas s/n, Universidad de Jaé

Memory and synaptic plasticity are impaired by dysregulated hippocampal O-GlcNAcylation

O-GlcNAcylated proteins are abundant in the brain and are associated with neuronal functions and neurodegenerative diseases. Although several studies have reported the effects of aberrant regulation of O-GlcNAcylation on brain function, the roles of O-GlcNAcylation in synaptic function remain unclear. To understand the effect of aberrant O-GlcNAcylation on the brain, we used Oga+/- mice which have an increased level of O-GlcNAcylation, and found that Oga+/- mice exhibited impaired spatial learning and memory. Consistent with this result, Oga+/- mice showed a defect in hippocampal synaptic plasticity. Oga heterozygosity causes impairment of both long-term potentiation and long-term depression due to dysregulation of AMPA receptor phosphorylation. These results demonstrate a role for hyper-O-GlcNAcylation in learning and memory.ope