Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation

Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30 to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE values preferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thereby enabling the putative glycan structure characterization. In addition, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. In contrast, HCD with higher NCE values preferentially fragmented the peptide backbone and, thus, provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N- and O-linked glycopeptides in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both the peptide and the glycan, thereby enabling comprehensive structural characterization of glycoproteins in a single LC–MS/MS analysis.     

Immobilization of Lactobacillus rhamnosus in polyvinyl alcohol/calcium alginate matrix for production of lactic acid

Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using “freezing–thawing” technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g⁻¹) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L⁻¹ h⁻¹, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L⁻¹ h⁻¹. This study suggested that the “freezing–thawing” technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.

     

Moving in the real world: tortoises take the plunge to cross steep steps

Despite exhibiting low velocity and limited agility, many tortoises undertake large scale movements and must overcome various obstacles, notably in populations living in hilly or rocky habitats. Although crucial, studies exploring how tortoises move in complex and irregular environments are scarce. In this context, we examined an important behavioural trait: how tortoises (Testudo hermanni) deal with step‐like obstacles. In their natural habitat, individuals were positioned in a challenging situation: they were placed on a bench approximately 50 cm high, and were observed over a 10‐min period. We compared the behaviour of the tortoises (taking a risk to ‘jump’ or waiting) from two populations living in contrasted habitats: flat versus rugged (crisscrossed by cliffs and rocky steps). Individuals from the flat habitat were reluctant to jump, whereas most tortoises from the rugged habitat jumped. Immature tortoises were less willing to jump compared to larger and more experienced adults. These results suggest that challenging habitats increase boldness. In addition to fundamental findings, these results may have conservation value and assist in improving translocation strategies for endangered tortoise populations. © 2013 The Linnean Society of London     

Loss of shear stress induces leukocyte-mediated cytokine release and blood-brain barrier failure in dynamic in vitro blood-brain barrier model

Brain ischemia is associated with an acute release of pro-inflammatory cytokines, notably TNF-alpha and IL-6 and failure of the blood-brain barrier. Shear stress, hypoxia-hypoglycemia, and blood leukocytes play a significant role in blood-brain barrier failure during transient or permanent ischemia. However, these mechanisms have not been studied as independent variables for in vitro ischemia. The present study, using a dynamic in vitro blood-brain barrier model, showed that flow cessation/reperfusion under normoxia-normoglycemia or hypoxia-hypoglycemia without blood leukocytes in the luminal perfusate had a modest, transient effect on cytokine release and blood-brain barrier permeability. By contrast, exposure to normoxic-normoglycemic flow cessation/reperfusion with blood leukocytes in the luminal perfusate led to a significant increase in TNF-alpha and IL-6, accompanied by biphasic blood-brain barrier opening. Enhanced permeability was partially prevented with an anti-TNF-alpha antibody. In leukocyte-free cartridges, the same levels of IL-6 had no effect, while TNF-alpha caused a moderate increase in blood-brain barrier permeability, suggesting that blood leukocytes are the prerequisite for cytokine release and blood-brain barrier failure during reduction or cessation of flow. These cells induce release of TNF-alpha early after ischemia/reperfusion; TNF-alpha triggers release of IL-6, since blockade of TNF-alpha prevents IL-6 release, whereas blockade of IL-6 induces TNF-alpha release. Pre-treatment of blood leukocytes with the cyclooxygenase (COX) inhibitor, ibuprofen, inhibited cytokine release and completely preserved blood-brain barrier permeability during the reperfusion period. In conclusion, loss of flow (flow cessation/reperfusion) independent of hypoxia-hypoglycemia plays a significant role in blood-brain barrier failure by stimulating leukocyte-mediated inflammatory mechanisms.     

Discovery and SAR study of novel dihydroquinoline containing glucocorticoid receptor ligands

We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.     

Hygromycin promotes somatic embryogenesis in spinach

Hygromycin (hyg) at low doses (0.5–1.0 mg l^?1) promoted somatic embryogenesis from apical sections of spinach lateral roots. The highest promoting effect on both the frequency of regeneration and the mean number of somatic embryos (SE) per explant was achieved at 0.5 mg l^?1 hyg. With increasing the concentration of hyg to 1 mg l^?1, the regeneration frequency decreased, while the mean SE number remained significantly higher than in control (hyg-free medium). Complete inhibition of SE regeneration started at 7.5 mg l^?1 hyg. Moreover, hyg efficiently promoted the process of secondary somatic embryogenesis. Compared to control, a 2.75-fold increase in the secondary somatic embryo (SSE) mean number was obtained at 0.5 mg l^?1 hyg, and the increment was still discernible at 1.0 and 2.5 mg l^?1 hyg. Both primary SE and SSE explants became completely necrotic at 12.5 mg l^?1 hyg. Since attempts with direct selection at 20 mg l^?1 hyg proved unsuccessful, the results obtained in this study suggest that a stepwise selection procedure is suitable, starting with selection at 0.5 mg l^?1 hyg, to exploit the promoting effect of low hyg doses on SE regeneration from transformed cells, then gradually increasing the hyg concentration to 20 mg l^?1 for final selection. Complete SE and SSE explant mortality at hyg above 12.5 mg l^?1 guarantees a low possibility of escape during the selection process. This study will be useful for increasing the efficiency of transgenic plant regeneration following genetic transformation in spinach.     

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.     

Morpho-anatomical differentiation of Suaeda maritima (L.) Dumort. 1827. (Chenopodiaceae) populations from inland and maritime saline area

Morphological analysis of four populations of Suaeda maritima was undertaken in order to examine the variation between populations from inland and maritime saline area, as well as between the two subspecies. Variability and significance of morpho-anatomical differentiation were examined using principal component analysis (PCA), discriminant component analysis (DCA) and cluster analysis. Plants of each population exhibited halomorphic and xeromorphic characteristics. The results of PCA and DCA showed that S. maritima subsp. prostrata and S. maritima subsp. maritima could be clearly separated based on their quantitative anatomical characteristics. Based on our analysis, climate and the amount of salt and ions in the soil, are important factors that enhance the adaptive potential of S. maritima.