The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation

Initiation of the development of the anterior-posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, preimplantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early periimplantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, whilst it is reinforced in the distal-lateral domain. Our time-lapse studies demonstrate that this distal-lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior-posterior axis is already anticipated before AVE migration.     

Expression of the RAI gene is conducive to apoptosis: studies of induction and interference

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shown that the region encompassing RAI is important for the development of cancer in young and middle-aged persons. It has been speculated that the RAI product induces apoptosis by blocking NF-kappaB or inhibits apoptosis by blocking p53. Either way the gene could influence the survival of precancerous lesions. Here we report that the expression of RAI mRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergo apoptosis by various means, such as treatment with etoposide, calcium ions, or interleukin-2 and/or serum deprivation. Treatment with etoposide increased the content of RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAI expression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatment with the NF-kappaB-inhibiting substance sulfasalazine relieved this dependence. In the transformed cell line HEK-293 the association between RAI induction and apoptosis seemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficient for apoptosis induction in non-transformed cells. Our results could be explained by a NF-kappaB mediated mechanism.     

Ticks and mosquitoes as vectors of Borrelia burgdorferi s. l. in the forested areas of Szczecin

The aim of the study was to determine the infection level of adult forms and larvae of ticks and mosquitoes with Borrelia burgdorferi in the forested areas of Szczecin. A total of 1699 ticks Ixodes ricinus, including 1422 nymphs, 277 adult forms and 2862 mosquito females representing the genera Aedes (89.6%) and Culex (10.4%) were collected between the years 2004 and 2005. A further 3746 larvae and 1596 pupae of Culex pipiens pipiens were colleted from water bodies. Borrelia burgdorferi s. l. was detected in the arthropods by the method of indirect immunofluorescence assay (IFA). A positive immunological reaction was detected in 16.6% of the adult forms and in 16.5% of the nymphs of Ixodes ricinus. Spirochetes were also detected in 1.7% of mosquito females, 3.2% of larvae and in 1.6% of pupae of Culex pipiens pipiens. The results of the present study confirm that contact with ticks constitutes the main risk of contracting Lyme disease, although mosquitoes play a role as vectors as well.     

Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.     

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.     

The studies on substrate,product and inhibitor binding to a wild-type and neuronopathic form of human acid-β-glucosidase

Gaucher disease is a lysosomal storage disorder caused by deficiency of human acid β-glucosidase. Recent x-ray structural elucidation of the enzyme alone and in the presence of its inhibitor was done, which provided an excellent template for further studies on the binding of substrate, product and inhibitor. To draw correlations between the clinical manifestation of the disease driven by point mutations, L444P and L444R, and the placement and function of putative S-binding sites, the presented theoretical studies were undertaken, which comprised of molecular dynamics and molecular docking methods. The obtained results indicate the D443 and D445 residues as extremely important for physiological functionality of an enzyme. They also show, although indirectly, that binding of the substrate is influenced by an interplay of E235 and E334 residues, constituting putative substrate binding site, and the region flanked by D435 and D445 residues. Figure The binding of an arbitrarily chosen structure of glucosylceramide (A), conduritol-β-epoxide (B), glucose (C) to the active site D443/D445 (A1, B1, C1) and E320/E340 (A2, B2, C2) of the wild-type structure of human acid-β-glucosidase. A1, B1, C1 blue mask represents the residues D443-D445; red mask represents the residue D444; A2, B2, C2 blue mask represents loop1 (Ser³⁴⁵-Glu³⁴⁹) and loop2 (Val³⁹⁴-Asp³⁹⁹), whereas red mask the residues E235 and 340     

Improving the proteome coverage of Daphnia magna - implications for future ecotoxicoproteomics studies

Aquatic pollution is an increasing problem and requires extensive research efforts to understand associated consequences and to find suitable solutions. The crustacean Daphnia is a keystone species in lacustrine ecosystems by connecting primary producers with higher trophic levels. Therefore, Daphnia is perfectly suitable to investigate biological effects of freshwater pollution and is frequently used as an important model organism in ecotoxicology. The field of ecotoxicoproteomics has become increasingly prevalent, as proteins are important for an organism's physiology and respond rapidly to changing environmental conditions. However, one obstacle in proteome analysis of Daphnia is highly abundant proteins like vitellogenin, decreasing the analytical depth of proteome analysis. To improve proteome coverage in Daphnia, we established an easy-to-use procedure based on the LC-MS/MS of whole daphnids and the dissected Daphnia gut, which is the main tissue getting in contact with soluble and particulate pollutants, separately. Using a comprehensive spectral library, generated by gas-phase fractionation and a data-independent acquisition method, we identified 4621 and 5233 protein groups at high confidence (false discovery rate < 0.01) in Daphnia and Daphnia gut samples, respectively. By combining both datasets, a proteome coverage of 6027 proteins was achieved, demonstrating the effectiveness of our approach.