Structure elucidation of the core regions from Citrobacter O4 and O36 lipopolysaccharides by chemical and enzymatic methods, gas chromatography/mass spectrometry, and NMR spectroscopy at 500 MHz

Novel enterobacterial core oligosaccharides were isolated from Citrobacter O4 and O36 lipopolysaccharides, and their structures were determined by methylation analysis, Smith degradation and enzymatic degradations, gas chromatography/mass spectrometry, and two-dimensional phase-sensitive correlated, relayed coherence transfer, double-quantum, triple-quantum-filtered, and nuclear Overhauser effect (NOE) 1H NMR spectroscopy at 500 MHz. In the formulas, all hexose residues are D-hexopyranoses, and heptoses are L-glycero-D-manno-heptopyranoses; the alternative locations of the side-chain heptose and pyrophosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (formula; see text) Along with these complete cores, incomplete ones, lacking the hexosamine trisaccharides, occur in the lipopolysaccharides of both types. Qualitative NOE data were in good agreement with the minimum energy conformation of the above O36 oligosaccharide, calculated with the aid of the SUGAR program [Sundin, A., Carter, R. E., & Liljefors, T. (1988) J. Mol. Graphics (in press)].     

Verleihung des Braun-Blanquet Preises an Herrn Prof,Dr. H. Dierschke in münster 1978

Ohne Zusammenfassung     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of alpha-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the alpha-amino group to glutamate. Measurements of synthesis of (14)C-glutamate and of (14)C-glutamine from H(14)CO(3) have shown that (14)C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The specific radioactivity of (14)C-glutamine indicated that approximately 30% of glutamine was derived from (14)CO(2) fixation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of (14)C-glutamate and (14)C-glutamine from H(14)CO(3)(-) was inhibited by 31%. These results suggest that transamination of alpha-ketoglutarate to glutamate in Müller cells is slow, the supply of branched-chain amino acids may limit flux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H(14)CO(3)(-). (14)C-metabolites in brain extracts were measured to determine net incorporation of (14)CO(2) and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from (14)CO(2) fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for approximately 60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.     

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.     

Rapid generation of a representative ensemble of N-glycan conformations

Glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces where their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events. Glycans constitute highly flexible molecules. Only a small number of glycan X-ray structures is available for which sufficient electron density for an entire oligosaccharide chain has been observed. An unambiguous structure determination based on NMR-derived geometric constraints alone is often not possible. Time consuming computational approaches such as Monte Carlo calculations and molecular dynamics simulations have been widely used to explore the conformational space accessible to complex carbohydrates. The generation of a comprehensive data base for N-glycan fragments based on long time molecular dynamics simulations is presented. The fragments are chosen in such a way that the effects of branched N-glycan structures are taken into account. The prediction database constitutes the basis of a procedure to generate a complete set of all possible conformations for a given N-glycan. The constructed conformations are ranked according to their energy content. The resulting conformations are in reasonable agreement with experimental data. A web interface has been established (http://www.dkfz.de/spec/glydict/), which enables to input any N-glycan of interest and to receive an ensemble of generated conformations within a few minutes.     

Characterization of the L1-neurocan-binding site. Implications for L1-L1 homophilic binding

The L1 adhesion molecule is a 200-220-kDa membrane glycoprotein of the Ig superfamily implicated in important neural processes including neuronal cell migration, axon outgrowth, learning, and memory formation. L1 supports homophilic L1-L1 binding that involves several Ig domains but can also bind with high affinity to the proteoglycan neurocan. It has been reported that neurocan can block homophilic binding; however, the mechanism of inhibition and the precise binding sites in both molecules have not been determined. By using fusion proteins, site-directed mutagenesis, and peptide blocking experiments, we have characterized the neurocan-binding site in the first Ig-like domain of human L1. Results from molecular modeling suggest that the sequences involved in neurocan binding are localized on the surface of the first Ig domain and largely overlap with the G-F-C beta-strands proposed to interact with the fourth Ig domain during homophilic binding. This suggests that neurocan may sterically hinder a proper alignment of L1 domains. We find that the C-terminal portion of neurocan is sufficient to mediate binding to the first Ig domain of L1, and we suggest that the sushi domain cooperates with a glycosaminoglycan side chain in forming the binding site for L1.     

EUROCarbDB: An open-access platform for glycoinformatics

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.