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)].     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

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

Ohne Zusammenfassung     

The Influence of Antimicrobial Peptides and Mucolytics on the Integrity of Biofilms Consisting of Bacteria and Yeasts as Affecting Voice Prosthetic Air Flow Resistances

The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech. Biofilm integrity is ensured by extracellular polymeric substances (EPS). This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms. Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan). Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses after biofilm formation. Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance. Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.     

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.