97 research outputs found
Technology transfer: Transportation
The application of NASA derived technology in solving problems related to highways, railroads, and other rapid systems is described. Additional areas/are identified where space technology may be utilized to meet requirements related to waterways, law enforcement agencies, and the trucking and recreational vehicle industries
Diffusion on random site percolation clusters. Theory and NMR microscopy experiments with model objects
Quasi two-dimensional random site percolation model objects were fabricate
based on computer generated templates. Samples consisting of two compartments,
a reservoir of HO gel attached to a percolation model object which was
initially filled with DO, were examined with NMR (nuclear magnetic
resonance) microscopy for rendering proton spin density maps. The propagating
proton/deuteron inter-diffusion profiles were recorded and evaluated with
respect to anomalous diffusion parameters. The deviation of the concentration
profiles from those expected for unobstructed diffusion directly reflects the
anomaly of the propagator for diffusion on a percolation cluster. The fractal
dimension of the random walk, , evaluated from the diffusion measurements
on the one hand and the fractal dimension, , deduced from the spin density
map of the percolation object on the other permits one to experimentally
compare dynamical and static exponents. Approximate calculations of the
propagator are given on the basis of the fractional diffusion equation.
Furthermore, the ordinary diffusion equation was solved numerically for the
corresponding initial and boundary conditions for comparison. The anomalous
diffusion constant was evaluated and is compared to the Brownian case. Some ad
hoc correction of the propagator is shown to pay tribute to the finiteness of
the system. In this way, anomalous solutions of the fractional diffusion
equation could experimentally be verified for the first time.Comment: REVTeX, 12 figures in GIF forma
Analysis of congenital disorder of glycosylation-Id in a yeast model system shows diverse site-specific under-glycosylation of glycoproteins
Asparagine-linked glycosylation is a common post translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site specific glycosylation analysis in many model systems and clinical applications
Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis
Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.National Institutes of Health (U.S.) (Grant GM039334
Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.
N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system
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