SummaryType-1 fimbriae are important virulence factors for the establishment of Escherichia coli urinary tract infections. Bacterial adhesion to the highmannosylated uroplakin Ia glycoprotein receptors of bladder epithelium is mediated by the FimH adhesin. Previous studies have attributed differences in mannose-sensitive adhesion phenotypes between faecal and uropathogenic E. coli to sequence variation in the FimH receptor-binding domain. We find that FimH variants from uropathogenic, faecal and enterohaemorrhagic isolates express the same specificities and affinities for high-mannose structures. The only exceptions are FimHs from O157 strains that carry a mutation (Asn135Lys) in the mannose-binding pocket that abolishes all binding. A high-mannose microarray shows that all substructures are bound by FimH and that the largest oligomannose is not necessarily the best binder. Affinity measurements demonstrate a strong preference towards oligomannosides exposing Mana1-3Man at their non-reducing end. Binding is further enhanced by the b1-4-linkage to GlcNAc, where binding is 100-fold better than that of a-D-mannose. Mana1-3Manb1-4GlcNAc, a major oligosaccharide present in the urine of a-mannosidosis patients, thus constitutes a well-defined FimH epitope. Differences in affinities for high-mannose structures are at least 10-fold larger than differences in numbers of adherent bacteria between faecal and uropathogenic strains. Our results imply that the carbohydrate expression profile of targeted host tissues and of natural inhibitors in urine, such as TammHorsfall protein, are stronger determinants of adhesion than FimH variation.
The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.
Plants produce a unique peroxisomal short chain-specific acyl-CoA oxidase (ACX4) for -oxidation of lipids. The short chain-specific oxidase has little resemblance to other peroxisomal acyl-CoA oxidases but has an ϳ30% sequence identity to mitochondrial acyl-CoA dehydrogenases. Two biochemical features have been linked to structural properties by comparing the structures of short chain-specific Arabidopsis thaliana ACX4 with and without a substrate analogue bound in the active site to known acyl-CoA oxidases and dehydrogenase structures: (i) a solvent-accessible acyl binding pocket is not required for oxygen reactivity, and (ii) the oligomeric state plays a role in substrate pocket architecture but is not linked to oxygen reactivity. The structures indicate that the acyl-CoA oxidases may encapsulate the electrons for transfer to molecular oxygen by blocking the dehydrogenase substrate interaction site with structural extensions. A small binding pocket observed adjoining the flavin adenine dinucleotide N5 and C4a atoms could increase the number of productive encounters between flavin adenine dinucleotide and O 2 .The central pathway for fatty acid breakdown in higher plants is via peroxisomal -oxidation. Fatty acids enter the cycle in the form of acyl-CoA thioesters, and during one round of -oxidation, the acyl chain is shortened by a two-carbon unit and one acetyl-CoA molecule is produced. The first step in the peroxisomal -oxidation cycle is the introduction of a double bond into the acyl-CoA substrate, resulting in the formation of 2-trans-enoyl-CoA. This reaction is a two-step reaction catalyzed by the family of acyl-CoA oxidases (ACXs) 2 requiring flavin adenine dinucleotide (FAD) as a cofactor. The cofactor gets reduced to FADH Ϫ in the first half-reaction concomitant with acyl-CoA oxidation. FADH Ϫ is reoxidized by molecular oxygen in the second step, thereby generating H 2 O 2 , an intracellular signaling molecule.Unlike plants, two parallel and distinct -oxidation pathways exist in mammals, the peroxisomal -oxidation pathway and a mitochondrial -oxidation pathway. In mitochondrial -oxidation, the first step is catalyzed by the acyl-CoA dehydrogenase family (ACD) (1), which is related to ACXs. The FADH Ϫ in ACDs is not, however, reoxidized by molecular oxygen but rather by another flavoprotein, the electron transfer flavoprotein, which transfers electrons to the electron transport chain. As a result, the oxidation of fatty acids/amino acids and the generation of ATP molecules are linked in mitochondrial -oxidation (2). It is puzzling that natural selection has not forced plants to utilize the mitochondrial electron transport chain for general lipid oxidation despite the apparent ATP advantage of this pathway. This might reflect less stress on the ATP requirement and a higher demand for lipid turnover and excess oxygen management in plants.Six genes for ACX isozymes have been identified in Arabidopsis thaliana. Five encode for proteins of ϳ75 kDa (3-5). The proteins have different but overlapping s...
A Silyl Carborane Containing the closo-1,10-C2B8H8 Fragment. -In view of the interest in developing routes to silicon-linked carborane units as precursors to silicon carbide/boron carbide composites, the compound (III) (space group P21/c; Z = 2) is prepared. It contains three carboranyl units linked in a zig-zag chain via two dimethylsilyl groups. -(GETMAN, T. D.; GARRETT, P. M.; KNOBLER, C. B.; HAWTHORNE, M. F.; THORNE, K.; MACKENZIE, J. D.; Organometallics 11 (1992) 7, 2723-2725; Dep. Chem.
l~eferate 67 wurde durchgefShrt ffir den SpeziMfall charakteristiseher Werte, die die folgende Uugleichung erfiillen: ~pLp-n.< 2A~-n, so dal~ J (~0Zvm) sich direk~ aus expertmentell bestir~maten Werten yon ¢ (w) ermittein li~l~t. W (M) erhi~lt man darm aus ] (w 2~-m) durch eine L6sung der Integralgleichung.~. ~Jhlein (Frankfurt a. M.)Quinn, J. A., u. P. G. Jeannin (Univ. of Illinois, Urbana, Ill., USA). Grenztliiehenwiderstand: Diffusion in einem laminaren Fliissigflliissig-Strahl. (Chem. Engng. Sci. 15, 253-250, 1961.) Die Diffusionsgeschwindigkeiten durch eine Flfissig/flfissig-Grenzfl~che wurden nnter Verwendung eines laminaren Strahles bestimm$. Dus System Isobutanol/Wasser zeigt bei einer nominellcn Kontuktzeig yon 0,05 his 0,5 Sekmaden einen anscheinend kleinen Grenzfli~ehenwiderstand yon armghernd 80 sec/cm. Durch die Stabilit~t des Strahles ist die Auswahl der zu untersuehenden Flfissigkeitspaare begrenzt. Es wird jedoch angenommen, dal~ sich diese ~V[ethode bei der Untersuchung yon Grenzfl/~chenerscheinungen im Zusammenhang mit der Diffusion durch eine Flfissigflfissig-Grenzfl~che bew/~hren wird.E. Uhlein (Frankfurt a. M.)
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