Extended type-1 chain glycosphingolipid antigens. Isolation and characterization of trifucosyl-Leb antigen (III4V4VI2Fuc3Lc6).

A Lewis-b-active glycosphingolipid containing a repetitive type-1 chain carbohydrate core was isolated from human colonic adenocarcinoma cell line Colo205. This glycosphingolipid was purified by HPLC and preparative high-performance thin-layer chromatography and its structure elucidated by positive-ion fast-atom-bombardment mass spectrometry with collision-induced disassociation, 1H-NMR spectroscopy and methylation analysis. The glycosphingolipid was found to be a trifucosylated derivative of this novel carbohydrate core, having the following structure: [formula; see text].     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.     

Numerical simulation of saccular aneurysm hemodynamics: influence of morphology on rupture risk

The governing equations for pulsatile fluid flow were solved in their finite volume formulation in order to simulate blood flow in a variety of three-dimensional aneurysm geometries. The influence of geometric factors on flow patterns and fluid mechanical forces was studied with the goal of identifying the risk of aneurysm rupture. Aneurysm morphology was characterized by quantitative shape indices reflecting the three dimensionality of the vasculature derived from clinical studies. Recirculation zones and secondary flows were observed in aneurysms and arteries. Regions of extreme and alternating shear stress were observed and identified as sites for potential aneurysm rupture. The ellipticity of an aneurysm was observed to be strongly correlated with wall shear stress at the aneurysm fundus, while its non-sphericity, volume, and degree of undulation were more weakly correlated.