Structural determination of the capsular polysaccharide of Neisseria meningitidis group I: a two-dimensional NMR analysis

The capsular polysaccharide antigen of Neisseria meningitidis group I was isolated by Cetavlon precipitation and purified by ion-exchange chromatography. The structure of the I polysaccharide was determined largely by comprehensive proton and carbon-13 nuclear magnetic resonance studies in which both one-dimensional and two-dimensional experiments were carried out directly on the I polysaccharide. The I polysaccharide is composed of the repeating unit----4)alpha-L-GulpNAcA(1----3)[4-OAc]beta-D-ManpNA-cA(-- --in which the former residue adopts the 4C1 (L) conformation and the latter residue adopts the 4C1 (D) conformation. The one-bond coupling between the anomeric carbon and proton (1J13C,H) of the 2-acetamido-2-deoxy-beta-D-mannuronopyranosyl residue is not consistent with its beta-D configuration. This anomalous value of 1J13C,H for this residue is due to through-space anisotropy effects on its anomeric proton, generated by the proximity of the carboxyl group of the neighboring 2-acetamido-2-deoxy-alpha-L-guluronopyranosyl residue. The O-acetyl substituents of the I polysaccharide are essential for its antigenicity to group I polysaccharide-specific antibodies.     

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene

Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim     

Concurrent synthesis and degradation of alcohol dehydrogenase in elicitor-treated and wounded potato tubers

The accumulation of alcohol dehydrogenase (ADH) in arachidonic acid-elicited potato (Solanum tuberosum L.) tuber discs was studied. In accordance with our previous report of the accumulation of Adh mRNA beginning 2 hours after elicitor treatment (DP Matton, CP Constabel, N Brisson [1990] Plant Mol Biol 14: 775-783), immunoprecipitation of ADH from in vivo labeled discs indicated that ADH synthesis occurred as early as 12 hours after treatment. However, levels of ADH activity and protein, as shown by enzyme assay and immunoblot, did not rise in parallel but decreased during the first 24 hours of treatment. After 24 hours, ADH activity and protein began to increase, reaching a several-fold increase at 96 hours after elicitation. Water-treated control discs showed a similar though delayed and less pronounced pattern. These results imply a turnover of ADH following elicitor treatment of potato tuber discs. As shown by nondenaturing gel electrophoresis, the synthesis and degradation involved the same ADH isozyme.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.     

Blood banking-induced senescent modifications on red blood cells.

In the past few years significant progress has been made in the extension of storage time for red blood cells (RBCs). Albeit this, membrane still undergoes damage during conservation under blood bank conditions, reducing red cell viability following transfusion. Consequently in this study, we evaluated the presence of senescence indices (appearing on RBCs during in vivo aging) on blood bank stored RBCs, more precisely the 4.1a/4.1b ratio and the binding of autologous immunoglobulins. Although not significant, a slight increase (0.995 +/- 0.070 to 1.008 +/- 0.058) was observed in the 4.1a/4.1b ratio during the first three weeks of storage. A rapid and significant (p less than 0.001) increase in the number of cell-bound IgGs (91 +/- 22 to 913 +/- 92), following incubation with purified autologous IgGs, was observed in the first days of storage. Thus, following transfusion, binding of autologous IgGs to stored RBCs could influence post-transfusion viability.     

The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.     

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewisx bound to E-selectin

The conformation of the sialyl Lewis ^x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewis^x in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewis^x/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewis^x/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewis^x/E-selectin complex.     

Population density of the Réunion Grey White-eye Zosterops borbonicus within the summit ecosystems of Réunion,Mascarene Islands

Assessing population density is crucial for studying the ecology and evolutionary biology of species as well as for conservation purposes. Here we used point count methods to infer population density in a single-island endemic passerine bird, the Réunion Grey White-eye Zosterops borbonicus, that displays striking evidence of differentiation at a small spatial scale. Population density was estimated at 5.17 birds ha^?1 (CL 4.85–5.50), a value somewhat higher than previously believed. This estimation provides the first detailed estimation of bird population density in the vulnerable summit ecosystems of Réunion and will possibly allow a better understanding of the evolutionary causes of this plumage colour variation.     

Oxidation of a glucose polymer during exercise: comparison with glucose and fructose

The purpose of this study was to compare the oxidation of 13C-labeled glucose, fructose, and glucose polymer ingested (1.33 g.kg-1 in 19 ml.kg-1 water) during cycle exercise (120 min, 53 +/- 2% maximal O2 uptake) in six healthy male subjects. Oxidation of exogenous glucose and glucose polymer (72 +/- 15 and 65 +/- 18%, respectively, of the 98.9 +/- 4.7 g ingested) was similar and significantly greater than exogenous fructose oxidation (54 +/- 13%). A transient rise in plasma glucose concentration was observed with glucose ingestion only. However, plasma insulin levels were similar with glucose and glucose polymer ingestions and significantly higher than with water or fructose ingestion. Plasma free fatty acid and glycerol responses to exercise were blunted with carbohydrate ingestion. However, fat utilization was not significantly different with water (82 +/- 14 g), glucose (60 +/- 3 g), fructose (59 +/- 11 g), or glucose polymer ingestion (60 +/- 8 g). Endogenous carbohydrate utilization was significantly lower with glucose (184 +/- 22 g), glucose polymer (187 +/- 31 g), and fructose (211 +/- 18 g) than with water (239 +/- 30 g) ingestion. Plasma volume slightly increased with water ingestion (7.4 +/- 4.5%), but the decrease was similar with glucose (-7.6 +/- 5.1%) and glucose polymer (-8.2 +/- 4.6%), suggesting that the rate of water delivery to plasma was similar with the two carbohydrates.(ABSTRACT TRUNCATED AT 250 WORDS)