Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.     

HGH20k species and variability of GH responses to long-duration exercise in male cyclists fed different food supplements.

Exercise studies dealing with hGH have always considered this hormone as a unique molecular entity. We postulated that the well-known variability in blood total hGH response could possibly be explained, at least in part, by concomitant changes in blood hGH20k levels, variant form possibly expressing some of the hGH anti-insulinic properties. Six male trained cyclists were imposed a 2-hr long ergocycle exercise. Food supplements were given prior to and/or during exertion to exacerbate a possible contribution from hGH20k to total hGH variability by modification of substrate availability. Both blood total hGH and hGH20k levels increased with exercise, the largest increases being observed in absence of supplementation. Large variability of responses were observed in both blood total hGH and hGH20k levels, the latter variant contributing minimally to total blood hGH response (4.3 +/- 0.8%), and being closely associated with the main species (r = 0.90; p less than 0.001). It was concluded that variations associated with hGH20k increases observed in response to prolonged exercise cannot explain the large intra-and inter-individual variability measured in blood total hGH response.     

Influence of the membrane surface on glycolipid conformation and dynamics. An interpretation of NMR results using conformational energy calculations.

Glycolipids constitute an important class of biomolecules that are involved in biomolecular recognition. The importance of carbohydrate head group conformation in such processes is well recognized. Glycolipids typically occur as minor components of the complex heterogeneous matrix of a biological membrane. As a result, the membrane surface may not only influence head group conformation but also serves as a spatial frame in which the glycolipid is oriented and recognized. In this study, conformational energy calculations have been used to assess the conformational space available to the glucose head group of 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in a liquid-crystalline membrane matrix. 2H NMR quadrupolar splittings are calculated and compared with those observed experimentally. This study demonstrates the importance of including surface interactions when considering the conformational space accessible to cell surface carbohydrates. The empirical approach taken here provides considerable insight at the molecular level, and offers the possibility of exploring even more complex systems.     

Combined effect of 2-deoxy-D-glucose and exercise on plasma catecholamine response.

2-Deoxy-D-glucose (2-DG) is a nonmetabolizable analogue of glucose that, by competitive inhibition of glucose utilization, produces a central neuroglucopenia and a peripheral hyperglycemia. This glucopenic agent was used to gain more insight into the combined effects of central glucopenia and exercise on plasma catecholamine response. This was carried out by comparing one group of exercising (26 m/min, 0% grade) rats injected with 2-DG (2-DG-EX; 250 mg/kg iv) with two control groups: one group of exercising rats injected with a saline solution (SAL-EX) and one group of resting rats injected with 2-DG (2-DG-RE). Significant (P less than 0.05) increases in blood glucose levels were observed 10 min after administration of 2-DG (7.2-13.8 and 7.3-12.4 mmol/l in 2-DG-EX and 2-DG-RE groups, respectively). These elevated blood glucose levels were maintained throughout the experiment in the 2-DG-RE condition but decreased in 2-DG-EX rats to levels observed in the SAL-EX group after 45 min of running (13.8-8.0 mmol/l). The combination of 2-DG-induced neuroglucopenia and exercise resulted in an additive response of norepinephrine (0.59 vs. 0.34 and 0.34 ng/ml; t = 12 min) and an amplified epinephrine response (1.4 vs. 0.37 and 0.31 ng/ml; t = 12 min) compared with the responses to each stimulus alone (2-DG-EX vs. 2-DG-RE and SAL-EX, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of exogenous medium-chain free fatty acids during prolonged exercise: comparison with glucose.

The purpose of this study was to compare the oxidation rate of exogenous 13C-labeled medium-chain triacylglycerols (MCT) with that of an isocaloric amount of exogenous [13C]glucose and to evaluate their respective effects on endocrine and metabolic responses to moderate prolonged exercise. To take into account changes in isotopic composition of 13CO2 arising from oxidation of endogenous substrates because of exercise and/or substrate ingestion that overestimates the oxidation rate of exogenous substrates, two levels of 13C enrichment were used for each substrate. Six young healthy males (20-26 yr of age) completed five 2-h periods of exercise at 65 +/- 3% maximal O2 uptake (VO2max) on a cycle ergometer at 7-day intervals: one control exercise with water ingestion, two trials with ingestion of 25 g of [13C]MCT (trioctanoate) 1 h before exercise, and two trials with 57 g of [13C]glucose (dissolved in 1,000 ml of water) ingested during exercise. Exogenous MCT and glucose began to be oxidized within the first 30 min of exercise, and the oxidation rate increased progressively until the end of exercise for both substrates. Over the 2-h period of exercise, 13.6 +/- 3.5 g of ingested MCT and 36.4 +/- 8.2 g of exogenous glucose were oxidized, which represent 54 and 64%, respectively, of the total amount ingested. The contribution of MCT (119 +/- 31 kcal) and glucose (140 +/- 36 kcal) was not significantly different and represented 7 and 8.5%, respectively, of the total energy expenditure.(ABSTRACT TRUNCATED AT 250 WORDS)     

6-OHDA sympathectomy and exercise performance in the rat.

6-hydroxydopamine (6-OHDA) was utilised for the study of the sympathetic nervous system in the resting rats and rats submitted to prolonged exercise. In order to reduce the acute physiological stress associated with an injection of 6-OHDA, beta-1 and alpha-1 adrenoceptors were blocked before the treatment leading to sympathectomy. Sympathectomised rats were divided in two groups: one sacrificed at rest, 24 hours after the treatment. The other group was sacrificed after a treadmill exercise to exhaustion. Running time to exhaustion was 36.0 +/- 4.5 min (mean +/- S.E.M.). This group ran significantly less than a control group brought to exhaustion in 73.7 +/- 10.0 min of exercise (P < 0.05). In order to make appropriate comparisons, another control group was run for 36 min. Some differences were observed between corresponding control and sympathectomized groups. At rest: 1) a lower plasma level of insulin, and 2) a higher plasma free fatty acid concentration were observed in sympathectomized rats. After 36 min of exercise: 1) a lower plasma concentration of norepinephrine, 2) no decrease of the plasma level of insulin, 3) no increase in the plasma glucagon concentration, and 4) a higher plasma glucose level were observed in sympathectomised rats when compared to control rats running for the same time. The lower plasma norepinephrine concentration in exercised sympathectomised rats suggests a lower sympathetic nervous activity in these animals than in control rats. The absence of a decrease of plasma insulin concentration and of an increase in glucagon can be attributed to this lower sympathetic activity in sympathectomised rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

Structure of soluble and membrane-bound human annexin V.

Annexins are a family of water-soluble proteins that bind to membranes in a calcium-dependent manner. Some members have been shown to exhibit voltage-dependent calcium channel activity, a property characteristic of integral membrane proteins. The structures of human annexin V in crystals obtained from aqueous solution and in two-dimensional crystals when bound to phospholipid layers have been determined by X-ray and electron crystallography, respectively. They are compared here. Both structures show close correspondence, suggesting that annexins attach to phospholipid membranes without substantial structural change. These observations, together with biochemical data, lead to the conclusion that annexin V interacts with phospholipid membranes with its convex face. We propose that binding is mediated by direct interaction between the phosphoryl headgroups and the calcium bound to polypeptide loops protruding from the convex face. The membrane area covered by annexin may thus become disordered and permeable allowing calcium flux through the membrane and the central channel-like structure found in annexin molecules.