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Biomedical subjects

J Bouckaert

Publications and source records attributed to J Bouckaert.

At least 19 recordsLinked to original sources

Legume lectin structure.

The legume lectins are a large family of homologous carbohydrate binding proteins that are found mainly in the seeds of most legume plants. Despite their strong similarity on the level of their amino acid sequences and tertiary structures, their carbohydrate specificities and quaternary structures vary widely. In this review we will focus on the structural features of legume lectins and their complexes with carbohydrates. These will be discussed in the light of recent mutagenesis results when appropriate. Monosaccharide specificity seems to be achieved by the use of a conserved core of residues that hydrogen bond to the sugar, and a variable loop that determines the exact shape of the monosaccharide binding site. The higher affinity for particular oligosaccharides and monosaccharides containing a hydrophobic aglycon results mainly from a few distinct subsites next to the monosaccharide binding site. These subsites consist of a small number of variable residues and are found in both the mannose and galactose specificity groups. The quaternary structures of these proteins form the basis of a higher level of specificity, where the spacing between individual epitopes of multivalent carbohydrates becomes important. This results in homogeneous cross-linked lattices even in mixed precipitation systems, and is of relevance for their effects on the biological activities of cells such as mitogenic responses. Quaternary structure is also thought to play an important role in the high affinity interaction between some legume lectins and adenine and a series of adenine-derived plant hormones. The molecular basis of the variation in quaternary structure in this group of proteins is poorly understood.

Carbohydrate Sequence

The influence of exercise and dehydration on postural stability.

The aim of this study was to investigate the effects of exercise-induced and thermal dehydration on postural balance. Eight male subjects cycled for 2 h at a power output equal to 57-63% VO2max on two different occasions: once without drinking (NF) and once with intake of 1.9 l of a carbohydrate-electrolyte solution (FR). Before and after the exercise test, the velocity of the centre of pressure (COP) excursion was measured on a force platform during 30-s bipedal standing in normal position, feet side by side, and tandem position, feet heel to toe. On another occasion, eight subjects underwent seven consecutive sauna sessions (85 degrees C, 50% rh) of 15 min duration with no fluid replacement (S) to induce thermal dehydration. Mean fluid loss was 2.7 (+/- 0.4)%, 0.5 (+/- 0.5)% and 3.0 (+/- 0.6)% of body mass after NF, FR and S, respectively. Mean velocity of COP excursion after the exercise test was significantly higher in the NF than in the FR trial (p < 0.05). Postural stability was not influenced by S. In conclusion, prolonged exercise without fluid ingestion seems to negatively affect postural stability, whereas no effect is observed after exercise with fluid replacement or after thermal dehydration.

Adult

Gender differences in blood ammonia response during exercise.

In order to test for possible gender differences in blood ammonia accumulation during exercise, groups of young, physically active women (n = 8) and men (n = 8) performed an incremental load (until exhaustion) and a constant load (30 min at 75% VO2 peak) treadmill exercise test. The groups were matched for physical activity habits. VO2 peak was significantly higher in males (55.8 +/- 2.6 ml/kg/min) than in females (44.5 +/- 2.8 ml/kg/min). During the incremental exercise test to exhaustion blood ammonia concentration progressively increased with increasing workload in both groups. Blood ammonia concentration was significantly higher in males at 70, 80 and 90% of VO2 peak. Peak blood ammonia concentration was higher in males than in females (155 +/- 35 vs 136 +/- 67 mumol/l, respectively), but the difference did not reach the level of statistical significance. During the constant load exercise test blood ammonia concentration increased in both groups. At the end of the exercise test blood ammonia concentration was significantly higher in males than in females (137 +/- 42 vs 91 +/- 24 mumol/l, respectively). In conclusion, the blood ammonia concentration during submaximal exercise is dependent on gender, the males showing higher ammonia concentrations compared to females at the same relative workload (percent of VO2 peak).

Adult

Structure of chymopapain at 1.7 A resolution.

The X-ray structure of chymopapain, a cysteine proteinase isolated from the latex of the fruits of Carica papaya L., has been determined by molecular replacement methods and refined to a conventional R factor of 0.19 for all observed reflections in the range from 9.5 to 1.7 A resolution. The crystals used in this study contained a unique molecular species of chymopapain with two moles of thiomethyl attached to the two free cysteines per mole of enzyme. A comparison is made with the other known papaya proteinase X-ray structures: papain, caricain, and glycyl endopeptidase. Their backbone conformations are extremely similar except for two loop regions. Both regions are located at the surface of the protein and far away of the active site cleft. In each X-ray structure the same water network was found at the interface between the two domains of the enzyme. A close examination of the active site groove showed that the specificity restrictions dictated by the S2 subsite did not differ significantly among the four proteinases.

Amino Acid Sequence

A structure of the complex between concanavalin A and methyl-3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside reveals two binding modes.

The structure of concanavalin A in complex with the trimannoside methyl-3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside has been determined in a novel space group. In three of the four subunits of the concanavalin A tetramer, the interactions between the protein and the bound saccharide are essentially identical to those reported previously by other authors (Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976). In the fourth subunit, however, the alpha1-->3 linkage has a different conformation, resulting in a different part of the alpha1-->3-linked mannose interacting with essentially the same surface of the protein. Furthermore, significant differences are observed in the quaternary associations of the subunits compared with the saccharide-free structures and other carbohydrate complexes, suggesting that the concanavalin A tetramer is a rather flexible entity.

Concanavalin A

Sequential structural changes upon zinc and calcium binding to metal-free concanavalin A.

The lectin concanavalin A (ConA) sequentially binds a transition metal ion in the metal-binding site S1 and a calcium ion in the metal-binding site S2 to form its saccharide-binding site. Metal-free ConA crystals soaked with either Zn2+ (apoZn-ConA) or Co2+ (apoCo-ConA) display partial binding of these ions in the proto-transition metal-binding site, but no further conformational changes are observed. These structures can represent the very first step in going from metal-free ConA toward the holoprotein. In the co-crystals of metal-free ConA with Zn2+ (Zn-ConA), the zinc ion can fully occupy the S1 site. The positions of the carboxylate ligands Asp10 and Asp19 that bridge the S1 and S2 sites are affected. The ligation to Zn2+ orients Asp10 optimally for calcium ligation and stabilizes Asp19 by a hydrogen bond to one of its water ligands. The neutralizing and stabilizing effect of the binding of Zn2+ in S1 is necessary to allow for subsequent Ca2+ binding in the S2 site. However, the S2 site of monometallized ConA is still disrupted. The co-crystals of metal-free ConA with both Zn2+ and Ca2+ contain the active holoprotein (ConA ZnCa). Ca2+ has induced large conformational changes to stabilize its hepta-coordination in the S2 site, which comprise the trans to cis isomerization of the Ala207-Asp208 peptide bond accompanied by the formation of the saccharide-binding site. The Zn2+ ligation in ConA ZnCa is similar to Mn2+, Cd2+, Co2+, or Ni2+ ligation in the S1 site, in disagreement with earlier extended x-ray absorption fine structure results that suggested a lower coordination number for Zn2+.

Amino Acid Sequence

Crystallographic structure of metal-free concanavalin A at 2.5 A resolution.

The three-dimensional structure of demetallized concanavalin A has been determined at 2.5 A resolution and refined to a crystallographic R-factor of 18%. The lectin activity of concanavalin A requires the binding of both a transition metal ion, generally Mn2+, and a Ca2+ ion in two neighboring sites in close proximity to the carbohydrate binding site. Large structural differences between the native and the metal-free lectin are observed in the metal-binding region and consequently for the residues involved in the specific binding of saccharides. The demetallization invokes a series of conformational changes in the protein backbone, apparently initiated mainly by the loss of the calcium ion. Most of the Mn2+ ligands retain their position, but the Ca2+ binding site is destroyed. The Ala207-Asp208 peptide bond, in the beta-strand neighboring the metal-binding sites, undergoes a cis to trans isomerization. The cis conformation for this bond is a highly conserved feature among the leguminous lectins and is critically maintained by the Ca2+ ion in metal-bound concanavalin A. A further and major change adjacent to the isomerized bond is an expansion of the loop containing the monosaccharide ligand residues Leu99 and Tyr100. The dispersion of the ligand residues for the monosaccharide binding site (Asn14, Agr228, Asp208, Leu99, and Tyr100) in metal-free concanavalin A abolishes the lectin's ability to bind saccharides. Since the quaternary structure of legume lectins is essential to their biological role, the tetramer formation was analyzed. In the crystal (pH 5), the metal-free concanavalin A dimers associate into a tetramer that is similar to the native one, but with a drastically reduced number of inter-dimer interactions. This explains the tetramer dissociation into dimers below pH values of 6.5.

Amino Acid Sequence

Blood ammonia response to treadmill and bicycle exercise in man.

Nine male healthy and physically active volunteers performed four different exercise tests: an incremental load exercise test to exhaustion and a constant load exercise test of 15 min at 65% VO2max, both on the bicycle ergometer and on the treadmill. During the incremental exercise test, blood ammonia levels were significantly higher on the bicycle ergometer as compared to the treadmill at the same submaximal VO2 (p < 0.05 at 80% of VO2max) and at the VO2max, which was identical in the two modes of exercise. Plasma lactate levels were also significantly higher on bicycle ergometer at high submaximal exercise intensity but not at VO2max. During the constant load exercise test blood ammonia levels increased continuously and showed no differences between cycling and running, in contrast to plasma lactate accumulation, which was higher on the bicycle ergometer. The finding that blood ammonia accumulation during exercise is critically dependent upon the test procedure has to be taken into account whenever blood ammonia measurements are used in the physiological monitoring of athletes.

Adult

Effect of growth hormone administration on the fatty acid composition of adipose tissue in growth-hormone-deficient men.

In adult patients with growth hormone deficiency, the fatty acid composition of abdominal and gluteal fat tissues was determined prior to and at several time points after administration of recombinant human growth hormone. Values obtained before growth hormone treatment were not different from those seen in a normal population. However, as in healthy individuals, significant differences were found in the composition of the fat sampled at the different sites. Administration of growth hormone had no effect on the composition. It is concluded that a change in fatty acid composition of fat tissue is unlikely to be a factor contributing to the reported increased cardiovascular mortality in hypopituitarism and that treatment with recombinant human growth hormone has no effect on that potential cardiovascular risk factor.

Adipose Tissue

The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study.

The X-ray crystal structure of lentil lectin in complex with alpha-D-glucopyranose has been determined by molecular replacement and refined to an R-value of 0.20 at 3.0 A resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I from Lathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the alpha-D-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123 beta. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin. Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)-alpha-D-mannopyranoside and Me-3-O-(m-nitro-benzyl)-alpha-D-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97 beta and Asn125 beta, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100 beta and Trp128 beta.

Binding Sites

Urinary excretion of ephedrine after nasal application in healthy volunteers.

The urinary excretion of ephedrine after intranasal administration of the drug was studied in 8 healthy volunteers. Ephedrine (6 drops of a commercial 0.75% nasal ephedrine solution in each nasal cavity) was administered 4 times at intervals of 2 h (total amount applied equivalent to approximately 14 mg ephedrine), and urine was collected each hour for 10 h; the volunteers exercised on a bicycle ergometer at 50% of their VO2max for 2 h after the last ephedrine application. Ephedrine was detected in all urine samples. The urinary ephedrine concentration ranged from 0.9 to 16.5 micrograms mL-1; the number of urine samples with an ephedrine concentration exceeding 5 micrograms mL-1 ranged from 1/10 (volunteer 2) to 9/10 (volunteers 1 and 3). The mean percentage of dose recovered within 10 h was 33% (range 23-50%). There was a weak but significant negative correlation between urinary pH and amount of ephedrine in the urine; exercise did not consistently influence the urinary amount. These results illustrate the systemic availability of ephedrine upon intranasal administration and show that the therapeutic use of a nasal ephedrine formulation by an athlete on the day of a competition can lead to a urinary ephedrine concentration above 5 micrograms mL-1, which is considered positive in current doping regulations of the International Union of Cyclists.

Administration, Intranasal

Effect of specific test procedures on plasma lactate concentration and peak oxygen uptake in endurance athletes.

Plasma lactate accumulation during submaximal work and VO2peak were determined on the bicycle ergometer and on the treadmill in all round trained control subjects (n = 9) and in specifically trained athletes such as distance runners (n = 9) and racing cyclists (n = 8). Plasma lactate concentration at equivalent levels of oxygen uptake was lower during treadmill exercise than during bicycle exercise in the controls and in the runners, but the difference between the two work modalities was more pronounced in the runners. In contrast to the runners, the cyclists had a higher plasma lactate concentration on the treadmill. VO2peak values on the two ergometers were similar in the controls, while the specifically trained athletes reached the highest value when they were tested with the exercise test which is specific to the training conducted. In the runners VO2peak was 14% higher on the treadmill than on the bicycle ergometer and in the cyclists VO2peak was 11% higher on the bicycle ergometer. It is concluded that the difference in plasma lactate concentration during submaximal work and in VO2peak between treadmill and bicycle exercise can be affected by the training conditions of the subjects.

Adult

Effects of diltiazem and atenolol on exercise performance in man.

The effects of diltiazem and atenolol on exercise performance were studied in 9 healthy and physically fit volunteers according to a double-blind cross-over design. All subjects performed, with an interval of 1 week, 3 exercise tests on a treadmill with stepwise increase of the workload until exhaustion. Two hours prior to each exercise test they received in a randomised order placebo, diltiazem 120 mg or atenolol 100 mg. Running time and VO2peak were not influenced by diltiazem, while running time was significantly reduced (-10%) after atenolol. The reduction of VO2peak (-9%) after atenolol did not reach statistical significance. Both diltiazem and atenolol significantly decreased heart rate at peak effort but the decrease was much more pronounced after atenolol (-52 b.min-1) than after diltiazem (-6 b.min-1). At submaximal level VO2 was not influenced by diltiazem, but significantly lowered (-6%) after atenolol. Submaximal heart rate was decreased and plasma lactate concentration was increased by both diltiazem and atenolol, but the effect of atenolol was more pronounced. The study shows that maximal work performance of young healthy subjects is not affected by diltiazem 120 mg, in contrast to atenolol 100 mg which decreases maximal work performance in the same subjects.

Adult

Influence of exercise on plasma concentrations of isosorbide dinitrate.

As the fall in hepatic blood flow caused by exercise is expected to decrease the elimination of high extraction drugs, the influence of exercise on the fate of the anti-anginal drug isosorbide dinitrate has been assessed. Isosorbide dinitrate 10 mg was given orally, after an overnight fast, on 2 different days to 7 healthy volunteers and the plasma concentrations of the parent compound and its mononitrate metabolites were measured. Heavy bicycle exercise from the 45th to the 105th min after intake of the drug on one of the experimental days did not change the plasma isosorbide dinitrate concentrations as compared to those on the rest day. The concentrations of both mononitrates were lower on the exercise day, but the difference was already present before the exercise started.

Administration, Oral

Cardiorespiratory response to bicycle and rowing ergometer exercise in oarsmen.

Maximal aerobic power and related variables during submaximal work were determined on the bicycle and on the rowing ergometer in 9 oarsmen and in 9 control subjects. During submaximal work, heart rate and pulmonary ventilation were similar with the two exercise procedures in each group, but the oarsmen had lower values than the control subjects. Oxygen uptake at a given workload was higher on the rowing ergometer than on the bicycle ergometer for both the oarsmen and the control subjects. During maximal exercise, the control subjects reached significantly lower values on the rowing ergometer than on the bicycle ergometer for maximal oxygen uptake and maximal oxygen pulse. The oarsmen however attained practically the same values on the two ergometers, with a rank difference correlation coefficient of 0.94. The difference between the results obtained in control subjects and in oarsmen supports the concept of training specificity.

Adult

Effects of training on maximal working capacity and haemodynamic response during arm and leg-exercise in a group of paddlers.

Maximal oxygen uptake and circulatory adaptation to work with legs and arms were studied in a group of 5 paddlers members of the Belgian national squad and a control-group of 9 trained subjects. The results showed that the specific armtraining of paddlers induced changes in the arm-to-leg ration of physiological parameters at submaximal and maximal work. In the group of paddlers maximal oxygen intake and workload during arm-exercise averaged respectively 88.6% and 80.3% of the scores obtained with leg-exercise. In the control group the arm to leg ratio varied between 81.2% and 65.2%. At a submaximal load of 100 W the difference in heartfrequency was 21 beats/min in the canoe group and 35 beats/min in the control group. Oxygen consumption and ventilation during work with the arms was lower in the group of paddlers. The data of our study suggest that the specific training of paddlers do result in a effect on the haemodynamic adaptations to arm work.

Adaptation, Physiological