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Biomedical subjects

J Wouters

Publications and source records attributed to J Wouters.

At least 19 recordsLinked to original sources
PubMed2001

Structure determination and comparison of BM567, a sulfonylurea, with terbogrel, two compounds with dual action, thromboxane receptor antagonism and thromboxane synthase inhibition.

BM567, a sulfonylurea compound whose crystal structure is here discussed and terbogrel, are both thromboxane receptor antagonists and thromboxane synthase inhibitors. In this paper, their crystallographic and electronic structures are compared and lead to new synthesis prospects among the sulfonylurea series.

Crystallography↗
PubMed2001

Expression, purification, crystallization and preliminary X-ray analysis of the native class C beta-lactamase from Enterobacter cloacae 908R and two mutants.

Crystals have been obtained of the Enterobacter cloacae 908R beta-lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (M(r) = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P2(1)2(1)2, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31 A. In the best cases, the crystals diffract to about 2.1 A resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C beta-lactamases.

Crystallization↗
PubMed2001

Coding of the fundamental frequency in continuous interleaved sampling processors for cochlear implants.

In this study the perception of the fundamental frequency (F0) of periodic stimuli by cochlear implant users is investigated. A widely used speech processor is the Continuous Interleaved Sampling (CIS) processor, for which the fundamental frequency appears as temporal fluctuations in the envelopes at the output. Three experiments with four users of the LAURA (Registered trade mark of Philips Hearing Implants, now Cochlear Technology Centre Europe) cochlear implant were carried out to examine the influence of the modulation depth of these envelope fluctuations on pitch discrimination. In the first experiment, the subjects were asked to discriminate between two SAM (sinusoidally amplitude modulated) pulse trains on a single electrode channel differing in modulation frequency ( deltaf = 20%). As expected, the results showed a decrease in the performance for smaller modulation depths. Optimal performance was reached for modulation depths between 20% and 99%, depending on subject, electrode channel, and modulation frequency. In the second experiment, the smallest noticeable difference in F0 of synthetic vowels was measured for three algorithms that differed in the obtained modulation depth at the output: the default CIS strategy, the CIS strategy in which the F0 fluctuations in the envelope were removed (FLAT CIS), and a third CIS strategy, which was especially designed to control and increase the depth of these fluctuations (F0 CIS). In general, performance was poorest for the FLAT CIS strategy, where changes in F0 are only apparent as changes of the average amplitude in the channel outputs. This emphasizes the importance of temporal coding of F0 in the speech envelope for pitch perception. No significantly better results were obtained for the F0 CIS strategy compared to the default CIS strategy, although the latter results in envelope modulation depths at which sub-optimal scores were obtained in some cases of the first experiment. This indicates that less modulation is needed if all channels are stimulated with synchronous F0 fluctuations. This hypothesis is confirmed in a third experiment where subjects performed significantly better in a pitch discrimination task with SAM pulse trains, if three channels were stimulated concurrently, as opposed to only one.

Acoustic Stimulation↗
PubMed2000

Purification, cloning, and three-dimensional structure prediction of Micrococcus luteus FAD-containing tyramine oxidase.

The FAD-containing tyramine oxidase enzyme and gene from the Gram (+) bacterium Micrococcus luteus were isolated, and computer prediction was used to propose a preliminary 3D model of the protein. A 2.8-kb Sau3AI fragment containing the structural gene of tyramine oxidase was cloned from a M. luteus genomic DNA library. The 1332 bp gene encodes a protein of 443 amino acids, with a calculated molecular mass of 49.1 kDa. The enzyme was found to be a homodimer with a molecular weight of 49,000. It oxidizes tyramine, adrenaline, 3-hydroxytyramine, dopamine, and noradrenaline, and was reversibly inhibited by FAD-containing monoamine oxidase A and B specific inhibitors. Sequence comparison show that tyramine oxidase is smaller than other FAD-amine oxidases but that it contains well-conserved amino acid residues reported in all other FAD-amine oxidases. A hypothetical three-dimensional structure of tyramine oxidase has also been proposed based on secondary structure predictions, threading, and comparative modeling.

Amino Acid Sequence↗
PubMed2000

Isosterism among analogues of torasemide: conformational, electronic and lipophilic properties.

The structures, electronic (charges, molecular electrostatic potential, molecular orbitals) and lipophilic properties of three isostere analogues of torasemide were determined and the influence of the replacement of the sulfonyl urea group on the conformation and electronic properties of the molecules is discussed. Lipophilicity of the compounds seems to be the most discriminating property along the series and affects their pharmacological activities.

Animals↗
PubMed2000

Detection of small across-channel timing differences by cochlear implantees.

Five post-lingually deafened users of the LAURA cochlear implant were presented with two trains of biphasic pulses applied concurrently to two widely separated channels. They could all discriminate between stimuli where pulses on the two channels were nearly synchronous (inter-channel delay=0.1 ms) and those where there was a longer delay applied to one channel. All showed an asymmetry, being more sensitive when the longer delay was on either the more basal or, depending on the listener, the more apical channel. For four out of the five listeners this asymmetry could be at least partly attributed to one stimulus, with a 0.1-ms delay in either the apical (three listeners) or basal (one listener) channel, sounding markedly different from all other stimuli used in the experiment. Both the overall sensitivity of listeners and the general pattern of results survived the presentation of maskers on intermediate channels, and did not vary markedly with changes in the polarity of the pulses applied to one channel. Although the results varied substantially across listeners, it is concluded that they demonstrate a genuine sensitivity to the relative timing of stimulation applied to discrete populations of auditory nerve fibers.

Acoustic Stimulation↗
PubMed2000

Coumarinic derivatives as mechanism-based inhibitors of alpha-chymotrypsin and human leukocyte elastase.

Novel coumarinic derivatives were synthesized and tested for their inhibitory potency toward alpha-CT and HLE. Cycloalkyl esters and amides were found to be essentially inactive on both enzymes. On the opposite, aromatic esters strongly inactivated alpha-CT whereas HLE was less efficiently inhibited with dichlorophenyl ester derivatives (kinact/K(I) = 4000 M(-1) s(-1) for 36). Representative examples of amide, ester, thioester and ketone derivatives were prepared in order to evaluate the influence of the link between the coumarinic ring and the phenyl side chain. The irreversible inactivation of alpha-CT by 6-chloromethyl derivatives should be due to alkylation of a histidine residue as suggested by the amino acid analysis of the modified chymotrypsin. Conversely the inhibition of HLE was transient. Intrinsic reactivity of coumarins has been calculated using a model of a nucleophilic reaction between the ligand and the couple methanol-water. From this calculation, it appears that differences in the inhibitory potency expressed by these molecules cannot only be explained by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack.

Chymotrypsin↗
PubMed2000

Topology prediction of Brucella abortus Omp2b and Omp2a porins after critical assessment of transmembrane beta strands prediction by several secondary structure prediction methods.

In order to propose a reliable model for Brucella porin topology, several structure prediction methods were evaluated in their ability to predict porin topology. Four porins of known structure were selected as test-cases and their secondary structure delineated. The specificity and sensitivity of 11 methods were separately evaluated. Our critical assessment shows that some secondary structure prediction methods (PHD, Dsc, Sopma) originally designed to predict globular protein structure are useful on porin topology prediction. The overall best prediction is obtained by combining these three "generalist" methods with a transmembrane beta strand prediction technique. This "consensus" method was applied to Brucella porins Omp2b and Omp2a, sharing no sequence homology with any other porin. The predicted topology is a 16-stranded antiparallel beta barrel with Omp2a showing a higher number of negatively charged residue in the exposed loops than Omp2b. Experiments are in progress to validate the proposed topology and the functional hypotheses. The ability of the proposed consensus method to predict topology of complex outer membrane protein is briefly discussed.

Amino Acid Sequence↗
PubMed2000

Terbogrel, a dual-acting agent for thromboxane receptor antagonism and thromboxane synthase inhibition.

Terbogrel, (E)-6-[4-(3-tert-butyl-2-cyanoguanidino)phenyl]-6-(3-pyridyl)hex-5 -enoic acid, C(23)H(27)N(5)O(2), a mixed thromboxane A(2) receptor antagonist and thromboxane A(2) synthase inhibitor, shows a hairpin-like conformation stabilized by an intramolecular hydrogen bond. A structural feature characteristic of the thromboxane A(2) synthase inhibitor mode is observed: a distance of 8.4257 (19) A between the pyridine N atom and the carboxyl group.

Crystallography, X-Ray↗
PubMed2000

Identification of a potential metal cation-pi binding site in the structure of a thermophilic Bacillus stearothermophilus triosephosphate isomerase mutant.

A potential metal cation-pi interaction between a sodium cation (Na(+)) and the indole ring of a tryptophan residue was detected in the crystallographic structure (2 A) of the thermophilic Bacillus stearothermophilus triosephosphate isomerase H12N/K13G mutant (bTIMmut). The cation-pi binding site is located near the surface of the protein, the alkali metal ion facing the benzo ring of Trp9. The presence of Phe21 and Glu17 close to Trp9 could indicate an additional role for those residues in the stability of the sodium-indole interaction. The sodium cation lies in a position that is occupied by CE and NZ of Lys13 in the wild-type structure.

Bacterial Proteins↗
PubMed2000

A concept for a research tool for experiments with cochlear implant users.

APEX, an acronym for computer Application for Psycho-Electrical eXperiments, is a user friendly tool used to conduct psychophysical experiments and to investigate new speech coding algorithms with cochlear implant users. Most common psychophysical experiments can be easily programmed and all stimuli can be easily created without any knowledge of computer programing. The pulsatile stimuli are composed off-line using custom-made MATLAB (Registered trademark of The Mathworks, Inc., http://www.mathworks.com) functions and are stored on hard disk or CD ROM. These functions convert either a speech signal into a pulse sequence or generate any sequence of pulses based on the parameters specified by the experimenter. The APEX personal computer (PC) software reads a text file which specifies the experiment and the stimuli, controls the experiment, delivers the stimuli to the subject through a digital signal processor (DSP) board, collects the responses via a computer mouse or a graphics tablet, and writes the results to the same file. At present, the APEX system is implemented for the LAURA (Registered trademark of Philips Hearing Implants) cochlear implant. However, the concept-and many parts of the system-is portable to any other device. Also, psycho-acoustical experiments can be conducted by presenting the stimuli acoustically through a sound card.

Cochlear Implants↗
PubMed2000

Molecular interaction between reversible MAO-A inhibitors and the enzyme. Application to aryloxazolidinone, a prototype series.

Among the various chemical classes of monoamine oxidase A inhibitors, phenyloxazolidinone represent one of the major series. The purpose of this paper is to review the experimental (X-ray diffraction, NMR, electronic absorption spectroscopy, lipophilicity studies) and theoretical (quantum chemistry, molecular mechanics, molecular dynamics) studies which have led to the description of the mode of interaction between phenyloxazolidinone inhibitors and the MAO-A enzyme.

Animals↗
PubMed2000

Structural approach of human MAO-A using fold recognition (threading) techniques.

The major goal of the present work is to further approach the structure of human monoamine oxidase A (MAO-A). A first partial three-dimensional model of human MAO-A has already been established using secondary structure predictions and fold recognition methods [Wouters and Baudoux, 1998]. In this modeled structure, a segment of the sequence (residues 369-393) located near the covalent linkage to the essential flavin cofactor, and potentially involved in the structure of the active site of the protein, could not be modeled. We here propose a possible fold for that segment, based on threading techniques. The identification of regions of the protein potentially involved in its dimerization was also undertaken by studying hydrophobic areas present at the surface of the structure.

Humans↗
PubMed1999

Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus beta-lactamase II: A perturbed angular correlation of gamma-rays spectroscopic study.

Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectroscopy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn(2)E, ZnE, Cd(2)E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.

Alanine↗
PubMed1999

Antagonism of the TXA2 receptor by seratrodast: a structural approach.

The crystal structure of seratrodast (AA-2414), a potent thromboxane A2 (TXA2) receptor antagonist, served as starting point to docking studies with the modeled human TXA2 receptor. This structural approach provides rational basis for the design of new antagonists within the aryl sulfonamide family.

Animals↗
PubMed1999

Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures.

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.

Adaptation, Physiological↗
PubMed1999

5-Substituted pyrimidine 1,5-anhydrohexitols: conformational analysis and interaction with viral thymidine kinase.

Conformational analysis of anhydrohexitol nucleosides using a combination of experimental (X-ray crystallography) and computational methods indicates that those antiviral compounds occur in an equilibrium between two forms, one conformation being adopted in solid phase and in solution, the other found when the nucleosides are in complex with HSV-1 thymidine kinase. The conformational change induced by the enzyme has been investigated.

Crystallography↗