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J Wouters

Publications and source records attributed to J Wouters.

At least 19 recordsLinked to original sources
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

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

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↗
PubMed1999

A reversible monoamine oxidase A inhibitor, befloxatone: structural approach of its mechanism of action.

Experimental and theoretical physico-chemical methods were used to investigate the interaction between several reversible monoamine oxidase A inhibitors in the oxazolidinone series and the active site of the enzyme. Phenyloxazolidinones include toloxatone and analogues, among which befloxatone was selected as drug candidate for the treatment of depression. Identification of the forces responsible for the crystal cohesion of befloxatone reveals functional groups that could interact with monoamine oxidase. Calculation of electronic properties of those compounds using ab initio molecular orbital methods lead to a description of the mode of interaction between befloxatone and the cofactor of the enzyme. Electronic absorption spectroscopy measurements confirm the hypothesis of a privileged interaction of phenyloxazolidinone-type inhibitors with the flavin cofactor of MAO. Additional sites of interaction with the protein core of MAO A are also examined with regard to the primary structure of the enzyme. As a result of this work, a model is proposed for the reversible inhibition of MAO A by befloxatone via long distance, reversible interactions with the flavin adenine dinucleotide (FAD) cofactor of the enzyme and with specific amino acids of the active site. This model is partially corroborated by experimental evidence and should be helpful in designing new potent inhibitors of monoamine oxidase.

Animals↗
PubMed1999

Structure and function prediction of the Brucella abortus P39 protein by comparative modeling with marginal sequence similarities.

A methodology is proposed to solve a difficult modeling problem related to the recently sequenced P39 protein. This sequence shares no similarity with any known 3D structure, but a fold is proposed by several threading tools. The difficulty in aligning the target sequence on one of the proposed template structures is overcome by combining the results of several available prediction methods and by refining a rational consensus between them. In silico validation of the obtained model and a preliminary cross-check with experimental features allow us to state that this borderline prediction is at least reasonable. This model raises relevant hypotheses on the main structural features of the protein and allows the design of site-directed mutations. Knowing the genetic context of the P39 reading frame, we are now able to suggest a function for the P39 protein: it would act as a periplasmic substrate-binding protein.

Amino Acid Sequence↗
PubMed1999

Natural vowel and consonant recognition by Laura cochlear implantees.

OBJECTIVE: This study was performed to examine whether spectral and temporal properties of speech sounds are transferred effectively by the Laura cochlear implant device, and to determine whether listeners with different levels of performance use the same type of information to understand speech. DESIGN: Twenty-five adult Laura cochlear implantees identified 12 repetitions of 10 vowels in /hVt/ utterances and 16 consonants placed in intervocalic /a/ context. The stimulus-response confusion matrices were analyzed in terms of relative information transmission scores (Miller & Nicely, 1955), and they were subjected to multidimensional INDSCAL analyses to examine whether a similar pattern of results would emerge when no prior assumptions were made about the division of categories. RESULTS: To examine perception for different levels of performance, the vowel and consonant confusion data were divided into three groups of subjects: the better, intermediate, and poorer performers. In general, the INDSCAL analyses confirmed the results obtained from the information transmission analyses. However, they also supplemented it with other perceptually relevant cues for cochlear implantees, as well as with the weightings over different perceptual dimensions for different types of performers. These analyses suggest that although all subjects use the same type of information, the better performers are more capable of using these different cues than poor performers.

Adolescent↗
PubMed1999

Enhancing the speech envelope of continuous interleaved sampling processors for cochlear implants.

A new method to code the speech envelope in continuous interleaved sampling (CIS) processors for cochlear implants is proposed. In this enhanced envelope, the rapid adaptation seen in the response of auditory nerves to sound stimuli is incorporated. Two strategies, one using the standard envelope (CIS) and one using the enhanced envelope (EECIS), were tested perceptually with six postlingually deafened users of the LAURA cochlear implant. The tests included identification of stop consonants in three different vowel contexts and monosyllabic consonant-vowel-consonant (CVC) words. Significant improvements in correct identification scores were observed for stop consonants in intervocalic /a/ context (p = 0.026): average results varied from 46% correct for CIS to 55% for EECIS. This improvement was mainly due to the better transmission of place of articulation. The differences in identification scores for stop consonants in /i/ and /u/ context were not significant. The identification scores for the medial vowels of the CVC words were significantly higher when the EECIS strategy was used: average results increased from 39% correct to 46% correct (p = 0.018). No significant differences were observed between the results for initial and final consonants of the CVC words. The present results demonstrate that the inclusion of the rapid adaptation in the speech processing for cochlear implants can improve speech intelligibility.

Adolescent↗
PubMed1999

Gap detection in single- and multiple-channel stimuli by LAURA cochlear implantees.

Gap-detection thresholds were determined for different complex patterns of electrical stimulation in four postlingually deafened LAURA cochlear implantees, to examine the nature of within- and across-channel auditory processes in more detail. Gap detectability was examined as a function of stimulus complexity (one, two, or three channels), channel distance within and across multichannel pre- and post-gap markers, stimulus asymmetry, and pulse rate. All markers roved in duration from 200 to 500 ms to ensure that subjects were not using overall stimulus duration as a cue. Gap-detection thresholds for all subjects were short (< 5 ms) when the pre- and post-gap markers stimulated the same single or multiple channels, even when the distance between simultaneously stimulated channels was large (exp. 1). For some subjects, gap detectability was more difficult in the across-channel condition, when the pre- and post-gap markers each stimulated different channels, although performance improved substantially in most subjects after extensive training (exp. 2). Additional tests with random maskers also suggest that neural interaction only affects performance mildly, and that the magnitude of the gap-detection threshold probably depends more on the subject's cognitive (in)ability to attend to the temporal gap than on the temporal acuity of their auditory system. Other stimulus conditions showed a difference in performance related to the order of the markers: gap thresholds were longer when the pre-gap marker stimulated one channel and the post-gap marker stimulated two or more channels, than vice versa (exp. 3). In addition, gap thresholds of three of the subjects increased with decreasing pulse rate from 1250 to 400 pps, a finding which may be related to the rate of the speech processing strategies used by each individual implantee (exp. 4).

Acoustic Stimulation↗