Biomedical subjects

X De Bolle

Publications and source records attributed to X De Bolle.

8 recordsLinked to original sources

PubMed1998

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4.

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

Animals↗

PubMed1998

Comparative analysis of seven multiple protein sequence alignment servers: clues to enhance reliability of predictions.

MOTIVATION: The prediction reliability of seven multiple alignment servers currently available on the Internet (ClustalW, MAP, PIMA, Block Maker, MSA, MEME and Match-Box) has been evaluated in terms of power (sensitivity) and confidence (selectivity). Therefore, the alignments obtained have been respectively compared to refined structural alignments for 20 families of related proteins with low levels of identity. RESULTS: Results clearly show that any powerful method remains reliable when the rate of identity falls. For some methods, power and confidence decrease linearly with the rate of identity, while other methods emphasize reliability at the cost of a lower power. Increasing the number of related sequences included in the alignment may either improve or decrease the quality of the predictions substantially. For some methods, the gain in power or in confidence is quite systematic; for others, the effect of the addition of homologous sequences is highly unpredictable. Extracting the consensus between two different methods may increase the overall confidence of the predictions tremendously. Our conclusions induce users of sequence alignment methods on the Internet to select the most suitable technique according to their requirements in terms of selectivity and sensitivity. AVAILABILITY: The aligned sequences of the 20 alignments of structure can be obtained automatically by sending the message 'send: cabios_tests.txt' by e-mail to 'matchbox@biq.fundp.ac.be'. CONTACT: eric.depiereux@fundp.ac.be

Computer Communication Networks↗

PubMed1997

Bivalent cations stabilize yeast alcohol dehydrogenase I.

The thermostability of yeast alcohol dehydrogenase (ADH) I is strongly dependent on the presence of NaCl, a salt that is almost neutral on the Hofmeister scale, which suggests that solvent-accessible electrostatic repulsion might play a role in the inactivation of the enzyme. Moreover, CaCl2 and MgCl2 are able to stabilize the enzyme at millimolar concentrations. Ca2+ stabilizes yeast ADH I by preventing the dissociation of the reduced form of the enzyme and by preventing the unfolding of the oxidized form of the enzyme. An analysis of several chimaeric ADHs suggests that Ca2+ is fixed by the Asp-236 and Glu-101 side chains in yeast ADH I, but that Ca2+ can be displaced by replacing Met-168 by an Arg residue, as suggested by a three-dimensional model of the enzyme structure. These results indicate that electrostatic repulsion can cause protein unfolding and/or dissociation. It is proposed that yeast ADH I binds Mg2+ in vivo.

Alcohol Dehydrogenase↗

PubMed1997

Match-Box_server: a multiple sequence alignment tool placing emphasis on reliability.

MOTIVATION: The Match-Box software comprises protein sequence alignment tools based on strict statistical thresholds of similarity between protein segments. The method circumvents the gap penalty requirement: gaps being the result of the alignment and not a governing parameter of the procedure. The reliable conserved regions outlined by Match-Box are particularly relevant for homology modelling of protein structures, prediction of essential residues for site-directed mutagenesis and oligonucleotide design for cloning homologous genes by polymerase chain reaction (PCR). RESULTS: The method produces reliable results, as assessed by tests performed on protein families of known structures and of low sequence similarity. A reliability score is computed in relation to a threshold of similarity progressively raised to extend the aligned regions to their maximal length, up to the significance limit of matching segments. The score obtained at each position is printed below the sequences and allows a discriminant reading of each aligned region. AVAILABILITY: Sequences may be submitted to a Web server at http://www.fundp.ac.be/sciences/biologie/bms/+ ++matchbox_submit.html or sent by e-mail to matchbox/biq.fundp.ac.be (help available by just mailing help).

Algorithms↗

PubMed1995

Identification of residues potentially involved in the interactions between subunits in yeast alcohol dehydrogenases.

The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH II isozymes as well as of several chimeric (ADH I-ADH II) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and II. The first group contains a Cys277-->Ser substitution, located at the interface between subunits in a three-dimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-->Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Met168-->Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.

Alcohol Dehydrogenase↗

PubMed1995

Knowledge-based modeling of the D-lactate dehydrogenase three-dimensional structure.

A three-dimensional structure of the NAD-dependent D-lactate dehydrogenase of Lactobacillus bulgaricus is modeled using the structure of the formate dehydrogenase of Pseudomonas sp. as template. Both sequences share only 22% of identical residues. Regions for knowledge-based modeling are defined from the structurally conserved regions predicted by multiple alignment of a set of related protein sequences with low homology. The model of the D-LDH subunit shows, as for the formate dehydrogenase, an alpha/beta structure, with a catalytic domain and a coenzyme binding domain. It points out the catalytic histidine (His-296) and supports the hypothetical catalytic mechanism. It also suggests that the other residues involved in the active site are Arg-235, possibly involved in the binding of the carboxyl group of the pyruvate, and Phe-299, a candidate for stabilizing the methyl group of the substrate.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.

Amino Acid Sequence↗

PubMed1993

Similarities between alanine dehydrogenase and the N-terminal part of pyridine nucleotide transhydrogenase and their possible implication in the virulence mechanism of Mycobacterium tuberculosis.

Recent developments in simultaneous multiple alignment methods of protein sequences allow prediction of structural similarity in related proteins. Alanine dehydrogenase and the N-terminal sequence of pyridine nucleotide transhydrogenase were compared for their sequences. High similarities of sequences were observed especially in their NAD(H)-binding sites. These similarities suggest that antibodies which recognized the alanine dehydrogenase of Mycobacterium tuberculosis can also be directed against the membrane bound pyridine nucleotide transhydrogenase. If this is the case, the virulent property of this pathogen could be linked to its higher synthesis of NADPH necessary for its anabolism.

Alanine Dehydrogenase↗