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G Leblanc

Publications and source records attributed to G Leblanc.

At least 19 recordsLinked to original sources
PubMed1995

Melibiose permease of Escherichia coli: substrate-induced conformational changes monitored by tryptophan fluorescence spectroscopy.

Tryptophan fluorescence spectroscopy has been used to investigate the effects of sugars and coupling cations (H+, Na+, or Li+) on the conformational properties of purified melibiose permease after reconstitution in liposomes. Melibiose permease emission fluorescence is selectively enhanced by sugars, which serve as substrates for the symport reaction, alpha-galactosides producing larger variations (13-17%) than beta-galactosides (7%). Moreover, the sugar-dependent fluorescence increase is specifically potentiated by NaCl and LiCl (5-7 times), which are well-established activators of sugar binding and transport by the permease. The potentiation effect is greater in the presence of LiCl than NaCl. On their own, sodium and lithium ions produce quenching of the fluorescence signal (2%). Evidence suggesting that sugars and cations compete for their respective binding sites is also given. Both the sugar-induced fluorescence variation and the NaCl(or LiCl)-dependent potentiation effect exhibit saturation kinetics. In each ionic condition, the half-maximal fluorescence change is found at a sugar concentration corresponding to the sugar-binding constant. Also, half-maximal potentiation of the fluorescence change by sodium or lithium occurs at a concentration comparable to the activation constant of sugar binding by each ion. The sugar- and ion-dependent fluorescence variations still take place after selective inactivation of the permease substrate translocation capacity by N-ethylmaleimide. Taken together, the data suggest that the changes in permease fluorescence reflect conformational changes occurring upon the formation of ternary sugar/cation/permease complexes.

Biological Transport↗
PubMed1995

Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide.

As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin. After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence. Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide.

Amino Acid Sequence↗
PubMed1995

[The importance and significance of post-micturitional bladder residue in the evaluation of prostatism].

The presence of a significant post-voiding residue is often considered to be secondary to bladder neck obstruction and frequently constitutes the main indication for transurethral resection of the prostate. In order to validate this approach, the files of 350 patients presenting with symptoms of prostatism and assessed by urodynamic studies were analysed retrospectively. The results of the study demonstrated that, regardless of the value of the residue, approximately 30% of patients were not obstructed despite a high post-voiding residue (PVR), while approximately 70% of patients with minimal or no PVR were considered to be obstructed on the basis of urodynamic criteria. These results therefore demonstrate that the presence of a post-voiding residue is not indicative of bladder neck, but reflects a lesion of the detrusor. Considered alone, it therefore should not constitute an indication for transurethral resection of the prostate.

Humans↗
PubMed1992

Melibiose permease of Escherichia coli: mutation of histidine-94 alters expression and stability rather than catalytic activity.

Previous studies utilizing site-directed mutagenesis [Pourcher et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 468-472] indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important. The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg. Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane. Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type. Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C. Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature. The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se. Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.

Base Sequence↗
PubMed1992

Structure of human type II 5 alpha-reductase gene.

The best known activity of steroid 5 alpha-reductase is the transformation of testosterone into dihydrotestosterone, the most potent androgen. Two types of human steroid 5 alpha-reductase cDNAs and the type I gene have previously been isolated and characterized. This report describes the isolation and characterization of the human type II 5 alpha-reductase gene, the gene most likely responsible for male pseudohermaphroditism due to 5 alpha-reductase deficiency as well as the one presumed to be involved in a major androgen-related diseases such as prostate cancer and benign prostatic hyperplasia. The type II 5 alpha-reductase gene contains five exons of 352, 164, 102, 151 and 1695 bp, respectively, which share 43.8% to 64.1% homology with exons of the corresponding type I gene. These exons are separated by four introns of greater than 29, and approximately 2.3, 2.0 and 3.0 kb. Analysis of primer extension products by polyacrylamide gel electrophoresis as well as by subcloning and sequencing reveals a start site located 71 nucleotides upstream the ATG initiating codon.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices.

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the beta 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to alpha 1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, alpha 1 integrin and beta 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.

Animals↗
PubMed1991

Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions.

An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.

Aspartic Acid↗
PubMed1991

Structure of the human type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) gene: adrenal and gonadal specificity.

While classical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase deficiency (3 beta-HSD) is a known cause of adrenal hyperplasia resulting in ambiguous genitalia and adrenal insufficiency at birth, nonclassical or late-onset 3 beta-HSD deficiency is found in an important proportion of women with androgen excess. We have previously isolated and sequenced the cDNA and gene for the human type I 3 beta-HSD, which represents the main species expressed in the placenta and skin. Recently, we isolated, sequenced, and expressed the functional cDNA encoding type II 3 beta-HSD, which is the predominant 3 beta-HSD expressed in human adrenals and gonads. The present study describes the isolation and complete sequence of the corresponding type II 3 beta-HSD gene, which is the form most likely responsible for human 3 beta-HSD deficiency. The structural gene contains four exons of 57, 231, 165, and 1,214 bp, respectively, separated by introns of 128, 3,383, and 2,162 bp. DNA sequence analysis of the 5'-flanking region reveals the existence of two putative TATA boxes situated 28 and 140 nucleotides upstream from the transcription start site whereas two putative CAAT boxes are located 57 and 38 nucleotides upstream from the TATA boxes, respectively. A restriction fragment length pattern specific for each gene has been characterized. The present findings should provide the tools required for detailed analysis of the molecular basis of 3 beta-HSD deficiency as well as of normal sex steroid biosynthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) catalyze the oxidative conversion of delta 5-3 beta-hydroxysteroids to the delta 4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3 beta-HSD cDNA as probe, a human 3 beta-HSD gene was isolated from a lambda-EMBL3 library of leucocyte genomic DNA. A fragment of 3 beta-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3 beta-HSD gene contains a 5'-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5'-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3 beta-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3 beta-hydroxysteroid dehydrogenase and delta 5-delta 4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3 beta-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3 beta-HSD gene should permit investigation of the molecular defects responsible for 3 beta-HSD deficiency, the second most common cause of adrenal hyperplasia in children.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties.

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

Amino Acid Sequence↗
PubMed1990

Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system.

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].

Animals↗
PubMed1990

Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli.

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced with Arg. Replacement of His-213, His-442, or His-456 has no significant effect on permease activity, while permease with Arg in place of His-198, His-318, or His-357 retains more than 70% of wild-type activity. In striking contrast, replacement of His-94 with Arg causes a complete loss of sugar binding and transport, although the cells contain a normal complement of permease molecules. Thus, as shown previously with lac permease, only a single His residue is important for activity, but, in the case of mel permease, the critical His residue is present in the 3rd putative transmembrane helix rather than the 10th.

Arginine↗
PubMed1990

[Who are the patients treated in an outpatient clinic with high dosage neuroleptics?].

The use of high doses of neuroleptics (NL) in treatment of chronic psychosis is a controversial subject in the literature. In this context, it is surprising to note the lack of objective data about the prevalence and the consequences concerning this mode of prescription when treating people suffering from severe mental disorders. This study describes the clientele exposed to high doses of NL from an outpatient clinic of a psychiatric hospital. The equivalent of 18 mg or more of haloperidol per day was used as the high dose criterion. Overall, we observed the use of NL in all diagnostic categories and the frequent use of polypharmacy in patients treated with NL. Among the 435 patients receiving NL, 26.4% had high dose prescriptions (two men for every woman). Most of these high dose NL subjects had a diagnosis of schizophrenic disorder (87.9%). Fifty-one of them had been receiving high doses for six months or more and 39 of them agreed to meet our research team. The mean age of these subjects was 37.2 years and the mean dose was 63.5 mg haloperidol equivalent/day. Thirty-five subjects were diagnosed as chronic schizophrenic disorder and four as schizo-affective disorder. Nineteen patients had tardive dyskinesia. In two out of three cases the high dose prescription began during hospitalization and the main reason was presence of severe psychotic symptoms. Significant positive correlations were found between parkinsonian symptoms and negative symptoms of schizophrenia as well as psychosocial dysfunctions on 39 subjects. These findings support the hypothesis that the use of high doses of NL contribute to the negative symptoms and the psychosocial dysfunctions. The implications of these findings relating to assessment and treatment of schizophrenic outpatients are discussed.

Adult↗
PubMed1990

Structure of two in tandem human 17 beta-hydroxysteroid dehydrogenase genes.

Two human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes (h17 beta-HSDI and h17 beta-HSDII) included in tandem within an approximately 13 kilobase pair fragment were isolated from a genomic lambda EMBL3 DNA library using cDNA encoding human 17 beta-HSD (hpE2DH216) as probe. We have determined the complete exon and intron sequences of the two genes as well as their 5' and 3'-flanking regions. Human 17 beta-HSDII contains six exons and five short introns for a total length of 3250 base pairs. The exon sequence of h17 beta-HSDII is identical to the previously reported hpE2DH216 cDNA while the overlapping nucleotide sequences of the corresponding exons and introns of h17 beta-HSDI and h17 beta-HSDII show 89% homology. In addition, we have used the hpE2DH216 cDNA to demonstrate the widespread expression of 17 beta-HSD mRNAs in steroidogenic and peripheral target tissues. These new findings provide the basis for a better understanding of the molecular mechanisms involved in 17 beta-HSD deficiency and peripheral sex steroid metabolism.

17-Hydroxysteroid Dehydrogenases↗
PubMed1989

Molecular biology and bacterial secondary transporters.

Bacterial permeases form a family of membrane proteins that actively transport nutrients according to a cation-solute cotransport mechanism. The purpose of this review is to consider the many perspectives offered by in vitro recombinant DNA and molecular biology techniques for analysis of structure-function relationships. In the first part of this review, the kinetic parameters that permit characterization of either the partial steps or the overall transport reaction are summarized. We then list the molecular properties (protein sequence and composition, secondary structure, biochemical identification) of the carriers readily available on cloning a structural gene into plasmids. Finally, the usefulness of site-specific, oligonucleotide-directed mutagenesis for investigation of the functional importance of given residues (cysteins and histidines) or domains of the transport proteins is illustrated.

Amino Acid Sequence↗
PubMed1989

Role of transmembrane electrical potential on cadmium fixation by a marine pseudomonad.

The role of cellular energy, and mainly that of electrical transmembrane potential, in cadmium fixation by a marine pseudomonad suspended in a mineral medium was investigated by studying the effects of ionophores. Although fixation of cadmium by cells was generally less when respiratory activity was inhibited, it was not affected by a reduction of the transmembrane electrical potential delta psi in mureinoplasts. These observations strongly suggest that cadmium fixation in this isolate was not the result of a delta psi-dependent active transport.

Antineoplastic Agents↗