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G Leblanc

Publications and source records attributed to G Leblanc.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1989

Characterization of cDNAs for human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 5'-termini in human placenta.

Human placenta estradiol 17 beta-dehydrogenase (E2DH) cDNA clones were isolated from a lambda gt11 expression library by screening with 33 mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of E2DH and with polyclonal antibodies raised against the enzyme purified from human placenta. Using 32P-labeled fragments from the coding and 5'-untranslated regions, two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kilobases (kb) while a minor one is found at 2.2 kb. Primer extension analysis identifies the major mRNA as starting 9-10 nucleotides upstream from the in-frame ATG initiating codon while the longer mRNA has at least 814 noncoding nucleotides at its 5'-terminus. Sequence analysis of the longest cDNA clone (2092 base pairs) shows that this clone possesses identical coding and noncoding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5'-noncoding fragment hybridized only to the 2.2 kb band, thus providing additional evidence for the existence of two distinct mRNA species which differ only in their 5'-noncoding regions. Using hpE2DH36 cDNA as a probe for in situ hybridization, the human E2DH gene was localized to the q11-q12 region of chromosome 17. The cloned cDNAs encode E2DH, a 327-amino acid protein having a calculated molecular weight of 34,853. Since E2DH is the enzyme required for the formation of 17 beta-estradiol, the availability of the cDNA encoding the enzyme should permit a detailed investigation of the factors regulating the expression and activity of this crucial enzyme, in both normal and malignant tissues, especially breast cancer.

17-Hydroxysteroid Dehydrogenases↗
PubMed1989

Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase.

Polyclonal antibodies raised against 3 beta-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a lambda gt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3 beta-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Melibiose permease of Escherichia coli. Characteristics of co-substrates release during facilitated diffusion reactions.

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was investigated by further analyzing the Na+ (H+ or Li+)-coupled facilitated diffusion reactions catalyzed by the carrier in de-energized membrane vesicles, with particular emphasis on the reaction of sugar exchange at equilibrium. It is first shown that melibiose exchange at equilibrium proceeds without concomitant movement of Na+, i.e. the coupled cation is kinetically occluded during the melibiose exchange reaction. These results provide further experimental support for the model of Na+ sugar co-transport of the physiological substrate melibiose previously suggested (Bassilana, M., Pourcher, T., and Leblanc, G. (1987) J. Biol. Chem. 262, 16865-16870) in which: 1) the mechanisms of co-substrate binding to (or release from) the carrier are ordered processes on both the outer (Na+ first, sugar last) and inner membrane surfaces (sugar first, Na+ last) and give rise to a mirror-type model; 2) release of Na+ from the carrier on the inner membrane surface is very slow and rate-limiting for carrier cycling but is fast on the opposite side, contributing to the asymmetrical functioning of the permease. On the other hand, analysis of the exchange of identical sugars (homologous exchange) and different sugar analogs (heterologous exchange) indicates that the overall rate of sugar exchange reaction coupled to Na+ or Li+ is limited by the rate of one (or more) partial step(s) associated with the inflow of co-substrates and most probably by the rate of sugar release into the intravesicular medium. It is proposed that the variability of the facilitated diffusion reactions catalyzed by the carrier in the presence of different coupled cations and/or sugar analogs reflects variations in the rate of co-substrate release from the carrier on the inner membrane surface.

Cations, Monovalent↗
PubMed1987

Facilitated diffusion properties of melibiose permease in Escherichia coli membrane vesicles. Release of co-substrates is rate limiting for permease cycling.

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+). Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+. The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes. This suggests that the permease functions asymmetrically. Cross-comparison between the rates of net [3H]melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease. The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling. This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation. Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.

Diffusion↗
PubMed1986

Sugar binding properties of the melibiose permease in Escherichia coli membrane vesicles. Effects of Na+ and H+ concentrations.

The substrate binding reaction of the melibiose carrier was analyzed by studying [3H]p-nitrophenyl-alpha-D-galactopyranoside (Np alpha Gal) binding to de-energized membrane vesicles from Escherichia coli RA11 as a function of H+ and Na+ (or Li+) concentrations. The data indicate first that Na+ (or Li+) activates Np alpha Gal binding at all pH values tested between 5.5 and 7.5 and second that H+ inhibits the Na+ (or Li+)-dependent activating effect on Np alpha Gal binding. Similar conclusions were drawn for melibiose and methyl-1-thio-beta-D-galactoside binding activities. Unexpectedly, Np alpha Gal, melibiose, and methyl-1-thio-beta-D-galactoside binding activities are insensitive to a variety of SH reagents which completely block transport activity. Quantitative analysis of the effects of H+ and Na+ ions on the parameters of Np alpha Gal binding show that 1) the maximal number of binding sites is constant irrespective of the concentration of Na+ or Li+ in the range of pH between 6 and 7.5 and 2) the apparent dissociation constant for Np alpha Gal binding varies with both Na+ and H+ according to a relation described by a linear combination of the concentration of H+ and the reciprocal of Na+ concentration. These results can be accounted for by a model which assumes sequential binding of the cation and substrate in this order and competition between Na+ and H+ for a common cationic binding site on the porter. Predictions of the proposed binding model for a carrier mechanism catalyzing sugar transport according to a Na+ symport mode or a H+ symport mode are discussed.

Carbohydrate Metabolism↗
PubMed1986

Development of choline acetyltransferase (CAT) in the sympathetic innervation of rat sweat glands.

It has been postulated that the developing sympathetic innervation of rat eccrine sweat glands changes from adrenergic to cholinergic under the influence of its target. In agreement with previous evidence that the sympathetic innervation of adult rat sweat glands is cholinergic, we found that choline acetyltransferase (CAT)-immunoreactive nerve fibers are present in adult glands, and that gland-rich chunks of adult footpads contain CAT enzyme activity. We were therefore interested in determining when CAT activity is first expressed in the developing gland innervation. Low levels of acetylating activity were observed in rat footpads as early as postnatal day 4, when sympathetic fibers first contact the glands. A greater than fourfold increase in CAT specific activity occurred between postnatal days 11 and 21. Neonatal treatment of rats with the adrenergic neurotoxin 6-hydroxydopamine (6-OHDA) eliminated most of the CAT activity in 14 and 19 d footpads. In contrast, the acetylating activity observed prior to day 11 was unaffected by neonatal 6-OHDA treatment, and only slightly reduced by the selective CAT inhibitor, naphthylvinylpyridine. These results indicate that the sympathetic fibers that innervate rat sweat glands do not acquire detectable levels of CAT activity until a full week after they contact the glands.

Acetylcholine↗
PubMed1986

[Early surgery of intracranial aneurysms].

In spite of scientific progress during the last two decades, microsurgery, neuroanesthesiology, and a better knowledge of the pathophysiology of cerebral vasospasm, the outcome of subarachnoid hemorrhage (S.A.H.) patients remains very poor. Each year, 28,000 North Americans are afflicted. Eighteen thousand of these patients will either die or become severely debilitated, a mortality morbidity of 64%. Only one patient out of five may return to the premorbid state. International cooperative studies report that the highest rate of rebleeding occurs during the first 24 hours post S.A.H. There is no rebound phenomenon during the 7th-8th day post S.A.H. Cerebral vasospasm begins during the 2d-4th day post S.A.H. and reaches its peak around the 8th day post bleed. Antifibrinolytics like AMICAR (aminocaproic acid) do not reduce significantly the rebleeding rate. In most of the cases the therapeutic level of these drugs is reached only on the third or fourth day of treatment. Hence antifibrinolytics are inactive during the first crucial seventy two hours. Antifibrinolytics increase the incidence of vasospasm, hydrocephalus, and thromboembolic phenomenon. At Infant Jesus Hospital, Quebec City, S.A.H. patients are operated on early since more than two years. Patients included in this study were admitted between January 1983 and December 1984. One hundred and thirty six patients were operated upon, 22 patients operated on acutely, less than 72 hours post S.A.H. Evaluation of these patients included the Glasgow Coma Scale (G.C.S.), the grade according to Botterell classification, a CT Scan, and angiography. A preoperative evaluation included Botterell classification and G.C.S., a post operative evaluation was performed during the first and seventh post operative days.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1985

Effect of membrane potential on the kinetic parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles.

Comparison of the transport properties of the melibiose permease of E. coli acting as a H+-symport or a Na+-symport has been performed by measuring initial rates of [3H]-melibiose transport or its accumulation in isolated membrane vesicles. The results show strikingly that although the membrane potential primarily drives melibiose accumulation by both types of symport, it selectively affects the apparent affinity constant Kt of the H+-melibiose symport while it specifically changes the maximal rate of transport (Vmax) of the Na+-melibiose symport. It is suggested that modification(s) of some partial reaction constants of a given transport cycle might lead to important changes in the kinetic properties of this transport system.

Biological Transport, Active↗
PubMed1985

Chemical modifications of the Na+-H+ antiport in Escherichia coli membrane vesicles.

The effects of chemical modifications of the Na+-H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy-dependent, downhill Na+ efflux from membrane vesicles. The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by delta microH+ or its components. Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine. The data suggest that histidine residues are involved in the molecular mechanism of the Na+-H+ antiport. In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+-H+ exchange reaction has been obtained.

Carboxylic Acids↗
PubMed1985

Regulation of human peripheral blood BFU-E growth in vitro by leukaemic B-lymphocytes.

We report a stimulating effect of leukaemic B-lymphocytes from anaemic and non-anaemic patients with CLL on the proliferation of normal peripheral blood BFU-E. Coculture of leukaemic B-cells at various concentrations (2.5 X 10(3)-10(6)) with 2.5 X 10(5) mononuclear cells from normal peripheral blood increased the number of BFU-E derived erythroid colonies. The same effect was observed when the number of target cells was varied in the presence of a fixed number of B-lymphocytes, with a clear linear relationship. B-cell conditioned medium gave a similar increase when added to the culture instead of B-cells. At high concentration of B-cells from anaemic patients, the size of the colonies was increased and a large number of macroscopic colonies was seen. The place of the B-cells in the regulation of erythroid progenitors in relation to monocytes and T-lymphocytes has still to be established.

Anemia↗