Search PubMed⌕ Search

Biomedical subjects

G Leblanc

Publications and source records attributed to G Leblanc.

At least 19 recordsLinked to original sources
PubMed1996

Melibiose permease of Escherichia coli: structural organization of cosubstrate binding sites as deduced from tryptophan fluorescence analyses.

Binding of the coupling ion (Na+ or Li+) and sugars to the purified melibiose permease of Escherichia coli, reconstituted in proteoliposomes, produces selective and cooperative changes of the transporter tryptophan fluorescence. To assess the individual contribution of N- or C-terminal domains of the permease to these substrate-induced fluorescence variations, we replaced the two tryptophans located in its C-terminal half (W299 and W342) by a phenylalanine and compared the signal change in mutants and wild-type permease. None of the mutations significantly impairs transport activity. Persistence of the ion-induced signal quenching in a permease carrying only the six other tryptophans of the N-terminal domain is consistent with a previous suggestion that this domain accommodates the ion-binding site. On the other hand, the sugar-induced fluorescence increase varies from mutant to mutant in a sugar-specific fashion. While alpha-galactosides increase essentially the fluorescence of W299 and W342, beta-galactosides enhance the signal of W299 and of one (or more) of the N-terminal tryptophans but quench that of W342. Moreover, addition of sugars producers a 10 nm blue shift of both W299 and W342 emission spectra, suggesting reduced accessibility of these residues to solvent following substrate binding. These data suggest that W299 and W342 are at or close to the sugar binding site and that this latter is lined by the C-terminal helices IX and X. Moreover, as sugars with the beta-configuration also enhance the fluorescence of the N-terminal tryptophans, it is suggested that one (or more) helix of the N-terminal half may be also at or near the sugar binding site. This implies close proximity and/or tight functional linkage between some N-terminal helices and helices IX and X of the C-terminal domain of the transporter.

Amino Acid Sequence↗
PubMed1996

Unidirectional reconstitution into detergent-destabilized liposomes of the purified lactose transport system of Streptococcus thermophilus.

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-beta-D-glucoside, n-dodecyl-beta-D-maltoside, or C12E8. The LacS protein carrying a poly-histidine tag was purified in large quantities (approximately 5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography. The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured. Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-beta-D-glucoside. For reconstitutions mediated by n-dodecyl-beta-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization). Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide. The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane. Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

Amino Acid Sequence↗
PubMed1996

Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis.

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane. Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.

Alkaline Phosphatase↗
PubMed1996

Cation and sugar selectivity determinants in a novel family of transport proteins.

A new family of homologous membrane proteins that transport galactosides-pentoses-hexuronides (GPH) is described. By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins. Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity. Most of the mutations map in the amino-terminal region, in or near amphipathic alpha-helices II and IV, or in interhelix-loop 10-11 of the transport proteins. On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters. The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed.

Amino Acid Sequence↗
PubMed1996

[Percutaneous treatment of iliac aneurysms with endovascular stented grafts].

Two cases of iliac aneurysms treated percutaneously by endovascular stented graft are reported. The initial radiographic evaluation included arteriography and computed tomography with helical acquisition and three dimensional reconstruction. This enabled analysis of the flow path, the extent of the wall thrombus, the choice of stent graft's size. The procedure of implantation was technically trouble free. The post-procedure arteriography and 3D CT demonstrated occlusion of the aneurysm and restauration of a normal flow path. The three, six and twelve month check-ups confirm the good results. In weakened patients or those not requiring aorto-iliac surgery, percutaneous treatment of iliac aneurysm can be carried out easily. The contribution of 3D CT is essential in assessing the endo- and extra-luminal characteristics of aneurysm for indicating the percutaneous route and selecting size of the stented graft.

Aged↗
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↗
PubMed1994

Structure of human estrogen and aryl sulfotransferase gene. Two mRNA species issued from a single gene.

Estrone sulfate is the predominant form of estrogens found in the circulation in women and could thus serve as precursor for active estrogens in target tissues by removal of the sulfate group through the action of endogenous steroid sulfatase. Recently, we isolated a cDNA encoding human placental estrogen sulfotransferase that differs from brain aryl sulfotransferase only in the 5'-noncoding sequence. To increase our knowledge of the regulation and tissue-specific expression of sulfotransferase gene, we screened a lambda EMBL3 library of human leucocyte genomic DNA using the estrogen sulfotransferase cDNA as probe and isolated a clone containing almost the whole gene sequence. Sequencing of the gene indicates that it is included in approximately 7.7 kilobases and contains nine short exons separated by eight introns. The two first exons, named exon 1a and exon 1b, are noncoding and correspond to the 5'-untranslated sequences of human brain and human placental estrogen sulfotransferase cDNAs, respectively. Transfection of chloramphenicol acetyltransferase reporter gene vectors containing the 5'-flanking sequence upstream from exon 1a and exon 1b in human adrenal adenocarcinoma cells indicates that both sequences possess promoter activity. The present results thus indicate that brain aryl sulfotransferase and placental human placental estrogen sulfotransferase mRNA species are transcribed from a single gene by alternate exon 1a and exon 1b promoters, respectively. Using DNA from panels of human/rodent somatic cell hybrids and amplification of the gene by polymerase chain reaction, the human placental estrogen sulfotransferase gene was assigned to chromosome 16.

Adenocarcinoma↗
PubMed1994

Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

The suggestion that acidic residues in the hydrophobic NH2-terminal domains of Mel permease (Asp-31 in helix I, Asp-51 and Asp-55 in helix II, Asp-120 in helix IV) may be essential components of a coordination network involved in cation recognition (Pourcher, T., Zani, M.L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215) is further analyzed using site-directed mutagenesis. To study whether nearby polar residues also contribute to the cation recognition process, Tyr-24, Tyr-27 and Tyr-28 (aligned with Asp-31) and Tyr-109 and Tyr-116 (aligned with Asp-120) were individually converted into a phenylalanine. The effect of replacing Arg-48 (aligned with Asp-51 and Asp-55) or Asn-83 (in the middle of helix III) by an alanine was also studied. The importance of the position of the carboxylate of the residue at position 31, 51, 55, or 120 was next examined by replacing each Asp by a Glu residue. Sugar binding and/or transport activity measurements indicate that all polar-->apolar or Asp-->Glu mutants use Na+ or Li+ for active sugar transport. Moreover, two groups of mutants could be distinguished. One group, composed of Y27F, Y28F, D31E, and Y109F mutants, retains wild type permease properties. A second group (Y24F, N83A, and Y116F and also D51E, D55E, and D120E) exhibits concomitant reduction of affinity for sodium and sugars and altered sugar specificity but conserves wild type cation selectivity profile. The data reinforce the notion that Asp-51, Asp-55, and Asp-120 residues and the position of their carboxyl side chains are of primary importance for cation recognition. Finally, since Mel permease properties are predominantly modified by mutagenizing residues located in the cytoplasmic half of the permease, we propose that Mel permease has a well-like shape opened toward the periplasmic space and is closed at its cytoplasmic extremity by a gate.

Base Sequence↗
PubMed1994

Effects of neuroleptic reduction in schizophrenic outpatients receiving high doses.

This paper presents an open study which evaluated the clinical effects of a partial and progressive reduction in neuroleptic medication in 32 outpatients suffering from schizophrenia who were receiving high doses (equivalent of > or = 18 mg of oral haloperidol per day; EHL). After an observation period of twelve weeks, each subject's dose of neuroleptics was reduced by 50% at the rate of 10% every four weeks. Patients were receiving a mean of 62 mg per day EHL at the beginning of the study and 30 mg per day EHL at the completion of the study. After the reduction, the following was observed: 1. a significant but modest change in psychopathology: a decrease in negative symptoms and in the total score on Brief Psychiatric Rating Scale; and 2. a significant increase in tardive dyskinesia symptoms. Six subjects relapsed but five of them recovered without increasing their reduced medication. Results of this study are discussed in the context of trying to find a minimal maintenance dose in the treatment of schizophrenia. The relative paucity of change despite a large reduction in medication argues for reevaluation of dosage in patients on high or very high doses of neuroleptics. The results suggest that many patients taking high doses could be maintained on significantly lower doses of neuroleptics. With gradual reduction of medication it would seem that many patients who are receiving a high dose of neuroleptic can achieve a lower dose than their current maintenance level.

Adult↗
PubMed1993

Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. I. Effect on Na(+)-dependent transport and binding properties.

Four aspartic acids, distributed in different putative membrane-spanning segments of the NH2-terminal domain of melibiose (mel) permease (D31 in helix I, D51 and D55 in helix II, and D120 in helix IV) were individually replaced by either Asn or Cys using site-directed mutagenesis. mel permease with either neutral residues at position 51, 55, or 120 or permease with a Cys in place of D31 does not catalyze significant Na(+)-linked methyl-1-thio-beta-D-galactopyranoside (TMG) accumulation. Binding studies of a high affinity ligand (p-nitrophenyl-alpha-D-galactopyranoside (NPG)) on de-energized membrane vesicles indicate that these modified transporters (i) retain the ability to bind the alpha-galactosides NPG or melibiose and the beta-galactoside TMG and (ii) exhibit a Na(+)-independent sugar-binding phenotype. In contrast, mel permease with an Asn residue at position 31 mediates Na(+)-coupled TMG transport and displays a Na(+)-dependent sugar binding phenotype, but requires a higher concentration of sodium than wild-type permease to produce maximal stimulation of sugar binding. The observation that individual mutation of the Asp residue at position 31, 51, 55, or 120 systematically and selectively modifies the contribution of the coupling ion to the early step of the transport reaction, i.e. cosubstrate binding, raises the possibility that (i) these 4 aspartic residues are at or near the cationic binding site of mel permease, (ii) the NH2-terminal domain of mel permease in which they are distributed accommodates or is part of the cationic binding site, and (iii) the oxygen atoms of these Asp side chains contribute to coordination of the coupling ion.

Amino Acid Sequence↗
PubMed1993

Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. II. Effect on cationic selectivity and coupling properties.

Individual substitution of Cys or Asn for Asp-31, Asp-51, Asp-55, or Asp-120, distributed in different membrane spanning segments of the NH2-terminal domain of melibiose (mel) permease partially or completely inactivates Na(+)-linked sugar transport and stimulation of sugar binding on mel permease by Na+ ions (Pourcher, T., Zani, M.-L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215). To investigate further the effect of these substitutions on the cationic selectivity and coupling properties of mel permease, H(+)-melibiose coupled transport, coupling between H+ and melibiose movements, sugar counterflow, and zero-trans sugar efflux by the mutant permeases were analyzed. The results provide additional evidence indicating that manipulation of some of these Asp in the membrane-spanning segments of mel permease alters its cationic selectivity properties. The results also indicate that the individual mutations diversely affect mel permease-coupling properties. For example, only permease with Asn in place of Asp-31 or Cys in place of Asp-51 retains the capacity to actively transport melibiose. On the other hand, replacing Asp-55 by Cys produces uncoupling of cosubstrate flows by the carrier but does not hamper sugar translocation. These and other features of the mutant permeases are used to discuss the relative participation of Asp-31, Asp-51, Asp-55, or Asp-120 to the mel symport mechanism and to its ionic selectivity and also the existence of a possible gating mechanism that may contribute the obligatory coupling of cosubstrate flows by the symporter.

Amino Acid Sequence↗
PubMed1993

Study of the clinical utility of radioreceptor assay in outpatients with schizophrenia receiving high doses of neuroleptics.

A radioreceptor assay (RRA) was used to determine the neuroleptic plasma levels of 32 outpatients with schizophrenia receiving a high dose of neuroleptics (the equivalent of 18 mg or more of oral haloperidol per day) and undergoing a 50% partial and progressive reduction (ten percent each month for five months) in their medication. Plasma levels of neuroleptics were measured three times: before (T1) and immediately after the 50% reduction (T2) and five months later (T3). A linear correlation was observed between neuroleptic plasma levels obtained by RRA and the neuroleptic doses prescribed at T1 and T3. Furthermore, neuroleptic plasma levels were significantly lower at T3 than at T1. Concurrent evaluations of psychopathology were done using the Brief Psychiatric Rating Scale, and the results indicated that no correlation exists between neuroleptic plasma levels and the total rating scale scores at T1 but a significant correlation was observed at T3.

Adult↗