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Biomedical subjects

L Bertrand

Publications and source records attributed to L Bertrand.

At least 37 records · Page 2Linked to original sources
PubMed1997

Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.

Adenosine Triphosphate↗
PubMed1997

The early neuronal organization predicts the path followed by some major axonal bundles in the embryonic brainstem.

In the embryonic CNS, preformed pathways precede the growth of axonal fasciculi [Katz M. J. and Lasek R. J. (1980) Cell Motil. 1, 141-157; Katz M. J. et al. (1980) Neuroscience 5, 821-833]. What are the developmental events that lead to the elaboration of these preformed pathways? To answer this question, we investigated the organization of the primitive neural tube and more particularly the arrangement of the early-generated cells using [3H]thymidine autoradiography or bromodeoxyuridine. Our data suggest that the position of early-generated cells might be involved in the setting of such pathways. In the brain stem of E12(0) (12 days and 0 h) and E12(15) rat embryos, the first-generated cells were organized into three longitudinal columns associated with glycoconjugate-rich extracellular spaces in the adjacent primitive marginal layer. Also, axons traced with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were contiguous to the early-generated cellular columns and represented the primordium of the medial longitudinal fasciculus, the lateral longitudinal tract and the mesencephalic trigeminal tract. Our results show a correlation between the organization of early-generated cells, likely neurons, and the pattern of extracellular spaces in the marginal layer where axons grow. It has been reported in the literature that neurons produce elements of the extracellular matrix such as growth-modulating molecules or space-creating molecules. We therefore suggest that the position of early-generated neurons could be involved in the elaboration of a template for the setting of some major longitudinal tracts during embryonic development of the brainstem.

Animals↗
PubMed1997

Sexual assault in school, mental health and suicidal behaviors in adolescent women in Canada.

Adolescent women (N = 1,025) in grades 7 through 12 in a stratified random sample of Alberta high schools completed measures of emotional problems and suicidal behavior in the past six months, and of frequency and type of sexual assault (including sexual harassment) experienced in school. It was found that 23% has experienced at least one event of assault (sexual touching, sexual threats or remarks, or an incident of indecent exposure); 4% had "often" experienced one or more of these assaults or harassments. Those experiencing a high number of sexual assaults or harassments were significantly more likely to have clinical profiles on the measures of emotional disorder; 15% of 38 women experiencing frequent, unwanted sexual touching had "often" made suicidal gestures or attempts in the previous six months, compared with 2% of 824 women with no experience of sexual assault.

Adolescent↗
PubMed1996

The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis.

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding. We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine. Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type. The mutations had no effect on fructose-2, 6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3', 5'-AMP-dependent protein kinase. Binding experiments with ATP, ADP, and their analogs (3'-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role. Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.

Adenosine Diphosphate↗
PubMed1996

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.

Adrenergic beta-Antagonists↗
PubMed1996

Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases.

The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization.

Adenylyl Cyclases↗
PubMed1995

Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis.

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

Animals↗
PubMed1995

Mental health profiles, suicidal behavior, and community sexual assault in 2112 Canadian adolescents.

1025 females and 1087 males in grades 7-12 in Alberta high schools completed measures of emotional and behavioral problems and suicidal behaviors, and of frequency of sexual assaults outside of school. Both males and females experiencing a high number of sexual assaults in the previous 6 months were significantly more likely to have clinical profiles on measures of conduct disorder, somatic disorder, and emotional disorder. 13.2% of 53 girls with frequent, unwanted sexual contact had made more than two suicidal gestures or suicide attempts in the previous 6 months, compared with 1.3% of 783 girls with no experience of sexual assault. 33.3% of 18 boys experiencing frequent sexual assault were suicidal, compared with 0.9% of 1064 boys with no recent experience of sexual assault. These results are in line with results of other recent Canadian surveys.

Adolescent↗
PubMed1994

Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain.

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

Amino Acid Sequence↗
PubMed1993

The disposition of early-generated neurons in the rat embryo predicts the pattern of major axonal tracts.

During embryogenesis, the fiber tracts grow in a highly stereotyped pattern. A very small number of predetermined paths, preceding the growth of fasciculi, are present in the young neural tube (10-12, 15). What is the origin of these substrate pathways defined by Katz et al. (16) as "... a set of similar guidance cues which are aligned in a continuous discrete pathway..."? Could the first neurons play a role in the guidance of early nerve fibers? Observations in the brain stem revealed the presence of two longitudinal columns of early-generated neurons. These longitudinal columns were associated with well-differentiated marginal zones, characterized by cell-free spaces and representing the prospective site of the medial longitudinal (mlf) and lateral longitudinal (llt) tracts. Nerve fibers were also traced in the brain stem of young embryos. Axons were seen to travel in the early mlf and llt, in close proximity to the regions of early-generated neuronal columns. The data suggest that the precocious neurons that are organized in a definite pattern could somehow be involved in the guidance of some longitudinal axonal tracts, either by directly promoting the formation of an adequate terrain in the marginal layer, or by inducing other cells to do so.

Animals↗
PubMed1993

Evaluation of two cross-linked collagen gels implanted in the transected spinal cord.

In previous experiments, we have shown that spinal axons grow into a collagen matrix implanted between the stumps of a transected spinal cord. However, the matrix became denatured after 2 to 3 months. To improve the stability and the durability of the collagen gel implants, collagen was coprecipitated with chondroitin-6-sulfate (C-6-S) or chemically cross-linked with carbodiimide (CD). The spinal cords were taken out after 3 days, 1, 3, or 6 months and analyzed using different histological and tracing techniques. The cross-linked collagen matrices underwent major structural changes. Cross-linking treatments improved the stability of collagen implants which withstood at least 6 months. Axons revealed with DiI or silver staining crossed the proximal interface and grew into the bioimplants. Some axons were also followed across the distal bioimplant-spinal interface in DiI treated tissues. This study suggests that cross-linking the collagen hydrogel has improved the mechanical properties of the matrix, modified the normal scarring process, and favored axonal regeneration.

Animals↗
PubMed1991

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron-containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium-dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.

Amino Acid Sequence↗
PubMed1991

Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.

Amino Acid Sequence↗
PubMed1991

Mouse fetal kidneys in serum-free organ culture: effects of epidermal growth factor and hydrocortisone.

1. The present study was undertaken to determine whether epidermal growth factor (EGF, 100 ng/ml) or hydrocortisone (HC, 10(-8)-10(-5) M) directly influence proliferation and differentiation of mouse fetal kidney maturing in serum-free organ culture. 2. Addition of EGF to the medium significantly stimulated DNA synthesis after 2 and 5 days of culture. Labelled nuclei were mainly localized in the mesenchymal tissue. Protein synthesis remained unchanged. Activities of three hydrolases, markers of brush border differentiation, were reduced. 3. Hydrocortisone (HC), at all concentrations used, significantly inhibited DNA synthesis. Labelled nuclei were distributed in various cell populations of both control and treated explants. Protein synthesis was stimulated by 10(-7) M after 5 days of culture. Hydrolase activities were slightly modified by HC treatment. 4. The present results indicate that EGF stimulates whereas HC decreases proliferation. Both factors have regulatory effects on brush border maturation. 5. Thus, this culture model is a valuable tool for the study of nephrogenesis.

Animals↗
PubMed1989

Developmental profile of DNA synthesis and hydrolase activities in human fetal kidney.

This study provides original data on human fetal kidney developing during the 13th to 18th week of gestation. The parameters evaluated were DNA synthesis and the activities of 5 hydrolases which are considered as good markers of the brush border membrane differentiation. The conclusions are that DNA synthesis decreased slightly from the 16th to 18th week. The activities of maltase, trehalase, alkaline phosphatase and leucylnapthylamidase remained nearly stable during the studied period. Only the gamma-glutamyltransferase activity decreased significantly between the 15th and 16th week, then it returned close to the 13th week value. The current results suggest that during the 13-18 week period of gestation, cell proliferation is slowed down while maturation of some enzymic activities of the brush border are not importantly modified. The present basic data might be used as reference standards by investigators in the field of human nephrogenesis.

Alkaline Phosphatase↗