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L Beaudet

Publications and source records attributed to L Beaudet.

At least 19 recordsLinked to original sources
PubMed2001

Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen.

AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with < 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 microL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.

Alleles↗
PubMed1998

Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity.

The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.

Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

Cone photoreceptor topography in the retina of sexually mature Pacific salmonid fishes.

We examined the retinal cone topography in sexually mature individuals from four species of Pacific salmonid fishes by using semithin plastic sections. We identified variations in cone density and cone arrangements and noted the presence of putative ultraviolet (UV) cones. Putative UV cones were found over an area extending dorsotemporally from the center of the retina. Because most of the putative UV cones are believed to disappear in early ontogeny, their presence over a large proportion (15-20%) of the surface area of the adult retina suggests that they may be reincorporated prior to or at sexual maturity, at least in rainbow trout. Cone density varied across the retina, with highest values at the peripheral margin. Relatively high densities were observed ventrotemporally (in all specimens) and, to a lesser extent, dorsonasally (7 of 11 specimens). The higher cone density in the ventrotemporal retina may represent a retinal specialization in the part of the visual field located above and in front of the animal. Lowest cone densities were found dorsocentrally and coincided approximately with the distribution of putative UV cones, raising the possibility that these cones may not be used in visual tasks requiring the higher visual acuity normally associated with higher cone densities. We also report a novel cone arrangement that consists of rows of double cones inserted between rows composed of single-double cone pairs alternating in position.

Animals↗
PubMed1996

[The impact of a child's death on the family].

The death of a child has major repercussions on the family system. To develop a reference framework for intervention by health care professionals, the authors describe their research on Quebec families who have lost a child. Semi-structured interviews and a questionnaire were used to gather data from families who had experienced the death of a child up to age 18 during the past six years. The final sample consisted of 76 participants. Analysis of the data highlights the impact of the death on the family's functioning and structure. Adjustment to the absence of the deceased member is a major cause of stress that effectively redefines the role of every family member. Their social network undergoes transformations and family members experience a period of great emotional vulnerability. The authors believe that specific, structured and appropriate support can make the difference between an extended period of psychosocial distress or greater family cohesion. Thus, a nurse who wishes to intervene with families in mourning following a perinatal death, a sudden death or an anticipated death, can use the intervention plan the authors developed for this purpose.

Adaptation, Psychological↗
PubMed1995

Functional dissection of P-glycoprotein nucleotide-binding domains in chimeric and mutant proteins. Modulation of drug resistance profiles.

We wished to determine if the two nucleotide-binding domains (NBD) of P-glycoprotein are functionally equivalent and interchangeable, and if not, which segments and amino acids are important for proper function of each NBD within the context of the C- or N-terminal P-glycoprotien halves. For this, we constructed and tested the biological activity in yeast and mammalian cells of a series of chimeric mdr3 cDNAs in which discrete domains of the N-terminal NBD (NBD1) were replaced by the homologous segments of the C-terminal NBD (NBD2). Although most NBD1 segments could be replaced without loss of P-glycoprotein function, exchange of small segments near the Walker B motif caused a dramatic reduction in Adriamycin, actinomycin D, and colchicine resistance in LR73 cells, as well as in FK506 resistance and STE6 complementation in yeast. Site-directed mutagenesis identified amino acid positions 522-525 (ERGA-->DKGT) and 578 (Thr-->Cys) as essential for proper function of NBD1 in the context of the N-terminal half P-glycoprotein. In addition, the observed phenotype of the mutants (altered drug resistance profile) suggests that these residues may participate directly or indirectly in substrate interactions and are possibly implicated in signal transduction from NBDs to transmembrane domains, the primary sites of drug binding in P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Pathologic alterations in pre- and postsynaptic elements in aged mouse sympathetic ganglia.

Dysfunction of the sympathetic autonomic nervous system is an increasingly recognized, although poorly understood, complication of increasing age in experimental animals and man. In this study of young adult (4-6 months old) and aged (12-24 months old) mice we have examined the ultrastructural appearance of perikarya, dendritic processes, preterminal axons, and synapses in selected sympathetic ganglia as well as the three-dimensional structure of the dendritic arborizations of principal sympathetic neurons using intracellular injections of Lucifer Yellow. Ultrastructural examination demonstrated numerous markedly enlarged presynaptic terminal axons and synapses which distorted the contours of perikarya and dendrites of neurons within the prevertebral celiac/superior mesenteric and paravertebral superior cervical and stellate sympathetic ganglia of aged mice. Dilated preterminal axons had the distinctive ultrastructural appearance of neuroaxonal dystrophy, a pathologic process described in a wide variety of clinical and experimental entities. Dystrophic axons were identical in ultrastructural appearance in young and old animals, differing only in frequency. A distinctive type of ultrastructural alteration, characterized by markedly distended neurites containing numerous vacuoles, was confined to the superior cervical ganglia and also increased in frequency with aging. Although many intraganglionic vacuolated processes disappeared with surgical interruption of the cervical sympathetic trunk, which contains the preganglionic axons innervating the superior cervical ganglia, others persisted. In addition, the presence in some processes of admixed ribosomes, lipofuscin, or continuity with the cell body indicated that numerous neuritic alterations within aged sympathetic ganglia were likely of dendritic origin. Intracellular injections of Lucifer Yellow into principal sympathetic neurons demonstrated that the dendritic arborizations of the celiac/superior mesenteric ganglia neurons of young adult mice were significantly more complex and extensive than those of the superior cervical ganglia. Sympathetic neurons of aged superior cervical ganglia, but not superior mesenteric ganglia, appeared significantly smaller with regard to total dendritic length, extent, and branching when compared to those of young animals. In the aged superior cervical ganglia, short, stunted dendritic processes also exhibited large, focal, often multiple, swellings, a phenomenon infrequently observed in the superior cervical ganglia of young animals. The celiac/superior mesenteric ganglia of aged or young adult mouse failed to exhibit comparable dendritic swellings.

Aging↗
PubMed1993

Optic nerve response and retinal structure in rainbow trout of different sizes.

This study presents evidence of ultraviolet (UV) sensitive, ON center ganglion cells in the fish retina. We determined the spectral sensitivity of ON and OFF responses from the optic nerve mass potential in small (18.0 - 28.5 g) and large (59.5-835 g) rainbow trout, with special reference to UV sensitivity. Under a mid+long-wavelength adapting background, the ON response of small fish revealed the presence of a UV cone mechanism (lambda max 390 nm) which was absent in large specimens. Under similar background conditions, the OFF response of both small and large fish showed one sensitivity peak, dominated by inputs from an M-cone mechanism. An almost complete absence of the accessory corner cones from the retinal mosaic was correlated with the loss of UV sensitivity.

Action Potentials↗
PubMed1993

Different posttranscriptional controls for the human neurofilament light and heavy genes in transgenic mice.

To investigate the mechanisms regulating neurofilament gene expression, we generated transgenic mice with high copy number of the intact human neurofilament light (NF-L) and heavy (NF-H) genes. Overexpression in transgenic mice of NF-L mRNA from 3- to 5-fold in different regions of the central nervous system (CNS) resulted only in a mild increase of 10-50% in the levels of NF-L proteins. The failure to enhance NF-L protein content was not due to interspecies differences in posttranscriptional NF-L regulation. For instance, based on specific immunodetection, it is estimated that human NF-L proteins composed 80% of total NF-L content in the spinal cord of transgenics. In contrast to the situation with NF-L, the CNS of transgenic mice bearing multiple copies of the human NF-H gene showed comparable increases in the levels of NF-H mRNA and proteins. These results suggest that the NF-L and NF-H genes are subject to different posttranscriptional regulation in the CNS. In vivo labeling of newly synthesized proteins by injection of [35S]methionine in the spinal cords of normal and transgenic mice provided evidence that the posttranscriptional regulation of NF-L expression in the CNS must occur, at least in part, at the level of translation.

Animals↗
PubMed1993

[Resources for preventing professional burnout].

Burnout in the nursing profession has been reported in the literature for the past decade. Yet few studies have identified the causative factors or the recommended resources for prevention and treatment. An exhaustive literature review by the authors produced over 300 articles--40 of which were selected and systematically analyzed. It became apparent that the contributing factors to nursing burnout include stressors relating to the nurse's role, tasks and interpersonal conflicts, and in particular, the lack of management and peer support. The nurse's susceptibility to burnout varied considerably and was dependent upon individual character traits such as hardiness. Hardy individuals demonstrated an ability to deal with life through qualities such as their ability to manage change; their sense of commitment, and their sense of control over their lives. According to the literature, these individuals are less susceptible to burnout manifestations. The article also identifies potential resources available to nurses and positive factors that could improve their adaptation to stressful situations or environments. In light of dwindling financial resources and increased nursing responsibilities, the authors advise steering nurses in a direction that will unite them with their peers, management, and their profession.

Adaptation, Psychological↗
PubMed1992

Intragenic regulatory elements contribute to transcriptional control of the neurofilament light gene.

To date, no DNA regions involved in the neuron-specific expression of the neurofilament light gene (NF-L) have been defined using transfection assays in cultured cells. To identify those regulatory regions in the human NF-L gene, we generated transgenic mice with a construct containing the basal NF-L promoter (-292 to +15) fused to the cat gene and with three DNA fragments of 21.5, 7.6 and 4.9 kb each, including NF-L with different lengths of either 5'- or 3'-flanking sequences. We show that the proximal NF-L 5' region (0.3 kb) constitutes a weak promoter and that it lacks information to confer neural specificity. However, appropriate expression in the nervous system occurred when this minimal promoter was combined with either 7.3 or 4.6 kb of NF-L sequences downstream from the transcription start point. We conclude that the intragenic NF-L region contains cis-acting elements conferring cell-type-specific regulation on the basal activity of the NF-L promoter. Interestingly, AP-2 motifs were found within homologously placed introns of all three NF genes, as well as in the promoter regulatory regions of many neuronal genes. We propose that the acquisition of introns by an ancestral intronless IF gene may have contributed to the emergence of a lineage of IF genes expressed in the nervous system.

Animals↗
PubMed1992

Origin of the two mRNA species for the human neurofilament light gene.

The human neurofilament light (hNF-L) gene yields two major species of mRNAs of 2.4 and 3.8 kilobases (kb) in size. To investigate the origin of these two mRNAs, we have analyzed in transgenic mice the expression of hNF-L DNA fragments including different lengths of 5'-flanking regions. The finding that the 3.8-kb mRNA species is produced by a hNF-L transgene that includes only the proximal promoter region (-0.3 kb) demonstrates that both the 2.4- and 3.8-kb mRNAs are derived from the same site of transcription initiation. Sequencing of the 3' untranslated region of the hNF-L gene revealed the presence of multiple AATAAA polyadenylation signals. We conclude from Northern blotting experiments using probes spanning various regions of the hNF-L gene that the 2.4- and 3.8-kb mRNAs originate from the selective use of polyadenylation signals located 1.4 kb apart.

Animals↗
PubMed1992

Effects of dilation with a balloon catheter on the endothelium of the internal thoracic artery.

Controlled dilation of the internal thoracic artery with a balloon catheter has been reported to effectively treat intraoperative arterial spasm. It has been shown in laboratory animals that dilation of the internal thoracic artery at prescribed shear force levels will not cause intimal damage. Using scanning electron and light microscopy, we have examined the effects of calibrated balloon dilation on the endothelium of the human internal thoracic artery. In 10 patients with bilateral internal thoracic artery grafting, the artery was dilated with a Fogarty IMAG balloon catheter (Baxter Healthcare Corporation, Edwards Division, Santa Ana, Calif.) that was withdrawn at tensions of 20 or 30 gm. Arterial segments and nondilated control specimens were prepared for scanning electron microscopy. The intimal surface of each internal thoracic artery was evaluated by assigning a score (from 0 to 3) to 10 examined scanning electron microscopy fields; subsequently the arterial tissue was viewed by light microscopy with paraffin-embedded sections stained for elastic tissue. Arteries were obtained from three additional patients so that the microscopic appearance of the arteries could be observed after rough manipulation or removal of the balloon without shearing. The results of this study are as follows: (1) By scanning electron microscopy, dilated internal thoracic arteries yielded consistently higher scores than the control arteries, reflecting severe, tension-dependent alterations of the endothelium, which included marked desquamation of endothelial cells, with extensive areas of complete denudation and pronounced attachment of platelets to these areas; (2) endothelial injury occurred by inflation alone, without shearing by the inflated balloon; (3) by light microscopy, the internal thoracic arteries showed (a) fenestrations of the internal elastic lamina with occasional transmigration of smooth muscle cells through these gaps and (b) foci of intimal thickening without overt atherosclerotic lesions. We conclude that the endothelium of human internal thoracic arteries is highly vulnerable to balloon dilation, which can severely injure the intimal surface. For this reason we prefer not to include this procedure in our protocol for preparing the internal thoracic artery.

Aged↗
PubMed1990

Neurofilament gene expression in transgenic mice.

1. DNA fragments that include the human neurofilament NF-L gene was found to be correctly expressed in the majority of neurons in transgenic mice. 2. The NF-L transgene product, which is detectable in situ with a species-specific monoclonal antibody, provides a powerful genotype marking system applicable to developmental and regeneration studies of the mammalian nervous system. 3. The proximal 5'-flanking region of the NF-L gene is sufficient to direct expression of a heterologous gene in the mouse nervous system.

Animals↗