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A Doucet

Publications and source records attributed to A Doucet.

At least 19 recordsLinked to original sources
PubMed2002

Design of high essential amino acid proteins: two design strategies for improving protease resistance of the nutritious MB-1 protein.

Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations we face in this endeavour is achieving stable proteins despite a highly biased amino acid content. We report here the synthesis and characterisation of two mutants derived from our MB-1 designer protein. The first mutant contains a disulphide bridge designed to cross-link remote segments of the polypeptide chain. The second one is a Tyr62-Trp mutant, where position 62 is buried in the core of the protein. Both mutants were found to be largely helical as per design, and based on thermal denaturation experiments, were substantially more stable than the MB-1 parent molecule. Enhancement of conformational stability in MB-1Trp translated into an impressive improvement of its ability to resist proteolytic degradation. Furthermore, digestion experiments intended to model degradation of proteins in a cow's rumen revealed that MB-1Trp's resistance to degradation compared to that of cytochrome c. Design strategies used for these mutants are discussed with regards to their applicability in creating efficient nutritional proteins.

Amino Acid Sequence↗
PubMed2002

[Primary molecular changes and secondary biological problems in Bartter and Gitelman syndrome].

Bartter syndrome and Gitelman syndrome are primary hereditary diseases characterized by hypokaliemia, alkalosis, hypertrophy of the juxtaglomerular complex with secondary hyperaldoteronism and normal blood pressure. They result from molecular disorders leading to a defect of sodium reabsorption in respectively the Henle's loop and the distal convoluted tubule. Biological adaptations of downstream tubular segments, i.e. distal convoluted tubule and collecting duct, are responsible for hypokaliemia, alkalosis, renin-aldosterone activation, prostaglandins hypersecretion and dysregulation of the urinary excretion of calcium and magnesium, illustrating the close integration of the regulation of different solutes in the distal tubular structures.

Bartter Syndrome↗
PubMed2001

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Aldosterone↗
PubMed2001

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells.

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Animals↗
PubMed2001

Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control.

Tubular reabsorption of filtered sodium is quantitatively the main contribution of kidneys to salt and water homeostasis. The transcellular reabsorption of sodium proceeds by a two-step mechanism: Na(+)-K(+)-ATPase-energized basolateral active extrusion of sodium permits passive apical entry through various sodium transport systems. In the past 15 years, most of the renal sodium transport systems (Na(+)-K(+)-ATPase, channels, cotransporters, and exchangers) have been characterized at a molecular level. Coupled to the methods developed during the 1965-1985 decades to circumvent kidney heterogeneity and analyze sodium transport at the level of single nephron segments, cloning of the transporters allowed us to move our understanding of hormone regulation of sodium transport from a cellular to a molecular level. The main purpose of this review is to analyze how molecular events at the transporter level account for the physiological changes in tubular handling of sodium promoted by hormones. In recent years, it also became obvious that intracellular signaling pathways interacted with each other, leading to synergisms or antagonisms. A second aim of this review is therefore to analyze the integrated network of signaling pathways underlying hormone action. Given the central role of Na(+)-K(+)-ATPase in sodium reabsorption, the first part of this review focuses on its structural and functional properties, with a special mention of the specificity of Na(+)-K(+)-ATPase expressed in renal tubule. In a second part, the general mechanisms of hormone signaling are briefly introduced before a more detailed discussion of the nephron segment-specific expression of hormone receptors and signaling pathways. The three following parts integrate the molecular and physiological aspects of the hormonal regulation of sodium transport processes in three nephron segments: the proximal tubule, the thick ascending limb of Henle's loop, and the collecting duct.

Angiotensin II↗
PubMed2001

Robust full Bayesian learning for radial basis networks.

We propose a hierarchical full Bayesian model for radial basis networks. This model treats the model dimension (number of neurons), model parameters, regularization parameters, and noise parameters as unknown random variables. We develop a reversible-jump Markov chain Monte Carlo (MCMC) method to perform the Bayesian computation. We find that the results obtained using this method are not only better than the ones reported previously, but also appear to be robust with respect to the prior specification. In addition, we propose a novel and computationally efficient reversible-jump MCMC simulated annealing algorithm to optimize neural networks. This algorithm enables us to maximize the joint posterior distribution of the network parameters and the number of basis function. It performs a global search in the joint space of the parameters and number of parameters, thereby surmounting the problem of local minima to a large extent. We show that by calibrating the full hierarchical Bayesian prior, we can obtain the classical Akaike information criterion, Bayesian information criterion, and minimum description length model selection criteria within a penalized likelihood framework. Finally, we present a geometric convergence theorem for the algorithm with homogeneous transition kernel and a convergence theorem for the reversible-jump MCMC simulated annealing method.

Journal Article↗
PubMed2000

Replacement of tyr62 by trp in the designer protein milk bundle-1 results in significant improvement of conformational stability.

Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content, having potential application in animal nutrition. One of the limitations we face in this endeavor is the achievement of stable proteins in spite of a highly biased amino acid content. We report here the synthesis and characterization of MB-1Trp, a protein with a tailored content in selected essential amino acids. The protein is a Tyr62-Trp mutant of the parent molecule MB-1 described earlier. The new protein is largely helical as per design, is well folded, and has a melting temperature of 55 degrees C. Its resistance to proteolytic degradation compares to that of cytochrome c, a protein of similar size. Design strategy used for MB-1Trp is discussed with regards to its applicability toward the creation of efficient nutritional proteins.

Amino Acid Substitution↗
PubMed2000

Cellular origin and hormonal regulation of K(+)-ATPase activities sensitive to Sch-28080 in rat collecting duct.

Rat collecting ducts exhibit type I or type III K(+)-ATPase activities when animals are fed a normal (NK) or a K(+)-depleted diet (LK). This study aimed at determining functionally the cell origin of these two K(+)-ATPases. For this purpose, we searched for an effect on K(+)-ATPases of hormones that trigger cAMP production in a cell-specific fashion. The effects of 1-deamino-8-D-arginine vasopressin (dD-AVP), calcitonin, and isoproterenol in principal cells, alpha-intercalated cells, and beta-intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and alpha-intercalated cells of outer medullary collecting duct (OMCD), respectively, were examined. In CCDs, K(+)-ATPase was stimulated by calcitonin and isoproterenol in NK rats (type I K(+)-ATPase) and by dD-AVP in LK rats (type III K(+)-ATPase). In OMCDs, dD-AVP and glucagon stimulated type III but not type I K(+)-ATPase. These hormone effects were mimicked by the cAMP-permeant analog dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I K(+)-ATPase activity in alpha- and beta-intercalated CCD cells, whereas in LK rats it stimulates type III K(+)-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells.

Adenosine Triphosphatases↗
PubMed2000

A repression-derepression mechanism regulating the transcription of human immunodeficiency virus type 1 in primary T cells.

BACKGROUND: Despite some controversy regarding the preferential infection and replication of human immunodeficiency virus type 1 (HIV-1), it appears that primary T lymphocytes, in their quiescent state, are nonpermissive for viral expression and propagation. Massive activation of viral gene expression occurs only when the host lymphocyte is activated. These observations prompted us to investigate the transcriptional regulation of HIV-1 in resting or activated T cells that were isolated from cord blood or adult peripheral blood. MATERIALS AND METHODS: To this end, we employed cellular purification and phenotyping techniques, in vitro protein-DNA binding studies, functional transactivation assays using proteins isolated from cord blood or adult peripheral blood T lymphocytes, and transfection experiments in primary T cells. RESULTS: We showed that transcription from the HIV-1 long terminal repeat is repressed in resting naive T lymphocytes; whereas, mitogenically stimulated CD4+ cells form an activator that derepresses transcription. Negative and positive regulation act through a repressor-activator target sequence (RATS), which shares homology with the interleukin-2 (IL-2) purine-rich response element, through the adjacent binding site of the nuclear factor of activated T cells (NFAT), and weakly, through the KB region. CONCLUSIONS: This regulation exerted by cellular transcription factors can account for several important features of HIV-1 expression in primary CD4+ cells. Tight repression in resting naive T helper cells may be a main cause of viral latency and transcriptional activation accounts for massive viral production in activated T lymphocytes.

Adult↗
PubMed1999

Serial microanalysis of renal transcriptomes.

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1, 000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

Animals↗
PubMed1999

Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle.

BACKGROUND: In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. METHODS: Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. RESULTS: cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. CONCLUSION: In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Benefit of prestorage leukocyte depletion of single-donor platelet concentrates.

BACKGROUND AND OBJECTIVES: The presence of contaminating white blood cells (WBCs) in platelet concentrates is associated with transfusion reactions and may adversely alter the quality of platelets during storage. Leukocyte depletion by filtration of platelets has been increasingly used to avoid these complications. However, the best time for filtration and the benefits of filtering single-donor platelet concentrates (thrombapheresis, TH) have yet to be clearly defined. METHODS: In a randomized study of 202 TH collected with an Autopheresis C system, we determined whether prestorage filtration (preSF) of WBCs from TH as compared with poststorage (bedside) filtration (postSF) resulted in a better product. Levels of cytokines and C3a accumulating in the medium during storage, platelet activation state, in vivo platelet recovery, and transfusion reactions were compared in pre- and poststorage products. RESULTS: As compared to preSF, significantly more postSF TH had detectable levels of tumor necrosis factor-alpha (TNF-alpha; 47 vs. 15%; p<0.0001) and interleukin 6 (13 vs. 3%; p = 0.02), lower pH (p<0.0001) and decreased levels of C3a (910 vs. 2,000 pg/ml; p<0. 0001). Furthermore, platelet activation was increased in postSF TH (p = 0.022). PostSF TH tended to plug the bedside filter (27% of postSF TH delivered) from day 3 onward. There was also a significant difference in platelet recovery, postSF TH showing a lower corrected count increment (CCI; p = 0.0055) when taking into account the postSF TH that plugged filters (CCI = 0), but no difference when plugged TH were excluded. A correlation could be established between TNF-alpha levels and poor in vivo recovery (p<0.0001). Febrile nonhemolytic transfusion reactions were low in both groups (4 and 9%). CONCLUSION: These results indicate a benefit of preSF TH as compared with postSF TH based on the following parameters: decrease in cytokine levels, less platelet activation, maintenance of higher pH, and more efficient use of stored platelets (27% of postSF TH were lost because of plugging of filters). These results apply particularly to the Autopheresis C systems with its high initial WBC content.

Blood Component Removal↗
PubMed1999

Collecting duct adaptation to potassium depletion.

Kidneys are the main effectors of the maintenance of potassium balance, under both normal and altered conditions of dietary potassium uptake. The collecting duct system plays a major role in this control of potassium homeostasis because most of the filtered potassium is reabsorbed between the glomerulus and the distal convoluted tubule: under normal physiological conditions or in response to potassium loading, collecting ducts adjust their rate of secretion of potassium into urine so as it matches the dietary daily intake, whereas in response to restriction of potassium ingestion, this secretion process is mostly curtailed, and a reabsorptive mechanism appears. In this short review, we analyzed the cellular and molecular mechanisms underlying transepithelial transport of potassium in the collecting duct and their adaptation in response to potassium depletion. A special emphasis is given on the axial and cellular heterogeneity of the collecting duct with regard to potassium transport and its adaptation. We also discuss the factors controlling duct hypertrophy and hyperplasia during potassium depletion and their possible relationship with the control of potassium conservation.

Absorption↗
PubMed1999

[Renal K-ATPases: structure, function and dysfunction].

Na,K-ATPase and H,K-ATPase consist of two transmembrane proteins, the larger of which (catalytic subunit) exchanges extracellular K+ against intracellular Na+ or proton, at the expense of ATP hydrolysis. Cloning of four isoforms of Na,K-ATPase and two isoforms of H,K-ATPase has provided a molecular basis to the functional heterogeneity of these ATPases. Besides its house keeping functions, renal Na,K-ATPase energizes most solute and water transports along the whole nephron. For this purpose, it utilizes about 80% of renal metabolic energy. H,K-ATPase, which is restricted to the renal collecting duct, has a more limited role: it energizes K+ reabsorption during hypokalemia and, along with H-ATPase, participates to acid/base homeostasis. Dysregulation of tubular Na,K-ATPase and H,K-ATPase are involved in physiopathological alterations. For examples, results are presented which show the relationships that exist between a) Na+ retention during experimental nephrotic syndrome and stimulation of collecting duct Na,K-ATPase, and b) kaliuretic effect of loop diuretics and inhibition of collecting duct H,K-ATPase.

Animals↗
PubMed1998

Quality evaluation of plateletpheresis using the new AMICUS (Baxter) cell separator: evolution of CD 62 expression.

The purpose of this study was to evaluate the new AMICUS (Baxter-Fenwal Division) cell separator in terms of donor safety, efficiency, and quality of the product obtained. One hundred eighty-three single-donor plateletpheresis procedures were performed, using a collection of 4-4.5 x 10(11) platelets as endpoint. During the first part of the study, the mean volume processed was 3,225 ml and the mean procedure duration 69.5 min. During the second part, after a software change, the mean volume and mean procedure time were 3,071 ml and 68.3 min, respectively. According to local policy, every collection bag was separated into two therapeutic units each containing a mean of 1.87 (1.83) x 10(11) platelets. The white blood cell (WBC) contamination per therapeutic unit was less than 5 x 10(6) in 91% of phereses performed in part one of the study and in 98% of phereses performed in part two. During the recommended 5 days storage, sequential in vitro analyses were performed in 27 units, showing limited platelet activation according to CD62 expression and morphological changes on electron microscopy (EM). Furthermore, there was a correlation between CD62 expression and the degree of WBC contamination (P = 0.03). In conclusion, platelet collection with the new Amicus allows for high platelet yields of adequate quality as judged by WBC content, CD62 expression, and electron microscopic morphological changes.

Humans↗
PubMed1998

Regulation of Na+, K(+)-ATPase in the rat outer medullary collecting duct during potassium depletion.

Because in outer medullary collecting ducts (OMCD) of K(+)-depleted rats, K+ secretion is abolished, whereas Na+, K(+)-ATPase, which energizes this secretion, is markedly stimulated, it has been proposed that Na+, K(+)-ATPase was mislocated to the apical cell membrane and energized K+ reabsorption. This hypothesis has been supported by paradoxical effects of ouabain in K(+)-depleted compared with normal rats. However, we have recently shown that ouabain inhibits not only Na+, K(+)-ATPase but also apical H+, K(+)-ATPase in the OMCD of K(+)-depleted rats. Therefore, this study was designed to evaluate whether previous observations were accounted for by Na+, K(+)-ATPase or by ouabain-sensitive H+, K(+)-ATPase. Na+, K(+)-ATPase was distinguished from H+, K(+)-ATPase by its insensitivity to Sch-28080. Results indicate that the hydrolytic and transport activities of Na+, K(+)-ATPase, the number of its functional units, and the expression of mRNA of its alpha 1 and beta 1 subunits were increased threefold or more in the OMCD of rats fed a K(+)-depleted diet for 2 wk. By immunofluorescence, Na+, K(+)-ATPase staining was strongly increased in K(+)-depleted rats but remained localized to the basolateral pole of OMCD principal cells. In conclusion, K+ depletion is associated with marked induction of functional Na+, K+ pumps at the basolateral pole of rat OMCD. Therefore, reduced K+ secretion might result from inhibition of apical K+ conductances and stimulation of basolateral K+ recycling. It is proposed that increased Na+, K(+)-ATPase participates in the increased Na+ reabsorption prevailing in collecting ducts of K(+)-depleted rats.

Animals↗