Étude biophysique des facteurs influençant l'activité des toxines du bacille de Thuringe

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac

AbstractAfter binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed

Eicosapentaenoic acid decreases postprandial beta-hydroxybutyrate and free fatty acid responses in healthy young and elderly

Objectives: We investigated whether a dietary supplement rich in eicosapentaenoic acid (EPA) increases fasting plasma ketones or postprandial ketone responses in healthy young and elderly subjects. Methods: Ten young (22 ± 1 y old) and 10 elderly (75 ± 1 y old) subjects were recruited and participated in two identical study days, one before and one 6 wk after providing an EPA-enriched supplement (1.4 g/d of EPA and 0.2 g/d of docosahexaenoic acid). On the study days, blood samples were collected at fasting and every hour for 6 h after giving a breakfast. Fasting and postprandial plasma β-hydroxybutyrate (β-OHB), free fatty acid (FFA), triacylglycerol, glucose, and insulin responses were measured. Fatty acid profiles were assessed in fasting plasma samples before and after the EPA supplement. Results: After the EPA supplement, postprandial plasma β-OHB responses decreased by 44% in the young and by 24% in the elderly subjects, in addition to 20% and 34% lower FFA responses in the young and elderly adults, respectively. β-OHB and FFAs were positively and significantly correlated in young but not in elderly subjects before and after the EPA supplement. In both groups, postprandial plasma triacylglycerols, glucose, and insulin were not significantly different after the intake of the EPA supplement. Before and after the EPA supplement, fasting plasma EPA was 50% higher in the elderly but increased by about five times in both groups after intake of the EPA supplement. Conclusion: Contrary to our expectations, EPA supplementation lowered postprandial β-OHB response and, in the elderly subjects, the concentration of postprandial β-OHB was not lowered after intake of the EPA supplement

Plasma n-3 fatty acids in the elderly

The elderly reportedly have a significantly higher % of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma and red cell lipids. However, these observations are from a few small studies and the health status of the elderly in these studies is for the most part unclear. Since the elderly are susceptible to cardiovascular and neurological illnesses that seem to be related in part to lower intake of n-3 fatty acids it seems paradoxical that their blood levels of EPA and DHA would be higher than in young adults. We report here plasma fatty acid profiles and their response to supplementation with two types of fish oils from several of our recent studies in the moderately healthy elderly. We define the moderately healthy elderly as those who were in good physical condition, had no cognitive decline and, if present, in whom hypothyroidism, hyperlipidemia and/or hypertension were well-controlled. As shown previously, we confirm the higher % EPA and % total n-3 fatty acids (but not DHA) in fasting plasma and extend these findings to include higher plasma concentrations (mg/L) of n-3 fatty acids as well. The EPA-predominant supplement raised DHA only in the young, whereas the DHA-predominant supplement raised EPA more in the young than in the elderly. The moderately healthy elderly clearly have higher plasma n-3 fatty acids but whether this reflects differences in intake versus aging-related changes in n-3 fatty acid metabolism remains to be elucidated

Plasma response to fish oil in the elderly

Little information is available concerning whether incorporation of dietary omega-3 fatty acids into plasma lipids changes during healthy aging. Elderly (74 ± 4 years old) and young (24 ± 2 years old) adults were given a fish oil supplement for 3 weeks that provided 680 mg/day of docosahexaenoic acid and 320 mg/day of eicosapentaenoic acid, followed by a 2 week wash-out period. Compliance was monitored by spiking the capsules with carbon-13 glucose, the excretion of which was measured in breath CO2. In response to the supplement, plasma docosahexaenoic acid rose 42% more in the elderly but eicosapentaenoic responded similarly in both groups. Despite raising docosahexaenoic acid intake by five to tenfold, the supplement did not raise plasma free docosahexaenoic acid (% or mg/dL) in either group. We conclude that healthy aging is accompanied by subtle but significant changes in DHA incorporation into plasma lipids

Plasma Ketone and Medium Chain Fatty Acid Response in Humans Consuming Different Medium Chain Triglycerides During a Metabolic Study Day

Background: Medium chain triglycerides (MCT) are ketogenic but the relationship between the change in plasma ketones and the change plasma medium chain fatty acids (MCFA)—octanoate, decanoate, or dodecanoate—after an oral dose of MCT is not well-known. An 8 h metabolic study day is a suitable model to assess the acute effects on plasma ketones and MCFA after a dose of tricaprylin (C8), tricaprin (C10), trilaurin (C12) or mixed MCT (C8C10).Objective: To assess in healthy humans the relationship between the change in plasma ketones, and octanoate, decanoate and dodecanoate in plasma total lipids during an 8 h metabolic study day in which a first 20 ml dose of the homogenized test oil is taken with breakfast and a second 20 ml dose is taken 4 h later without an accompanying meal.Results: The change in plasma acetoacetate, β-hydroxybutyrate and total ketones was highest after C8 (0.5 to 3 h post-dose) and was lower during tests in which octanoate was absent or was diluted by C10 in the test oil. The plasma ketone response was also about 2 fold higher without an accompanying meal (P = 0.012). However, except during the pure C10 test, the response of octanoate, decanoate or dodecanoate in plasma total lipids to the test oils was not affected by consuming an accompanying meal. Except with C12, the 4 h area-under-the-curve of plasma β-hydroxybutyrate/acetoacetate was 2–3 fold higher when no meal was consumed (P < 0.04).Conclusion: C8 was about three times more ketogenic than C10 and about six times more ketogenic than C12 under these acute metabolic test conditions, an effect related to the post-dose increase in octanoate in plasma total lipids

Cardiorenal ketone metabolism: a positron emission tomography study in healthy humans

Ketones are alternative energy substrates for the heart and kidney but no studies have investigated their metabolism simultaneously in both organs in humans. The present double tracer positron emission tomography (PET) study evaluated the organ distribution and basal kinetic rates of the radiolabeled ketone, 11C-acetoacetate (11C-AcAc), in the heart and kidney compared to 11C-acetate (11C-Ac), which is a well-validated metabolic radiotracer. Both tracers were highly metabolized by the left ventricle and the renal cortex. In the heart, kinetic rates were similar for both tracers. But in the renal cortex, uptake of 11C-Ac was higher compared to 11C-AcAc, while the reverse was observed for the clearance. Interestingly, infusion of 11C-AcAc led to a significantly delayed release of radioactivity in the renal medulla and pelvis, a phenomenon not observed with 11C-Ac. This suggests an equilibrium of 11C-AcAc with the other ketone, 11C-D-beta-hydroxybutyrate, and a different clearance profile. Overall, this suggests that in the kidney, the absorption and metabolism of 11C-AcAc is different compared to 11C-Ac. This dual tracer PET protocol provides the opportunity to explore the relative importance of ketone metabolism in cardiac and renal diseases, and to improve our mechanistic understanding of new metabolic interventions targeting these two organs

Kinetics of 13C-DHA before and during fish-oil supplementation in healthy older individuals

Background: Docosahexaenoic acid (DHA) kinetics appear to change with intake, which is an effect that we studied in an older population by using uniformly carbon-13–labeled DHA (13C-DHA). Objective: We evaluated the influence of a fish-oil supplement over 5 mo on the kinetics of 13C-DHA in older persons. Design: Thirty-four healthy, cognitively normal participants (12 men, 22 women) aged between 52 and 90 y were recruited. Two identical kinetic studies were performed, each with the use of a single oral dose of 40 mg 13C-DHA. The first kinetic study was performed before participants started taking a 5-mo supplementation that provided 1.4 g DHA/d plus 1.8 g eicosapentaenoic acid (EPA)/d (baseline); the second study was performed during the final month of supplementation (supplement). In both kinetic studies, blood and breath samples were collected ≤8 h and weekly over 4 wk to analyze 13C enrichment. Results: The time × supplement interaction for 13C-DHA in the plasma was not significant, but there were separate time and supplement effects (P < 0.0001). The area under the curve for plasma 13C-DHA was 60% lower while subjects were taking the supplement than at baseline (P < 0.0001). The uniformly carbon-13–labeled EPA concentration was 2.6 times as high 1 d posttracer while patients were taking the supplement as it was at baseline. The mean (±SEM) plasma 13C-DHA half-life was 4.5 ± 0.4 d at baseline compared with 3.0 ± 0.2 d while taking the supplement (P < 0.0001). Compared with baseline, the mean whole-body half-life was 61% lower while subjects were taking the supplement. The loss of 13C-DHA through β-oxidation to carbon dioxide labeled with carbon-13 increased from 0.085% of dose/h at baseline to 0.208% of dose/h while subjects were taking the supplement. Conclusions: In older persons, a supplement of 3.2 g EPA + DHA/d increased β-oxidation of 13C-DHA and shortened the plasma 13C-DHA half-life. Therefore, when circulating concentrations of EPA and DHA are increased, more DHA is available for β-oxidation. This trial was registered at clinicaltrials.gov as NCT01577004

Green Edge ice camp campaigns : understanding the processes controlling the under-ice Arctic phytoplankton spring bloom

The Green Edge initiative was developed to investigate the processes controlling the primary productivity and fate of organic matter produced during the Arctic phytoplankton spring bloom (PSB) and to determine its role in the ecosystem. Two field campaigns were conducted in 2015 and 2016 at an ice camp located on landfast sea ice southeast of Qikiqtarjuaq Island in Baffin Bay (67.4797∘ N, 63.7895∘ W). During both expeditions, a large suite of physical, chemical and biological variables was measured beneath a consolidated sea-ice cover from the surface to the bottom (at 360 m depth) to better understand the factors driving the PSB. Key variables, such as conservative temperature, absolute salinity, radiance, irradiance, nutrient concentrations, chlorophyll a concentration, bacteria, phytoplankton and zooplankton abundance and taxonomy, and carbon stocks and fluxes were routinely measured at the ice camp. Meteorological and snow-relevant variables were also monitored. Here, we present the results of a joint effort to tidy and standardize the collected datasets, which will facilitate their reuse in other Arctic studies

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity