Relations between membrane lipid structures and enzyme activities in mitochondria of oyster Crassostrea gigas

Tout d’abord considérés comme simples composants d’une barrière imperméable, il a été démontré que les lipides membranaires auraient en fait un rôle biologique bien plus important, pouvant modifier l’environnement des enzymes membranaires et moduler l’activité de ces dernières. Dans la thèse présentée ici, ces relations ont été étudiées dans les mitochondries de l’huître creuse Crassostrea gigas. Les bivalves subissent d’importants changements environnementaux et l’adaptation à ces changements peut passer par un remodelage des membranes, ce qui fait de ces animaux des modèles intéressants pour les études des relations entre la structure des membranes et les activités d’enzymes associées. Des huîtres ont été nourries en écloserie avec deux régimes d’algues monospécifiques, T-Iso et Chaetoceros gracilis, et un mélange équilibré de ces deux algues. Malgré d’importantes modifications de composition en acides gras induites par les différents régimes alimentaires, une grande stabilité des processus membranaires mitochondriaux a été observée. D’un autre côté, la comparaison entre des huîtres élevées en écloserie et des huîtres élevées dans leur milieu naturel a révélé d’importantes modifications de capacités mitochondriales, qui pourraient être liées à une modulation des classes de phospholipides et de leur insaturation. Ces différences ne peuvent pas s’expliquer par une influence des cycles tidaux dans la mesure où, malgré un changement de production d’ATP, l’activité des mitochondries a été montrée comme étant similaire chez les huîtres collectées en émersion et en immersion. L’homéostasie mitochondriale observée dans cette étude pourrait être un moyen pour les huîtres de faire face aux variations biotiques (disponibilité en nourriture) et abiotiques (disponibilité en oxygène) de l’environnement naturel de C. gigas, et de maintenir leurs fonctions physiologiques malgré ces variations.First considered as simple components of an impermeable barrier, it has been shown that membrane lipids would have a more important biological role. These lipids could modify the environment of membrane enzymes and modulate their activity. In this thesis, these relationships have been studied in mitochondria of the oyster Crassostrea gigas. Bivalves undergo major environmental changes and adaptation to these changes may require a membrane remodelling, which makes these animals interesting models to study the relationship between membrane structure and membrane processes. In this study oysters were fed in hatchery with two monospecific algal diets, T-Iso and Chaetoceros gracilis, and an equilibrated mix of both algae. Despite significant changes in fatty acid composition induced by these diets, mitochondrial capacities remained stable. On the other hand, the comparison between hatchery-reared oysters and oysters reared in their natural environment revealed significant changes in mitochondrial capacity, which could be related to modulation of phospholipid class composition and unsaturation. These differences can not be explained by the influence of tidal cycles. Indeed, despite a change in ATP production, mitochondrial activity was shown to be similar in oysters collected during emersion and immersion.Mitochondrial homeostasis observed in this study could be a way for oysters to cope with biotic (food availability) and abiotic (oxygen availability) variations in the natural environment of C. gigas, and to maintain their physiological functions despite these variations

Relations entre la structure des lipides membranaires de mitochondries et l'activité d'enzymes associées chez l'huître creuse Crassostrea gigas

First considered as simple components of an impermeable barrier, it has been shown that membrane lipids would have a more important biological role. These lipids could modify the environment of membrane enzymes and modulate their activity. In this thesis, these relationships have been studied in mitochondria of the oyster Crassostrea gigas. Bivalves undergo major environmental changes and adaptation to these changes may require a membrane remodelling, which makes these animals interesting models to study the relationship between membrane structure and membrane processes. In this study oysters were fed in hatchery with two monospecific algal diets, T-Iso and Chaetoceros gracilis, and an equilibrated mix of both algae. Despite significant changes in fatty acid composition induced by these diets, mitochondrial capacities remained stable. On the other hand, the comparison between hatchery-reared oysters and oysters reared in their natural environment revealed significant changes in mitochondrial capacity, which could be related to modulation of phospholipid class composition and unsaturation. These differences can not be explained by the influence of tidal cycles. Indeed, despite a change in ATP production, mitochondrial activity was shown to be similar in oysters collected during emersion and immersion.Mitochondrial homeostasis observed in this study could be a way for oysters to cope with biotic (food availability) and abiotic (oxygen availability) variations in the natural environment of C. gigas, and to maintain their physiological functions despite these variations.Tout d’abord considérés comme simples composants d’une barrière imperméable, il a été démontré que les lipides membranaires auraient en fait un rôle biologique bien plus important, pouvant modifier l’environnement des enzymes membranaires et moduler l’activité de ces dernières. Dans la thèse présentée ici, ces relations ont été étudiées dans les mitochondries de l’huître creuse Crassostrea gigas. Les bivalves subissent d’importants changements environnementaux et l’adaptation à ces changements peut passer par un remodelage des membranes, ce qui fait de ces animaux des modèles intéressants pour les études des relations entre la structure des membranes et les activités d’enzymes associées. Des huîtres ont été nourries en écloserie avec deux régimes d’algues monospécifiques, T-Iso et Chaetoceros gracilis, et un mélange équilibré de ces deux algues. Malgré d’importantes modifications de composition en acides gras induites par les différents régimes alimentaires, une grande stabilité des processus membranaires mitochondriaux a été observée. D’un autre côté, la comparaison entre des huîtres élevées en écloserie et des huîtres élevées dans leur milieu naturel a révélé d’importantes modifications de capacités mitochondriales, qui pourraient être liées à une modulation des classes de phospholipides et de leur insaturation. Ces différences ne peuvent pas s’expliquer par une influence des cycles tidaux dans la mesure où, malgré un changement de production d’ATP, l’activité des mitochondries a été montrée comme étant similaire chez les huîtres collectées en émersion et en immersion. L’homéostasie mitochondriale observée dans cette étude pourrait être un moyen pour les huîtres de faire face aux variations biotiques (disponibilité en nourriture) et abiotiques (disponibilité en oxygène) de l’environnement naturel de C. gigas, et de maintenir leurs fonctions physiologiques malgré ces variations

Rapid mitochondrial adjustments in response to short-term hypoxia and re-oxygenation in the Pacific oyster, Crassostrea gigas.

International audienceAs oxygen concentrations in marine coastal habitats can fluctuate rapidly and drastically, sessile marine organisms such as the oyster Crassostrea gigas can experience marked and rapid oxygen variations. In this study, we investigated the responses of oyster gill mitochondria to short-term hypoxia (3 and 12 h, at 1.7 mg O2 l(-1)) and subsequent re-oxygenation. Mitochondrial respiratory rates (states 3 and 4 stimulated by glutamate) and phosphorylation efficiency [respiratory control ratio (RCR) and the relationship between ADP and oxygen consumption (ADP/O)] were measured. Cytochrome c oxidase (CCO) activity and cytochrome concentrations (a, b, c1 and c) were measured to investigate the rearrangements of respiratory chain subunits. The potential implication of an alternative oxidase (AOX) was investigated using an inhibitor of the respiratory chain (antimycin A) and through gene expression analysis in gills and digestive gland. Results indicate a downregulation of mitochondrial capacity, with 60% inhibition of respiratory rates after 12 h of hypoxia. RCR remained stable, while ADP/O increased after 12 h of hypoxia and 1 h of re-oxygenation, suggesting increased phosphorylation efficiency. CCO showed a fast and remarkable increase of its catalytic activity only after 3 h of hypoxia. AOX mRNA levels showed similar patterns in gills and digestive gland, and were upregulated after 12 and 24 h of hypoxia and during re-oxygenation. Results suggest a set of controls regulating mitochondrial functions in response to oxygen fluctuations, and demonstrate the fast and extreme plasticity of oyster mitochondria in response to oxygen variations

Functional capacities of gill mitochondria in oyster Crassostrea gigas during an emersion/immersion tidal cycle

Sessile animals that live on the foreshore undergo tidal cycles, and have to face variations in physical and chemical parameters such as oxygen concentration. During emersion, availability of dissolved oxygen can be lowered for bivalves, which have only a small reserve of seawater inside their closed shell. Differences in oxygen concentration are thus expected to lead to modifications of the metabolism, including changes in mitochondrial activity. Previous studies investigated air exposure under extreme conditions, which do not always reflect environmental conditions these invertebrates have to cope with. In this study, oxidative capacities of gill mitochondria of the oyster Crassostrea gigas were studied during a tidal cycle period, by comparing oysters collected after emersion and immersion. Only minor differences were found in state 3 (oxidative phosphorylation) or state 4 (non-phosphorylating oxygen consumption) rates between the two conditions. Similarly, no difference was observed in cytochrome c oxidase activity or in oxygen consumption related to maximal electron flux through complexes I-IV, II-IV and IV. While capacities of substrate oxidation were maintained in both emersion and immersion conditions, capacity of mitochondria to produce adenosine triphosphate (ATP) was significantly lower in oysters sampled during emersion. These results suggest that although C. gigas could maintain aerobic metabolism during emersion period within a tidal cycle in its environment, energy producing mechanisms are affected

Mitochondrial activity, hemocyte parameters and lipid composition modulation by dietary conditioning in the Pacific oyster Crassostrea gigas.

International audienceSeveral parameters can affect membrane lipid composition in bivalves, including diet. Although two fatty acids (FA) 22:6n-3 and 20:5n-3 are essential membrane components, they are sparingly synthesized by bivalves and must be obtained from their diet. Here, effects of dietary modifications of membrane lipid composition were studied at both cellular and subcellular levels in the oyster Crassostrea gigas. To this end, we compared oysters fed two monoalgal diets that differed markedly in their FA composition and a mix of both. As expected, algae impacted phospholipids, in particular 22:6n-3 and 20:5n-3, reflecting differences of dietary microalgae FA composition. Meantime, total saturated FA, total monounsaturated FA, total polyunsaturated FA and total non-methylene-interrupted FA varied little and phospholipid class composition was only slightly affected by diets. Measures made in hemocytes indicated that only mitochondrial membrane potential was affected by diets. Total ROS production as well as mitochondrial superoxide production did not differ with diet. There was no difference in phosphorylating (state 3) and non-phosphorylating (state 4) rates of oxygen consumption rates or in cytochrome c oxidase activity of mitochondria isolated from gills between the three diets. Similarly, neither cytochromes a, b, c or c 1 content nor citrate synthase activities were changed, suggesting that number and morphology of mitochondria were not affected by dietary treatment. These results suggest that oysters could possess high homeostatic capabilities, at both cellular and subcellular levels, to minimize the effect of dietary FA and related membrane lipid FA modifications on mitochondrial functions. These capabilities could be a means to face variations in diet composition in their natural environment and to preserve important oyster physiological functions such as growth and reproduction

Laboratory conditioning modifies properties of gills mitochondria from the Pacific oyster Crassostrea gigas

Although laboratory experiments allow greater control of environmental conditions than field studies, they have several drawbacks. To analyze physiological responses to forcing environmental variables, experimental conditions should mimic natural conditions as closely as possible. For filter-feeding organisms in particular, diet quality and quantity is one of the environmental parameters that can differ markedly between experimental and field conditions. In the hatchery, Pacific oysters, Crassostrea gigas, commonly show good physiological performance and growth on a mixed algal diet of Tisochrysis lutea, formerly Isochrysis aff. galbana clone Tahiti (T-Iso), and Chaetoceros calcitrans, presumably as it provides a good supply of essential polyunsaturated fatty acids (PUFA) as 20:4n-6, 20:5n-3 and 22:6n-3. The present study tests whether the fluctuating biotic and abiotic conditions in the field modify the structure and function of oyster mitochondria. One group of oysters was maintained in the intertidal zone, and the other group was fed the mixed diet in a nearby experimental hatchery under salinity and temperature conditions equivalent to those in the field. After 4 weeks of conditioning, the functional capacities and membrane lipid composition of gill mitochondria were measured. For essential polyunsaturated fatty acids, only the proportion of 20:5n-3 differed between field and laboratory oysters, and confirmed the capacity of the mixed diet T-Iso + C. gracilis, to provide the essential PUFA. Nevertheless, proportions of other FA (e.g., 22:5n-6 and non-methylene-interrupted FA) differed markedly between laboratory and field-conditioned oysters. Mitochondrial oxygen uptake, cytochrome c oxidase activity, content of cardiolipin and concentration of cytochrome b were significantly lower in laboratory-conditioned than in field-conditioned oysters. These results indicate that laboratory conditioning, although allowing similar growth and gonad maturation, only partially mimics conditions that allow C. gigas to maintain mitochondrial function. Although our experimental design cannot ascertain what difference between experimental laboratory and field conditions led to changes in membrane composition and mitochondrial function, differences in nutritional quality (other than known essential PUFA) and abiotic factors (e.g., oxygen availability, emersion or daily temperature fluctuations) had a major impact on mitochondrial properties in oysters

Influence of the Duplication of CFTR Exon 9 and Its Flanking Sequences on Diagnosis of Cystic Fibrosis Mutations

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact, present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions

Ibuprofen-loaded poly(epsilon-caprolactone) layered silicate nanocomposites prepared by hot melt extrusion

Ibuprofen loaded poly(epsilon-caprolactone) (PCL) layered silicate nanocomposites were prepared by hot-melt extrusion. The morphology and extent of dispersion of ibuprofen and layered silicate was studied using a combination of wide-angle X-ray diffraction (WAXD), field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM). Exhaustive examination across the length scales revealed the composite to have both an intercalated and exfoliated morphology. The ibuprofen was well dispersed and distributed throughout the PCL matrix. Most significantly, the static tensile and dynamic mechanical properties of PCL can be manipulated as a function of nanoclay loading and is dependent on the aspect ratio of clay platelets. The glass transition of PCL increased by up to 16A degrees C on addition of nanoclay, as determined from dynamic mechanical thermal analysis (DMTA). This behaviour was attributed to the constrained mobility of PCL chains intercalated between clay platelets and to the tethering of PCL chains by hydrogen bonding with platelet edges. As a consequence, PCL crystallisation was inhibited and confirmed from non-isothermal crystallisation experiments using differential scanning calorimetry (DSC). The fraction of PCL that was crystalline (X-c) decreased by 15% on addition of ibuprofen and nanoclay, although the temperature of crystallisation (T-c) did not change significantly. The dissolution of ibuprofen from PCL can be retarded by addition of layered silicates (nanoclays) to the polymer matrix