Éditeur(s) : HAL CCSD American Society for Biochemistry and Molecular Biology
Résumé : International audience
Background: We used evolutionary analysis of alkaline phosphatase (tissue nonspecific alkaline phosphatase, TNSALP) to predict missense mutations leading to hypophosphatasia. Results: We found 469 sensitive positions and validated 99% of the 204 known mutations. Conclusion: It is a more powerful method than in silico models to validate missense mutations in TNSALP. Significance: Such an approach should be widely used to support genetic diagnostics. ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, ...). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction.
ISSN: 0021-9258

hal-01545479
https://hal.archives-ouvertes.fr/hal-01545479
DOI : 10.1074/jbc.M114.576843

Éditeur(s) : Université de la Méditerranée
Résumé : This study was supported by the Atlantic working site of the « Programme National d’Océanographie Côtière » (PNOC, PNEC since 1999). The objectives were (1) to check the existence and to understand the mechanisms of winter phytoplankton blooms in the bay of Biscay influenced by the Gironde waters (2) to determine the limiting nutrient of the primary production associated to these blooms and to follow the seasonal evolution of the limiting nutrient, (3) to understand the consequences of this early limitation on the structuration of algal populations in spring. Since phosphorus was supposed to be the major limiting nutrient of algal growth in the Gironde plume, the different forms of the phosphorus cycle as well as the bacterial compartment, central in this cycle, were also studied in term of stocks and fluxes. The sampling strategy consisted in three cruises in 1998, BIOMET 2 (January), BIOMET 3 (beginning of March), PEGASE (June) and six cruises in 1999, PLAGIA 1 to 6 (late February, late April, late May, late June, the middle of July and the beginning of October). Winter algal blooms, essentially diatoms, were actually observed both in the typical waters of the plume (S < 34.5) and at the limit of the plume and adjacent atlantic oceanic waters (S > 34.5) in the successives years 1998 and 1999. These blooms were initiated by the occurrence of short anticyclonic windows in winter, acting on physical processes which dominate the evolution and characteristics of the Gironde plume (runoff, wind direction and speed) and, ultimately, on the evolution of the depth of the mixed layer (haline stratification) and available light (decrease of turbidity). The algal growth was actually phosphorus limited in the typical waters of the plume at the end of the winter bloom, in spring and became nitrogen and phosphorus limited in summer until the first autumn gales restored the nutritive balance. More oceanic waters (S > 34.5) were probably N+P limited from the end of the winter-the beginning of spring to autumn. Phosphorus limitation of winter blooms, associated with girondine unbalanced nutritive supplies (high NO3/PO4 ratios) favoured the development of small cells at the beginning of spring and the later presence of spring blooms (June 1998, late May 1999) composed of pico and nanophytoplankton (nanoflagellates), instead of typical diatom spring blooms for temperate waters. This size reduction of algal communities is strongthened by the competition between phytoplankton and bacteria for the phosphate uptake, bacteria being more competitives at low concentrations and being phosphorus limited sometimes in spring. The decreasing size of algal cells probably acted on the structuration of the whole food web. The study of the different forms of phosphorus displayed the importance of the Dissolved Organic Phosphorus (DOP) pool. The potential biological lability and the capacity of algae and bacteria to use the DOP in spring when phosphate are undetectable was shown by the very high specific activities of alkaline phosphatases and the very rapid cycling of phosphate monoesters. According to the few estimations of actual phosphatasic activity and phosphate assimilation fluxes, the hydrolysis of DOP associated with the coupled assimilation of liberated phosphate would represent 91 to 99 % of the phosphorus fluxes into algal and bacterial cells when phosphate were exhausted. In such conditions DOP could satisfy most of microorganisms phosphorus needs.
Cette thèse s’inscrit dans le cadre du Chantier Atlantique du Programme National d’Océanographie Côtière (PNOC, PNEC depuis 1999). Les objectifs de ce travail étaient (1) de vérifier l’existence et comprendre le déterminisme de blooms phytoplanctoniques hivernaux dans les eaux du Golfe de Gascogne influencées par la Gironde, (2) de préciser le facteur limitant de la production primaire associée à ces blooms et de suivre l’évolution saisonnière de ce facteur et (3) de comprendre les conséquences de cette limitation précoce sur la structuration des populations algales au printemps. Le phosphore étant supposé être le principal élément limitant de la croissance algale dans le panache de la Gironde, les différentes composantes du cycle du phosphore ainsi que le compartiment bactérien, central dans ce cycle, ont été étudiés en terme de stock et de flux. La stratégie d’échantillonnage a consisté en trois campagnes en 1998, BIOMET 2 (janvier), BIOMET 3 (début mars), PEGASE (juin) et six campagnes en 1999, PLAGIA 1 à 6 (fin février, fin avril, fin mai, fin juin, mi-juillet et début octobre). Des efflorescences algales hivernales, constituées essentiellement de diatomées, ont effectivement été observées de façon récurrente dans les eaux typiques du panache (S < 34,5) et à la limite du panache et des eaux océaniques atlantiques adjacentes (S > 34,5) au cours des deux années successives 1998 et 1999. Ces blooms ont été engendrés par l’apparition de courtes fenêtres anticycloniques en période hivernale, ces fenêtres interférant sur les processus physiques qui prévalent sur l’évolution et les caractéristiques du panache de la Gironde (régime des débits, orientation et force du vent) et, de façon ultime, sur l’évolution de la profondeur de la couche de mélange (halostratification) et la disponibilité en lumière (diminution de la turbidité). La croissance algale était limitée par le phosphore dans les eaux typiques du panache à la fin du bloom hivernal, au printemps et devint limitée en azote et en phosphore en période estivale et ceci jusqu’à ce que les premières tempêtes automnales rétablissent l’équilibre nutritif des eaux. Les eaux à caractère plus océanique (S > 34,5) étaient plus probablement limitées par N+P dès la fin de l’hiverdébut du printemps et ce jusqu’à l’automne. La limitation des blooms hivernaux par le phosphore, associée à des apports nutritifs girondins déséquilibrés en phosphate (rapports NO3/PO4 élevés) a favorisé le développement de cellules de petite taille au début du printemps et la présence ultérieure de blooms printaniers (juin 1998, fin mai 1999) composés de pico- et de nanophytoplancton (nanoflagellés), blooms « atypiques » pour des eaux tempérées à cette saison. Cette structuration des communautés algales est renforcée par la compétition entre le phytoplancton et les bactéries pour l’acquisition du phosphate, les bactéries étant plus compétitives aux faibles teneurs en phosphate et pouvant être elles-mêmes limitées par le phosphore à certains moments du printemps. La diminution de taille des cellules algales a probablement une incidence sur la structure de taille de l’ensemble du réseau trophique. L’étude des différentes composantes du phosphore montre l’importance du pool de Phosphore Organique Dissous (POD), sa labilité biologique potentielle et la capacité des communautés algales et bactériennes à utiliser ce pool au printemps quand les phosphates sont indétectables (activités spécifiques des phosphatases alcalines très élevées, recyclage très rapide des monoesters de phosphate). Au vu des quelques estimations de l’activité phosphatasique réelle et des flux d’assimilation de phosphate, l’hydrolyse du POD associée à l’assimilation couplée du phosphate libéré représenterait 91 à 99 % des flux de phosphore vers les cellules algales et bactériennes, quand les phosphates du milieu sont déficients. Dans de telles conditions, le POD pourrait satisfaire l’essentiel des besoins des microorganismes en phosphore.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/00013/12463/9298.pdf
http://archimer.ifremer.fr/doc/00013/12463/

Éditeur(s) : Elsevier
Résumé : Previous studies conducted on the continental shelf in the Southeast Bay of Biscay influenced by Gironde waters (one of the two largest rivers on the French Atlantic coast) showed the occurrence of late winter phytoplankton blooms and phosphorus limitation of algal growth thereafter. In this context, the importance of dissolved organic phosphorus (DOP) for both algae and bacteria was investigated in 1998 and 1999 in terms of stocks and fluxes. Within the mixed layer, although phosphate decreased until exhaustion from winter to spring, DOP remained high and phosphate monoesters made up between I I to 65% of this pool. Total alkaline phosphatase activity (APA, V-max) rose gradually from winter (2-8 nM h(-1)) to late spring (100-400 nM h-1), which was mainly due to an increase in specific phytoplankton (from 0.02 to 3.0 nmol mu gC(-1) h(-1)) and bacterial APA (from 0.04 to 4.0 nmol mu gC(-1) h(-1)), a strategy to compensate for the lack of phosphate. At each season, both communities had equal competitive abilities to exploit DOP but, taking into account biomass, the phytoplankton community activity always dominated (57-63% of total APA) that of bacterial community (9-11%). The dissolved APA represented a significant contribution. In situ regulation of phytoplanktonic APA by phosphate (induction or inversely repression of enzyme synthesis) was confirmed by simultaneously conducted phosphate-enrichment bioassays. Such changes recorded at a time scale of a few days could partly explain the seasonal response of phytoplankton communities to phosphate depletion.
Journal of Experimental Marine Biology and Ecology (0022-0981) (Elsevier), 2005-05 , Vol. 318 , N. 2 , P. 213-225

Droits : 2005 Elsevier B.V. All rights reserved
http://archimer.ifremer.fr/doc/2005/publication-608.pdf
DOI:10.1016/j.jembe.2004.12.017
http://archimer.ifremer.fr/doc/00000/608/

Éditeur(s) : Co-tutelle Al Centro de Investigaciones Biológicas del Noroeste - Université de Bretagne occidentale
Résumé : The interest in probiotics for aquaculture is increasing rapidly. In the present work we evaluated the potential of some yeast to induce the larvae gut maturation in sea bass larvae Dicentrarchus labrax. The production of polyamines putrescine, spermidine and spermine was quantified in ten yeast strains using high performance liquid chromatography (HPLC). Afterwards, fluorescently labelled yeast adhesion to the intestine of sea bass larvae and the spotted sand bass (Paralabrax maculatofasciatus) was evaluated by fluorescent microscopy. Two of them, CBS 8339 strain (Debaryomyces hansenii) and X2180 strain (Saccharomyces cerevisiae) produce the polyamines of our interest. The same strains showed the best adhering capabilities to the sea bass and spotted sand bass intestine. Both strains were selected and introduced by pulverization into the diet of larvae. The larvae fed with CBS 8339 strain, increase survival in 8.3% but not in their weight. The secretion of trypsin and amylase as well as those from the brush border membrane: alkaline phosphatase, maltase and leucine aminopeptidase, are incresed at 27 days after hatching (dah). At 42 dah difference in the enzyme activity of the different groups does not exist, suggesting a maturation of the digestive system in early stages of larval development. A second experiment was carried out to know the effect of different levels of incorporation into the diet of CBS 8339 strain: 1.1% and 5.7%. The minimum level, 1.1% of yeast produce best growth and survival rates, as well as lower deformed larvae than those obtained by 5.7% and the control one. Both level of yeast inclusion favored the trypsin secretion and lipase activity at 26 dah. Whereas mRNA levels for trypsin and amylase were influenced by both yeast concentrations at 26 and 36 dah, which suggest a development of digestive capacity of larvae as a result of the presence of polyamines secreted by the yeast.
Les probiotiques ont été définis comme des préparations microbiennes vivantes utilisées comme additif alimentaire, et qui ont une action bénéfique sur l'animal hôte en améliorant la digestion et l'hygiène intestinale. L'application de tels traitements aux productions aquacoles suscite un intérêt qui s'accroît rapidement. Dans ce travail, nous avons étudié l'effet de souches de levures sur la maturation de l'intestin des larves du bar Dicentrarchus labrax. Ces souches produisent des polyamines qui sont impliquées dans la réplication et la différentiation cellulaire, ainsi que dans la synthèse des protéines et des acides nucléiques. Dix souches de levures ont été testées pour leur production de polyamines (putrescine, spermidine et spermine), et pour leur adhérence à la paroi intestinale du bar et la cabrilla (Paralabrax maculatofasciatus). La production des polyamines a été analysée en utilisant la chromatographie liquide à haute performance (HPLC). Les levures selectionées ont été marquées avec un composé fluorescent pour tester leur adhérence a l'intestin de larves de bar et de juvéniles de cabrilla. L'adhérence a pu ainsi être evaluée par observation microscopique en épifluorescence. Les souches CBS 8339 (Debaryomyces hansenii) et X2180 (Saccharomyces cerevisiae) ont été choisies pour leur forte production de spermidine et de spermine, respectivement, et pour leur capacité d'adhérence à l'intestin du bar et de la cabrilla. Les deux souches ont été ajoutées par pulvérisation au régime des larves. Nous avons observé une augmentation de 8.3% du taux de survie avec les larves alimentées avec la souche CBS 8339, mais le poids moyen de ces larves était inférieur à celui obtenu sans ajout de levure. Une augmentation de l'activité de la trypsine et de l'amylase, aussi bien que des activités enzymatiques dans la bordure en brosse, aminopeptidase, phosphatase alkaline et maltase, ont été observées dans les larves âgées de 27 jours alimentées avec la souche CBS 8339. Au jour 42, il n'existait plus de différence d'activité enzymatique entre les larves traitées ou non avec la levure. Ces résultats suggèrent que la maturation du système digestif a été accélérée chez les larves nourries avec la levure, la maturation étant achevée chez les larves de 42 jours, qu'elles aient reçu l'un ou l'autre régime. Une deuxième expérience a été effectuée pour étudier l'effet de différents niveaux d'incorporation dans le régime contenant 1,1% et 5,7% de levure CBS 8339. L'incorporation de 1,1% a induit une augmentation du poids moyen et du taux de survie des larves, ainsi qu'une réduction du taux de malformation de la colonne vertébrale, par rapport aux résultats obtenus avec 0 ou 5,7% de levure. Les deux niveaux d'incorporation de la levure ont favorisé l'activité de la trypsine et de la lipase au jour 26. Les niveaux d'ARNm codant pour la synthèse de la trypsine et de l'amylase ont été influencés par les deux doses de levure aux jours 26 et 36. Ces résultats suggèrent qu'une faible dose de levure améliore le développement et la capacité digestive des larves, et nous formulons l'hypothèse que cette efficacité est due à la production de polyamines in situ. Le rôle des polyamines est particulièrement important pendant les phases de forte croissance comme les stades larvaires, mais elles peuvent être toxiques à dose trop élevée. L'incorporation de levure dans l'aliment destiné aux larves semble donc une excellente méthode pour délivrer les polyamines chez l'animal hôte.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/2002/these-103.pdf
http://archimer.ifremer.fr/doc/00000/103/

Éditeur(s) : Elsevier
Résumé : Marine fish larvae undergo major morphological and cellular changes during the first month of life. The ontogeny of the gastrointestinal tract combines these two aspects of the larval development and is very interesting in that the timing of functional changes appears genetically hard-wired. The goal of this paper is to give an overview of the gastrointestinal development process in marine fish larvae, with particular attention to three species: sea bass; red drum; and sole, since the description of gut maturation in fish larvae was initiated during the last decade with these species. During the early stages, marine fish larvae exhibit particular digestive features. Concerning the exocrine pancreas, amylase expression decreases with age from the third week post-hatching in sea bass and red drum (approximately 400 degree days), whereas expression of other enzymes (trypsin, lipase, phospholipase A2...) increases until the end of the larva period. Moreover, secretory function of the exocrine pancreas progressively develops and becomes efficient after the third week of life. Concerning the intestine, enzymes of the enterocyte cytosol (in particular peptidase) have higher activity in young larvae than in older. Approximately in the fourth week of post-hatching development in sea bass, red drum and sole larvae, the cytosolic activities dramatically decline concurrently with a sharp increase in membranous enzyme activities of the brush border, such as alkaline phosphatase, aminopeptidase N, maltase... This process characterises the normal maturation of enterocytes in developing fish larvae and also in other vertebrates' species. The establishment of an efficient brush border membrane digestion represents the adult mode of digestion of enterocytes. This paper also describes the role of diet on the development of the gastrointestinal tract. Indeed, the maturational process of digestive enzyme can be enhanced, stopped, or delayed depending on the composition of the diet.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology (1532-0456) (Elsevier), 2001-12 , Vol. 130 , N. 4 , P. 477-487

Droits : 2001 Elsevier Science Inc. All rights reserved
http://archimer.ifremer.fr/doc/2001/publication-650.pdf
DOI:10.1016/S1532-0456(01)00274-5
http://archimer.ifremer.fr/doc/00000/650/

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT-and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.
ISSN: 1932-6203

hal-01142641
http://hal.upmc.fr/hal-01142641
http://hal.upmc.fr/hal-01142641/document
http://hal.upmc.fr/hal-01142641/file/Silvent_2013_Collagen.pdf
DOI : 10.1371/journal.pone.0057344