Exhaled nitric oxide in high-altitude pulmonary edema: role in the regulation of pulmonary vascular tone and evidence for a role against inflammation.

High-altitude pulmonary edema (HAPE) is a life-threatening condition occurring in predisposed subjects at altitudes above 2,500 m. It is not clear whether, in addition to hemodynamic factors and defective alveolar fluid clearance, inflammation plays a pathogenic role in HAPE. We therefore made serial measurements of exhaled pulmonary nitric oxide (NO), a marker of airway inflammation, in 28 HAPE-prone and 24 control subjects during high-altitude exposure (4,559 m). To examine the relationship between pulmonary NO synthesis and pulmonary vascular tone, we also measured systolic pulmonary artery pressure (Ppa). In the 13 subjects who developed HAPE, exhaled NO did not show any tendency to increase during the development of lung edema. Throughout the entire sojourn at high altitude, pulmonary exhaled NO was roughly 30% lower in HAPE-prone than in control subjects, and there existed an inverse relationship between Ppa and exhaled NO (r = -0.51, p < 0.001). These findings suggest that HAPE is not preceded by airway inflammation. Reduced exhaled NO may be related to altered pulmonary NO synthesis and/or transport and clearance, and the data in our study could be consistent with the novel concept that in HAPE-prone subjects, a defect in pulmonary epithelial NO synthesis may contribute to exaggerated hypoxic pulmonary vasoconstriction and in turn to pulmonary edema.

Cross-talk between glutamate and potassium regulation at the astrocyte membrane

Astrocytes play a central role in the brain by regulating glutamate and extracellular potassium concentrations ([K+]0), both released by neurons into the extracellular space during neuronal activity. Glutamate uptake is driven by the inwardly directed sodium gradient across the astrocyte membrane and involves the influx of three sodium ions and one proton and the efflux of one K+ ion per glutamate molecule. The glutamate transport induced rise in intracellular sodium stimulates the Na+/K+-ATPase which leads to significant energetic costs in astrocytes. To evaluate how these two fundamental functions of astrocytes, namely glutamate transport and K+ buffering, which are directly associated with neuronal activity, coexist and if they influence each other, in this thesis work we examined different cellular parameters of astrocytes. We therefore investigated the impact of altered [K+]0 on glutamate transporter activity. To assess this question we measured intracellular sodium fluctuations in mouse primary cultured astrocytes using dynamic fluorescence imaging. We found that glutamate uptake was tightly modulated both in amplitude and kinetics by [K+]0. Elevated [K+]0 strongly decreased glutamate transporter activity, with significant consequences on the cells energy metabolism. To ultimately evaluate potential effects of [K+]0 and glutamate on the astrocyte mitochondrial energy production we extended these studies by investigating their impact on the cytosolic and mitochondrial pH. We found that both [K+],, and glutamate strongly influenced cytosolic and mitochondrial pH, but in opposite directions. The effect of a simultaneous application of K+ and glutamate, however, did not fit with the arithmetical sum of each individual effects, suggesting that an additional non¬linear process is involved. We also investigated the impact of [K+]0 and glutamate transport, respectively, on intracellular potassium concentrations ([K+]0 in cultured astrocytes by characterizing and applying a newly developed Insensitive fluorescent dye. We observed that [K+]i followed [K+]0 changes in a nearly proportional way and that glutamate superfusion caused a reversible, glutamate-concentration dependent drop of [K+],, Our study shows the powerful influence of [K+]u on glutamate capture. These findings have strong implications for our understanding of the tightly regulated interplay between astrocytes and neurons in situations where [K+]0 undergoes large activity-dependent fluctuations. However, depending on the extent of K+ versus glutamate extracellular rise, energy metabolism in astrocytes will be differently regulated. Moreover, the novel insights obtained during this thesis work help understanding some of the underlying processes that prevail in certain pathologies of central nervous system, such as epilepsy and stroke. These results will possibly provide a basis for the development of novel therapeutic strategies. -- Les astrocytes jouent un rôle central dans le cerveau en régulant les concentrations de potassium (K+) et de glutamate, qui sont relâchés par les neurones dans l'espace extracellulaire lorsque ceux- ci sont actifs. La capture par les astrocytes du glutamate est un processus secondairement actif qui implique l'influx d'ions sodium (Na+) et d'un proton, ainsi que l'efflux d'ions K+, ce processus entraîne un coût métabolique important. Nous avons évalué comment ces fonctions fondamentales des astrocytes, la régulation du glutamate et du K+ extracellulaire, qui sont directement associés à l'activité neuronale, coexistent et si elles interagissent, en examinant différents paramètres cellulaires. Dans ce projet de thèse nous avons évalué l'impact des modifications de la concentration de potassium extracellulaire ([K+],,) sur le transport du glutamate. Nous avons mesuré le transport du glutamate par le biais des fluctuations internes de Na+ grâce à un colorant fluorescent en utilisant de l'imagerie à fluorescence dynamique sur des cultures primaires d'astrocytes. Nous avons trouvé que la capture du glutamate était étroitement régulée par [K+]0 aussi bien dans son amplitude que dans sa cinétique. Par la suite, nous avons porté notre attention sur l'impact de [K+]0 et du glutamate sur le pH cytosolique et mitochondrial de l'astrocyte dans le but, in fine, d'évaluer les effets potentiels sur la production d'énergie par la mitochondrie. Nous avons trouvé qu'autant le K+ que le glutamate, de manière individuelle, influençaient fortement le pH, cependant dans des directions opposées. Leurs effets individuels, ne peuvent toutefois pas être additionnés ce qui suggère qu'un processus additionnel non-linéaire est impliqué. En appliquant une nouvelle approche pour suivre et quantifier la concentration intracellulaire de potassium ([K+]0 par imagerie à fluorescence, nous avons observé que [K+]i suivait les changements de [K+]0 de manière quasiment proportionnelle et que la superfusion de glutamate induisait un décroissement rapide et réversible de [K+]i, qui dépend de la concentration de glutamate. Notre étude démontre l'influence de [K+]0 sur la capture du glutamate. Ces résultats permettent d'améliorer notre compréhension de l'interaction entre astrocytes et neurones dans des situations où [K+]0 fluctue en fonction de l'activité neuronale. Cependant, en fonction de l'importance de l'augmentation extracellulaire du K+ versus le glutamate, le métabolisme énergétique des astrocytes va être régulé de manière différente. De plus, les informations nouvelles que nous avons obtenues durant ce travail de thèse nous aident à comprendre quelques- uns des processus sous-jacents qui prévalent dans certaines pathologies du système nerveux central, comme par exemple l'épilepsie ou l'accident vasculaire cérébral. Ces informations pourront être importantes à intégrer dans la cadre du développement de nouvelles stratégies thérapeutiques.

CYP3A5 and ABCB1 genes influence blood pressure and response to treatment, and their effect is modified by salt.

The permeability-glycoprotein efflux-transporter encoded by the multidrug resistance 1 (ABCB1) gene and the cytochromes P450 3A4/5 encoded by the CYP3A4/5 genes are known to interact in the transport and metabolism of many drugs. Recent data have shown that the CYP3A5 genotypes influence blood pressure and that permeability-glycoprotein activity might influence the activity of the renin-angiotensin system. Hence, these 2 genes may contribute to blood pressure regulation in humans. We analyzed the association of variants of the ABCB1 and CYP3A5 genes with ambulatory blood pressure, plasma renin activity, plasma aldosterone, endogenous lithium clearance, and blood pressure response to treatment in 72 families (373 individuals; 55% women; mean age: 46 years) of East African descent. The ABCB1 and CYP3A5 genes interact with urinary sodium excretion in their effect on ambulatory blood pressure (daytime systolic: P=0.05; nighttime systolic and diastolic: P<0.01), suggesting a gene-gene-environment interaction. The combined action of these genes is also associated with postproximal tubular sodium reabsorption, plasma renin activity, plasma aldosterone, and with an altered blood pressure response to the angiotensin-converting enzyme inhibitor lisinopril (P<0.05). This is the first reported association of the ABCB1 gene with blood pressure in humans and demonstration that genes encoding for proteins metabolizing and transporting drugs and endogenous substrates contribute to blood pressure regulation.

Unraveling the complex network of cuticular structure and function.

A hydrophobic cuticle is deposited at the outermost extracellular matrix of the epidermis in primary tissues of terrestrial plants. Besides forming a protective shield against the environment, the cuticle is potentially involved in several developmental processes during plant growth. A high degree of variation in cuticle composition and structure exists between different plant species and tissues. Lots of progress has been made recently in understanding the different steps of biosynthesis, transport, and deposition of cuticular components. However, the molecular mechanisms that underlie cuticular function remain largely elusive.

Impaired expression of NADH dehydrogenase subunit 1 and PPARgamma coactivator-1 in skeletal muscle of ZDF rats: restoration by troglitazone.

Type 2 diabetes has been related to a decrease of mitochondrial DNA (mtDNA) content. In this study, we show increased expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) and its target genes involved in fatty acid metabolism in skeletal muscle of Zucker Diabetic Fatty (ZDF) (fa/fa) rats. In contrast, the mRNA levels of genes involved in glucose transport and utilization (GLUT4 and phosphofructokinase) were decreased, whereas the expression of pyruvate dehydrogenase kinase 4 (PDK-4), which suppresses glucose oxidation, was increased. The shift from glucose to fatty acids as the source of energy in skeletal muscle of ZDF rats was accompanied by a reduction of subunit 1 of complex I (NADH dehydrogenase subunit 1, ND1) and subunit II of complex IV (cytochrome c oxidase II, COII), two genes of the electronic transport chain encoded by mtDNA. The transcript levels of PPARgamma Coactivator 1 (PGC-1) showed a significant reduction. Treatment with troglitazone (30 mg/kg/day) for 15 days reduced insulin values and reversed the increase in PDK-4 mRNA levels, suggesting improved insulin sensitivity. In addition, troglitazone treatment restored ND1 and PGC-1 expression in skeletal muscle. These results suggest that troglitazone may avoid mitochondrial metabolic derangement during the development of diabetes mellitus 2 in skeletal muscle.

Fracture imaging within a granitic rock aquifer using multiple-offset single-hole and cross-hole GPR reflection data

The sparsely spaced highly permeable fractures of the granitic rock aquifer at Stang-er-Brune (Brittany, France) form a well-connected fracture network of high permeability but unknown geometry. Previous work based on optical and acoustic logging together with single-hole and cross-hole flowmeter data acquired in 3 neighbouring boreholes (70-100 m deep) has identified the most important permeable fractures crossing the boreholes and their hydraulic connections. To constrain possible flow paths by estimating the geometries of known and previously unknown fractures, we have acquired, processed and interpreted multifold, single- and cross-hole GPR data using 100 and 250 MHz antennas. The GPR data processing scheme consisting of timezero corrections, scaling, bandpass filtering and F-X deconvolution, eigenvector filtering, muting, pre-stack Kirchhoff depth migration and stacking was used to differentiate fluid-filled fracture reflections from source generated noise. The final stacked and pre-stack depth-migrated GPR sections provide high-resolution images of individual fractures (dipping 30-90°) in the surroundings (2-20 m for the 100 MHz antennas; 2-12 m for the 250 MHz antennas) of each borehole in a 2D plane projection that are of superior quality to those obtained from single-offset sections. Most fractures previously identified from hydraulic testing can be correlated to reflections in the single-hole data. Several previously unknown major near vertical fractures have also been identified away from the boreholes.

Genetic and phenotypic population divergence on a microgeographic scale in brown trout.

Salmonid populations of many rivers are rapidly declining. One possible explanation is that habitat fragmentation increases genetic drift and reduces the populations' potential to adapt to changing environmental conditions. We measured the genetic and eco-morphological diversity of brown trout (Salmo trutta) in a Swiss stream system, using multivariate statistics and Bayesian clustering. We found large genetic and phenotypic variation within only 40 km of stream length. Eighty-eight percent of all pairwise F(ST) comparisons and 50% of the population comparisons in body shape were significant. High success rates of population assignment tests confirmed the distinctiveness of populations in both genotype and phenotype. Spatial analysis revealed that divergence increased with waterway distance, the number of weirs, and stretches of poor habitat between sampling locations, but effects of isolation-by-distance and habitat fragmentation could not be fully disentangled. Stocking intensity varied between streams but did not appear to erode genetic diversity within populations. A lack of association between phenotypic and genetic divergence points to a role of local adaptation or phenotypically plastic responses to habitat heterogeneity. Indeed, body shape could be largely explained by topographic stream slope, and variation in overall phenotype matched the flow regimes of the respective habitats.

Metabolic Flux and Compartmentation Analysis in the Brain In vivo.

Through significant developments and progresses in the last two decades, in vivo localized nuclear magnetic resonance spectroscopy (MRS) became a method of choice to probe brain metabolic pathways in a non-invasive way. Beside the measurement of the total concentration of more than 20 metabolites, (1)H MRS can be used to quantify the dynamics of substrate transport across the blood-brain barrier by varying the plasma substrate level. On the other hand, (13)C MRS with the infusion of (13)C-enriched substrates enables the characterization of brain oxidative metabolism and neurotransmission by incorporation of (13)C in the different carbon positions of amino acid neurotransmitters. The quantitative determination of the biochemical reactions involved in these processes requires the use of appropriate metabolic models, whose level of details is strongly related to the amount of data accessible with in vivo MRS. In the present work, we present the different steps involved in the elaboration of a mathematical model of a given brain metabolic process and its application to the experimental data in order to extract quantitative brain metabolic rates. We review the recent advances in the localized measurement of brain glucose transport and compartmentalized brain energy metabolism, and how these reveal mechanistic details on glial support to glutamatergic and GABAergic neurons.

Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects.

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.

Caution on the storage of waters and aqueous solutions in plastic containers for hydrogen and oxygen stable isotope analysis

RATIONALE The choice of containers for storage of aqueous samples between their collection, transport and water hydrogen (2H) and oxygen (18O) stable isotope analysis is a topic of concern for a wide range of fields in environmental, geological, biomedical, food, and forensic sciences. The transport and separation of water molecules during water vapor or liquid uptake by sorption or solution and the diffusive transport of water molecules through organic polymer material by permeation or pervaporation may entail an isotopic fractionation. An experiment was conducted to evaluate the extent of such fractionation. METHODS Sixteen bottle-like containers of eleven different organic polymers, including low and high density polyethylene (LDPE and HDPE), polypropylene (PP), polycarbonate (PC), polyethylene terephthalate (PET), and perfluoroalkoxy-Teflon (PFA), of different wall thickness and size were completely filled with the same mineral water and stored for 659?days under the same conditions of temperature and humidity. Particular care was exercised to keep the bottles tightly closed and prevent loss of water vapor through the seals. RESULTS Changes of up to +5 parts per thousand for d2H values and +2.0 parts per thousand for d18O values were measured for water after more than 1?year of storage within a plastic container, with the magnitude of change depending mainly on the type of organic polymer, wall thickness, and container size. The most important variations were measured for the PET and PC bottles. Waters stored in glass bottles with Polyseal (TM) cone-lined PP screw caps and thick-walled HDPE or PFA containers with linerless screw caps having an integrally molded inner sealing ring preserved their original d2H and d18O values. The carbon, hydrogen, and oxygen stable isotope compositions of the organic polymeric materials were also determined. CONCLUSIONS The results of this study clearly show that for precise and accurate measurements of the water stable isotope composition in aqueous solutions, rigorous sampling and storage procedures are needed both for laboratory standards and for unknown samples. Copyright (c) 2012 John Wiley & Sons, Ltd.

Effects of mineralocorticoid and K+ concentration on K+ secretion and ROMK channel expression in a mouse cortical collecting duct cell line.

The cortical collecting duct (CCD) plays a key role in regulated K(+) secretion, which is mediated mainly through renal outer medullary K(+) (ROMK) channels located in the apical membrane. However, the mechanisms of the regulation of urinary K(+) excretion with regard to K(+) balance are not well known. We took advantage of a recently established mouse CCD cell line (mCCD(cl1)) to investigate the regulation of K(+) secretion by mineralocorticoid and K(+) concentration. We show that this cell line expresses ROMK mRNA and a barium-sensitive K(+) conductance in its apical membrane. As this conductance is sensitive to tertiapin-Q, with an apparent affinity of 6 nM, and to intracellular acidification, it is probably mediated by ROMK. Overnight exposure to 100 nM aldosterone did not significantly change the K(+) conductance, while it increased the amiloride-sensitive Na(+) transport. Overnight exposure to a high K(+) (7 mM) concentration produced a small but significant increase in the apical membrane barium-sensitive K(+) conductance. The mRNA levels of all ROMK isoforms measured by qRT-PCR were not changed by altering the basolateral K(+) concentration but were decreased by 15-45% upon treatment with aldosterone (0.3 or 300 nM for 1 and 3 h). The paradoxical response of ROMK expression to aldosterone could possibly work as a preventative mechanism to avoid excessive K(+) loss which would otherwise result from the increased electrogenic Na(+) transport and associated depolarization of the apical membrane in the CCD. In conclusion, mCCD(cl1) cells demonstrate a significant K(+) secretion, probably mediated by ROMK, which is not stimulated by aldosterone but increased by overnight exposure to a high K(+) concentration.

Dispersion of natural arsenic in the Malcantone watershed, Southern Switzerland: field evidence for repeated sorption-desorption and oxidation-reduction processes

In recent years, elevated arsenic concentrations have been found in waters and soils of many, countries, often resulting in a health threat for the local population. Switzerland is not an exception and this paper deals with the release and subsequent fate of arsenic in a 200-km(2) mountainous watershed, characterized by crystalline silicate rocks (gneisses, schists, amphibolites) that contain abundant As-bearing sulfide ore deposits, some of which have been mined for iron and gold in the past. Using analytical methods common for mineralogical, ground water and soil studies (XRD, XRF, XAS-XANES and -EXAFS, electron microprobe, extraction, ICP, AAS with hydride generator, ion chromatography), seven different field situations and related dispersion processes of natural arsenic have been studied: (1) release by rock weathering, (2) transport and deposition by water and ice; (3) release of As to the ground and surface water due to increasing pH; (4) accumulation in humic soil horizons; (5) remobilization by reduction in water-saturated soils and stagnant ground waters; (6) remobilization by using P-rich fertilizers or dung and (7) oxidation, precipitation and dilution in surface waters. Comparison of the results with experimental adsorption studies and speciation diagrams from the literature allows us to reconstruct and identify the typical behavior of arsenic in a natural environment under temperate climatic conditions. The main parameters identified are: (a) once liberated from the primary minerals, sorption processes on Fe-oxy-hydroxides dominate over Al-phases, such as Al-hydroxides or clay minerals and limit the As concentrations in the spring and well waters between 20 and 300 mug/l. (b) Precipitation as secondary minerals is limited to the weathering domain, where the As concentrations are still high and not yet too diluted by rain and soils waters. (c) Although neutral and alkaline pH conditions clearly increase the mobility of As, the main factor to mobilize As is a low redox potential (Eh close or below 0 mV), which favors the dissolution of the Fe-oxy-hydroxides on which the As is sorbed. (d) X-ray absorption spectroscopy (XAS) of As in water-logged humic forest soils indicates that the reduction to As III only occurs at the solid-water interface and that the solid contains As as As V (e) A and Bh horizons of humic cambisols can effectively capture As when As-rich waters flow through them. Complex spatial and temporal variation of the various parameters in a watershed results in repeated mobilization and immobilization of As, which continuously transports As from the upper to the lower part of a watershed and ultimately to the ocean. (C) 2004 Elsevier B.V. All rights reserved.

Characterization of PHO1 and PHO1;H1 gene function in Arabidopsis thaliana

Summary Phosphorus is one of the major macronutrients required for plant growth and development. Plant roots acquire phosphorus as inorganic phosphate (Pi), which is further distributed to the shoot, via the transpiration stream and root pressure, where Pi is imported again into cells. PHO1 in Arabidopsis has been identified as a protein involved in the loading of Pi into the root xylem. PHO1 does not have any homology to described Pi transporters including the Pht1 family of H+/ Pi cotransporters. PHO1 bears two domains, SPX and EXS domains, previously identified in Saccharomyces cerevisiae proteins involved in Pi transport and/or sensing, or in sorting proteins to endomembranes. Phylogenetic analysis of the PHO1 gene family revealed the presence of three clusters, with PHO1 and PHO1;H1 forming one cluster. The biological significance behind this cluster was demonstrated by the complementation of the pho1 mutant with only PHO1 and PHO1;H1, of all the PHO1 family members, when expressed under the PHO1 promoter. PHO1 has been shown to be expressed mostly in the root vascular cylinder and at low level in the shoot. PHO1;H1 had a different expression pattern, being expressed in both root and shoot vascular cylinder to the same level, with the levels in leaves increasing with the leaf maturity, suggesting additional role of PHO1;H1 in the Pi mobilization in leaves. In order to further explore the role of PHO1, Pi dynamics was studied on plants expressing PHO1 at different levels compared to the wild type: PHO1 overexpressors, PHO1 underexpressors and the pho1 mutant. Overexpression of the PHO1 protein in the shoot vascular tissue was shown to lead to increased Pi efflux out of the leaf cells and Pi accumulation in the shoot xylem apoplast compared to wild type, confirming the hypothesized role of PHO1 in xylem loading with Pi. The overexpression of PHO1 in the shoot was responsible far both changed Pi dynamic and stunted growth of PHO1 overexpressors, as shown by grafting experiments between wild type and PHO1 overexpressor. We found a ca. 2 fold decrease of shoot phosphorus and a 5-10 fold decrease in vacuolar Pi content in the PHO1 underexpressors and the pho1 null mutant compared to wild type, consistent with the role of PHO1 in the transfer of Pi from the root to the shoot. Shoot Pi deficiency results in a poor growth of the pho1 mutant. Grafting experiments between pho1 and wild type confirmed that both Pi deficiency and stunt growth of the pho1 mutant were dependent on the pho1 root, further supporting the importance of PHO1 in the root xylem loading with Pi. The pho1 mutant and the PHO1 underexpressors accumulated 8-15 fold more Pi in the root relative to wild type. In contrast to the pho1 mutant, the growth of PHO1 underexpressors was not impaired by the low shoat Pi content. This finding suggests that either PHO1 protein or root Pi concentration is important in Pi signaling and development of Pi deficiency symptoms leading to reduced growth. Résumé Le phosphore est l'un des nutriments essentiels à la croissance et au développement des plantes. Les racines absorbent le phosphore sous forme de phosphate inorganique (Pi) qui est dirigé, par la transpiration et la pression de la racine, vers les feuilles où le phosphate est acquis par les cellules. La protéine PHO1 a été démontrée indispensable au chargement du Pi dans le xylème des racines d'Arabidopsis. PHO1 ne démontre pas d'homologie aux transporteurs de Pi connus, incluant la famille Pht1 de cotransporteurs H+/Pi qui ont comme fonction le transport du phosphate à l'intérieur de la cellule. PHO1 contient deux domaines, SPX et EXS, aussi présents dans des protéines de Saccharomyces cerevisiae impliquées dans le transport ou la perception du phosphate, ou dans la localisation des protéines vers différentes membranes. Le génome d'Arabidopsis contient onze gènes homologues à PHO1. Neuf de ces homologues sont répartis en trois groupes. PHO1 et PHO1;H1 forment un de ces groupes. Nos travaux ont démontré que seuls PHO1;H1 et PHO1, sous contrôle du promoteur PHO1, peuvent complémenter le mutant pho1. PHO1 est exprimé principalement dans le cylindre vasculaire de la racine et faiblement dans la partie aérienne. Le degré d'expression de PHO1;H1 est similaire dans le cylindre vasculaire de la racine et des feuilles. Ceci suggère que PHO1;H1 est aussi impliqué dans la mobilisation du Pi dans les feuilles, en plus de son rôle dans le transfert du Pi dans le xylème des racines. Afin de mieux explorer le rôle de PHO1, la dynamique du phosphate a été observée dans trois lignées de plantes transgéniques: un sur-expresseur de PHO1, un sous-expresseur de PHO1 et le mutant pho1. La sur-expression de PHO1 dans le tissue vasculaire des feuilles a provoqué l'efflux du Pi vers l'espace apoplastic du xylème, ce qui confirme le rôle de PHO1 dans le chargement du Pi dans le xylème. La sur-expressìon de PHO1 dans la rosette est responsable d'un changement de la dynamique du Pi et de la diminution de la croissance, ce qui fut démontré par une expérience de greffe de la rosette du sur-expresseur de PHO1 sur les racines du sauvage. On a observé pour le sous-expresseur de PHO1 et le mutant pho1 une diminution du phosphore d'environ 2 fais au niveau des feuilles, et une diminution de 5-10 fois du Pi dans les vacuoles des feuilles, par rapport au sauvage. Ceci confirme le rôle proposé de PHO1 dans le transfert du Pi des racines aux feuilles. La carence de Pi chez pho1 implique une diminution de la taille de la rosette. Pour expliquer ce phénotype une autre expérience de greffe démontra que la cause de ce changement provenait des racines. Ceci renforce l'hypothèse de l'importance du rôle de PHO1 dans le xylème de la racine pour le chargement du Pi. Le mutant phot et le sous-expresseur de PHO1 accumulent 8-15 fois plus de Pi dans leurs racines comparé au sauvage. Cependant, contrairement au phot mutant, le sous-expresseur de PHO1 avait une croissance comparable au sauvage malgré le niveau bas du Pi dans les feuilles. Ceci suggère que la taille de la rosette lors d'une carence en Pi chez Arabidopsis serait la conséquence d'un changement de concentration de Pi dans les racines ou d'une influence de la protéine PHO1.

PH01 gene family in Arabidopsis thaliana and Physcomitrella patens

Summary Inorganic phosphate (Pi) is a main limiting nutrient to the growth and production yield of plants in many agro-ecosystems. Plants have evolved a series of metabolic and developmental adaptations to cope with low Pi availability. PH01 has been identified as a protein involved in the loading of Pi into the xylem of roots in Arabidopsis. In this study, the PHO1 gene family in both higher plant Arabidopsis and lower plant Physcomitrella was characterized. Additional ten PHO1 homologues in Arabidopsis and three homologues in Physcomitrella were identified. All proteins harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the highly conserved C-terminal hydrophobic portion. RT-PCR analysis of the Arabidopsis PHO1 genes revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers, indicating their potential roles not only in Pi transport but also in Pi homeostasis within the Arabidopsis plant. The regulation of gene expression by different nutrient-starvations showed that some genes are strongly up-regulated by elements other than Pi, e.g. by NO3, Mg, and Zn starvation. Northern blot and RT-PCR analysis showed distinct expression patterns of the three Physcomitrella PHO1 genes. The investigation of Pi starvation effects on some Pi-deprivation responsive genes demonstrates that Physcomitrella has evolved a similar mechanism as higher plants to respond to Pi deficiency. Promoter activity analysis for the Physcomitrella PHO1 family genes using promoter-GUS fusions revealed their expression in protonemata and gametophores but at different levels and with different patterns, suggesting these genes may play distinct roles in Pi transport and/or Pi homeostasis in the moss plant. Single knockout mutants of the three genes were generated by gene targeting and one of them displayed a reduced Pi content in the protonemata under Pi starvation. The evolution of the PHO1 family in land plants was also discussed. Together, these findings indicate that the PHO1 family genes, present in a broad range of plant species from lower plants to flowering plants, play important roles in Pi transport and homeostasis. Résumé Le phosphate inorganique (Pi) est un nutriment essentiel à la croissance des plantes et au rendement de la production végétale. Dans beaucoup d'agro-écosystèmes, ce nutriment est limitant. Les plantes ont développé des adaptations métaboliques et développementales pour palier à la faible disponibilité du Pi. Il a été démontré que la protéine PHOI est indispensable au transfert du Pi dans le xylème des racines d' Arabidopsis. Cette étude porte sur la famille de gènes définie par PHO1 ; ceci, dans deux organismes modèles : la plante Arabidopsis pour les végétaux supérieurs, et la mousse Physcomitrella pour les végétaux inférieurs. Dix homologues à PHOI dans Arabidopsis et trois homologues dans Physcomitrella ont été identifiés. Toutes les protéines encodées présentent un domaine tripartite SPX dans leur partie N terminale hydrophile et un domaine EXS dans la partie C terminale hydrophobe hautement conservée d'entre eux. L'analyse par RT-PCR de l'expression des gènes PHO1 dans Arabidopsis révèle une expression ectopique pour la plupart, à l'exception de deux gènes dont l'expression est uniquement florale ; ceci suggère l'implication de cette famille non seulement dans le transport mais aussi dans l'homéostasie du Pi dans Arabidopsis. L'observation de l'expression de ces gènes en fonction de l'absence de différents nutriments montre que certains gènes sont fortement régulés lors de carences en NO3, Mg et Zn. L'analyse par northern blot et RT-PCR met en évidence des profils d'expression distincts pour les trois gènes de Physcomitrella. Les effets de la carence en Pi sur Physcomitrella ont été étudiés par le biais de gènes dépendants connus pour Arabidopsis, les résultats suggèrent un mode de réponse à cette carence conservé entre les végétaux inférieurs et supérieurs. La localisation tissulaire de l'expression de la famille PHO1 dans la mousse a été étudiée au moyen du gène rapporteur GUS fusionné aux différents promoteurs. Ceci a révélé leur expression dans les protonemata et les gametophores, mais à des intensités et avec des profils différents, ce qui suggère des implications distinctes dans le transport et/ou l'homéostasie du Pi dans la mousse. Des simples mutants knockout ont été générés pour chaque gène de mousse ; l'un d'eux présente une diminution du contenu protonemal en Pi lorsque soumis à une carence en Pi. L'évolution de la famille PHO1 dans les plantes terrestres est également discutée. Ensemble, ces résultats indiquent que les gènes de la famille PHO1 sont présents dans une large gamme de plantes allant des végétaux inférieurs aux supérieurs, et cette étude démontre que leur conservation se justifie potentiellement par le fait qu'ils sont probablement impliqués dans des mécanismes conservés de transport et d'homéostasie du Pi.

Pattern recognition ecological niche models fit to presence-only and presence-absence data

1. Identifying the boundary of a species' niche from observational and environmental data is a common problem in ecology and conservation biology and a variety of techniques have been developed or applied to model niches and predict distributions. Here, we examine the performance of some pattern-recognition methods as ecological niche models (ENMs). Particularly, one-class pattern recognition is a flexible and seldom used methodology for modelling ecological niches and distributions from presence-only data. The development of one-class methods that perform comparably to two-class methods (for presence/absence data) would remove modelling decisions about sampling pseudo-absences or background data points when absence points are unavailable. 2. We studied nine methods for one-class classification and seven methods for two-class classification (five common to both), all primarily used in pattern recognition and therefore not common in species distribution and ecological niche modelling, across a set of 106 mountain plant species for which presence-absence data was available. We assessed accuracy using standard metrics and compared trade-offs in omission and commission errors between classification groups as well as effects of prevalence and spatial autocorrelation on accuracy. 3. One-class models fit to presence-only data were comparable to two-class models fit to presence-absence data when performance was evaluated with a measure weighting omission and commission errors equally. One-class models were superior for reducing omission errors (i.e. yielding higher sensitivity), and two-classes models were superior for reducing commission errors (i.e. yielding higher specificity). For these methods, spatial autocorrelation was only influential when prevalence was low. 4. These results differ from previous efforts to evaluate alternative modelling approaches to build ENM and are particularly noteworthy because data are from exhaustively sampled populations minimizing false absence records. Accurate, transferable models of species' ecological niches and distributions are needed to advance ecological research and are crucial for effective environmental planning and conservation; the pattern-recognition approaches studied here show good potential for future modelling studies. This study also provides an introduction to promising methods for ecological modelling inherited from the pattern-recognition discipline.