CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

EFFECT OF ALTERNATIVE MULTINUTRIENT SOURCES ON SOIL CHEMICAL PROPERTIES

The current high price of potassium chloride and the dependence of Brazil on imported materials to supply the domestic demand call for studies evaluating the efficiency of alternative sources of nutrients. The aim of this work was to evaluate the effect of silicate rock powder and a manganese mining by-product, and secondary materials originated from these two materials, on soil chemical properties and on brachiaria production. This greenhouse experiment was conducted in pots with 5 kg of soil (Latossolo Vermelho-Amarelo distrófico - Oxisol). The alternative nutrient sources were: verdete, verdete treated with NH4OH, phonolite, ultramafic rock, mining waste and the proportion of 75 % of these K fertilizers and 25 % lime. Mixtures containing 25 % of lime were heated at 800 ºC for 1 h. These sources were applied at rates of 0, 150, 300, 450 and 600 kg ha-1 K2O, and incubated for 45 days. The mixtures of heated silicate rocks with lime promoted higher increases in soil pH in decreasing order: ultramafic rock>verdete>phonolite>mining waste. Applying the mining waste-lime mixture increased soil exchangeable K, and available P when ultramafic rock was incorporated. When ultramafic rock was applied, the release of Ca2+ increased significantly. Mining subproduct released the highest amount of Zn2+ and Mn2+ to the soil. The application of alternative sources of K, with variable chemical composition, altered the nutrient availability and soil chemical properties, improving mainly plant development and K plant uptake, and are important nutrient sources.

NUTRIENT DEMAND BY THE CARROT CROP IS INFLUENCED BY THE CULTIVAR

Farmers must carefully choose the cultivar to be grown for a successful carrot crop. The yield potential of the cultivar may influence nutrient demand and should be known to plan for fertilization application. The aim of this study was to evaluate the cultivar effect on carrot yield and on the nutrient content and quantities allocated to leaves and roots. Three experiments were set up in two crop seasons in Rio Paranaíba, MG, Brazil. In the first season, typical summer, 10 summer cultivars were sown. In the second season, summer-winter (transition), two experiments were set up, one with summer cultivars and the other with winter cultivars. The treatments consisted of the carrot cultivars distributed in randomized blocks with four replications. Fresh and dry matter of the roots and leaves was quantified. Yield was calculated based on fresh matter of the roots. The nutrient content in leaves and roots was determined at the time of harvest. These contents and the dry matter production of roots and leaves were used to calculate nutrient uptake and export. The greatest average for total and commercial yield occurred in the crop under summer conditions. Extraction of N and K for most of the cultivars in the three experiments went beyond the amounts applied through fertilizers. Thus, there was contribution of nutrients from the soil to obtain the yields observed. However, the amount of P taken up was considerably less than that applied. This implies that soil P fertility will increase after cropping. The crop season and the cultivars influenced yield, nutrient content in the leaves and roots, and extraction and export of nutrients by the carrot crop.

BRACHIARIA IN SELENIUM-CONTAMINATED SOIL UNDER SULPHUR SOURCE APPLICATIONS

ABSTRACT High contents of plant-available selenium in the soil in the form of selenate, resulting from natural or anthropogenic action, jeopardizes agricultural areas and requires research for solutions to establish or re-establish agricultural or livestock operation, avoiding the risk of poisoning of plants, animals and humans. The purpose was to evaluate sulfur sources in the form of sulfate, e.g., ammonium sulfate, calcium sulfate, ferric sulfate, in the remediation of tropical soils anthropogenically contaminated with Se under the tropical forage grass Brachiaria brizantha (Hochst. ex A. Rich.) Stapf cv. Marandu. More clayey soils are less able to supply plants with Se, which influences the effects of S sources, but it was found that high soil Se concentrations negatively affected forage biomass production, regardless of the soil. Of the tested S sources, the highly soluble ammonium sulfate and ferric sulfate reduced plant Se uptake and raised the available sulfur content in the soil.

Enantio-pyochelin: a novel siderophore produced by Pseudomonas fluorescens CHA0

RESUME Pour favoriser sa croissance en condition limitante de fer, le pathogène opportunistePseudomonas aeruginosa PAO1 sécrète un sidérophore nommé pyochéline. Celui-ci estproduit par un mécanisme de "thiotemplate", à partir de l'acide salicylique et de deuxmolécules de cystéine, et existe sous forme d'une paire de diastéréoisomèresinterconvertibles: pyochéline I (4'R, 2?R, 4?R) et pyochéline II (4'R, 2?S, 4?R). Deprécédentes études ont montré que la pyochéline induit l'expression de ses propres gènes debiosynthèse via le régulateur transcriptionnel PchR qui appartient à la famille AraC/XylS. Lapyochéline est donc non seulement un sidérophore mais également une molécule signale.Nous avons découvert que Pseudomonas fluorescens CHA0 sécrète une pyochélinestéréochimiquement distincte de celle produite par P. aeruginosa. Ce nouveau sidérophorefavorise la croissance de P. fluorescens en condition limitante en fer et induit l'expression deses propres gènes de biosynthèse. Cependant, cette molécule n'est pas reconnue commesidérophore ou molécule signale par P. aeruginosa. Réciproquement, la pyochéline estincapable de stimuler la croissance et la signalisation chez P. fluorescens. La structure dusiderophore de P. fluorescens CHA0 a été déterminée comme étant un antipode optique de lapyochéline et nommé énantio-pyochéline.La stéréospécificité de l'induction des gènes de biosynthèse de la pyochéline/énantiopyochélineest basée sur la stéréospécificité des protéines PchR de P. aeruginosa et P.fluorescens envers leur sidérophores-ligands respectifs. PchR est fonctionnel chez l'espècehétérologue, mais uniquement en présence de son propre ligand. Les récepteurs spécifiquesdes sidérophores pyochéline/enantio-pyochéline ne sont pas indispensables à la signalisationmais sont essentiels à l'incorporation du fer et à la croissance en carence de fer. Laconstruction de protéines hybrides et tronquées a révélé que le domaine N-terminal de PchRest l'élément déterminant pour la spécificité de la protéine vis-à-vis de son ligand. SUMMARY : The siderophore pyochelin is produced by the opportunistic pathogen Pseudomonas aeruginosa PAO1 and promotes growth under iron limitation. Pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine and exists as a pair of interconvertible diastereoisomers: pyochelin I (4'R, 2"R, 4"R) and pyochelin II (4'R, 2"S, 4"R). Pyochelin induces the expression of its biosynthesis and uptake genes via the transcriptional AraC/Xy1S family regulator PchR in a process termed pyochelin signaling. Pseudomonas fluorescens CHAO was found to make a stereochemically distinct pyochelin to P. aeruginosa. This siderophore promoted the growth of P. fluorescens under iron limitation and induced the expression of its biosynthesis genes but was not recognised as a siderophore or signaling molecule by P. aeruginosa. Reciprocally, pyochelin was unable to promote growth or signaling in P. fluorescens. The structure of the P. fluorescens CHAO siderophore was determined and found to be enantio-pyochelin, the optical antipode of pyochelin. Stereospecificity in induction of pyochelin/enantio-pyochelin biosynthesis genes was found to be due to stereospecificity of the homologous PchR proteins of P. aeruginosa and P. fluorescens towards their respective siderophore ligands. PchR was able to function in the heterologous species, but only if supplied with its native ligand. The pyochelin/enantiopyochelin receptors were not essential for signaling although both receptors are essential for iron uptake and growth under iron limitation. Construction of hybrid and truncated PchR proteins revealed that the N-terminal domain of PchR is responsible for siderophore recognition/stereospecificity.

Insulin-secreting beta-cell dysfunction induced by human lipoproteins.

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.

Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

Role of glutamate in neuron-glia metabolic coupling.

The coupling between synaptic activity and glucose utilization (neurometabolic coupling) is a central physiologic principle of brain function that has provided the basis for 2-deoxyglucose-based functional imaging with positron emission tomography. Approximately 10 y ago we provided experimental evidence that indicated a central role of glutamate signaling on astrocytes in neurometabolic coupling. The basic mechanism in neurometabolic coupling is the glutamate-stimulated aerobic glycolysis in astrocytes, such that the sodium-coupled reuptake of glutamate by astrocytes and the ensuing activation of the Na(+)-K(+) ATPase triggers glucose uptake and its glycolytic processing, which results in the release of lactate from astrocytes. Lactate can then contribute to the activity-dependent fueling of the neuronal energy demands associated with synaptic transmission. Analyses of this coupling have been extended in vivo and have defined the methods of coupling for inhibitory neurotransmission as well as its spatial extent in relation to the propagation of metabolic signals within the astrocytic syncytium. On the basis of a large body of experimental evidence, we proposed an operational model, "the astrocyte-neuron lactate shuttle." A series of results obtained by independent laboratories have provided further support for this model. This body of evidence provides a molecular and cellular basis for interpreting data that are obtained with functional brain imaging studies.

Iron supply for erythropoiesis in the rabbit

Marrow radioiron uptake and marrow blood flow were measured in order to evaluate iron supply for erythropoiesis. Normal, phenylhydrazine-treated and bled animals were studied. The plasma iron turnover of seven normal rabbits was 1.49 +/- 0.22 mg/dl whole blood per d, of 11 rabbits treated 4 d before with phenylhydrazine was 5.16 +/- 1.81, and of four bled animals the plasma iron turnover was 3.75 +/- 1.61. The cardiac output and the percentage of blood flow to the marrow was increased in phenylhydrazine-treated and bled animals. Marrow iron flow in phenylhydrazine-treated animals was 38.3 +/- 32.6 micrograms/min per kg as compared with control values of 7.0 +/- 1.3 (P less than 0.01). This was due to an increase in marrow flow, an increase in plasma iron, and an increase in plasmatocrit. In bled animals, in spite of an increased marrow blood flow, marrow iron flow of 7.3 +/- 2.2 was similar to that of control animals due to a lower plasma iron concentration. The calculated marrow iron extraction of 3.7 +/- 2.4% in phenylhydrazine-treated animals was not different from that of control animals of 4.3 +/- 1.1, whereas extraction was increased in bled animals to 7.9 +/- 1.3 (P less than 0.01). In additional studies of transfused animals, acutely induced anemia was associated with an increased cardiac output, but also with a relative decrease in marrow flow, which left marrow iron supply unaffected. It would appear from these studies that an important mechanism for meeting the increased iron requirement of the hyperplastic erythroid marrow is an increase in marrow blood flow.

Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability.

Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism in cultured astrocytes. Following Abeta(25-35) exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Abeta increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Abeta on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Abeta impair neuronal viability. The effects of the Abeta(25-35) fragment were reproduced by Abeta(1-42) but not by Abeta(1-40). Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Abeta aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

Cellular delivery of pyrenyl-arene ruthenium complexes by a water-soluble arene ruthenium metalla-cage.

Three pyrenyl-arene ruthenium complexes (M(1)-M(3)) of the general formula [Ru(η(6)-arene-pyrenyl)Cl(2)(pta)] (pta = 1,3,5-triaza-7-phosphaadamantane) have been synthesised and characterised. Prior to the coordination to ruthenium, pyrene was connected to the arene ligand via an alkane chain containing different functional groups: ester (L(1)), ether (L(2)) and amide (L(3)), respectively. Furthermore, the pyrenyl moieties of the M(n) complexes were encapsulated within the hydrophobic cavity of the water soluble metalla-cage, [Ru(6)(η(6)-p-cymene)(6)(tpt)(2)(donq)(3)](6+) (tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-triazine; donq = 5,8-dioxydo-1,4-naphthoquinonato), while the arene ruthenium end was pointing out of the cage, thus giving rise to the corresponding host-guest systems [M(n)⊂Ru(6)(η(6)-p-cymene)(6)(tpt)(2)(donq)(3)](6+) ([M(n)⊂cage](6+)). The antitumor activity of the pyrenyl-arene ruthenium complexes (M(n)) and the corresponding host-guest systems [M(n)⊂cage][CF(3)SO(3)](6) were evaluated in vitro in different types of human cancer cell lines (A549, A2780, A2780cisR, Me300 and HeLa). Complex M(2), which contains an ether group within the alkane chain, demonstrated at least a 10 times higher cytotoxicity than the reference compound [Ru(η(6)-p-cymene)Cl(2)(pta)] (RAPTA-C). All host-guest systems [M(n)⊂cage](6+) showed good anticancer activity with IC(50) values ranging from 2 to 8 μM after 72 h exposure. The fluorescence of the pyrenyl moiety allowed the monitoring of the cellular uptake and revealed an increase of uptake by a factor two of the M(2) complex when encapsulated in the metalla-cage [Ru(6)(η(6)-p-cymene)(6)(tpt)(2)(donq)(3)](6+).

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation.

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.

Estequiometria i metabolòmica d'Alopecurus pratensis i Holcus lanatus en condicions de sequera

La variabilitat de l’estequiometria elemental dels organismes a causa de l’ontogènia i dels canvis en les condicions ambientals està relacionada amb la variabilitat metabolòmica. Això és degut a que els elements operen majoritàriament com a parts de compostos moleculars. Així doncs, la hipòtesi realitzada per Rivas-Ubach et al., (2012), la qual postula que els estudis estequiomètrics i metabolòmics d’un conjunt d’espècies vegetals exposades a condicions ambientals diferents han de mostrar la flexibilitat que posseeix un organisme a l’hora de modular la seva estequiometria i el seu metaboloma per tal de mantenir la forma òptima sota condicions variants, esdevé la base que sustenta l’experiment EVENT II. A partir de l’estudi de les relacions estequiomètriques, -principalment C:N:P- i del metabolisme d’Alopecurus pratensis i Holcus lanatus en situacions simulades de sequera, s’han obtingut resultats que evidencien una clara diferenciació a nivell d’espècie, de part de la planta i de tractament. El metabolisme i l’estequiometria diferencial que presenten ambdues gramínies dóna suport a la hipòtesi del nínxol biogeoquímic. A nivell de parts de la planta, s’observa un clar augment de la relació C:nutrients a la part aèria, mentre que a les arrels, aquesta relació disminueix. La part aèria doncs, necessita més C per invertir en funcions estructurals, mentre que l’elevada concentració de nutrients i metabòlits a les arrels donen indicis de la presència de mecanismes osmòtics per a facilitar l’entrada d’aigua, i de creixement, per a la recerca de noves fonts d’aigua, observant-se una disminució de la relació part aèria:arrels. Un altre factor que demostra aquest creixement radicular són les baixes relacions N:P trobades, fet que dóna suport a la hipòtesi de la velocitat de creixement.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Characterization of cerebral glucose dynamics in vivo with a four-state conformational model of transport at the blood-brain barrier.

Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.