Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein.

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.

Retinoblastoma Protein Knockdown Favors Oxidative Metabolism and Glucose and Fatty Acid Disposal in Muscle Cells.

Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non-targeting RNAs, myotubes silenced for Rb (by 80-90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5-fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up-regulated levels of glucose transporter type 4 expression (∼5-fold increase), increased basal glucose uptake, and enhanced insulin-induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise. J. Cell. Physiol. 231: 708-718, 2016. © 2015 Wiley Periodicals, Inc.

Future perspectives in melanoma research: meeting report from the "Melanoma Bridge": Napoli, December 3rd-6th 2014.

The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Jasmonic acid and the THO/TREX complex participate in phosphate starvation response

All plants are typically confronted to simultaneous biotic and abiotic stress throughout their life cycle. Low inorganic phosphate (Pi) is the most common nutrient deficiency limiting plant growth in natural and agricultural ecosystems while insect herbivory accounts for major losses in plant productivity and impacts on ecological and evolutionary changes in plant populations. Here we report that plants experiencing Pi deficiency induce the jasmonic acid (JA) pathway and enhance their defence against insect herbivory. The phol mutant is impaired in the translocation of Pi from roots to shoots and shows the typical symptoms associated with Pi deficiency, including high anthocyanin and poor shoot growth. These phol shoot phenotypes were significantly attenuated by blocking the JA biosynthesis or signalling pathways. Wounded phol leaves hyper-accumulated JA in comparison to wild type, leading to increased resistance against the generalist herbivore Spodoptera littoralis. Pi deficiency also triggered enhanced resistance to herbivory in wild-type Arabidopsis as well as tomato and tobacco, revealing that the link between Pi deficiency and JA-mediated herbivory resistance is conserved in a diversity of plants, including crops. We performed a phol suppressor screen to identify new components involved in the adaptation of plants to Pi deficiency. We report that the THO RNA TRANSCRIPTION AND EXPORT (THO/TREX) complex is a crucial component involved in modulating the Pi- deficiency response. Knockout mutants of at least three members of the THO/TREX complex, including TEX1, HPR1, and TH06, can suppress the phol shoot phenotype. Grafting experiments showed that loss of function of TEX1 only in the root was sufficient to suppress the reduced shoot growth phenotype of phol while maintaining low Pi contents. This indicates that TEX1 is involved in a long distance root-to-shoot signalling component of the Pi-deficiency response. We identified a small MYB-like transcription factor, RAD LIKE 3 (RL3), as a potential downstream target of the THO/TREX complex. RL3 expression is induced in phol mutants but attenuated in phol-7 texl-4 double mutants. Identical to knockout mutants of the THO/TREX complex, rl3 mutants can suppress the phol shoot phenotypes. Interestingly, RL3 is induced during Pi deficiency and is described in the literature as likely being mobile. It is therefore a promising new candidate involved in the root-to-shoot Pi-deficiency signalling response. Finally, we report that PHOl and its homologue PH01:H3 are involved in the co-regulation of Pi and zinc (Zn) homeostasis. PH01;H3 is up-regulated in response to Zn deficiency and, like PHOl, is expressed in the root vascular cylinder and localizes to the Golgi when expressed transiently in tobacco cells. The phol;h3 mutant accumulates more Pi as compared to wild-type when grown in Zn-deficient medium, but this increase is abolished in the phol phol;h3 double mutant. These results suggest that PH01;H3 restricts the PHOl-mediated root-to-shoot Pi transfer in responsé to Zn deficiency. Résumé Au cours de leur cycle de vie, toutes les plantes sont généralement confrontées à divers stress biotiques et abiotiques. La carence nutritionnelle la plus fréquente, limitant la croissance des plantes dans les écosystèmes naturels et agricoles, est la faible teneur en phosphate inorganique (Pi). Au niveau des stress biotiques, les insectes herbivores sont responsables de pertes majeures de rendement et ont un impact considérable sur les changements écologiques et évolutifs dans les populations des plantes. Au cours de ce travail, nous avons mis en évidence que les plantes en situation de carence en Pi induisent la voie de l'acide jasmonique (JA) et augmentent leur défense contre les insectes herbivores. Le mutant phol est déficient dans le transport du phosphate des racines aux feuilles et démontre les symptômes typiques associés à la carence, tels que la forte concentration en anthocyane et une faible croissance foliaire. Ces phénotypes du mutant phol sont significativement atténués lors d'un blocage de la voie de la biosynthèse ou des voies de signalisation du JA. La blessure des feuilles induit une hyper-accumulation de JA chez phol, résultant en une augmentation de la résistance contre l'herbivore généraliste Spodoptera littoralis. Outre Arabidopsis, la carence en Pi induit une résistance accrue aux insectes herbivores aussi chez la tomate et le tabac. Cette découverte révèle que le lien entre la carence en Pi et la résistance aux insectes herbivores via le JA est conservé dans différentes espèces végétales, y compris les plantes de grandes cultures. Nous avons effectué un crible du suppresseur de phol afin d'identifier de nouveaux acteurs impliqués dans l'adaptation de la plante à la carence en Pi. Nous rapportons que le complexe nommé THO RNA TRANSCRIPTION AND EXPORT (THO/TREX) est un élément crucial participant à la réponse des feuilles à la carence en Pi. Les mutations d'au moins trois des membres que composent le complexe THO/TREX, incluant TEX1, HPR1 et 77/06, peuvent supprimer le phénotype de phol. Des expériences de greffes ont montré que la perte de fonction de TEX1, seulement dans la racine, est suffisante pour supprimer le phénotype de la croissance réduite des parties aériennes observé chez le mutant phol, tout en maintenant de faibles teneurs en Pi foliaire. Ceci indique que TEX1 est impliqué dans la signalisation longue distance entre les racines et les parties aériennes. Nous avons identifié un petit facteur de transcription proche de la famille des MYB, RAD LIKE 3 (RL3), comme une cible potentielle en aval du complexe THO / TREX. L'expression du gène RL3 est induite dans le mutant phol mais atténuée dans le double mutant phol-7 texl-4. Exactement comme les plantes mutées d'un des membres du complexe THO/TREX, le mutant rl3 peut supprimer le phénotype foliaire de phol. RL3 est induit au cours d'une carence en Pi et est décrit dans la littérature comme étant potentiellement mobile. Par conséquent, il serait un nouveau candidat potentiellement impliqué dans la réponse longue distance entre les racines et les parties aériennes lors d'un déficit en Pi. Enfin, nous reportons que PHOl et son homologue PHOl: H3 sont impliqués dans la co- régulation de l'homéostasie du Pi et du zinc (Zn). PHOl; H3 est sur-exprimé en réponse au déficit en Zn et, comme PHOl, est exprimé dans les tissus vasculaires des racines et se localise dans l'appareil de Golgi lorsqu'il est exprimé de manière transitoire dans des cellules de tabac. Le mutant phol; h3 accumule plus de Pi par rapport aux plantes sauvages lorsqu'il est cultivé sur un milieu déficient en Zn, mais cette augmentation en Pi est abolie dans le double mutant phol phol; h3. Ces résultats suggèrent qu'en réponse à une carence en Zn, PHOl; H3 limite l'action de PHOl et diminue le transfert du Pi des racines aux parties aériennes.

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart.

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-l-arginine and N-iminoethyl-l-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.

In vivo detection of brain Krebs cycle intermediate by hyperpolarized magnetic resonance.

The Krebs (or tricarboxylic acid (TCA)) cycle has a central role in the regulation of brain energy regulation and metabolism, yet brain TCA cycle intermediates have never been directly detected in vivo. This study reports the first direct in vivo observation of a TCA cycle intermediate in intact brain, namely, 2-oxoglutarate, a key biomolecule connecting metabolism to neuronal activity. Our observation reveals important information about in vivo biochemical processes hitherto considered undetectable. In particular, it provides direct evidence that transport across the inner mitochondria membrane is rate limiting in the brain. The hyperpolarized magnetic resonance protocol designed for this study opens the way to direct and real-time studies of TCA cycle kinetics.

Diethyldithiocarbamic acid inhibits the octadecanoid signaling pathway for the wound induction of proteinase-inhibitors in tomato leaves

The induction of proteinase inhibitor I synthesis in tomato (Lycopersicon esculentum) leaves in response to wounding is strongly inhibited by diethyldithiocarbamic acid (DIECA). DIECA also inhibits the induction of inhibitor I synthesis by the 18-amino acid polypeptide systemin, polygalacturonic acid (PCA), and linolenic acid, but not by jasmonic acid, suggesting that DIECA interferes with the octadecanoid signaling pathway. DIECA only weakly inhibited tomato lipoxygenase activity, indicating that DIECA action occurred at a step after the conversion of linolenic acid to 13(S)-hydroperoxylinolenic acid (HPOTrE). DIECA was shown to efficiently reduce HPOTrE to 13-hydroxylinolenic acid (HOTrE), which is not a signaling intermediate. Therefore, in vivo, DIECA is likely inhibiting the signaling pathway by shunting HPOTrE to HOTrE, thereby severely reducing the precursor pool leading to cyclization and eventual synthesis of jasmonic acid. Phenidone, an inhibitor of lipoxygenase, inhibited proteinase inhibitor I accumulation in response to wounding, further supporting a role for its substrate, linolenic acid, and its product, HPOTrE, as components of the signal-transduction pathway that induces proteinase inhibitor synthesis in response to wounding, systemin, and PCA.

In vivo quantification of neuro-glial metabolism and glial glutamate concentration using 1H-[13C] MRS at 14.1T.

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo (13) C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-(13) C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using (1) H-[(13) C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg ) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT ) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC ) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vx (g) and Vx (n) ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations. In this (13) C-acetate labeling study, we propose a refined two-compartment analysis of brain energy metabolism based on (13) C turnover curves of acetate, glutamate and glutamine measured with state of the art in vivo dynamic MRS at high magnetic field in rats, enabling a deeper understanding of the specific role of glial cells in brain oxidative metabolism. In addition, the robustness of the metabolic fluxes determination relative to MRS data quality was carefully studied.

Genome reconstruction and comparative genomics of pneumocystis jirovecii

Pneumocystis jirovecii is a fungus belonging to a basal lineage of the Ascomycotina, the Taphrinomycotina subphylum. It is a parasite specific to humans that dwells primarily in the lung and can cause severe pneumonia in individuals with debilitated immune system. Despite its clinical importance, many aspects of its biology remain poorly understood, at least in part because of the lack of a continuous in vitro cultivation system. The present thesis consists in the genome reconstruction and comparative genomics of P. jirovecii. It is made of three parts: (i) the de novo sequencing of P. jirovecii genome starting from a single broncho- alveolar lavage fluid of a single patient (ii) the de novo sequencing of the genome of the plant pathogen Taphrina deformans, a fungus closely related to P. jirovecii, and (iii) the genome scale comparison of P. jirovecii to other Taphrinomycotina members. Enrichment in P. jirovecii cells by immuno-precipitation, whole DNA random amplification, two complementary high throughput DNA sequencing methods, and in silico sorting and assembly of sequences were used for the de novo reconstruction of P. jirovecii genome from the microbiota of a single clinical specimen. An iterative ad hoc pipeline as well as numerical simulations was used to recover P. jirovecii sequences while purging out contaminants and assembly or amplification chimeras. This strategy produced a 8.1 Mb assembly, which encodes 3,898 genes. Homology searches, mapping on biochemical pathways atlases, and manual validations revealed that this genome lacks (i) most of the enzymes dedicated to the amino acids biosyntheses, and (ii) most virulence factors observed in other fungi, e.g. the glyoxylate shunt pathway and specific peptidases involved in the degradation of the host cell membrane. The same analyses applied to the available genomic sequences from Pneumocystis carinii the species infecting rats and Pneumocystis murina the species infecting mice revealed the same deficiencies. The genome sequencing of T. deformans yielded a 13 Mb assembly, which encodes 5,735 genes. T. deformans possesses enzymes involved plant cell wall degradation, secondary metabolism, the glyoxylate cycle, detoxification, sterol biosynthesis, as well as the biosyntheses of plant hormones such as abscisic acid or indole-3-acetic acid. T. deformans also harbors gene subsets that have counterparts in plant saprophytes or pathogens, which is consistent with its alternate saprophytic and pathogenic lifestyles. Mating genes were also identified. The homothallism of this fungus suggests a mating-type switching mechanism. Comparative analyses indicated that 81% of P. jirovecii genes are shared with eight other Taphrinomycotina members, including T. deformans, P. carinii and P. murina. These genes are mostly involved in housekeeping activities. The genes specific to the Pneumocystis genus represent 8%, and are involved in RNA metabolism and signaling. The signaling is known to be crucial for interaction of Pneumocystis spp with their environment. Eleven percent are unique to P. jirovecii and encode mostly proteins of unknown function. These genes in conjunction with other ones (e.g. the major surface glycoproteins) might govern the interaction of P. jirovecii with its human host cells, and potentially be responsible of the host specificity. P. jirovecii exhibits a reduced genome in size with a low GC content, and most probably scavenges vital compounds such as amino acids and cholesterol from human lungs. Consistently, its genome encodes a large set of transporters (ca. 22% of its genes), which may play a pivotal role in the acquisition of these compounds. All these features are generally observed in obligate parasite of various kingdoms (bacteria, protozoa, fungi). Moreover, epidemiological studies failed to evidence a free-living form of the fungus and Pneumocystis spp were shown to co-evolved with their hosts. Given also the lack of virulence factors, our observations strongly suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, and which causes disease only in individuals with compromised immune system. The same conclusion is most likely true for all other Pneumocystis spp in their respective mammalian host. - Pneumocystis jirovecii est un champignon appartenant à ine branche basale des Ascomycotina, le sous-embranchement des Taphrinomycotina. C'est un parasite spécifique aux humains qui réside principalement dans les poumons, et qui peut causer des pneumonies sévères chez des individus ayant un système immunitaire déficient. En dépit de son importance clinique, de nombreux aspects de sa biologie demeurent,largement méconnus, au moins en partie à cause de l'absence d'un système de culture in vitro continu. Cette thèse traite de la reconstruction du génome et de la génomique comparative de P. jirovecii. Elle comporte trois parties: (i) le séquençage de novo du génome de P. jirovecii à partir d'un lavage broncho-alvéolaire provenant d'un seul patient, (ii) le séquençage de novo du génome d'un champignon pathogène de plante Taphrina deformans qui est phylogénétiquement proche de P. jirovecii, et (iii) la comparaison du génome de P. jirovecii à celui d'autres membres du sous-embranchement des Taphrinomycotina. Un enrichissement en cellules de P. jirovecii par immuno-précipitation, une amplification aléatoire des molécules d'ADN, deux méthodes complémentaires de séquençage à haut débit, un tri in silico et un assemblage des séquences ont été utilisés pour reconstruire de novo le génome de P. jirovecii à partir du microbiote d'un seul échantillon clinique. Un pipeline spécifique ainsi que des simulations numériques ont été utilisés pour récupérer les séquences de P. jirovecii tout en éliminant les séquences contaminants et les chimères d'amplification ou d'assemblage. Cette stratégie a produit un assemblage de 8.1 Mb, qui contient 3898 gènes. Les recherches d'homologies, de cartographie des voies métaboliques et des validations manuelles ont révélé que ce génome est dépourvu (i) de la plupart des enzymes dédiées à la biosynthèse des acides aminés, et (ii) de la plupart des facteurs de virulence observés chez d'autres champignons, par exemple, le cycle du glyoxylate ainsi que des peptidases spécifiques impliquées dans la dégradation de la membrane de la cellule hôte. Les analyses appliquées aux données génomiques disponibles de Pneumocystis carinii, l'espèce infectant les rats, et de Pneumocystis murina, l'espèce infectant les souris, ont révélé les mêmes déficiences. Le séquençage du génome de T. deformans a généré un assemblage de 13.3 Mb qui contient 5735 gènes. T. deformans possède les gènes codant pour les enzymes impliquées dans la dégradation des parois cellulaires des plantes, le métabolisme secondaire, le cycle du glyoxylate, la détoxification, la biosynthèse des stérols ainsi que la biosynthèse d'hormones de plantes telles que l'acide abscissique ou l'acide indole 3-acétique. T. deformans possède également des sous-ensembles de gènes présents exclusivement chez des saprophytes ou des pathogènes de plantes, ce qui est consistent avec son mode de vie alternatif saprophyte et pathogène. Des gènes impliqués dans la conjugaison ont été identifiés. L'homothallisme de ce champignon suggère mécanisme de permutation du type conjuguant. Les analyses comparatives ont démontré que 81% des gènes de P. jirovecii sont présent chez les autres membres du sous-embranchement des Taphrinomycotina. Ces gènes sont essentiellement impliqués dans le métabolisme basai. Les gènes spécifiques au genre Pneumocystis représentent 8%, et sont impliqués dans le métabolisme de l'ARN et la signalisation. La signalisation est connue pour être cruciale pour l'interaction des espèces de Pneumocystis avec leur environnement. Les gènes propres à P. jirovecii représentent 11% et codent en majorité pour des protéines dont la fonction est inconnue. Ces gènes en conjonction avec d'autres (par exemple, les glycoprotéines de surface), pourraient être déterminants dans l'interaction de P. jirovecii avec les cellules de l'hôte humain, et être potentiellement responsable de la spécificité d'hôte. P. jirovecii possède un génome de taille réduite à faible pourcentage en GC et récupère très probablement des composés vitaux comme les acides aminés et le cholestérol à partir des poumons humains. De manière consistante, son génome code pour de nombreux transporteurs (22% de ses gènes), qui pourraient jouer un rôle essentiel dans l'acquisition de ces composés. Ces caractéristiques sont généralement observées chez les parasites obligatoires de plusieurs règnes (bactéries, protozoaires, champignons). De plus, les études épidémiologiques n'ont pas réussi à prouver l'existence d'ime forme vivant librement du champignon. Etant donné également l'absence de facteurs de virulence, nos observations suggèrent que P. jirovecii est un parasite obligatoire spécialisé dans la colonisation des poumons humains, ne causant une maladie que chez des individus ayant un système immunitaire compromis. La même conclusion est très probablement applicable à toutes les autres espèces de Pneumocystis dans leur hôte mammifère respectif.

n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.

We elucidated the mechanisms of action of two n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in Jurkat T-cells. Both DHA and EPA were principally incorporated into phospholipids in the following order: phosphatidylcholine < phosphatidylethanolamine < phosphatidylinositol/phosphatidylserine. Furthermore, two isoforms of phospholipase A(2) (i.e., calcium-dependent and calcium-independent) were implicated in the release of DHA and EPA, respectively, during activation of these cells. The two fatty acids inhibited the phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane translocation of protein kinase C (PKC)-alpha and -epsilon. The two n-3 PUFAs also inhibited the nuclear translocation of nuclear factor kappaB (NF-kappaB) and the transcription of the interleukin-2 (IL-2) gene in PMA-activated Jurkat T-cells. Together, these results demonstrate that DHA and EPA, being released by two isoforms of phospholipase A(2), modulate IL-2 gene expression by exerting their action on two PKC isoforms and NF-kappaB in Jurkat T-cells.

Glutamatergic and GABAergic energy metabolism measured in the rat brain by (13) C NMR spectroscopy at 14.1 T.

Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ-aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore, we measured (13) C incorporation into brain metabolites by dynamic (13) C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α-chloralose anaesthesia during infusion of [1,6-(13) C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of (13) C into the three aliphatic carbons of GABA non-invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11 ± 0.01 μmol/g/min. GABA-glutamine cycle was 0.053 ± 0.003 μmol/g/min and accounted for 22 ± 1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47 ± 0.02 μmol/g/min, of which 35 ± 1% and 7 ± 1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12 ± 1% of the TCA cycle flux was dedicated to oxidation of GABA. 16 ± 2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain. We performed (13) C NMR spectroscopy in vivo at high magnetic field (14.1 T) upon administration of [1,6-(13) C]glucose. This allowed to measure (13) C incorporation into the three aliphatic carbons of GABA in the rat brain, in addition to those of glutamate, glutamine and aspartate. These data were then modelled to determine fluxes of energy metabolism in GABAergic and glutamatergic neurons and glial cells.

PPARs: transcriptional effectors of fatty acids and their derivatives.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. These receptors stimulate transcription after activation by their cognate ligand and binding to the promoter of target genes. In this review, we discuss how fatty acids affect PPAR functions in the cell. We first describe the structural features of the ligand binding domains of PPARs, as defined by crystallographic analyses. We then present the ligand-binding characteristics of each of the three PPARs (alpha, beta/delta, gamma) and relate ligand activation to various cellular processes: (i) fatty acid catabolism and modulation of the inflammatory response for PPARalpha, (ii) embryo implantation, cell proliferation and apoptosis for PPARbeta, and (iii) adipocytic differentiation, monocytic differentiation and cell cycle withdrawal for PPARgamma. Finally, we present possible cross-talk between the PPAR pathway and different endocrine routes within the cell, including the thyroid hormone and retinoid pathways.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.