Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

Amyloid aggregation involves the spontaneous formation of fibers from misfolded proteins. This process requires low energy input, results in robust fibers, and is thus of interest from a materials manufacturing perspective. The effect of glutamine content and hydrophobicity of template peptides on amyloid aggregation of a template-peptide system involving myoglobin was studied at near-physiological conditions by Fourier transform infrared spectroscopy, atomic force microscopy, field emission scanning electron microscopy, and nanoindentation. Hydrophobic interactions were found to be important for controlled hierarchical fiber growth via a cooperative mechanism, with the largest effect in myoglobin mixtures. Hydrophobic packing increased for most systems as aggregation progressed. The largest changes in structure occurred upon drying. When myoglobin was present with the highest glutamine-containing template (P7), the high glutamine peptide was not effective as a template, since it appeared to prefer self-catalysis. A low level of glutamine in some unordered templates was insufficient for amyloid development. However, templating was more important in glutamine-free templates mixed with myoglobin, which formed fibers with a surprisingly high elastic modulus. This may have been due to template patterning. Nanoindentation results confirmed that glutamine blocks were not necessary for strong intermolecular interactions and cooperative fibril formation. / Master of Science

The gram-negative, gamma proteobacterium Pseudomonas aeruginosa demonstrates substantial metabolic flexibility, allowing it to survive and thrive in diverse environments. Indeed, its ability to carefully maintain a buffered intracellular oxidoreduction state permits it to maintain structural and functional stability in the face of both nutrient-poor and nutrient-rich conditions. It is clear that metabolism is simply central to the existence of this microbe, yet knowledge of the genetics and biochemistry underlying this deeply intricate world of metabolic regulation is fundamentally incomplete. Two critical metabolites, alpha-ketoglutarate and glutamine, appear at the intersection of carbon and nitrogen metabolism, and may represent the status of Pseudomonas’ intracellular carbon or nitrogen pools. However, the coordination of carbon and nitrogen assimilation, whether through PII proteins, the Ntr regulatory cascade, the CbrAB regulatory system, or the PTS-NTR system, as well as these pathways’ cross-talk and ability to control downstream processes based on nutrient availability, still remain to be elucidated. Further, a more comprehensive understanding of P. aeruginosa’s metabolic regulation could be significant for the development of new therapies that specifically target critical biochemical pathways involved in the metabolism of this organism and offer new hope to patients suffering from P. aeruginosa infections in the clinic. Thus, this review considers how, when, and why carbon and nitrogen metabolism may be regulated in Pseudomonas aeruginosa, and proposes there may be more interaction and cross-regulation between these two seemingly divergent metabolic arms than originally thought.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20239号 / 医博第4198号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The gene gdhA from Escherichia coli, that encodes a NADPH-dependent glutamate dehydrogenase (GDH), directs a novel pathway in transgenic plants that potentially allows an increase in ammonium assimilation. Glufosinate leads to plant death by the irreversible inhibition of glutamate synthetase (GS) leading to a disruption of subsequent GS-related processes resulting in elevated ammonium and disruption of photorespiration. Therefore, it was speculated that the gdhA-transformed plants may exhibit a novel mechanism of resistance to glufosinate by altered activity of the GDH pathway and subsequently related processes. Studies were conducted in the greenhouse to evaluate 1) whole plant tolerance to glufosinate, 2) changes in absorption, translocation and metabolism of glufosinate, and 3) metabolic fingerprint changes in response to glufosinate treatment in tobacco plants containing the gdhA gene. Whole plant tolerance experiments showed that tobacco transformed with the gdhA gene expressed up to six fold increased resistance (GR50) to glufosinate compared with the non-gdhA control line. GDH enzyme activity among gdhA-transformed tobacco lines was highly correlated (r2 = 0.9903) with the amount of herbicide resistance. Thus, use of the E. coli gdhA gene in plant transformations can provide an additional mechanism for resistance to glufosinate. Foliar absorption and translocation of 14C from glufosinate was not altered to any large extent in gdhA-transformed plants which suggests these factors cannot fully explain the mechanism for whole-plant resistance to glufosinate. However, the metabolic fingerprint resulting from glufosinate treatment was significantly altered in gdhA tobacco. It was also shown that metabolic perturbation induced by glufosinate was lower in the high GDH activity tobacco line, +gdhA 9, than in the non-gdhA control tobacco line as evidenced by the reduced number of altered peaks recorded in leaves of these two tobacco lines. Thus, gdhA-transformed tobacco plants with low and high expression of GDH activity, exhibited greater overall stability of metabolism following the application of glufosinate, than recorded in non-gdhA control plants. This greater metabolic stability during GS inhibition was likely due to the amelioration of amino acid production through the increased activity of GDH. Therefore, the hypothesized mechanism of increased resistance to glufosinate in gdhA-transformed tobacco lines is by maintenance of amino acid production and maintenance of photorespiratory activity.

The purpose of this study was to compare the efficacy of two dose levels of L-Alanyl-LGlutamine in a commercially available sports drink to the sports drink only on time to exhaustion, neuromuscular fatigue and physiological measures during prolonged endurance exercise. Twelve endurance-trained males (23.5±3.7 yrs; 175.5±5.4 cm; 70.7±7.6 kg) performed four trials, each consisting of 1 hr treadmill runs at 75% of VO2peak followed by a run to exhaustion at 90% of VO2peak. The trials differed in type of hydration. One trial consisted of no hydration (NHY), another required ingestion of only a sports drink (ET), and two trials required ingestion of a low dose (LD) (300 mg∙500 ml-1) and high dose (HD) of L-Alanyl-L-Glutamine (1 g∙500 ml-1) mixed in the sports drink. During the fluid ingestion trials 250 ml were consumed every 15 min. Plasma glutamine, glucose, electrolytes, and osmolality were measured prior to the run (PRE), and at 30, 45, and 60 min. VO2, RQ, and HR were measured every 15 min and surface electromyography (EMG) of the vastus lateralis and rectus femoris were measured every 10 min during the 1 hr run. Time to exhaustion was significantly longer during the LD and HD trials compared with NHY. Plasma glutamine concentrations were significantly elevated at 45 min in LD and HD trials, and remained elevated at 60 min during HD. Sodium concentrations increased with the beginning of exercise and remained stable for the duration of the 1 hr run. At 60 min plasma sodium was significantly lower in all trials compared with NHY. The results from this study indicated that ingestion of the alanine-glutamine dipeptide at either the low or high dose significantly improved time to exhaustion during high intensity exercise compared to a no hydration trial. These differences were not noted between ET and NHY.

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease (CVD) and stroke, however the cellular mechanisms by which diabetes contributes to vascular dysfunction are not fully understood. In recent decades, multiple molecular mechanisms have been implicated in hyperglycemia-associated vascular damage and CVD [1]. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway where the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) produces glucosamine-6-phosphate [2,3]. We have shown that elevated levels of intracellular glucosamine cause ER stress and activation of the UPR in multiple cell types [4]. Additionally, we have previously shown that ER stress is associated with lipid accumulation, activation of inflammatory pathways, and is associated with atherosclerotic plaque formation in hyperglycemic mice [ 4,5]. We hypothesize that the accumulation of intracellular glucosamine, observed in conditions of hyperglycemia, promotes atherogenesis via a mechanism that involves the hexosamine pathway, protein glycosylation and ER stress.</p> <p> Using in vitro over-expression studies, we investigated the role of GFAT in hyperglycemia-associated ER stress. We developed methods to increase GFAT expression in both HepG2 cells and HASMC. However, we found that GFAT over-expression is insufficient to induce an ER stress response. Further investigation of this system suggests that the over-expressed GFAT does not increase intracellular glucosamine levels to sufficiently promote ER stress.</p> <p> We have also investigated the role of protein glycosylation in glucosamine-induced ER stress. We have shown that O-linked glycosylation plays a role in ER stress induction. We have also shown that N-linked protein glycosylation is affected by elevated cellular glucosamine levels. Thus, dysregulated glycosylation of newly synthesized proteins may contribute to the accumulation of unfolded protein in the ER and lead to the activation of the UPR.</p> / Thesis / Master of Science (MSc)

No description available.

L’activité de croissance des plantes se trouve souvent limitée par les conditions contraignantes de l’environnement. La salinité du sol est l’une des majeures contraintes abiotiques qui ne cesse d’envahir les surfaces cultivés chaque année. Elle entraine chez les espèces glycophytes des perturbations d’ordre osmotique, nutritionnel et métaboliques. La nutrition et le métabolisme de l’azote minéral constituent des étapes primordiales dans la synthèse des acides aminés et des composés azotés indispensables chez les plantes. Par conséquent, l’étude de l’expression des enzymes impliquées dans l’assimilation d’azote telle que l’asparagine synthétase (AS, EC 6.3.5.4) chez l’arabette des dames (Arabidopsis thaliana) permet d’avancer nos connaissances sur la régulation transcriptionnelle du métabolisme azoté sous stress salin. Au cours des travaux de recherche entamés dans le cadre de cette thèse, un intérêt particulier est accordé au gène ASN2 chez Arabidopsis. Les résultats obtenus ont montré que la mutation ASN2 a accentué les effets du NaCl sur l’assimilation de l’ammonium. Le mutant asn2-1 se montre plus sensible au stress salin que le sauvage malgré que l’absence des transcrits du gène ASN2 est associé à une expression importante du gène ASN1. L’inhibition de l’activité glutamine synthétase (GS, EC 6.3.1.2), la faible activité aminatrice de la GDH (NADH-GDH, EC 1.4.1.2) sous stress salin ainsi que l’absence des transcrits ASN2 seraient à l’origine de l’accumulation de l’ammonium chez le mutant asn2-1. Toutefois, l’application exogène de l’ammonium nous a montré que l’action du NaCl sur l’expression de l’asparagine synthétase n’est pas directement liée à l’accumulation endogène d’ammonium. L’accumulation d’autres métabolites tels que l’asparagine, la glutamine et la glutamate pourrait être à l’origine des effets du sel sur l’expression des gènes ASN. / Plant growth activity is often limited by constraint environment conditions. Soil salinity is one of major abiotic stress which is becoming more problematic every year. In glycophytes species, it induced osmotic, nutritional and metabolic disturbances. The nitrogen nutrition and metabolism constitute an essential step in amino acid and nitrogen compounds synthesis in plants. Therefore, studying the expression of enzymes involved in nitrogen assimilation such as asparagine synthetase (AS, EC 6.3.5.4) in Arabidopsis thaliana will improve our knowledge on the transcriptional regulation of nitrogen metabolism under salt stress. In the present work of this thesis, a special attention was taken on AS gene (ASN2) wild type and mutants. Obtained results showed that ASN2 mutation accentuated the salt-induced effects on ammonium assimilation. The asn2-1 mutant was more sensitive to salt stress than the wild type, while the ASN2 transcript absence was associated with an important ASN1expression. The observed inhibition of glutamine synthetase (GS, EC 6.3.1.2) activity, the low aminatrice GDH (NADH-GDH, EC 1.4.1.2) activity under salt stress as well as the ASN2 transcript loss brought to an ammonium accumulation in asn2-1mutant. However, exogenous ammonium application showed that NaCl effect on asparagine synthetase expression was not directly related to the endogenous ammonium accumulation. Other metabolites accumulation such as asparagine, glutamine and glutamate could be involved in the obtained salt-effects on ASN expression in Arabidopsis.

L’orge (Hordeum vulgare L.) est l'une des céréales les plus importantes du monde et l’une des premières cultures domestiquées. Elle a été utilisée pendant des siècles pour l'alimentation humaine. Comme toutes les autres plantes, l'orge est dépendante de l'azote inorganique. L’efficacité de remobilisation de l'azote est donc très importante pour le remplissage des grains et pour la teneur en protéines du grain. L'objectif de ce travail est de donner une image du métabolisme des feuilles sénescence chez l'orge lorsque les plantes sont cultivées dans des conditions limitantes ou non en nitrates. Les analyses biochimiques, physiologiques et moléculaires de la sénescence des feuilles d'orge ont été réalisées. La gestion de l'azote pendant la sénescence des feuilles a été suivie par l'évolution des différents composés azotés au cours du vieillissement de la feuille. Une étude de profilage métabolique a été effectuée afin de déterminer les caractéristiques métaboliques de la sénescence des feuilles dans l'orge. En parallèle, les enzymes impliquées dans la remobilisation de l'azote ont été étudiées. Leurs activités et les niveaux de leurs transcripts ont été mesurés. Une attention particulière a été portée aux glutamine synthétases et asparagine synthétases et aux protéines de la machinerie de l'autophagie, processus connus pour jouer un rôle dans la remobilisation de l'azote pendant la sénescence des feuilles. A partir de toutes les données de séquences disponibles, ADNc, EST et séquences génomiques, cinq gènes codant pour les isoformes de glutamine synthétase cytosoliques (GS1), cinq gènes codant pour les isoformes d’asparagine synthétase (AS) isoformes et 19 gènes codant pour des protéines de la machinerie de l'autophagie ont été identifiés. Les expressions de tous les gènes identifiés ont été suivies au cours de la sénescence des feuilles et en fonction de l'alimentation en nitrates. La plupart de ces gènes sont sur-exprimés dans les feuilles sénescentes et de façon différentielle en fonction des conditions de nutrition. Toutes les données de séquences fournies par ce travail seront utiles à d'autres études translationelles et d'association génétique. / Barley (Hordeum vulgare L.) is one of the most important cereals in the world. It was one of the first domesticated crops and was used for centuries for human food. As all plants, barley has a fundamental dependence of inorganic nitrogen and nitrogen remobilization efficiency is very important for grain filling and grain protein content. The aim of this work was then to give a picture of the leaf-senescence metabolism in barley leaves when plants are grown under low or high nitrate conditions. Biochemical, physiological and molecular analyses of barley leaf senescence were performed. Nitrogen management during leaf senescence was monitored measuring changes in the different nitrogen pools during leaf ageing. In addition a large metabolite profiling study was performed in order to determine the metabolic hallmarks of leaf senescence in barley. In parallel enzymes involved in nitrogen remobilization were studied measuring their activity and the transcript levels of their coding genes. There was a special focus on glutamine synthetase and asparagine synthetase enzymes and for autophagy machinery that are known to play a role in nitrogen remobilisation during leaf senescence.From all the sequences data available, cDNA, EST and genomic sequences, we could identified five genes coding for cytosolic glutamine synthetase (GS1), five genes coding for asparagine synthetase (AS) and 19 genes coding for autophagy machinery proteins. Transcript levels of all the genes identified were monitored during leaf senescence and depending on nitrate nutrition. Most of these genes were over-expressed in senescing leaves and differentially expressed depending on nitrate conditions. In addition to the characterization of autophagy, GS1 and ASN genes, phylogenic and gene structures were analysed. All the sequences data provided by this work will be helpful to further translational and genetic association studies.