Resolving the Role of Plant Glutamate Dehydrogenase. I. in vivo Real Time Nuclear Magnetic Resonance Spectroscopy Experiments

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time 15N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [15N]Glu or 15NH4+ respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants

Etude du type métabolisme intégré entre embryons de colza natif et transgénique

COMPIEGNE-BU (601592101) / SudocSudocFranceF

Étude du métabolisme d'alcaloides N-Méthyles (Utilisation d'analogues et d'isotopes stables)

De par leurs propriétés pharmacologiques, les alcaloïdes nicotiniques et tropaniques suscitent un grand intérêt. Ces alcaloïdes sont essentiellement décrits chez les Solanacées pour lesquels leurs voies de biosynthèse sont désormais bien connues. Néanmoins, leur dégradation n'est pas clairement élucidée. Afin d'appréhender ce phénomène, les réactions de dégradation de la nicotine et de la tropanone ont été étudiées chez des plantes et des bactéries. D'une part, la N'-déméthylation de la nicotine a été étudiée dans une suspension cellulaire de Nicotiana plumbaginifolia. Il a été montré que les cellules pouvaient dégrader différents analogues pyridinniques et pipéridiniques de la nicotine et ce, avec une stéréosélectivité pour les isomères (R). L'utilisation de précurseurs marqués en isotopes stables sur ce même modèle a permis de démontrer que la N'-formylnornicotine, décrite comme intermédiaire potentiel de la déméthylation de la nicotine en nornicotine, dérive en fait de la nornicotine. D'autre part, la dégradation de la tropanone a été étudiée chez Solanum dulcamara et chez Pseudomonas AT3. Chez ces bactéries, il a pu être observé et quantifié in vivo la réduction de la tropanone en tropanol et pseudotropanol par RMN du proton. Des expériences de supplémentation avec de la tropanone marquée au carbone 13 ont également été réalisées sur ce même modèle ainsi que sur le modèle végétal. Le marquage a été retrouvé dans des acides aminés chez Solanum dulcamara, révélant une connexion étroite entre métabolisme secondaire et métabolisme primaire.Tobacco and tropane alkaloids are of interest as they have important pharmacological activities. They are produced in Solanaceous plants and their biosythetic pathways have been fairly well-studied. However their degradation in plants has not yet been fully elucidated. Hence, the aim of this thesis is to investigate the degradation mechanisms of nicotine and tropinone in plant and bacterial cultures. The N -demethylation of nicotine was studied in Nicotiana plumbaginifolia suspension cells by HPLC, GC and NMR. Firstly, the demethylating enzymes were found to accept a range of compounds structurally related to nicotine and to show stereoselectivity with the (R)-isomer being preferentially degraded compared to the (S)-isomer. Secondly, the hypothesis of a putative imtermediate, N -formylnornicotine, in the demethylation of nicotine to nornicotine was refuted; this compound was proved to be derived from nornicotine and formaldehyde. The degradation of tropinone was investigated in Pseudomonas AT3 and Solanum dulcamara. In Pseudomonas AT3, tropinone was observed to be reduced to tropine and pseudotropine. The kinetics of this degradation was successfully quantified directly in vivo by NMR. Experiments with labelled tropinone were performed on this model as well as the plant model of Solanum dulcamara. The label was detected in amino acids in Solanum dulcamara roots, hinting at correlation between secondary and primary metabolism.AMIENS-BU Sciences (800212103) / SudocSudocFranceF

In Vivo Use of 1D and 2D 1H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions

International audienceCell suspensions initiated from Duboisia myoporoides–a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids–previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and 1H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling

Unidirectional steady state rates of central metabolism enzymes measured simultaneously in a living plant tissue

International audienc

Quasi‐freestanding graphene on SiC(0001) by Ar‐mediated intercalation of antimony: a route toward intercalation of high‐vapor‐pressure elements

A novel strategy for the intercalation of antimony (Sb) under the (6√3×6√3)\u1d44530° reconstruction, also known as buffer layer, on SiC(0001) is reported. Using X‐ray photoelectron spectroscopy, low‐energy electron diffraction, and angle‐resolved photoelectron spectroscopy, it is demonstrated that, while the intercalation of the volatile Sb is not possible by annealing the Sb‐coated buffer layer in ultrahigh vacuum, it can be achieved by annealing the sample in an atmosphere of Ar, which suppresses Sb desorption. The intercalation leads to a decoupling of the buffer layer from the SiC(0001) surface and the formation of quasi‐freestanding graphene. The intercalation process paves the way for future studies of the formation of quasi‐freestanding graphene by intercalation of high‐vapor‐pressure elements, which are not accessible by previously known intercalation techniques, and thus provides new avenues for the manipulation of epitaxial graphene on SiC

A biomechanical comparison of a cement-augmented odontoid screw with a posterior-instrumented fusion in geriatric patients with an odontoid fracture type IIb

Purpose!#!Possible surgical therapies for odontoid fracture type IIb include odontoid screw osteosynthesis (OG) with preservation of mobility or dorsal C1/2 fusion with restriction of cervical rotation. In order to reduce material loosening in odontoid screw osteosynthesis in patients with low bone density, augmentation at the base of the axis using bone cement has been established as a suitable alternative. In this study, we compared cement-augmented OG and C1/2 fusion according to Harms (HG).!##!Methods!#!Body donor preparations of the 1st and 2nd cervical vertebrae were randomized in 2 groups (OG vs. HG). The range of motion (ROM) was determined in 3 principle motion plains. Subsequently, a cyclic loading test was performed. The decrease in height of the specimen and the double amplitude height were determined as absolute values as an indication of screw loosening. Afterward, the ROM was determined again and loosening of the screws was measured in a computed tomography.!##!Results!#!A total of 16 were included. Two groups of 8 specimens (OG vs. HG) from patients with a median age of 80 (interquartile range (IQ) 73.5-85) years and a reduced bone density of 87.2 (IQ 71.2-104.5) mg/cc dipotassium hydrogen phosphate were examined for their biomechanical properties. Before and after exposure, the OG preparations were significantly more mobile. At the time of loading, the OG had similar loading properties to HG decrease in height of the specimen and the double amplitude height. Computed tomography revealed similar outcomes with regard to the screw loosening rate (62.5 vs. 87.5%, p = 0.586).!##!Conclusion!#!In patients with an odontoid fracture type IIb and reduced bone density, cement-augmented odontoid screw yielded similar properties in the loading tests compared to the HG. It may, therefore, be considered as a primary alternative to preserve cervical mobility in these patients