Solution Structures of the cis- and trans-Pro30 Isomers of a Novel 38-Residue Toxin from the Venom of Hadronyche Infensa sp. that Contains a Cystine-Knot Motif within Its Four Disulfide Bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-HI:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-HI:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing, the presence of a triple-stranded antiparallel sheet consistent with the inhibitor cystine-knot (ICK) motif.

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Journal of Biological Inorganic Chemistry (2010)15: 271-281

Pesquisa de assinaturas de selecção de malária na região do gene humano TPI (triosefosfato isomerase)

A malária continua a ser a maior causa de doença e mortalidade no Mundo, sobretudo no continente Africano. Das cinco espécies do parasita causador de malária em humanos, Plasmodium falciparum é a mais letal. Em termos evolutivos a malária é um fenómeno recente com cerca de 10 000 anos, período onde tem atuado como importante pressão seletiva no genoma humano, contribuindo para a seleção de inúmeros polimorfismos que propiciam maior resistência ao protozoário parasita. Apesar da interação entre o parasita e o hospedeiro ser foco de inúmeros estudos, é bastante complexa e ainda está longe de ser totalmente compreendida, nomeadamente os fatores de suscetibilidade/resistência à infeção ou à doença. Este estudo teve como principal objetivo contribuir para o estudo dos polimorfismos eritrocitários associados à proteção contra a infeção malárica grave, centrando-se no gene TPI que codifica uma enzima glicolítica. Foi selecionada uma amostra populacional Africana (Moçambique) agrupada de acordo com a gravidade da doença (grupos clínicos). Fez-se o rastreio dos exões do gene TPI por SSCP, na busca de alterações no padrão de migração dos produtos amplificados, indiciadores de alterações na sequência nucleotídica que pudessem ser assinaturas de eventuais efeitos evolutivos exercidos pela malária; caracterizaram-se as variantes do promotor do gene TPI: -5A>G, -8G>A e -24T>G; e analisou-se o polimorfismo intrónico 2262 situado no intrão 5. Esta análise permitiu identificar o polimorfismo -8G>A como possível marcador associado à proteção contra a malária, observando-se diferenças estatisticamente significativas entre doentes com malária grave e não-grave [P = 0,032; OR = 0,230 (CI95%: 0,049-1,081)], quando associado ao haplótipo -5G/-8A/2262G. Através de um estudo de microssatélites nas regiões adjacentes ao gene TPI, estimou-se a antiguidade dos polimorfismos -5G>A e -8G>A, tendo-se obtido a idade de ~20 mil anos e ~14 mil anos, respetivamente. Ao nível bioquímico, o polimorfismo -8G>A poderá estar associado à acumulação celular de metilglioxal citotóxico, um potente inibidor da proliferação parasitária.

Pharmacokinetic aspects of Tert-Butylaminoethyl disulfide, an experimental drug against schistosomiasis in mice

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethyl disulfide was performed after administration of two different single doses (35 and 300 mg/kg) of either the cold or labelled drug. Plasma or blood samples were treated with dithiothreitol, perchloric acid, and, after filtration, submitted to further purification with anionic resein. In the final step, the drug was retained on a cationic resin column, eluted with NaCl 1M and detected according to the method of Ellman (1958). Alternatively, radioactive drug was detected by liquid scintillation counting. The results corresponding to the smaller dose of total drug suggested a pharmacokinetic behavior related to a one open compartment model with the following parameters: area under the intravenous curve (AUC i.v.):671 ± 14; AUC oral: 150 ± 40 µg.min. ml [raised to the power of -1]; elimination rate constant: 0.071 min [raised to the power of -1]; biological half life: 9.8 min; distribution volume: 0.74 ml/g. For the higher dose, the results seemed to obey a more complex undertermined model. Combining the results, the occurence of a dose-dependent pharmacokinetic behavior is suggested, the drug being rapidly absorbed and rapidly eliminated; the elimination process being related mainly to metabolization. The drug seems to be more toxic when administered I.V. because by this route it escapes first pass metabolism, while being quickly distributed to tissues. The maximum tolerated blood level seems to be around 16 µg/ml.

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids.

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Methylglyoxal Produced by Amyloid- Peptide-Induced Nitrotyrosination of Triosephosphate Isomerase Triggers Neuronal Death in Alzheimer’s Disease

Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.

Síntese e modificações de derivados heterocíclicos de D-arabinose: potenciais inibidores de glicose-6-fosfato isomerase e de glicosamina-6-fosfato sintase

The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(D-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from D-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethylphosphoryl chloride. The resulting 5-[D-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

EFEITO DA ASSOCIAÇÃO DE PECTINASE, INVERTASE E GLICOSE ISOMERASE NA QUALIDADE DO SUCO DE BANANA

O efeito do tratamento em que se associou as enzimas comerciais: 0,03 % v/p de pectinase (Clarex) a 0,6 % v/p de invertase (Invertase-S) e 0,5 % p/p de glicose-isomerase (Taka-sweet) sobre purê de banana (Musa cavendishii), em condições amenas de hidrólise (40o C, 15 min.) foi observado e comparado com o efeito de outros três tratamentos enzimáticos: 0,03 % v/p de pectinase (Clarex); 0,03 % v/p de pectinase (Clarex) associada à 0,6 % v/p de invertase (Invertase-S); e 0,03 % v/p de pectinase (Sigma) associada a 0,03 % v/p de celulase (Sigma), visando determinar a qualidade representada por um conjunto de propriedades físicas, fisico-químicas, químicas, microbiológicas e sensoriais dos sucos de banana obtidos. Essas propriedades não diferiram significativamente em função das pectinases e celulase empregadas. A adição de invertase provocou aumento de doçura e diminuição da viscosidade do suco. Por outro lado, a adição de glicose isomerase ao suco invertido não foi capaz de aumentar significativamente o teor de frutose.

Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20μM GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.

The role of the peptidyl prolyl isomerase Rrd1 in the transcriptional stress response

La régulation de la transcription est un processus complexe qui a évolué pendant des millions d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1 sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la régulation de la transcription via RNAPII en réponse au stress.

Investigating the role of the isomerase Rrd1/PTPA : from yeast to human

Chez Saccharomyces cerevisiae, les souches mutantes pour Rrd1, une protéine qui possède une activité de peptidyl prolyl cis/trans isomérase, montrent une résistance marquée à la rapamycine et sont sensibles au 4-nitroquinoline 1-oxide, un agent causant des dommages à l’ADN. PTPA, l’homologue de Rrd1 chez les mammifères, est reconnu en tant qu’activateur de protéine phosphatase 2A. Notre laboratoire a précédemment démontré que la surexpression de PTPA mène à l’apoptose de façon indépendante des protéines phosphatase 2A. La fonction moléculaire de Rrd1/PTPA était encore largement inconnue au départ de mon projet de doctorat. Mes recherches ont d’abord montré que Rrd1 est associé à la chromatine ainsi qu’à l’ARN polymérase II. L’analyse in vitro et in vivo par dichroïsme circulaire a révélé que Rrd1 est responsable de changements au niveau de la structure du domaine C-terminal de la grande sous-unité de l’ARN polymérase II, Rpb1, en réponse à la rapamycine et au 4-nitroquinoline 1-oxide. Nous avons également démontré que Rrd1 est requis pour modifier l’occupation de l’ARN polymérase II sur des gènes répondant à un traitement à la rapamycine. Finalement, nous avons montré que suite à un traitement avec la rapamycine, Rrd1 médie la dégradation de l’ARN polymérase II et que ce mécanisme est indépendant de l’ubiquitine. La dernière partie de mon projet était d’acquérir une meilleure connaissance de la fonction de PTPA, l’homologue de Rrd1 chez les mammifères. Nos résultats montrent que le «knockdown» de PTPA n’affecte pas la sensibilité des cellules à différentes drogues telles que la rapamycine, le 4-nitroquinoline 1-oxide ou le peroxyde d’hydrogène (H2O2). Nous avons également tenté d’identifier des partenaires protéiques pour PTPA grâce à la méthode TAP, mais nous ne sommes pas parvenus à identifier de partenaires stables. Nous avons démontré que la surexpression de la protéine PTPA catalytiquement inactive n’induisait pas l’apoptose indiquant que l’activité de PTPA est requise pour produire cet effet. Finalement, nous avons tenté d’étudier PTPA dans un modèle de souris. Dans un premier lieu, nous avons déterminé que PTPA était exprimé surtout au niveau des tissus suivants : la moelle osseuse, le thymus et le cerveau. Nous avons également généré avec succès plusieurs souris chimères dans le but de créer une souris «knockout» pour PTPA, mais l’allèle mutante ne s’est pas transférée au niveau des cellules germinales. Mes résultats ainsi que ceux obtenus par mon laboratoire sur la levure suggèrent un rôle général pour Rrd1 au niveau de la régulation des gènes. La question demeure toujours toutefois à savoir si PTPA peut effectuer un rôle similaire chez les mammifères et une vision différente pour déterminer la fonction de cette protéine sera requise pour adresser adéquatement cette question dans le futur.

Carbon metabolism in transgenic roots with altered levels of hexokinase and triosephosphate isomerase and growing under different nitrogen status

Ce projet a pour but d’évaluer la capacité de la voie des pentoses phosphates (VPP) dans les racines transgéniques de pomme de terre (Solanum tuberosum) modifiées pour exprimer différents niveaux de l'hexokinase (HK) et de la triosephosphate isomérase cytosolique (cTPI). Dans les racines, la VPP alimente la voie de l’assimilation de l’azote en equivalents réducteurs et permet donc la biosynthèse des acides aminés. Le glucose-6-phosphate produit par l’HK est consommé par la partie oxydative de la VPP catalysée par la glucose-6-phosphate déshydrogénase (G6PDH) et la 6-phosphogluconate déshydrogénase (6PGDH). Les changements dans l'expression de HK et cTPI peuvent affecter le fonctionnement de la VPP et les mécanismes qui sont liés à l’utilisation des équivalents réducteurs produits par la VPP, comme l'assimilation de l’azote et la synthèse des acides aminés. Afin d’évaluer l’effet des manipulations génétiques de l’HK et de la cTPI sur l’assimilation de l’azote, nous avons cultivé les racines transgéniques sur des milieux contenant des concentrations élevées (7 mM) ou basses (0,7 mM) de nitrate d’ammonium comme source d’azote. Les résultats montrent que la culture sur un milieu riche en azote induit les activités G6PDH et 6PGDH. Les données montrent que la capacité de la VPP est plus grande avec des niveaux élevés en HK ou en cTPI. Nous avons aussi pu démontrer une plus grande activité spécifique de l’HK dans les conditions pauvres en azote. Ces données ont été complémentées par des mesures des pools d’acides aminés dans les racines transgéniques cultivées sur différents niveaux d’azote. Aucune tendance notable des pools d’acides aminés n’a été remarquée dans les racines modifiées pour leur contenu en HK suggèrant que la manipulation de HK n’affecte pas l'assimilation de l’azote. Dans les racines transgéniques modifiées pour la cTPI, les ratios Gln/Glu et Asn/Asp sont plus élevés chez les clones antisens, indiquant une assimilation de l’azote plus élevée. Ces résultats ont démontré l'activation de l'assimilation de l’azote chez les clones antisens cTPI dans les conditions élevées et basses d’azote alors que la manipulation de l’HK n’affecte pas l’assimilation de l’azote.