ETUDE DES INTERACTIONS ENTRE PROTEINES ET LESIONS DE L'ADN PAR RESONANCE PLASMONIQUE DE SURFACE PAR IMAGERIE (SPRI)

DNA is the carrier of genetic information. DNA damage caused by various physical or chemical stresses is a challenge for cellular repair systems. These include the base excision repair system (BER) which involves several enzymes whose objectives are the recognition and removal of damaged bases, well-recognised functions for two glycosylases: prokaryotic Fpg and eukaryotic OGG1. Many approaches have been described to study DNA / protein interactions in vitro. With surface plasmon resonance imaging (SPRi), we have a real-time technique, without labeling, with which we can observe interactions in parallel for a single protein purified enzyme (Fpg, OGG1, EndoIV or Ape1) vis-à-vis various injuries to synthetic oligonucleotides immobilized on a gold surface. The damages studied were an oxidized base (8-oxoG), a cyclised base (cycloadenine) and analogues of abasic sites (THF and C3). We also studied the action of these enzymes on multiple lesions, in tandem, combining the 8-oxoG and 8-oxoA bases on the same strand of DNA. The originality of our system combines the direct analysis of the DNA / protein interaction with the indirect approach of observing its outcome by hybridization and amplification of the signal after a thermal ramp. The results obtained enable us to consider the use of our technique to observe the simultaneous repair of certain lesions by cell extracts for biochemical work, or by human tissue extracts for bio-medical work.L'ADN étant le support de l'information génétique, les lésions de l'ADN provoquées par différents stress physiques ou chimiques sont un défi pour les systèmes de réparation cellulaire. Parmi ceux-ci le système de réparation par excision de bases (BER) implique plusieurs enzymes dont les objectifs sont la reconnaissance et le retrait de la base lésée, fonctions bien connues pour deux glycosylases : Fpg Procaryote et OGG1 Eucaryote. De nombreuses approches ont été décrites pour étudier les interactions ADN/protéine in vitro. Avec la résonance plasmonique de surface par imagerie (SPRi), nous disposons d'une technique d'analyse en temps réel, sans marquage avec laquelle nous avons pu observer des interactions parallélisées d'une même protéine enzymatique purifiée (Fpg, OGG1, EndoIV ou Ape1) vis-à-vis de différentes lésions sur des oligonucléotides de synthèse immobilisés sur une surface d'or. Les dommages étudiés sont une base oxydée (8-oxoG), une base cyclisée (cycloadénine) et des analogues de sites abasiques (THF et C3). Nous avons également étudié l'action de ces mêmes enzymes sur des lésions multiples, en tandem, associant les bases 8-oxoG et 8-oxoA sur le même brin d'ADN. L'originalité de notre dispositif associe l'analyse directe de l'interaction ADN/protéine et l'approche indirecte de sa conséquence par une stratégie d'hybridation et d'amplification du signal après une rampe thermique. Les résultats obtenus permettent d'envisager l'utilisation de notre technique pour observer la réparation simultanée de certaines lésions par des extraits cellulaires pour des travaux de biochimie ou des extraits tissulaires humains pour des travaux de biologie médicale

ETUDE DES INTERACTIONS ENTRE PROTEINES ET LESIONS DE L'ADN PAR RESONANCE PLASMONIQUE DE SURFACE PAR IMAGERIE (SPRI)

DNA is the carrier of genetic information. DNA damage caused by various physical or chemical stresses is a challenge for cellular repair systems. These include the base excision repair system (BER) which involves several enzymes whose objectives are the recognition and removal of damaged bases, well-recognised functions for two glycosylases: prokaryotic Fpg and eukaryotic OGG1. Many approaches have been described to study DNA / protein interactions in vitro. With surface plasmon resonance imaging (SPRi), we have a real-time technique, without labeling, with which we can observe interactions in parallel for a single protein purified enzyme (Fpg, OGG1, EndoIV or Ape1) vis-à-vis various injuries to synthetic oligonucleotides immobilized on a gold surface. The damages studied were an oxidized base (8-oxoG), a cyclised base (cycloadenine) and analogues of abasic sites (THF and C3). We also studied the action of these enzymes on multiple lesions, in tandem, combining the 8-oxoG and 8-oxoA bases on the same strand of DNA. The originality of our system combines the direct analysis of the DNA / protein interaction with the indirect approach of observing its outcome by hybridization and amplification of the signal after a thermal ramp. The results obtained enable us to consider the use of our technique to observe the simultaneous repair of certain lesions by cell extracts for biochemical work, or by human tissue extracts for bio-medical work.L'ADN étant le support de l'information génétique, les lésions de l'ADN provoquées par différents stress physiques ou chimiques sont un défi pour les systèmes de réparation cellulaire. Parmi ceux-ci le système de réparation par excision de bases (BER) implique plusieurs enzymes dont les objectifs sont la reconnaissance et le retrait de la base lésée, fonctions bien connues pour deux glycosylases : Fpg Procaryote et OGG1 Eucaryote. De nombreuses approches ont été décrites pour étudier les interactions ADN/protéine in vitro. Avec la résonance plasmonique de surface par imagerie (SPRi), nous disposons d'une technique d'analyse en temps réel, sans marquage avec laquelle nous avons pu observer des interactions parallélisées d'une même protéine enzymatique purifiée (Fpg, OGG1, EndoIV ou Ape1) vis-à-vis de différentes lésions sur des oligonucléotides de synthèse immobilisés sur une surface d'or. Les dommages étudiés sont une base oxydée (8-oxoG), une base cyclisée (cycloadénine) et des analogues de sites abasiques (THF et C3). Nous avons également étudié l'action de ces mêmes enzymes sur des lésions multiples, en tandem, associant les bases 8-oxoG et 8-oxoA sur le même brin d'ADN. L'originalité de notre dispositif associe l'analyse directe de l'interaction ADN/protéine et l'approche indirecte de sa conséquence par une stratégie d'hybridation et d'amplification du signal après une rampe thermique. Les résultats obtenus permettent d'envisager l'utilisation de notre technique pour observer la réparation simultanée de certaines lésions par des extraits cellulaires pour des travaux de biochimie ou des extraits tissulaires humains pour des travaux de biologie médicale

Etude des interactions entre protéines et lésions de l'ADN par résonance plasmonique de surface par imagerie (SPRI)

L'ADN étant le support de l'information génétique, les lésions de l'ADN provoquées par différents stress physiques ou chimiques sont un défi pour les systèmes de réparation cellulaire. Parmi ceux-ci le système de réparation par excision de bases (BER) implique plusieurs enzymes dont les objectifs sont la reconnaissance et le retrait de la base lésée, fonctions bien connues pour deux glycosylases : Fpg Procaryote et OGG1 Eucaryote. De nombreuses approches ont été décrites pour étudier les interactions ADN/protéine in vitro. Avec la résonance plasmonique de surface par imagerie (SPRi), nous disposons d'une technique d'analyse en temps réel, sans marquage avec laquelle nous avons pu observer des interactions parallélisées d'une même protéine enzymatique purifiée (Fpg, OGG1, EndoIV ou Ape1) vis-à-vis de différentes lésions sur des oligonucléotides de synthèse immobilisés sur une surface d'or. Les dommages étudiés sont une base oxydée (8-oxoG), une base cyclisée (cycloadénine) et des analogues de sites abasiques (THF et C3). Nous avons également étudié l'action de ces mêmes enzymes sur des lésions multiples, en tandem, associant les bases 8-oxoG et 8-oxoA sur le même brin d'ADN. L'originalité de notre dispositif associe l'analyse directe de l'interaction ADN/protéine et l'approche indirecte de sa conséquence par une stratégie d'hybridation et d'amplification du signal après une rampe thermique. Les résultats obtenus permettent d'envisager l'utilisation de notre technique pour observer la réparation simultanée de certaines lésions par des extraits cellulaires pour des travaux de biochimie ou des extraits tissulaires humains pour des travaux de biologie médicale.DNA is the carrier of genetic information. DNA damage caused by various physical or chemical stresses is a challenge for cellular repair systems. These include the base excision repair system (BER) which involves several enzymes whose objectives are the recognition and removal of damaged bases, well-recognised functions for two glycosylases: prokaryotic Fpg and eukaryotic OGG1. Many approaches have been described to study DNA / protein interactions in vitro. With surface plasmon resonance imaging (SPRi), we have a real-time technique, without labeling, with which we can observe interactions in parallel for a single protein purified enzyme (Fpg, OGG1, EndoIV or Ape1) vis-à-vis various injuries to synthetic oligonucleotides immobilized on a gold surface. The damages studied were an oxidized base (8-oxoG), a cyclised base (cycloadenine) and analogues of abasic sites (THF and C3). We also studied the action of these enzymes on multiple lesions, in tandem, combining the 8-oxoG and 8-oxoA bases on the same strand of DNA. The originality of our system combines the direct analysis of the DNA / protein interaction with the indirect approach of observing its outcome by hybridization and amplification of the signal after a thermal ramp. The results obtained enable us to consider the use of our technique to observe the simultaneous repair of certain lesions by cell extracts for biochemical work, or by human tissue extracts for bio-medical work.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Détermination du profil des acylcarnitines plasmatiques par spectrométrie de masse en tandem (développement de la technique et intérêt pour le diagnostic de maladies héréditaires du métabolisme)

Les anomalies de la b-oxydation mitochondriale des acides gras constituent un groupe à part entière des maladies héréditaires du métabolisme. On connaît environ un vingtaine de déficits qui peuvent se manifester à tout âge et de diverses manières : hypoglycémie, myocardiopathie ou épisodes de rhabdomyolyse Leur diagnostic biochimique repose sur la détermination du profil des acylcarnitines. En effet la carnitine permet de détoxifier les acyl-coenzyme A qui s accumulent dans la mitochondrie, on les retrouve alors sous forme d acylcarnitines qui s accumulent en cas d anomalies de la b-oxydation. Nous avons mis en place au laboratoire, la détermination du profil des acylcarnitines plasmatiques par spectrométrie de masse en tandem. En effet, grâce à ses nombreux avantages, la spectrométrie de masse est devenue un outil diagnostique puissant indispensable à la biologie médicale actuelle. Les critères de validation de la méthode ont été analysés (stabilité, interférences analytiques, anticoagulant utilisé, répétabilité, reproductibilité ) et les limites de la technique concernant la validation biologique des profils ont également été étudiées. Ainsi, le CHU de Grenoble est désormais en mesure de proposer un bilan complet du métabolisme intermédiaire (chromatographie des acides organiques urinaires, chromatographie des acides aminés plasmatiques, profil des acylcarnitines plasmatiques) et peut répondre à l urgence diagnostique des pathologies héréditaires du métabolisme.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

Ornithine Transcarbamylase – From Structure to Metabolism: An Update

Ornithine transcarbamylase (OTC; EC 2.1.3.3) is a ubiquitous enzyme found in almost all organisms, including vertebrates, microorganisms, and plants. Anabolic, mostly trimeric OTCs catalyze the production of L-citrulline from L-ornithine which is a part of the urea cycle. In eukaryotes, such OTC localizes to the mitochondrial matrix, partially bound to the mitochondrial inner membrane and part of channeling multi-enzyme assemblies. In mammals, mainly two organs express OTC: the liver, where it is an integral part of the urea cycle, and the intestine, where it synthesizes citrulline for export and plays a major role in amino acid homeostasis, particularly of L-glutamine and L-arginine. Here, we give an overview on OTC genes and proteins, their tissue distribution, regulation, and physiological function, emphasizing the importance of OTC and urea cycle enzymes for metabolic regulation in human health and disease. Finally, we summarize the current knowledge of OTC deficiency, a rare X-linked human genetic disorder, and its emerging role in various chronic pathologies

An in vitro explant model for studies of intestinal amino acid metabolism

International audienceBackground & aimsThe main role of the small intestine to absorb the bulk of dietary nutrients, including proteins and amino acids (AA). Enterocytes have long been considered a major player in amino acid homeostasis, especially the glutamine–citrulline–arginine crossroads. In vivo intestinal epithelium cell lines for studies of nitrogen metabolism have now reached their limitations. To push research forward, an interesting approach could be to use the explant incubation model of intestinal tissue, involving in vitro incubation of duodenal tissue biopsy specimens taken from human subjects.MethodsEight duodenal biopsies were taken during endoscopy from patients without proven intestinal disease. Each biopsy was incubated in complete culture medium for 23 h then weighed, and cell viability was evaluated after 6 and 18 h. We then explored L-citrulline (L-CIT) production (reflecting metabolic activity) and the presence of the main enzymes involved in AA metabolism in the intestine (arginase II, glutaminase I, ornithine aminotransferase, and ornithine carbamoyltransferase).ResultsMean weight of biopsies was 12.36 ± 1.00 mg. Mortality was around 20% after 6 h and 50% after 18 h of incubation. CIT was amply produced by the biopsies, and enzymes implicated in intestinal AA metabolism were expressed in this model.ConclusionsThis in vitro explant model of intestinal tissue emerges as a reliable model for conducting ex vivo investigations on AA metabolism

J Cachexia Sarcopenia Muscle

Combating malnutrition and cachexia is a core challenge in oncology. To limit muscle mass loss, the use of proteins in cancer is encouraged by experts in the field, but it is still debated due to their antagonist effects. Indeed, a high protein intake could preserve lean body mass but may promote tumour growth, whereas a low-protein diet could reduce tumour size but without addressing cachexia. Here we used a realistic rodent model of cancer and chemotherapy to evaluate the influence of different protein intakes on cachexia, tumour response to chemotherapy and immune system response. The goal is to gain a closer understanding of the effect of protein intake in cancer patients undergoing chemotherapy. Female Fischer 344 rats were divided into six groups: five groups (n = 14 per group) with cancer (Ward colon tumour) and chemotherapy were fed with isocaloric diets with 8%, 12%, 16%, 24% or 32% of caloric intake from protein and one healthy control group (n = 8) fed a 16% protein diet, considered as a standard diet. Chemotherapy included two cycles, 1 week apart, each consisting of an injection of CPT-11 (50 mg/kg) followed by 5-fluorouracil (50 mg/kg) the day after. Food intake, body weight, and tumour size were measured daily. On day 9, the rats were euthanized and organs were weighed. Body composition was determined and protein content and protein synthesis (SUnSET method) were measured in the muscle, liver, intestine, and tumour. Immune function was explored by flow cytometry. Cancer and chemotherapy led to a decrease in body weight characterized by a decrease of both fat mass (-56 ± 3%, P < 0.05) and fat-free mass (-8 ± 1%, P < 0.05). Surprisingly, there was no effect of protein diet on body composition, muscle or tumour parameters (weight, protein content, or protein synthesis) but a high cumulative protein intake was positively associated with a high relative body weight and high fat-free mass. The immune system was impacted by cancer and chemotherapy but not by the different amount of protein intake. Using a realistic model of cancer and chemotherapy, we demonstrated for the first time that protein intake did not positively or negatively modulate tumour growth. Moreover, our results suggested that a high cumulative protein intake was able to improve moderately nutritional status in chemotherapy treated cancer rodents. Although this work cannot be evaluated clinically for ethical reasons, it nevertheless brings an essential contribution to nutrition management for cancer patients

Effects of acute nitric oxide precursor intake on peripheral and central fatigue during knee extensions in healthy men

International audienceNew findings: What is the central question of this study? What is the effect of acute NO precursor intake on vascular function, muscle and cerebral oxygenation and peripheral and central neuromuscular fatigue during knee-extension exercise? What is the main finding and its importance? Acute NO precursor ingestion increases the plasma concentrations of NO precursors (nitrate, arginine and citrulline) and enhances post-ischaemic vasodilatation, but has no significant effect on muscle and cerebral oxygenation, peripheral and central mechanisms of neuromuscular fatigue and, consequently, does not improve exercise performance.Abstract: Nitric oxide (NO) plays an important role in matching blood flow to oxygen demand in the brain and contracting muscles during exercise. Previous studies have shown that increasing NO bioavailability can improve muscle function. The aim of this study was to assess the effect of acute NO precursor intake on muscle and cerebral oxygenation and on peripheral and central neuromuscular fatigue during exercise. In four experimental sessions, 15 healthy men performed a thigh ischaemia-reperfusion test followed by submaximal isometric knee extensions (5 s on-4 s off; 45% of maximal voluntary contraction) until task failure. In each session, subjects drank a nitrate-rich beetroot juice containing 520 mg nitrate (N), N and citrulline (6 g; N+C), N and arginine (6 g; N+A) or a placebo (PLA). Prefrontal cortex and quadriceps near-infrared spectroscopy parameters were monitored continuously. Transcranial magnetic stimulation and femoral nerve electrical stimulation were used to assess central and peripheral determinants of fatigue. The post-ischaemic increase in thigh blood total haemoglobin concentration was larger in N (10.1 ± 3.7 mmol) and N+C (10.9 ± 3.3 mmol) compared with PLA (8.2 ± 2.7 mmol; P 0.05). In contrast to the post-ischaemic hyperaemic response, NO bioavailability in healthy subjects might not be the limiting factor for tissue perfusion and oxygenation during submaximal knee extensions to task failure

SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes

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Synergistic effects of citrulline supplementation and exercise on performance in male rats: evidence for implication of protein and energy metabolisms

International audienceBackground: Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Methods: Three-month-old healthy male rats were randomly assigned to be fed ad libitum for 4 weeks with either a citrulline-enriched diet (1 g·kg-1·day-1) (CIT) or an isonitrogenous standard diet (by addition of nonessential amino acid) (Ctrl) and trained (running on treadmill 5 days·week-1) (ex) or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored