Fonction surrénalienne après bolus d'étomidate en chirurgie cardiaque: une étude rétrospective [Adrenal function after induction of cardiac surgery patients with etomidate: a retrospective study]

OBJECTIVE: A single bolus dose of etomidate decreases cortisol synthesis by inhibiting the 11-beta hydroxylase, a mitochondrial enzyme in the final step of cortisol synthesis. In our institution, all the patients undergoing cardiac surgery receive etomidate at anesthesia induction. The purpose of this study was to assess the incidence of adrenocortical dysfunction after a single dose of etomidate in selected patients undergoing major cardiac surgery and requiring high-dose norepinephrine postoperatively. STUDY DESIGN: Retrospective descriptive study in the surgical ICU of a university hospital. PATIENTS AND METHODS: Sixty-three patients presented acute circulatory failure requiring norepinephrine (>0,2 microg/kg/min) during the 48 hours following cardiac surgery. Absolute adrenal insufficiency was defined as a basal cortisol below 414 nmo/l (15 microg/dl) and relative adrenal insufficiency as a basal plasma cortisol between 414 nmo/l (15 microg/dl) and 938 nmo/l (34 microg/dl) with an incremental response after 250 microg of synthetic corticotropin (measured at 60 minutes) below 250 nmol/l (9 microg/dl). RESULTS: Fourteen patients (22%) had normal corticotropin test results, 10 (16%) had absolute and 39 (62%) relative adrenal insufficiency. All patients received a low-dose steroid substitution after the corticotropin test. Substituted patients had similar clinical outcomes compared to patients with normal adrenal function. CONCLUSION: A high incidence of relative adrenal failure was observed in selected cardiac surgery patients with acute postoperative circulatory failure.

A phenotypical study of vascular smooth muscle cells in human arterial and venous stenosis

Abstract Introduction The primary function of the contractile vascular smooth muscle cells (cVSMCs) is the regulation of the vascular contractility which means the adaptation of the vascular tonus in response to the modulation of the blood pressure and blood flow. The cVSMCs are essentially quiescent, and therefore their synthesis rate is very limited. They are characterized by the expression of contractile proteins specific to the muscular tissue including myosin, h-­‐caldesmon and <-­‐smooth muscle actin (〈-­‐SMA). These contractile cells are strongly represented in the media layer of the arterial wall and, in a smaller proportion, of the vein wall. Their typical stretched-­‐out morphology allows recognizing them by a histological analysis. They do not produce any extracellular matrix (ECM), and do not migrate through the different layers of the vessel wall, and are not directly involved in the development of intimal hyperplasia (IH). Neointimal formation occurs after endothelial disruption leading to complex molecular and biological mechanisms. The de-­‐differentiation of cVSMCs into synthetic VSMCs (sVSMCs) is mentioned as a key element. These non mature cells are able to proliferate and produce ECM. The characterization of the vascular smooth muscle cells (VSMCs) from healthy and stenosed vascular tissues will contribue to the understanding of the different biological processes leading to IH and will be useful for the development of new therapies to interfere with the cVSMCs growth and migration. The aim of our research was to quantify the proportion of cVSMCs and sVSMCs into the healthy and pathologic human blood vessel wall and to characterize their phenotype. Methods We selected 23 specimens of arterial and venous segments from 18 patients. All these specimens were stored in the biobank from the thoracic and vascular surgery departement. 4 groups were designed (group 1 :arteries without lesions (n=3) ;group 2 : veins without lesions (n=1); group 3: arteries with stenosis (n=9); group 4: veins with stenosis (n=10)). Histology: 5µm-­‐sections were made from each sample embedded in paraffin wax and further stained with hematoxylin & eosin (HE), Van Gieson's stain (VGEL) and Masson's Trichrome (TMB). Pathologic tissues were defined using the label that was given to the macroscopic samples by the surgeon and also, based on the histological analysis with HE and VGEL evaluating the presence of a thickened intima. The same was done to the control samples evaluating the absence of thickening. Immunohistochemistry : The primary antibodies were used :〈-­‐SMA, vimentin, h-­‐ caldesmon, calponin, smooth muscle-myosin heavy chain (SM-­‐MHC), tropomyosin-­‐4, retinol binding protein-­‐1 (RBP-­‐1), nonmuscle-­‐myosin heavy chain-­‐B (NM-­‐MHC-­‐B), Von Willebrand factor (VWF). A semi-­‐quantitative assessment of the intensity of each sample stained was performed. Western Blot : Segments of arteries and veins were analyzed using the following primary antibodies :〈-­‐SMA, Calponin, SM-­‐MHC, NM-­‐MHC-­‐B. The given results were then normalized with tubulin. Results Our data showed that, when using immunohistochemistry analysis we found that〈-­‐SMA was mostly expressed in control arteries, whereas NM-­‐MHC-­‐B in the pathologic ones. Using SM-­‐MHC, calponin, vimentin and caldesmon we found no significative differences in the expression of these proteins in the control and in the pathologic samples. Western Blot analysis showed an inverse correlation between healthy and pathological samples as <-­‐ SMA was more expressed in the pathological samples, while NM-­‐MHC-­‐B in the control group; SM-­‐MHC and calponin were mostly expressed in the pathologic samples. Conclusion Our study showed no clear differences between stenotic and control arterial and venous segments using semi-­‐quantitative assessement by immunohistochemistry. Western Blot showed a significant increased expression of 〈-­‐SMA, calponin and SM-­‐MHC in the arteries with stenosis, while NM-­‐MHC-­‐B was mostly expressed in the arteries without lesions. Further studies are needed to track the lineage of VSMCs to understand the mechanisms leading toIH.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

Divinyl ether fatty acid synthesis in late blight-diseased potato leaves.

We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems.

Genetic dysregulation of glutathione synthesis predicts alteration of plasma thiol redox status in schizophrenia.

Abstract Genetic studies have shown an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL), the key enzyme for glutathione (GSH) synthesis. The present study was aimed at analyzing the influence of a GSH dysregulation of genetic origin on plasma thiols (total cysteine, homocysteine, and cysteine-glycine) and other free amino acid levels as well as fibroblast cultures GSH levels. Plasma thiols levels were also compared between patients and controls. As compared with patients with a low-risk GCLC GAG TNR genotype, patients with a high-risk genotype, having an impaired GSH synthesis, displayed a decrease of fibroblast GSH and plasma total cysteine levels, and an increase of the oxidized form of cysteine (cystine) content. Increased levels of plasma free serine, glutamine, citrulline, and arginine were also observed in the high-risk genotype. Taken together, the high-risk genotypes were associated with a subgroup of schizophrenia characterized by altered plasma thiols and free amino acid levels that reflect a dysregulation of redox control and an increased susceptibility to oxidative stress. This altered pattern potentially contributes to the development of a biomarker profile useful for early diagnosis and monitoring the effectiveness of novel drugs targeting redox dysregulation in schizophrenia. Antioxid. Redox Signal. 15, 2003-2010.

Computational fluid dynamics of the right ventricular outflow tract and of the pulmonary artery: a bench model of flow dynamics.

OBJECTIVES: The reconstruction of the right ventricular outflow tract (RVOT) with valved conduits remains a challenge. The reoperation rate at 5 years can be as high as 25% and depends on age, type of conduit, conduit diameter and principal heart malformation. The aim of this study is to provide a bench model with computer fluid dynamics to analyse the haemodynamics of the RVOT, pulmonary artery, its bifurcation, and left and right pulmonary arteries that in the future may serve as a tool for analysis and prediction of outcome following RVOT reconstruction. METHODS: Pressure, flow and diameter at the RVOT, pulmonary artery, bifurcation of the pulmonary artery, and left and right pulmonary arteries were measured in five normal pigs with a mean weight of 24.6 ± 0.89 kg. Data obtained were used for a 3D computer fluid-dynamics simulation of flow conditions, focusing on the pressure, flow and shear stress profile of the pulmonary trunk to the level of the left and right pulmonary arteries. RESULTS: Three inlet steady flow profiles were obtained at 0.2, 0.29 and 0.36 m/s that correspond to the flow rates of 1.5, 2.0 and 2.5 l/min flow at the RVOT. The flow velocity profile was constant at the RVOT down to the bifurcation and decreased at the left and right pulmonary arteries. In all three inlet velocity profiles, low sheer stress and low-velocity areas were detected along the left wall of the pulmonary artery, at the pulmonary artery bifurcation and at the ostia of both pulmonary arteries. CONCLUSIONS: This computed fluid real-time model provides us with a realistic picture of fluid dynamics in the pulmonary tract area. Deep shear stress areas correspond to a turbulent flow profile that is a predictive factor for the development of vessel wall arteriosclerosis. We believe that this bench model may be a useful tool for further evaluation of RVOT pathology following surgical reconstructions.

Long-term cerebral plasticity-related gene expression : a study of the respective role of CBP and CRTC1 in CREB transcriptional activation

1.1 AbstractThe treatment of memory disorders and cognitive deficits in various forms of mental retardation may greatly benefit from a better understanding of the molecular and cellular mechanisms of memory formation. Different forms of memory have distinct molecular requirements.Short-term memory (STM) is thought to be mediated by covalent modifications of existing synaptic molecules, such as phosphorylation or dephosphorylation of enzymes, receptors or ion channels. In contrast, long-term memoiy (LTM) is thought to be mediated by growth of new synapses and restructuring of existing synapses. There is extensive evidence that changes in gene expression and de novo protein synthesis are key processes for LTM formation. In this context, the transcription factor CREB (cAMP-response element-binding protein) was shown to be crucial. Activation of CREB requires phosphorylation of a serine residue (Ser-133), and the subsequent recruitment of a coactivator called CREB-binding protein (CBP). Moreover, we have recently shown that another coactivator called CREB Regulated Transcription Coactivator 1 (CRTC1) functions as a calcium- and cAMP-sensitive coincidence detector in neurons, and is involved in hippocampal long-term synaptic plasticity. Given the importance of cAMP and calcium signaling for plasticity-related gene expression in neurons and in astrocytes, we sought to determine the respective involvement of the CREB coactivators CBP and CRTC1 in CREB-mediated transcription.We developed various strategies to selectively interfere with these CREB coactivators in mouse primary neurons and in astrocytes in vitro. However, despite several pieces of evidence implicating CBP and/or CRTC1 in the regulation of neuronal plasticity genes, we could not clearly determine the respective requirement of these coactivators for the activation of these genes. Nevertheless, we showed that calcineurin activity, which is important for CRTC1 nuclear translocation, is necessary for the expression of some CREB-regulated plasticity genes. We associated this phenomena to physiopathological conditions observed in Down's syndrome. In addition, we demonstrated that in astrocytes, noradrenaline stimulates CREB-target gene expression through β-adrenergic receptor activation, intracellular cAMP pathway activation, and CRTC-induced CREB transactivation.Defining the respective role of CREB and its coactivators CBP and CRTC1 in neuronal and astrocytic cultures in vitro sets the stage for future in vivo studies and for the possible development of new therapeutic strategies to improve the treatment of memoiy and cognitive disorders.1.2 RésuméUne meilleure connaissance des mécanismes moléculaires et cellulaires responsables de la formation de la mémoire pourrait grandement améliorer le traitement des troubles de la mémoire ainsi que des déficits cognitifs observés dans différentes formes de pathologies psychiatriques telles que le retard mental. Les différentes formes de mémoire dépendent de processus moléculaires différents.La mémoire à court terme (STM) semble prendre forme suite à des modifications covalentes de molécules synaptiques préexistantes, telles que la phosphorylation ou la déphosphorylation d'enzymes, de récepteurs ou de canaux ioniques. En revanche, la mémoire à long terme (LTM) semble être due à la génération de nouvelles synapses et à la restructuration des synapses existantes. De nombreuses études ont permis de démontrer que les changements dans l'expression des gènes et la synthèse de protéine de novo sont des processus clés pour la formation de la LTM. Dans ce contexte, le facteur de transcription CREB (cAMP-response element-binding protein) s'est avéré être un élément crucial. L'activation de CREB nécessite la phosphorylation d'un résidu sérine (Ser-133), et le recrutement d'un coactivateur nommé CBP (CREB binding protein). En outre, nous avons récemment démontré qu'un autre coactivateur de CREB nommé CRTC1 (CREB Regulated Transcription Coactivator 1) agit comme un détecteur de coïncidence de l'AMP cyclique (AMPc) et du calcium dans les neurones et qu'il est impliqué dans la formation de la plasticité synaptique à long terme dans l'hippocampe. Etant donné l'importance des voies de l'AMPc et du calcium dans l'expression des gènes impliqués dans la plasticité cérébrale, nous voulions déterminer le rôle respectif des coactivateurs de CREB, CBP et CRTC1.Nous avons développé diverses stratégies pour interférer de façon sélective avec les coactivateurs de CREB dans les neurones et dans les astrocytes chez la souris in vitro. Nos résultats indiquent que CBP et CRTC1 sont tous deux impliqués dans la transcription dépendante de CREB induite par l'AMPc et le calcium dans les neurones. Cependant, malgré plusieurs évidences impliquant CBP et/ou CRTC1 dans l'expression de gènes de plasticité neuronale, nous n'avons pas pu déterminer clairement leur nécessité respective pour l'activation de ces gènes. Toutefois, nous avons montré que l'activité de la calcineurine, dont dépend la translocation nucléaire de CRTC1, est nécessaire à l'expression de certains de ces gènes. Nous avons pu associer ce phénomène à une condition physiopathologique observée dans le syndrome de Down. Nous avons également montré que dans les astrocytes, la noradrénaline stimule l'expression de gènes cibles de CREB par une activation des récepteurs β- adrénergiques, l'activation de la voie de l'AMPc et la transactivation de CREB par les CRTCs.Définir le rôle respectif de CREB et de ses coactivateurs CBP et CRTC1 dans les neurones et dans les astrocytes in vitro permettra d'acquérir les connaissances nécessaires à de futures études in vivo et, à plus long terme d'éventuellement développer des stratégies thérapeutiques pour améliorer les traitements des troubles cognitifs.

Impaired glutathione synthesis in schizophrenia: convergent genetic and functional evidence

Schizophrenia is a complex multifactorial brain disorder with a genetic component. Convergent evidence has implicated oxidative stress and glutathione (GSH) deficits in the pathogenesis of this disease. The aim of the present study was to test whether schizophrenia is associated with a deficit of GSH synthesis. Cultured skin fibroblasts from schizophrenia patients and control subjects were challenged with oxidative stress, and parameters of the rate-limiting enzyme for the GSH synthesis, the glutamate cysteine ligase (GCL), were measured. Stressed cells of patients had a 26% (P = 0.002) decreased GCL activity as compared with controls. This reduction correlated with a 29% (P < 0.001) decreased protein expression of the catalytic GCL subunit (GCLC). Genetic analysis of a trinucleotide repeat (TNR) polymorphism in the GCLC gene showed a significant association with schizophrenia in two independent case-control studies. The most common TNR genotype 7/7 was more frequent in controls [odds ratio (OR) = 0.6, P = 0.003], whereas the rarest TNR genotype 8/8 was three times more frequent in patients (OR = 3.0, P = 0.007). Moreover, subjects with disease-associated genotypes had lower GCLC protein expression (P = 0.017), GCL activity (P = 0.037), and GSH contents (P = 0.004) than subjects with genotypes that were more frequent in controls. Taken together, the study provides genetic and functional evidence that an impaired capacity to synthesize GSH under conditions of oxidative stress is a vulnerability factor for schizophrenia.

Molecular modeling study of the binding and signaling properties of the TCRpMHC complex

The activation of the specific immune response against tumor cells is based on the recognition by the CD8+ Cytotoxic Τ Lymphocytes (CTL), of antigenic peptides (p) presented at the surface of the cell by the class I major histocompatibility complex (MHC). The ability of the so-called T-Cell Receptors (TCR) to discriminate between self and non-self peptides constitutes the most important specific control mechanism against infected cells. The TCR/pMHC interaction has been the subject of much attention in cancer therapy since the design of the adoptive transfer approach, in which Τ lymphocytes presenting an interesting response against tumor cells are extracted from the patient, expanded in vitro, and reinfused after immunodepletion, possibly leading to cancer regression. In the last decade, major progress has been achieved by the introduction of engineered lypmhocytes. In the meantime, the understanding of the molecular aspects of the TCRpMHC interaction has become essential to guide in vitro and in vivo studies. In 1996, the determination of the first structure of a TCRpMHC complex by X-ray crystallography revealed the molecular basis of the interaction. Since then, molecular modeling techniques have taken advantage of crystal structures to study the conformational space of the complex, and understand the specificity of the recognition of the pMHC by the TCR. In the meantime, experimental techniques used to determine the sequences of TCR that bind to a pMHC complex have been used intensively, leading to the collection of large repertoires of TCR sequences that are specific for a given pMHC. There is a growing need for computational approaches capable of predicting the molecular interactions that occur upon TCR/pMHC binding without relying on the time consuming resolution of a crystal structure. This work presents new approaches to analyze the molecular principles that govern the recognition of the pMHC by the TCR and the subsequent activation of the T-cell. We first introduce TCRep 3D, a new method to model and study the structural properties of TCR repertoires, based on homology and ab initio modeling. We discuss the methodology in details, and demonstrate that it outperforms state of the art modeling methods in predicting relevant TCR conformations. Two successful applications of TCRep 3D that supported experimental studies on TCR repertoires are presented. Second, we present a rigid body study of TCRpMHC complexes that gives a fair insight on the TCR approach towards pMHC. We show that the binding mode of the TCR is correctly described by long-distance interactions. Finally, the last section is dedicated to a detailed analysis of an experimental hydrogen exchange study, which suggests that some regions of the constant domain of the TCR are subject to conformational changes upon binding to the pMHC. We propose a hypothesis of the structural signaling of TCR molecules leading to the activation of the T-cell. It is based on the analysis of correlated motions in the TCRpMHC structure. - L'activation de la réponse immunitaire spécifique dirigée contre les cellules tumorales est basée sur la reconnaissance par les Lymphocytes Τ Cytotoxiques (CTL), d'un peptide antigénique (p) présenté à la suface de la cellule par le complexe majeur d'histocompatibilité de classe I (MHC). La capacité des récepteurs des lymphocytes (TCR) à distinguer les peptides endogènes des peptides étrangers constitue le mécanisme de contrôle le plus important dirigé contre les cellules infectées. L'interaction entre le TCR et le pMHC est le sujet de beaucoup d'attention dans la thérapie du cancer, depuis la conception de la méthode de transfer adoptif: les lymphocytes capables d'une réponse importante contre les cellules tumorales sont extraits du patient, amplifiés in vitro, et réintroduits après immunosuppression. Il peut en résulter une régression du cancer. Ces dix dernières années, d'importants progrès ont été réalisés grâce à l'introduction de lymphocytes modifiés par génie génétique. En parallèle, la compréhension du TCRpMHC au niveau moléculaire est donc devenue essentielle pour soutenir les études in vitro et in vivo. En 1996, l'obtention de la première structure du complexe TCRpMHC à l'aide de la cristallographie par rayons X a révélé les bases moléculaires de l'interaction. Depuis lors, les techniques de modélisation moléculaire ont exploité les structures expérimentales pour comprendre la spécificité de la reconnaissance du pMHC par le TCR. Dans le même temps, de nouvelles techniques expérimentales permettant de déterminer la séquence de TCR spécifiques envers un pMHC donné, ont été largement exploitées. Ainsi, d'importants répertoires de TCR sont devenus disponibles, et il est plus que jamais nécessaire de développer des approches informatiques capables de prédire les interactions moléculaires qui ont lieu lors de la liaison du TCR au pMHC, et ce sans dépendre systématiquement de la résolution d'une structure cristalline. Ce mémoire présente une nouvelle approche pour analyser les principes moléculaires régissant la reconnaissance du pMHC par le TCR, et l'activation du lymphocyte qui en résulte. Dans un premier temps, nous présentons TCRep 3D, une nouvelle méthode basée sur les modélisations par homologie et ab initio, pour l'étude de propriétés structurales des répertoires de TCR. Le procédé est discuté en détails et comparé à des approches standard. Nous démontrons ainsi que TCRep 3D est le plus performant pour prédire des conformations pertinentes du TCR. Deux applications à des études expérimentales des répertoires TCR sont ensuite présentées. Dans la seconde partie de ce travail nous présentons une étude de complexes TCRpMHC qui donne un aperçu intéressant du mécanisme d'approche du pMHC par le TCR. Finalement, la dernière section se concentre sur l'analyse détaillée d'une étude expérimentale basée sur les échanges deuterium/hydrogène, dont les résultats révèlent que certaines régions clés du domaine constant du TCR sont sujettes à un changement conformationnel lors de la liaison au pMHC. Nous proposons une hypothèse pour la signalisation structurelle des TCR, menant à l'activation du lymphocyte. Celle-ci est basée sur l'analyse des mouvements corrélés observés dans la structure du TCRpMHC.

Is carbohydrate-deficient transferrin a specific marker for alcohol abuse? A study in patients with chronic viral hepatitis.

Carbohydrate-deficient transferrin, a transferrin isoform, is hailed as a new marker of chronic alcohol abuse, but its specificity is, however, not unequivocally accepted. The aim of the present study was therefore to determine carbohydrate-deficient transferrin levels in patients with chronic hepatitis B and C with or without documented chronic alcohol intake. Carbohydrate-deficient transferrin was measured using a double-antibody radioimmunoassay (CDTect, Pharmacia) in serum samples from 66 patients (45 males and 21 females; mean age: 39 years) with chronic viral hepatitis B (n = 20) or C (n = 46). Diagnosis of the underlying liver disease was established by liver biopsy. Carbohydrate-deficient transferrin levels were raised in 15 patients [23%; hepatitis B (n = 2) and hepatitis C (n = 13)]. In patients with chronic hepatitis B, the carbohydrate-deficient transferrin level was raised in two abstainers. In the 46 patients with chronic hepatitis C, 10 (22%) patients with an alcohol consumption of < 60 g/day for the men and 30 g/day for the women had raised carbohydrate-deficient transferrin levels. The overall specificity of carbohydrate-deficient transferrin for chronic alcohol abuse was thus 78%, suggesting an association between elevated carbohydrate-deficient transferrin levels and the presence of chronic viral hepatitis. Carbohydrate-deficient transferrin levels were not correlated with the histological grading or staging of chronic hepatitis B and C, or with biological markers of hepatic synthesis and cellular damage. Thus, an increased carbohydrate-deficient transferrin level may occur in patients with chronic viral hepatitis in the absence of chronic alcohol abuse. This fact should be kept in mind by physicians when using this marker to detect alcohol abuse.

Synthesis of polyhydroxyalkanoate in the peroxisome of Pichia pastoris.

Polyhydroxyalkanoates (PHAs) are polyesters naturally produced by bacteria that have properties of biodegradable plastics and elastomers. A PHA synthase from Pseudomonas aeruginosa modified at the carboxy-end for peroxisomal targeting was transformed in Pichia pastoris. The PHA synthase was expressed under the control of the promoter of the P. pastoris acyl-CoA oxidase gene. Synthesis of up to 1% medium-chain-length PHA per g dry weight was dependent on both the expression of the PHA synthase and the presence of oleic acid in the medium. PHA accumulated as inclusions within the peroxisomes. P. pastoris could be used as a model system to study how peroxisomal metabolism needs to be modified to increase PHA production in other eukaryotes, such as plants.

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.

Computational problems in perfect phylogeny haplotyping: typing without calling the allele.

A haplotype is an m-long binary vector. The XOR-genotype of two haplotypes is the m-vector of their coordinate-wise XOR. We study the following problem: Given a set of XOR-genotypes, reconstruct their haplotypes so that the set of resulting haplotypes can be mapped onto a perfect phylogeny (PP) tree. The question is motivated by studying population evolution in human genetics, and is a variant of the perfect phylogeny haplotyping problem that has received intensive attention recently. Unlike the latter problem, in which the input is "full" genotypes, here we assume less informative input, and so may be more economical to obtain experimentally. Building on ideas of Gusfield, we show how to solve the problem in polynomial time, by a reduction to the graph realization problem. The actual haplotypes are not uniquely determined by that tree they map onto, and the tree itself may or may not be unique. We show that tree uniqueness implies uniquely determined haplotypes, up to inherent degrees of freedom, and give a sufficient condition for the uniqueness. To actually determine the haplotypes given the tree, additional information is necessary. We show that two or three full genotypes suffice to reconstruct all the haplotypes, and present a linear algorithm for identifying those genotypes.

Glutamine synthesis rate in the hyperammonaemic rat brain using simultaneous localized in vivo H-1 and N-15 MRS

Objectives: Glutamine synthetase is a critical step in the glutamate-glutamine cycle, the major mechanism of glutamate neurotransmission and is implicated in the mechanism of ammonia toxicity. 15N MRS is an alternative approach to 13C MRS in studying glutamate- glutamine metabolism. 15N MRS studies allow to measure an apparent glutamine synthesis rate (Vsyn) which reflects a combination of the glutamate- glutamine cycle activity (Vnt) and net glutamine accumulation. The net glutamine synthesis (Vsyn-Vnt) can be directly measured from 1H NMR. Therefore, the aim of this study was to perform in vivo localized 1H MRS interleaved with 15N MRS to directly measure the net glutamine synthesis rate and the apparent glutamine synthesis rate under 15N labeled ammonia infusion in the rat brain, respectively. Methods: 1H and 15N MRS data were acquired interleaved on a 9.4T system (Varian/Magnex Scientific) using 5 rats. 15NH4Cl solution was infused continuously into the femoral vein for up to 10 h (4.5 mmol/h/kg).1 The plasma ammonia concentration was increased to 0.95±0.08 mmol/L (Analox GM7 analyzer). 1H spectra were acquired and quantified as described previously.2 15N unlocalized and localized spectra were acquired using the sequence;3 and quantified using AMARES and an external reference method.4 The metabolic model used to analyze the total Gln and 5-15N labeled Gln time courses is shown on Figure 1A. Results: Glutamine concentration increased from 2.5±0.3 to 15±3.3 mmol/kg whereas the total glutamate concentrations remained unchanged (Figure 1B). The linear fit of the time-evolution of the total Gln from the 1H spectra gave the net synthesis flux (Vsyn-Vnt), which was 0.021± 0.006 mmol/min per g (Figure 1D). The 5-15N Gln peak (_271 ppm) was visible in the first and all subsequent scans, whereas the 2-15N Gln/Glu peak (_342 ppm) appeared after B1.5 h (Figure 1C). From the in vivo 5-15N Gln time course, Vsyn = 0.29±0.1 mmol/min per g and a plasma NH3 fractional enrichment of 71%±6% were calculated. Vnt was 0.26±0.1 mmol/min/g, obtained assuming a negligible Gln efflux.5 Vsyn and Vnt were within the range of 13C NMR measurements.6 Conclusion: The combination of 1H and 15N NMR allowed for the first time a direct and localized measurement of Vnt and apparent glutamine synthesis rate. Vnt is approximately one order of magnitude faster than the net glutamine accumulation.