Peroxisomal beta-oxidation--a metabolic pathway with multiple functions.

Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes.

The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L.

In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations.

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.

The lack of effect of insulin on luteinizing hormone pulsatility in healthy male volunteers provides evidence of a sexual dimorphism in the metabolic regulation of reproductive hormones.

BACKGROUND: The activity of the neuroendocrine reproductive axis is closely related to nutritional status. This link is particularly important in healthy women, in whom insulin is a positive signal for the reproductive system. In contrast, very little is known regarding this relation in men. OBJECTIVES: This study was designed to evaluate the effect of insulin on the reproductive axis of young male volunteers and to study the effect of short-term hypercaloric feeding on this modulation. DESIGN: The activity of the neuroendocrine reproductive axis was characterized by the pattern of endogenous luteinizing hormone (LH) secretion on the basis of frequent blood sampling protocols. The effect of insulin was tested by comparing the LH secretion pattern between a baseline study and a hyperinsulinemic euglycemic clamp. These studies were performed first in subjects fed a controlled isocaloric diet for 6 d (calculated as 1.5 times their resting metabolic rate) then in the same subjects fed a controlled hypercaloric diet in which 30% extra calories were provided as fat and fructose (3 g · kg(-1) · d(-1)) before undergoing identical protocols. Serum gonadotropins, sex steroids, glucose, insulin, ghrelin, and leptin concentrations were assessed, and the HOMA-IR was calculated. RESULTS: The LH secretion pattern was not affected by insulin or by hypercaloric feeding. Insulin decreased ghrelin and increased leptin concentrations but had no additional effect of hypercaloric feeding despite significantly lower HOMA-IR indexes. CONCLUSIONS: Our data indicate that neither insulin nor short-term hypercaloric feeding has any effect on the activity of the male reproductive axis. They also further support the association between ghrelin and insulin and glucose metabolism. This trial was registered at clinicaltrials.gov as NCT01058681.

Effects of gastric bypass and gastric banding on glucose kinetics and gut hormone release.

BACKGROUND: Bariatric surgery markedly improves glucose homeostasis in patients with type 2 diabetes even before any significant weight loss is achieved. Procedures that involve bypassing the proximal small bowel, such as Roux-en-Y gastric bypass (RYGBP), are more efficient than gastric restriction procedures such as gastric banding (GB). OBJECTIVE: To evaluate the effects of RYGBP and GB on postprandial glucose kinetics and gastro-intestinal hormone secretion after an oral glucose load. METHODS AND PROCEDURES: This study was a cross-sectional comparison among non-diabetic, weight-stable women who had undergone RYGBP (n = 8) between 9 and 48 months earlier or GB (n = 6) from 25 to 85 months earlier, and weight- and age-matched control subjects (n = 8). The women were studied over 4 h following ingestion of an oral glucose load. Total glucose and meal glucose kinetics were assessed using glucose tracers and plasma insulin, and gut hormone concentrations were simultaneously monitored. RESULTS: Patients who had undergone RYGBP showed a a more rapid appearance of exogenous glucose in the systemic circulation and a shorter duration of postprandial hyperglycemia than patients who had undergone GB and C. The response in RYGBP patients was characterized by early and accentuated insulin response, enhanced postprandial levels of glucagon-like peptide-1 (GLP-1) and polypeptide YY (PYY), and greater postprandial suppression of ghrelin. DISCUSSION: These findings indicate that RYGBP is associated with alterations in glucose kinetics and glucoregulatory hormone secretion. These alterations are probably secondary to the anatomic rearrangement of the foregut, given the fact that they are not observed after GB. Increased PYY and GLP-1 concentrations and enhanced ghrelin suppression are compatible with reduced food intake after RYGBP.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism.

OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

Gut-derived signaling molecules and vagal afferents in the control of glucose and energy homeostasis.

PURPOSE OF REVIEW: The control of glucose and energy homeostasis, including feeding behaviour, is tightly regulated by gut-derived peptidic and nonpeptidic endocrine mediators, autonomic nervous signals, as well as nutrients such as glucose. We will review recent findings on the role of the gastrointestinal tract innervation and of portal vein glucose sensors; we will review selected data on the action of gastrointestinally released hormones. RECENT FINDINGS: The involvement of mechanosensory vagal afferents in postprandial meal termination has been clarified using mouse models with selective impairments of genes required for development of mechanosensory fibres. These activate central glucogen-like peptide-1/glucogen-like peptide-2 containing ascending pathways linking the visceroceptive brainstem neurons to hypothalamic nuclei. Mucosal terminals comprise the chemosensory vagal afferents responsive to postprandially released gastrointestinal hormones. The mechanism by which the hepatoportal glucose sensor stimulates glucose utilization by muscles was demonstrated, using genetically modified mice, to be insulin-independent but to require GLUT4 and AMP-kinase. This sensor is a key site of glucogen-like peptide-1 action and plays a critical role in triggering first phase insulin secretion. PeptideYY and ghrelin target intracerebral receptors as they are bidirectionally transported across the blood brain barrier. The anorectic functions of peripherally released peptideYY may however be mediated both via vagal afferents and intracerebral Y2 receptors in the brainstem and arcuate nucleus. SUMMARY: These recent findings demonstrate that the use of improved anatomical and physiological techniques and animal models with targeted gene modifications lead to an improved understanding of the complex role of gastrointestinal signals in the control of energy homeostasis.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway.

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.

CHARACTERISATION OF ARABIDOPSIS THALIANA PERMEABLE CUTICLE MUTANTS SHOWING A STRONG RESISTANCE TO BOTRYTIS CINEREA

La cuticule des plantes, composée de cutine, un polyester lipidique complexe et de cires cuticulaires, couvre l'épiderme de la plupart des parties aériennes des plantes. Elle est constituée d'une barrière hydrophobique primaire qui minimise les pertes en eau et en soluté et protège l'organisme de différents stress environnementaux tels que les rayons UV, la dessiccation et l'infection par des pathogènes. Elle est aussi impliquée dans la délimitation des organes durant le développement. La cutine est un polyester qui, dans la plupart des espèces végétales, est principalement composé d'acides gras ω-hydroxylés composé de 16 à 18 carbones. Cependant, la cutine des feuilles d'Arabidopsis a une composition différente et est principalement constituée d'acides dicarboxyliques à 16-18 carbones. Les cires sont présentes dans le polyester de la cutine ou le recouvrent. Chez Arabidopsis, un nombre de mutants, tel que 1er, bdg, hth, att1, wbc11, et des plantes transgéniques avec différents changement dans la structure de la cuticule dans les feuilles et la tige, ont récemment été décrits et servent d'outils pour étudier la relation entre la structure et la fonction de la cuticule.7 mutants d'Arabidopsis ont été isolés par une méthode de coloration qui permet de détecter une augmentation dans la perméabilité cuticulaire. Ces mutants ont été appelés pec pour permeable cuticle.Pour la première partie de mon projet, j'ai principalement travaillé avec pec9/bre1 (permeable cuticle 9/botrytis resistance 1). PEC9/BRE1 a été identifié comme étant LACS2 (LONG CHAIN ACYL-CoA SYNTHETASE 2). Dans ce mutant, la cuticule n'est pas visible sous microscopie électronique et la quantité en acides gras omega- hydroxylés et en leurs dérivés est fortement réduite. Ces altérations conduisent à une plus grande perméabilité de la cuticule qui est mise en évidence par une plus grande sensibilité à la sécheresse et aux xénobiotiques et une coloration plus rapide par bleu de toluidine. Le mutant Iacs2 démontre aussi une grande capacité de résistance à l'infection du champignon nécrotrophique B. cinerea. Cette résistance est due à l'extrusion sur les feuilles d'un composé antifongique durant l'infection. Ce travail a été publié dans EMBO journal (Bessire et al., 2007, EMBO Journal).Mon second projet était principalement concentré sur pec1, un autre mutant isolé par le premier crible. La caractérisation de pec1 a révélé des phénotypes similaires à ceux de Iacs2, mais à chaque fois dans des proportions moindres : sensibilité accrue à la sécheresse et aux herbicides, plus grande perméabilité au bleu de toluidine et au calcofluor white, altération de la structure cuticulaire et résistance à B. cinerea à travers la même activité antifongique. PEC1 a été identifié comme étant AtPDR4. Ce gène code pour un transporteur ABC de la famille PDR ("Pleiotropic Drugs Resistance") qui sont des transporteurs ayants un large spectre de substrats. Le mutant se différencie de Iacs2, en cela que la composition en acides gras de la cuticule n'est pas autant altérée. C'est principalement le dihydroxypalmitate des fleurs dont la quantité est réduite. L'expression du gène marqué avec une GFP sous le contrôle du promoteur endogène a permis de localiser le transporteur au niveau de la membrane plasmique des cellules de l'épiderme, de manière polaire. En effet, la protéine est principalement dirigée vers l'extérieure de la plante, là où se trouve la cuticule, suggérant une implication d'AtPDR4 dans le transport de composants de la cuticule. Ce travail est actuellement soumis à Plant Cell.Une étude phylogénétique a aussi montré qu'AtPDR4 était très proche d'OsPDR6 du riz. Le mutant du riz a d'ailleurs montré des phénotypes de nanisme et de perméabilité similaire au mutant chez Arabidopsis.AbstractThe cuticle, consisting principally of cutin and cuticular waxes, is a hydrophobic layer of lipidic nature, which covers all aerial parts of plants and protects them from different abiotic and biotic stresses. Recently, the research in this area has given us a better understanding of the structure and the formation of the cuticle. The Arabidopsis mutants permeable cuticle 1 (peel) and botrytis resistance 1 (brel) were identified in two screens to identify permeable cuticles. The screens used the fluorescent dye calcofluor to measure permeability and also resistance to the fungal pathogen Botrytis. These mutants have highly permeable cuticle characteristics such as higher water loss, intake of chemicals through the cuticle, higher resistance to Botrytis cinerea infection, and organ fusion.BRE1 was cloned and found to be LACS2, a gene previously identified which is important in the formation and biosynthetic pathway of the cuticle. In brel, the amount of the major component of cutin in Arabidopsis leaves and stems, dicarboxylic acids, is five times lower than in the wild type. Moreover, the permeability of the cuticle allows the release of antifungal compounds at the leaf surface that inhibits the growth of two necrotrophic fungi: Botrytis cinerea and Sclerotinia sclerotiorum.PEC1 was identified as AtPDR4, a gene that codes for a plasma membrane transporter of the Pleiotropic Drug Resistance family, a sub-family of the ABC- transporters. AtPDR4 is strongly expressed in the epidermis of expanding tissues. In the epidermis it is located in a polar manner on the external plasma membrane, facing the cuticle. Analysis of the monomer composition of the cutin reveals that in this mutant the amount of hydroxy-acids and dihydroxy-palmitate is 2-3 times lower in flowers, in which organ these cutin monomers are the major components. Thus AtPDR4 is thought to function as a putative cutin monomer transporter.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (&lt; or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Molecular dissection of Arabidopsis thaliana endodermis development, structure and function

In vascular plants, the endodermis establishes a protective diffusion barrier surrounding the vasculature preventing the passive, uncontrolled entry of nutrients absorbed by the plant. It does so by means of a differentiation feature, the "Casparian Strip" (CS), a highly localized cell wall impregnation made of lignin, which seals the extracellular space. Although the existence of this differentiation feature has been intensively described, the mechanisms establishing this hallmark remain obscure. In this work I report, the developmental sequence of events that leads to a differentiated endodermis, in the plant model Arabidopsis thaliana. In addition, my descriptive approach gave important insights as to how these cells define membrane domains involved in the directional transport of nutrients. I also participated in characterizing a new transmembrane protein family, the CASPs, localized to the membrane domain underlying the CS, which we accordingly named the Casparian Strip membrane Domain (CSD). Our molecular analysis indicates that these proteins drive CS establishment. To identify more molecular factors of CS establishment, I performed a forward genetic screen. This screen led to the identification of 11 endodermis permissive mutants, which we named schengen (sgn) mutants. The causative mutations have been mapped to 5 independent loci: SGN1 to SGN5. SGN1 and SGN3 encode Receptor Like Kinases involved in the correct establishment of the CSD. A lack of those kinases leads to an incomplete CSD, which gives rise to interrupted CS barriers. Interestingly, SGN1 seems to also regulate CSD positioning to the middle of endodermal transversal walls. SGN4 encodes an NADPH oxidase involved in lignin polymerization essential for CS formation. The sgn5 mutant induces extra divisions of cortical cells strongly affecting the cell identity, but also leading to incorrect differentiation. A thorough characterization of the sgn2 mutant will follow elsewhere, yet preliminary results indicate that SGN2 encodes an Acyl-CoA N-acyltransferase. . In summary, with my work I have contributed a first set of molecular players of Casparian strip formation and initiated their characterization. Eventually, this might lead to an understanding of the molecular mechanisms of CS establishment in A.thaliana . This in turn will hopefully help to better understand nutrient uptake in higher plants and their response to environmental stresses. - Au sein des plantes vasculaires, l'endoderme représente un tissu protecteur mettant en place une barrière imperméable, empêchant n'importe quel élément de rejoindre les tissus conducteurs par simple diffusion. Cette barrière, appelée « Cadre de Caspary », correspond à une lignification de la paroi de l'endoderme et donne lieu à un cloisonnement de l'espace intercellulaire. Bien que cet élément de différenciation soit décrit en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements aboutissant à l'établissement du cadre de Caspary chez la plante modèle Arabidopsis thaliana. De plus, ce travail apporte de nouvelles connaissances expliquant comment ces cellules définissent des domaines membranaires importants pour le transport des nutriments. Nous décrivons une nouvelle famille de protéines membranaires, les CASPs (« CAparian Strip membrane domain Proteins »), localisées dans un domaine membranaire longeant le cadre de Caspary : le domaine de Caspary (CSD). L'analyse moléculaire des CASPs indique qu'elles dirigent la formation du cadre de Caspary. Par ailleurs, une approche génétique directe nous a permis d'identifier 11 mutants ayant un endoderme perméable. Nous avons nommé ces mutants Schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) qui participent à la délimitation du CSD. L'absence de ces kinases aboutit à un domaine CSD incomplet, se traduisant par un cadre de Caspary discontinu. De plus, SGN1 semble réguler le positionnement du CSD au milieu de la paroi transversale de l'endoderme. SGN4 produit une enzyme de type NADPH oxydase impliquée dans la polymérisation du cadre de Caspary. Dans le mutant sgn5, on observe une division anormale des cellules du cortex créant ainsi une nouvelle couche cellulaire incapable d'achever sa différenciation en endoderme. Quant à la mutation sgn2, bien que nous pensons qu'elle affecte une Acyl-CoA N-acyltransferase, sa caractérisation ne sera réalisée que prochainement. Au final, ce travail procure de nouveaux éléments sur l'établissement du cadre de Caspary qui pourraient être importants afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles. - De par leur immobilité, les plantes terrestres n'ont pas d'autre choix que de puiser leurs ressources dans leur environnement direct. La plante extrait du sol les nutriments qui lui sont nécessaires et les redistribue grâce à des tissus conducteurs. Afin de ne pas s'intoxiquer, il est donc essentiel de pouvoir sélectionner les éléments entrant dans la racine. Etonnement, ce n'est pas la surface des racines qui permet ce contrôle mais un tissu interne appelé endoderme. Ce dernier forme une barrière imperméable qui entoure chaque cellule et crée une jointure permettant de bloquer le passage des éléments entre les cellules. Cette structure, appelée « cadre de Caspary », oblige les éléments à entrer dans les cellules de l'endoderme et à être ainsi sélectionnés. Bien que cette structure soit décrite en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements qui aboutit à la formation du cadre de Caspary chez la plante modèle Arabidopsis thaliana. Ce travail apporte également de nouvelles connaissances expliquant comment ces cellules définissent, organisent et dirigent le transport des nutriments. Nous décrivons comment certains éléments de la cellule, les protéines CASPs (CAsparian Strip membrane domain Proteins), sont organisées un domaine particulier des membranes afin de créer une plateforme de construction longeant le cadre de Caspary : le domaine de Caspary (CSD). Afin de déterminer ce qu'il se passerait si une plante ne possédait pas de cadre de Caspary, nous avons réalisé une mutagénèse, ou approche génétique directe, et identifié 11 mutants (individu ayant un gène défectueux conduisant à la perte d'une fonction) ayant un endoderme perméable. Nous avons nommé ces mutants schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. Les gènes SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) servant à l'établissement de la plateforme de construction. L'absence de ces kinases aboutit à une base incomplète, se traduisant par un cadre de Caspary discontinu. Qui plus est, la kinase SGN1 semble réguler le positionnement de la plateforme au milieu de l'endoderme. Le gène SGN4 est par contre, impliqué dans la construction à proprement dite du cadre de Caspary. Dans le mutant sgn5, on observe une nouvelle couche de cellules ressemblant à de l'endoderme mais incapable de former correctement une barrière identique au cadre de Caspary. Quant au dernier mutant, sgn2, bien que cette étude fournisse des indices permettant de comprendre pourquoi le mutant sgn2 est défectueux, nous n'expliquerons ce cas que prochainement. En résumé, ce travail procure de nouvelles connaissances sur l'établissement du cadre de Caspary qui pourraient être importantes afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles.

Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans?

The purpose of the workshop "Do Peroxisome Proliferating Compounds Pose a Hepatocarcinogenic Hazard to Humans?" was to provide a review of the current state of the science on the relationship between peroxisome proliferation and hepatocarcinogenesis. There has been much debate regarding the mechanism by which peroxisome proliferators may induce liver tumors in rats and mice and whether these events occur in humans. A primary goal of the workshop was to determine where consensus might be reached regarding the interpretation of these data relative to the assessment of potential human risks. A core set of biochemical and cellular events has been identified in the rodent strains that are susceptible to the hepatocarcinogenic effects of peroxisome proliferators, including peroxisome proliferation, increases in fatty acyl-CoA oxidase levels, microsomal fatty acid oxidation, excess production of hydrogen peroxide, increases in rates of cell proliferation, and expression and activation of the alpha subtype of the peroxisome proliferator-activated receptor (PPAR-alpha). Such effects have not been identified clinically in liver biopsies from humans exposed to peroxisome proliferators or in in vitro studies with human hepatocytes, although PPAR-alpha is expressed at a very low level in human liver. Consensus was reached regarding the significant intermediary roles of cell proliferation and PPAR-alpha receptor expression and activation in tumor formation. Information considered necessary for characterizing a compound as a peroxisome proliferating hepatocarcinogen include hepatomegaly, enhanced cell proliferation, and an increase in hepatic acyl-CoA oxidase and/or palmitoyl-CoA oxidation levels. Given the lack of genotoxic potential of most peroxisome proliferating agents, and since humans appear likely to be refractive or insensitive to the tumorigenic response, risk assessments based on tumor data may not be appropriate. However, nontumor data on intermediate endpoints would provide appropriate toxicological endpoints to determine a point of departure such as the LED10 or NOAEL which would be the basis for a margin-of-exposure (MOE) risk assessment approach. Pertinent factors to be considered in the MOE evaluation would include the slope of the dose-response curve at the point of departure, the background exposure levels, and variability in the human response.