Developmental change in isoproterenol-mediated relaxation of pulmonary veins of fetal and newborn lambs.

beta-Adrenergic agonists are important regulators of perinatal pulmonary circulation. They cause vasodilation primarily via the adenyl cyclase-adenosine 3',5'-cyclic monophosphate (cAMP) pathway. We examined the responses of isolated fourth-generation pulmonary veins of term fetal (145 +/- 2 days gestation) and newborn (10 +/- 1 days) lambs to isoproterenol, a beta-adrenergic agonist. In vessels preconstricted with U-46619 (a thromboxane A2 analog), isoproterenol induced greater relaxation in pulmonary veins of newborn lambs than in those of fetal lambs. The relaxation was eliminated by propranolol, a beta-adrenergic antagonist. Forskolin, an activator of adenyl cyclase, also caused greater relaxation of veins of newborn than those of fetal lambs. 8-Bromoadenosine 3',5'-cyclic monophosphate, a cell membrane-permeable analog of cAMP, induced a similar relaxation of all vessels. Biochemical studies show that isoproterenol and forskolin induced a greater increase in cAMP content and in adenyl cyclase activity of pulmonary veins in the newborn than in the fetal lamb. These results demonstrate that beta-adrenergic-agonist-mediated relaxation of pulmonary veins increases with maturation. An increase in the activity of adenyl cyclase may contribute to the change.

Nitric oxide activity through guanylate cyclase and phosphodiesterase modulation is impaired in fetal lambs with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3'-5'-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3'-5'-guanylosine monophosphate. METHODS: Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels. RESULTS: In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5 trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5'hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1-induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032. CONCLUSIONS: Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.

New insights into the role of microRNAs in pancreatic ß-cell maturation and plasticity

Le diabète est une maladie chronique caractérisée par une élévation du taux de sucre dans le sang aussi appelé « glycémie » reflétant un état pathologique. L'élévation de la glycémie au long cours a des répercussions délétères sur nombreux de nos tissus et organes d'où l'apparition de complications sévères chez les sujets diabétiques pouvant atteindre les yeux, les reins, le système nerveux, le système cardiovasculaire et les membres inférieurs. La carence en une hormone essentielle à notre organisme, l'insuline, est au coeur du développement de la maladie. L'insuline induit la captation du glucose circulant dans le sang en excès suite à une prise alimentaire riche en glucides et favorise son utilisation et éventuellement son stockage dans les tissus tels que le foie, le tissu adipeux et les muscles. Ainsi, l'insuline est vitale pour réguler et maintenir stable notre niveau de glycémie. Les cellules bêta du pancréas sont les seules entités de notre corps capables de produire de l'insuline et une perte de fonctionnalité associée à leur destruction ont été mises en cause dans le processus pathologique du diabète de type 2. Cependant la pleine fonctionnalité et la maturation des cellules bêta n'apparaissent qu'après la naissance lorsque le pancréas en développement a atteint sa masse adulte définitive. Enfin, une fois la masse des cellules bêta définitive établie, leur nombre et volume restent relativement constants au cours de la vie adulte chez un sujet sain. Néanmoins, au cours de périodes critiques les besoins en insuline sont augmentés tel qu'observé chez les femmes enceintes et les personnes obèses qui ont une perte de sensibilité à l'insuline qui se traduit par la nécessité de sécréter plus d'insuline afin de maintenir une glycémie normale. Dans l'hypothèse où la compensation n'a pas lieu ou n'est pas aboutie, le diabète se développe. Le processus de maturation postnatale ainsi que les événements compensatoires sont donc des étapes essentielles et de nombreuses questions sont encore non résolues concernant l'identification des mécanismes les régulant. Parmi les acteurs potentiels figurent de petites molécules d'ARN découvertes récemment appelées microARNs et qui ont été rapidement suggérées très prometteuses dans l'identification de nouvelles cibles thérapeutiques dans le cadre du diabète et d'autres pathologies. Les microARNs vont réguler l'expression de notre génome sans en modifier la séquence, phénomène également appelé épigénétique, ce qui résulte en des différences de comportement et de fonction cellulaires. Les microARNs sont donc susceptibles de jouer un rôle clé dans l'ensemble des processus biologiques et notre environnement associé à nos prédispositions génétiques peuvent grandement modifier leur niveau et donc leur action, qui à son tour se répercutera sur notre état physiologique. En effet nous avons identifié des changements de microARNs dans les cellules d'îlots pancréatiques de modèles animaux (rats et souris) associés à un état de résistance à l'insuline (grossesse et obésité). Par le biais d'expériences in vitro sur des cellules bêta extraites de rats et conservées en culture, nous avons pu analyser de plus près l'implication des microARNs dans la capacité des cellules bêta à sécréter de l'insuline mais aussi à se multiplier et à survivre au sein d'un environnement toxique. Ainsi, nous avons identifié des microARNs qui participent positivement à la compensation des cellules bêta, sous la direction d'hormones telles les estrogènes ou d'une hormone libérée par l'intestin au cours de la digestion (l'inerétine GLP1) et qui est largement utilisée comme agent thérapeutique dans la médication contre le diabète. Dans un second temps nous avons utilisé une stratégie similaire afin de déterminer le rôle de microARNs préalablement détectés comme étant changés au cours du développement postnatal des cellules bêta chez le rat. Cette étude a également mené à l'identification de microARNs participant à la maturation et à l'expansion de la masse des cellules bêta sous l'influence de la composition du régime alimentaire et des besoins en insuline adéquats qui en dépendent. Ces études apportent la vision de nouveaux mécanismes moléculaires impliquant les microARNs et démontrant leur importance pour le bon fonctionnement des cellules bêta et leur capacité d'adaptation à l'environnement. -- Les cellules bêta sont une composante des îlots pancréatiques de Langerhans et sont des cellules hautement différenciées qui ont l'unique capacité de sécréter de l'insuline sous l'influence des nutriments suite à une prise alimentaire. L'insuline facilite l'incorporation de glucose dans ses tissus cibles tels le foie, le tissu adipeux et les muscles. Bien que les besoins en insuline soient relativement constants au cours de la vie d'un individu sain, certaines conditions associées à un état de résistance à l'insuline, telles la grossesse ou l'obésité, requièrent une libération d'insuline majorée. En cas de résistance à l'insuline, une dysfonction des cellules bêta plus ou moins associée à leur mort cellulaire, conduisent à une sécrétion d'insuline insuffisante et au développement d'une hyperglycémie chronique, caractéristique du diabète de type 2. Jusqu'à présent, les mécanismes moléculaires sous- jacents à la compensation des cellules bêta ou encore menant à leur dysfonction restent peu connus. Découverts récemment, les petits ARNs non-codant appelés microARNs (miARNs), suscitent un intérêt grandissant de par leur potentiel thérapeutique pour la prise en charge et le traitement du diabète. Les miARNs sont de puissants régulateurs de l'expression génique qui lient directement le 3'UTR de leurs ARN messagers cibles afin d'inhiber leur traduction ou d'induire leur dégradation, ce qui leur permet de contrôler des fonctions biologiques multiples. Ainsi, nous avons pris pour hypothèse que les miARNs pourraient jouer un rôle essentiel en maintenant la fonction des cellules bêta et des processus compensatoires afin de prévenir le développement du diabète. Lors d'une première étude, une analyse transcriptomique a permis l'identification de miARNs différemment exprimés au sein d'îlots pancréatiques de rattes gestantes. Parmi eux, le miR-338-3p a démontré la capacité de promouvoir la prolifération et la survie des cellules bêta exposées à des acides gras saturés et des cytokines pro-inflammatoires, sans altérer leur propriété sécrétrice d'insuline. Nous avons également identifié deux hormones reconnues pour leurs propriétés bénéfiques pour la physiologie de la cellule bêta, l'estradiol et l'incrétine GLP1, qui régulent les niveaux du miR-338-3p. Ce miARN intègre parfaitement les voies de signalisation de ces deux hormones dépendantes de l'AMP cyclique, afin de contrôler l'expression de nombreux gènes conduisant à son action biologique. Dans un projet ultérieur, notre objectif était de déterminer la contribution de miARNs dans l'acquisition de l'identité fonctionnelle des cellules bêta en période postnatale. En effet, directement après la naissance les cellules bêta sont reconnues pour être encore immatures et incapables de sécréter de l'insuline spécifiquement en réponse à l'élévation de la glycémie. Au contraire, la réponse insulinique induite par les acides aminés ainsi que la biosynthèse d'insuline sont déjà fonctionnelles. Nos recherches ont permis de montrer que les changements de miARNs corrélés avec l'apparition du phénotype sécrétoire en réponse au glucose, sont régis par la composition nutritionnelle du régime alimentaire et des besoins en insuline qui en découlent. En parallèle, le taux de prolifération des cellules bêta est considérablement réduit. Les miARNs que nous avons étudiés coordonnent des changements d'expression de gènes clés impliqués dans l'acquisition de propriétés vitales de la cellule bêta et dans la maintenancé de son identité propre. Enfin, ces études ont permis de clairement démontrer l'importance des miARNs dans la régulation de la fonction des cellules bêta pancréatiques. -- Beta-cells are highly differentiated cells localized in the pancreatic islets and are characterized by the unique property of secreting insulin in response to nutrient stimulation after meal intake. Insulin is then in charge of facilitating glucose uptake by insulin target tissues such as liver, adipose tissue and muscles. Despite insulin needs stay more or less constant throughout life of healthy individuals, there are circumstances such as during pregnancy or obesity which are associated to insulin resistance, where insulin needs are increased. In this context, defects in beta-cell function, sometimes associated with beta-cell loss, may result in the release of inappropriate amounts of insulin leading to chronic hyperglycemia, properly defined as type 2 diabetes mellitus. So far, the mechanisms underlying beta- cell compensation as well as beta-cell failure remain to be established. The recently discovered small non-coding RNAs called microRNAs (miRNAs) are emerging as interesting therapeutic targets and are bringing new hope for the treatment of diabetes. miRNAs display a massive potential in regulating gene expression by directly binding to the 3'UTR of messenger RNAs and by inhibiting their translation and/or stability, enabling them to modify a wide range of biological functions. In view of this, we hypothesized that miRNAs may play an essential role in preserving the functional beta-cell mass and permitting to fight against beta-cell exhaustion and decompensation that can lead to diabetes development. In a first study, global profiling in pancreatic islets of pregnant rats, a model of insulin resistance, led to the identification of a set of differentially expressed miRNAs. Among them, miR-338- 3p was found to promote beta-cell proliferation and survival upon exposure of islet cells to pro- apoptotic stimuli such as saturated fatty acids or pro-inflammatory cytokines, without impairment in their capacity to release insulin. We also discovered that miR-338-3p changes are driven by two hormones, the estradiol and the incretin GLP1, both well known for their beneficial impact on beta- cell physiology. Consistently, we found that miR-338-3p integrates the cAMP-dependent signaling pathways regulated by these two hormones in order to control the expression of numerous genes and execute its biological functions. In a second project, we aimed at determining whether miRNAs contribute to the acquisition of beta-cell identity. Indeed, we confirmed that right after birth beta-cells are still immature and are unable to secrete insulin specifically in response to elevated concentrations of glucose. In contrast, amino acid-stimulated insulin release as well as insulin biosynthesis are already fully functional. In parallel, newborn beta-cells are proliferating intensively within the expanding pancreas. Interestingly, we demonstrated that the miRNA changes and the subsequent acquisition of glucose responsiveness is influenced by the diet composition and the resulting insulin needs. At the same time, beta-cell proliferation declines. The miRNAs that we have identified orchestrate expression changes of essential genes involved in the acquisition of specific beta-cell properties and in the maintenance of a mature beta-cell identity. Altogether, these studies clearly demonstrate that miRNAs play important roles in the regulation of beta-cell function.

Impact of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry on the Clinical Management of Patients With Gram-negative Bacteremia: A Prospective Observational Study.

Background. Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. Methods. We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. Results. Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. Conclusions. In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Nouveaux traitements de l'hépatite C: aspects pharmacologiques et potentiel d'interaction [New hepatitis C treatments: pharmacological considerations and potential for drug interactions].

Progress in the understanding of the hepatitis C virus life cycle allowed the development of new, very promising antiviral therapies. Although these new drugs have a favourable profile in terms of efficacy, tolerance and interaction potential, their prescription in the setting of comedication and impaired renal or hepatic function remains a challenge. Here, we provide a summary of pharmacological considerations, focusing on sofosbuvir, simeprevir and daclatasvir. A better understanding of their metabolic pathways and transporters may help the prescriber to identify and manage drug interactions especially in patients under immunosuppressive or anti-HIV therapy. Recommendations for the prescription of these drugs in specific situations are also discussed.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family.

Sequence data from regions of five vertebrate vitellogenin genes were used to examine the frequency, distribution, and mutability of the dinucleotide CpG, the preferred modification site for eukaryotic DNA methyltransferases. The observed level of the CpG dinucleotide in all five genes was markedly lower than that expected from the known mononucleotide frequencies. CpG suppression was greater in introns than in exons. CpG-containing codons were found to be avoided in the vitellogenin genes, but not completely despite the redundancy of the genetic code. Frequency and distribution patterns of this dinucleotide varied dramatically among these otherwise closely related genes. Dense clusters of CpG dinucleotides tended to appear in regions of either functional or structural interest (e.g., in the transposon-like Vi-element of Xenopus) and these clusters contained 5-methylcytosine (5 mC). 5 mC is known to undergo deamination to form thymidine, but the extent to which this transition occurs in the heavily methylated genomes of vertebrates and its contribution to CpG suppression are still unclear. Sequence comparison of the methylated vitellogenin gene regions identified C----T and G----A substitutions that were found to occur at relatively high frequencies. The predicted products of CpG deamination, TpG and CpA, were elevated. These findings are consistent with the view that CpG distribution and methylation are interdependent and that deamination of 5 mC plays an important role in promoting evolutionary change at the nucleotide sequence level.

Control of transepithelial Na+ transport and Na-K-ATPase by oxytocin and aldosterone.

Short- and long-term effect of oxytocin on Na+ transport and Na-K-ATPase biosynthesis in the toad bladder, and the potential interaction of this hormone with aldosterone have been studied, leading to the following observations. An early Na+ transport response (oxytocin, 50 mU/ml) peaked at 10-15 min of hormone addition. At maximal stimulation a three- to fourfold increase in Na+ transport was observed, a sustained Na+ transport response (about two-fold control base line) was observed as long as the hormone was present in the medium and for up to 20 h of incubation. Pretreatment for 30 min with actinomycin D (2 micrograms/ml) did not inhibit the early response, but significantly impaired the sustained response, suggesting that de novo protein synthesis was required. The simultaneous addition of the two hormones led within 60 min to a marked potentiation of the action on Na+ transport. This synergism could be mimicked by exogenous cyclic adenosine monophosphate (cAMP). Oxytocin alone (18 h exposure, 50 mU/ml) increased the relative rate of synthesis of both alpha and beta subunits of Na-K-ATPase (1.9- and 1.6-fold, respectively; P less than 0.05), whereas aldosterone (80 nM) increased the relative rate of synthesis of the same subunits (2.6- and 2.2-fold, respectively; P less than 0.02). Finally, in contrast to what was observed at the physiological level, the interaction of oxytocin and aldosterone did not lead to a similar potentiation at the biochemical level, i.e., induction of Na-K-ATPase biosynthesis (2.7- and 2.9-fold, for alpha and beta subunits, respectively; P less than 0.025).

Nitric oxide synthase (NOS) inhibition for one week improves renal sodium and water excretion in cirrhotic rats with ascites

Normalization of the increased vascular nitric oxide (NO) generation with low doses of NG-nitro-L-arginine methyl ester (L-NAME) corrects the hemodynamic abnormalities of cirrhotic rats with ascites. We have undertaken this study to investigate the effect of the normalization of vascular NO production, as estimated by aortic cyclic guanosine monophosphate (cGMP) concentration and endothelial nitric oxide synthase (eNOS) protein expression in the aorta and mesenteric artery, on sodium and water excretion. Rats with carbon tetrachloride-induced cirrhosis and ascites were investigated using balance studies. The cirrhotic rats were separated into two groups, one receiving 0.5 mg/kg per day of L-NAME (CIR-NAME) during 7 d, whereas the other group (CIR) was administrated the same volume of vehicle. Two other groups of rats were used as controls, one group treated with L-NAME and another group receiving the same volume of vehicle. Sodium and water excretion was measured on days 0 and 7. On day 8, blood samples were collected for electrolyte and hormone measurements, and aorta and mesenteric arteries were harvested for cGMP determination and nitric oxide synthase (NOS) immunoblotting. Aortic cGMP and eNOS protein expression in the aorta and mesenteric artery were increased in CIR as compared with CIR-NAME. Both cirrhotic groups had a similar decrease in sodium excretion on day 0 (0.7 versus 0.6 mmol per day, NS) and a positive sodium balance (+0.9 versus +1.2 mmol per day, NS). On day 7, CIR-NAME rats had an increase in sodium excretion as compared with the CIR rats (sodium excretion: 2.4 versus 0.7 mmol per day, P < 0.001) and a negative sodium balance (-0.5 versus +0.8 mmol per day, P < 0.001). The excretion of a water load was also increased after L-NAME administration (from 28+/-5% to 65+/-7, P < 0.05). Plasma renin activity, aldosterone and arginine vasopressin were also significantly decreased in the CIR-NAME, as compared with the CIR rats. The results thus indicate that normalization of aortic cGMP and eNOS protein expression in vascular tissue is associated with increased sodium and water excretion in cirrhotic rats with ascites.

Effects of SCH 34826, an orally active inhibitor of atrial natriuretic peptide degradation, in healthy volunteers.

Atrial natriuretic peptide is cleared from plasma by clearance receptors and by enzymatic degradation by way of a neutral metalloendopeptidase. Inhibition of neutral metalloendopeptidase activity appears to provide an interesting approach to interfere with metabolism of atrial natriuretic peptide to enhance the renal and haemodynamic effects of endogenous atrial natriuretic peptide. In this study, the effects of SCH 34826, a new orally active neutral metalloendopeptidase inhibitor, have been evaluated in a single-blind, placebo-controlled study involving eight healthy volunteers who had maintained a high sodium intake for 5 days. SCH 34826 had no effect on blood pressure or heart rate in these normotensive subjects. SCH 34826 promoted significant increases in excretion of urinary sodium, phosphate, and calcium. The cumulative 5-hour urinary sodium excretion was 15.7 +/- 7.3 mmol for the placebo and 22.9 +/- 5, 26.7 +/- 6 (p less than 0.05), and 30.9 +/- 6.8 mmol (p less than 0.01) for the 400, 800, and 1600 mg SCH 34826 doses, respectively. During the same time interval, the cumulative urinary phosphate excretion increased by 0.3 +/- 0.4 mmol after placebo and by 1.5 +/- 0.3 (p less than 0.01), 1.95 +/- 0.3 (p less than 0.01), and 2.4 +/- 0.4 mmol (p less than 0.001) after 400, 800, and 1600 mg SCH 34826, respectively. There was no change in diuresis or excretion of urinary potassium and uric acid. The natriuretic response to SCH 34826 occurred in the absence of any change in plasma atrial natriuretic peptide levels but was associated with a dose-dependent elevation of urinary atrial natriuretic peptide and cyclic guanosine monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2.

Several adenosine 3',5'-cyclic monophosphate (cAMP)-hydrolyzing phosphodiesterase isozymes are present in the pulmonary vasculature. The present study was designed to determine the effect of selective inhibitors of phosphodiesterase subtypes on prostaglandin E2 (PGE2)-induced relaxation of isolated fourth-generation pulmonary arteries of newborn lambs. PGE2 and forskolin caused pulmonary arteries to relax and induced an increase in the intracellular cAMP content in the vessels. The relaxation and change in cAMP content were augmented by milrinone and rolipram, inhibitors of phosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. The augmentation in relaxation and the increase in cAMP content caused by milrinone plus rolipram was greater than the sum of the responses caused by either of the inhibitors alone. 8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation and change in cAMP induced by PGE2 and forskolin. Acetylcholine alone had no effect on cAMP content in the vessels but augmented the relaxation and the increase in cAMP induced by PGE2 and forskolin in arteries with endothelium. This effect was not observed in arteries without endothelium or in arteries with endothelium treated with NG-nitro-L-arginine. These results suggest that PDE3 and PDE4 are the primary enzymes hydrolyzing cAMP of pulmonary arteries of newborn lambs and that an inhibition of both PDE3 and PDE4 would result in a greater effect than that caused by inhibition of either one of the subtype isozymes alone. Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediated relaxation by inhibition of PDE3.

Deletion of CREB-Regulated Transcription Coactivator 1 Induces Pathological Aggression, Depression-Related Behaviors, and Neuroplasticity Genes Dysregulation in Mice.

BACKGROUND: Mood disorders are polygenic disorders in which the alteration of several susceptibility genes results in dysfunctional mood regulation. However, the molecular mechanisms underlying their transcriptional dysregulation are still unclear. The transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and the neurotrophin brain-derived neurotrophic factor (BDNF) have been implicated in rodent models of depression. We previously provided evidence that Bdnf expression critically rely on a potent CREB coactivator called CREB-regulated transcription coactivator 1 (CRTC1). METHODS: To further evaluate the role of CRTC1 in the brain, we generated a knockout mouse line and analyzed its behavioral and molecular phenotype. RESULTS: We found that mice lacking CRTC1 associate neurobehavioral endophenotypes related to mood disorders. Crtc1(-/-) mice exhibit impulsive aggressiveness, social withdrawal, and decreased sexual motivation, together with increased behavioral despair, anhedonia, and anxiety-related behavior in the novelty-induced hypophagia test. They also present psychomotor retardation as well as increased emotional response to stressful events. Crtc1(-/-) mice have a blunted response to the antidepressant fluoxetine in behavioral despair paradigms, whereas fluoxetine normalizes their aggressiveness and their behavioral response in the novelty-induced hypophagia test. Crtc1(-/-) mice strikingly show, in addition to a reduced dopamine and serotonin turnover in the prefrontal cortex, a concomitant decreased expression of several susceptibility genes involved in neuroplasticity, including Bdnf, its receptor TrkB, the nuclear receptors Nr4a1-3, and several other CREB-regulated genes. CONCLUSIONS: Collectively, these findings support a role for the CRTC1-CREB pathway in mood disorders etiology and behavioral response to antidepressants and identify CRTC1 as an essential coactivator of genes involved in mood regulation.

Qualitative and quantitative analysis of thalamo-cortical axons in the somatosensory cortex of normal and barrelless mice

Résumé Les rongeurs utilisent leurs moustaches (vibrisses) pour explorer le milieu environnant. Chaque moustache est mue par un système des muscles. Les récepteurs situés à sa base transmettent les informations au système nerveux central. La transmission vers l'écorce se fait via trois neurones de relais qui se trouvent au niveau du ganglion trigéminé, du tronc cérébral et du thalamus. La représentation corticale d'une vibrisse est une concentration des axones thalamo-corticaux (ATC) autour desquelles s'organisent leurs cibles, les cellules de la couche IV. La structure peut être identifiée histologiquement en coupes tangentielles et porte le nom de « barrel » (« tonneau »). Cette correspondance vibrisse - barrel fait de ce système un model idéal pour étudier l'influence de l'activité périphérique sur l'établissement et le maintien des cartes somatotopiques. Notre laboratoire dispose d'une souche de souris qui a subi une mutation spontanée pour le gène codant l'adenylyl cyclase I (ACI). Cette enzyme membranaire catalyse la formation de l'AMPc et joue un rôle important dans le guidage axonal, la libération des neurotransmetteurs et l'intégration des signaux postsynaptiques. Nous avons démontré dans un premier temps que cette souris adulte ne développe pas de barrels. Cela est dû à un manque d'organisation des ATC et aussi des cellules de la couche IV. De plus, les résultats électrophysiologiques montrent que les informations venant des vibrisses adjacentes ne sont pas intégrées d'une manière normale. Dans ce travail de thèse, j'ai analysé la morphologie des ATC révélés individuellement avec de la biocytine. L'analyse quantitative des ATC a mis en évidence les points suivants: 1. Les axones de la souris normale (NOR) quittent le thalamus, traversent la capsule interne et la substance blanche sous-corticale et pénètrent dans le cortex somato-sensoriel primaire. A l'intérieur de l'écorce ils traversent au maximum 3 colonnes corticales adjacentes dont une contient le barrel cible. En passant à travers les couches VI et V, ces axones arborisent et convergent progressivement vers le barrel dans lequel ils forment une riche arborisation. Un petit nombre des branches « errantes », pleines de boutons synaptiques, pénètrent dans les barrels voisins. Deux axones NOR provenant de corps cellulaires très proches dans le thalamus peuvent avoir un cheminement très divergent lors de la traversée de la capsule interne et de la substance blanche sous-corticale mais, à leur entrée dans le cortex, ils sont distants d'au maximum 2 colonnes corticales de la colonne qui contient le barrel cible et ils convergent progressivement vers ce barrel. 2. Les axones de la souris mutante (BRL) ont le même trajet sous-cortical que les axones NOR, mais leur entrée dans le cortex somato-sensoriel primaire est aléatoire. A l'interface entre la substance blanche sous-corticale et le cortex, l'axone principal se divise rapidement en troncs axonaux qui traversent les couches VI et V d'une manière divergente pour arriver dans la couche IV. Cela contraste beaucoup avec la trajectoire des NOR qui convergent graduellement vers leur barrel cible. Le nombre de branches radiales que les axones BRL utilisent pour entrer dans le cortex et dans la couche IV est double par rapport aux axones NOR. Parmi ces branches, seules quelques-unes donnent des arborisations, les autres ne sont pas développées et leur morphologie est semblable à celle des branches formées par les axones de la souris normale lors du développement. Deux axones BRL issus de corps cellulaires proches dans le thalamus peuvent avoir une trajectoire très divergente jusqu'à leur entrée dans la couche IV, mais à ce niveau ils sont réorientés pour se retrouver et faire un nombre maximal de branches et boutons synaptiques dans la même région corticale. Dans un cas extrême, un des axones observés est entré dans le cortex à la limite entre l'aire somatosensorielle primaire et secondaire et a parcouru une distance de 2 mm pour retrouver son partenaire thalamique et donner avec celui-ci un nombre maximal de branches dans la même région de la couche IV. 3. Les mesures quantitatives ont montré que les arborisations corticales des axones NOR ont une longueur moyenne de 18mm et sont formées par 200 segments qui portent 1200 boutons synaptiques. Par rapport à la souris NOR, les axones BRL ont en moyenne la même longueur, le même nombre de segments et boutons synaptiques, mais donnent deux fois plus de branches radiales. La surface tangentielle occupée par les arborisations BRL dans la couche IV est 2 fois plus grande que celle des NOR. Cela signifie que les 1000 boutons synaptiques qui caractérisent les arborisations NOR et BRL dans la couche IV sont disséminés sur une surface tangentielle double chez les derniers, et donc que la densité des boutons par unité de surface corticale est en moyenne plus faible. En effet, l'augmentation de la surface corticale tangentielle des BRL est due aux surfaces de faible et moyenne densité synaptique (0 - 8 boutons / 400pn2) qui augmentent 2 fois tandis que les surfaces de haute densité synaptiques (8 - 64 boutons / 4001.tm2) sont les mêmes. Nous émettons l'hypothèse selon laquelle, durant le développement, les ATC de la souris BRL divergent et forment un nombre exubérant de branches. Grâce à cette divergence et aux branches supranuméraires, ils trouvent l'endroit de l'écorce où se trouvent leurs voisins thalamiques et arborisent abondamment dans cette région. Cependant, le déficit en AGI ne leurs permet pas par la suite, sous influence de l'activité périphérique, de retirer les branches qui se trouvent dans les endroits inappropriés de l'écorce, avec de possibles conséquences sur la discrimination tactile.

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.

Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1.

Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.