In vivo characterization of the circadian clock output gene usp2

Life on earth is subject to the repeated change between day and night periods. All organisms that undergo these alterations have to anticipate consequently the adaptation of their physiology and possess an endogenous periodicity of about 24 hours called circadian rhythm from the Latin circa (about) and diem (day). At the molecular level, virtually all cells of an organism possess a molecular clock which drives rhythmic gene expression and output functions. Besides altered rhythmicity in constant conditions, impaired clock function causes pathophysiological conditions such as diabetes or hypertension. These data unveil a part of the mechanisms underlying the well-described epidemiology of shift work and highlight the function of clock-driven regulatory mechanisms. The post-translational modification of proteins by the ubiquitin polypeptide is a central mechanism to regulate their stability and activity and is capital for clock function. Similarly to the majority of biological processes, it is reversible. Deubiquitylation is carried out by a wide variety of about ninety deubiquitylating enzymes and their function remains poorly understood, especially in vivo. This class of proteolytic enzymes is parted into five families including the Ubiquitin-Specific Proteases (USP), which is the most important with about sixty members. Among them, the Ubiquitin-Specific Protease 2 (Usp2) gene encodes two protein isoforms, USP2-45 and USP2-69. The first is ubiquitously expressed under the control of the circadian clock and displays all features of core clock genes or its closest outputs effectors. Additionally, Usp2-45 was also found to be induced by the mineralocorticoid hormone aldosterone and thought to participate in Na+ reabsorption and blood pressure regulation by Epithelial Na+ Channel ENaC in the kidneys. During my thesis, I aimed to characterize the role of Usp2 in vivo with respect to these two areas, by taking advantage of a total constitutive knockout mouse model. In the first project I aimed to validate the role of USP2-45 in Na+ homeostasis and blood pressure regulation by the kidneys. I found no significant alterations of diurnal Na+ homeostasis and blood pressure in these mice, indicating that Usp2 does not play a substantial role in this process. In urine analyses, we found that our Usp2-KO mice are actually hypercalciuric. In a second project, I aimed to understand the causes of this phenotype. I found that the observed hypercalciuria results essentially from intestinal hyperabsorption. These data reveal a new role for Usp2 as an output effector of the circadian clock in dietary Ca2+ metabolism in the intestine.

Use of levosimendan as rescue therapy in children with low cardiac output syndrome.

Objective: Aim of post operative treatments after cardiac surgery is to avoid low cardiac output syndrome (LCOS). Levosimendan, a new inotrope agent, has been demonstrated in adult patient to be an effective treatment for this purpose when classical therapy is not effective. It shows a positive effect on cardiac output, with fewer adverse effects and lower mortality than with dopamine. There is very few data on its benefit in the paediatric population. The aim of this study is to evaluate the effect of levosimendan in cardiac children with LCOS.Methods: Retrospective analysis of 25 children hospitalised in our PICU after cardiac surgery that demonstrated LCOS not responding to classical catecholamine therapy and who received levosimendan as rescue. LCOS parameters like urine output, mixed venous oxygen saturation (SvO2), arterio-venous differences in CO2 (AVCO2) and plasmatic lactate were compared before therapy and at 12, 24, 48 and 72 hours after the beginning of the levosimendan infusion. We also analyzed the effect on the utilisation of amines (amine score), adverse events and mortality.Results: After the beginning of levosimendan infusion, urine output (3.1 vs 5.3ml/kg/h, p=0.003) and SVO2 (56 vs 64mmHg, p=0.001) increase significantly during first 72 hours and at the same time plasmatic lactate (2.6 vs 1.4 mmole/l, p<0.001), AVCO2 (11 vs 8 mmHg, p=0.002) and amine score (63 vs 39, p=0.007) decrease significantly. No side effects were noted during administration of levosimendan. In this group of patients, mortality was 0%.Conclusion: Levosimendan is an effective treatment in children after congenital heart surgery. Our study, with a greater sample of patient than other studies, confirms the improvement of cardiac output already shown in other paediatric studies.

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Television before TV : a transnational history of an experimental medium on display, 1928 - 1939

Dès le milieu des années 1920 et le développement des premiers prototypes fonctionnels, la télévision se fait objet d'exposition. La nouvelle technologie est montrée à des foires industrielles et dans des grands magasins, à des expositions universelles et nationales : exposée, la télévision devient un mass media avant même qu'elle ne diffuse des émissions régulières. En étudiant les présentations publiques de la télévision en Allemagne, en Grande-Bretagne et aux Etats-Unis entre 1928 - date de l'ouverture des premières expositions annuelles dans les trois pays - et 1939 - moment de l'interruption de celles-ci suite à l'éclatement de la Deuxième Guerre mondiale - Television before TV montre que les lieux où le médium rencontre son premier public constituent les mêmes espaces qui, rétrospectivement, permettent de comprendre son avènement. Cette recherche propose ainsi de nouvelles pistes pour l'histoire des médias en interrogeant la définition de la télévision en tant que médium du privé. Elle rappelle l'importance historique de l'espace public pour la « vision à distance » et, dépassant la perspective nationale, reconstitue les différences et similarités techniques, médiatiques et institutionnelles de la télévision allemande, américaine et britannique dans l'entre-deux-guerres. Développée dans un contexte qui est simultanément nationaliste et nourri d'échanges et de compétitions internationales, la télévision sert de comparatif dans les rivalités entre pays, mais stimule également la circulation de savoir-faire et de personnes. Sa forte valeur symbolique comme emblème du progrès scientifique et, dans le cas allemand, comme preuve de la modernité du régime national-socialiste se traduit dans les salles d'expositions par des scénographies souvent innovatrices, dont les photographies et descriptions se propagent au-delà de la presse locale. Au centre d'un réseau discursif et représentationnel, les expositions jouent alors un rôle essentiel pour la construction sociale, politique et culturelle de la télévision. Remplaçant l'analyse de textes audiovisuels par l'étude de la machine exposée, ma recherche reformule la question bazinienne de qu'est-ce que la télévision ? pour demander où le médium se situe-t-il ? Cette prémisse méthodologique permet de contourner une évaluation qualitative de la technologie qui déplorerait ses multiples imperfections, et éclaire d'un nouveau jour des dispositifs télévisuels qui, même sans programme, intègrent un univers voué à la culture de consommation et du loisir.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

Structured output SVM for remote sensing image classification

In the recent years, kernel methods have revealed very powerful tools in many application domains in general and in remote sensing image classification in particular. The special characteristics of remote sensing images (high dimension, few labeled samples and different noise sources) are efficiently dealt with kernel machines. In this paper, we propose the use of structured output learning to improve remote sensing image classification based on kernels. Structured output learning is concerned with the design of machine learning algorithms that not only implement input-output mapping, but also take into account the relations between output labels, thus generalizing unstructured kernel methods. We analyze the framework and introduce it to the remote sensing community. Output similarity is here encoded into SVM classifiers by modifying the model loss function and the kernel function either independently or jointly. Experiments on a very high resolution (VHR) image classification problem shows promising results and opens a wide field of research with structured output kernel methods.

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Physiological function of PARbZip circadian clock-controlled transcription factors.

PARbZip proteins (proline and acidic amino acid-rich basic leucine zipper) represent a subfamily of circadian transcription factors belonging to the bZip family. They are transcriptionally controlled by the circadian molecular oscillator and are suspected to accomplish output functions of the clock. In turn, PARbZip proteins control expression of genes coding for enzymes involved in metabolism, but also expression of transcription factors which control the expression of these enzymes. For example, these transcription factors control vitamin B6 metabolism, which influences neurotransmitter homeostasis in the brain, and loss of PARbZip function leads to spontaneous and sound-induced epilepsy that are frequently lethal. In liver, kidney, and small intestine, PAR bZip transcription factors regulate phase I, II, and III detoxifying enzymes in addition to the constitutive androstane receptor (CAR), one of the principal sensors of xenobiotics. Indeed, knockout mice for the three PARbZip transcription factors are deficient in xenobiotic detoxification and display high morbidity, high mortality, and accelerated aging. Finally, less than 20% of these animals reach an age of 1 year. Accumulated evidences suggest that PARbZip transcription factors play a role of relay, coupling circadian metabolism of xenobiotic and probably endobiotic substances to the core clock circuitry of local circadian oscillators.

Mice carrying ubiquitin-specific protease 2 (Usp2) gene inactivation maintain normal sodium balance and blood pressure.

Ubiquitylation plays an important role in the control of Na⁺ homeostasis by the kidney. It is well established that the epithelial Na⁺ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney and is also a circadian output gene. In heterologous expression systems, USP2-45 binds to ENaC, deubiquitylates it, and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na⁺ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO mice did not show any differences from wild-type littermates with respect to the diurnal control of Na⁺ or K⁺ urinary excretion and plasma levels either on a standard diet or after acute and chronic changes to low- and high-Na⁺ diets, respectively. Moreover, they had similar aldosterone levels on either a low- or high-Na⁺ diet. Blood pressure measurements using telemetry did not reveal variations compared with control mice. Usp2-KO mice did not display alterations in expression of genes involved in sodium homeostasis or the ubiquitin system, as evidenced by transcriptome analysis in the kidney. Our data suggest that USP2 does not play a primary role in the control of Na⁺ balance or blood pressure.

Inner-membrane transporters for the siderophores pyochelin in Pseudomonas aeruginosa and enantio-pyochelin in Pseudomonas fluorescens display different enantioselectivities.

Iron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch (ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two AraC-type regulators PchR, which are activated by their cognate siderophore. Here, stereospecific siderophore recognition is shown to occur at the inner membrane also. In P. aeruginosa, translocation of ferriPch across the inner membrane is carried out by the single-subunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational fetA-gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination with either of the two siderophore receptors FetA and FptA in a siderophore-negative P. aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific siderophore recognition occurs at the inner membrane by the FetCDE transporter.