O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver

International audienceOBJECTIVE Carbohydrate-responsive element–binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction. RESEARCH DESIGN AND METHODS O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBPOG in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice. RESULTS Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBPOG in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice. CONCLUSIONS Taken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions

Cardiac Transcription Factor Nkx2.5 Is Downregulated under Excessive O-GlcNAcylation Condition

Post-translational modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is linked the development of diabetic cardiomyopathy. We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc. Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag). Co-immunoprecipitation analysis revealed that myc-Nkx2.5 and OGT-flag proteins interacted and myc-Nkx2.5 proteins were modified by O-GlcNAc. In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart. Thus, excessive O-GlcNAcylation causes downregulation of Nkx2.5, which may be an underlying contributing factor for the development of diabetic cardiomyopathy

Proposal for a method to estimate nutrient shock effects in bacteria

Plating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested. Findings To estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA). The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon. This work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration. <br/

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes

Kollaborative klinische Simulation in kardiologischen Notfallszenarien für Medizinstudenten/-studentinnen. Eine explorative Studie über die Anwendbarkeit von Modellen und Bewertungsinstrumenten

Aims: This paper evaluates the feasibility of piloting the collaborative clinical simulation (CCS) model and its assessment instruments applicability for measuring interpersonal, collaborative, and clinical competencies in cardiologic emergency scenarios for medical students. The CCS model is a structured learning model for the acquisition and assessment of clinical competencies through small groups working collaboratively to design and perform in simulated environments supported by technology.Methods: Fifty-five students were allocated in five sessions (one weekly session) conducted with the CCS model within the course Cardiovascular Diseases. The applied practice aimed at the diagnosis and treatment of tachyarrhythmias in a simulated emergency department. In addition to the theoretical classes four weeks before the simulation sessions, students were sent a study guide that summarized the Guide to the European Society of Cardiology. For each simulation session, one clinical simulation instructor, one cardiologist teacher, and the principal investigator participated. Students were divided into three groups (3-5 students) for each-session. They designed, performed, role-played, and debriefed three different diagnoses.Three instruments to assess each group's performance were applied: peer assessment used by groups, performance assessment, created and applied by the cardiologist teacher, and individual satisfaction questionnaire for students. Results: The applicability of the CCS model was satisfactory for both students and teachers. The assessment instruments' internal reliability was good, as was internal consistency with a Cronbach Alpha of 0.7, 0.4, and 0.8 for each section (Interpersonal, Clinical, and Collaborative competencies, respectively). The performance group's evaluation was 0.8 for the two competencies assessed (Tachyarrhythmia and Electrical Cardioversion) and 0.8 for the satisfaction questionnaire's reliability. Conclusions: The CCS model for teaching emergency tachyarrhythmias to medical students was applicable and well accepted. The internal reliability of the assessment instruments was considered satisfactory by measuring satisfaction and performance in the exploratory study.Ziele: In dieser Arbeit wird die Durchführbarkeit der Pilotumsetzung des Modells für kollaborative klinische Simulation (KKS) und dessen Bewertungsinstrumenten für die Messung der interpersonellen, kollaborativen und klinischen Kompetenzen in kardiologischen Notfallszenarien für Medizinstudenten/-studentinnen ausgewertet. Das KKS-Modell ist ein strukturiertes Lernmodell für den Erwerb und die Bewertung klinischer Kompetenzen in kleinen Gruppen, in denen die Studenten/Studentinnen zusammenarbeiten und dabei die simulierten Umgebungen mithilfe von Technologie entwickeln und anwenden.Methoden: Fünfundfünfzig Studenten/Studentinnen wurden fünf Sitzungen zugeordnet (eine Sitzung pro Woche), die unter Anwendung des KKS-Modells im Rahmen des Seminars Kardiovaskuläre Erkrankungen durchführt wurden. Dabei sollten die Diagnostik und Behandlung von Tachyarrhythmien in einer simulierten Notaufnahme geübt werden. Zusätzlich zum theoretischen Unterricht wurde den Studenten/Studentinnen vier Wochen vor Beginn der Simulationssitzungen ein Lernleitfaden mit einer Zusammenfassung der Leitlinien der European Society of Cardiology zugesendet. An den Simulationssitzungen nahmen jeweils ein Anleiter/eine Anleiterin im Bereich klinische Simulation, eine Lehrkraft für Kardiologie und der Studienleiter teil. Die Studenten/Studentinnen wurden für jede Sitzung in drei Gruppen (3-5 Studenten/Studentinnen) aufgeteilt. Sie entwarfen drei unterschiedliche Diagnosen, führten sie durch, wandten Rollenspiele an und führten ein Debriefing durch.Zur Bewertung der Leistung jeder Gruppe wurden drei Instrumente angewendet: Peer-Bewertung durch die Gruppen, Leistungsbewertung, erstellt und angewendet durch die Lehrkraft für Kardiologie, und Einzelfragebogen zur Zufriedenheit für die Studenten/Studentinnen. Ergebnisse: Die Anwendbarkeit des KKS-Modells war sowohl für die Studenten/Studentinnen als auch die Lehrer/-innen zufriedenstellend. Die interne Reliabilität der Bewertungsinstrumente war gut, genau wie die interne Konsistenz mit einem Cronbachs Alpha von 0,7, 0,4 und 0,8 für jeden Bereich (jeweils Interpersonelle, Klinische und Kollaborative Kompetenzen). Der Wert für die Beurteilung der durchführenden Gruppe war 0,8 für die beiden bewerteten Kompetenzen (Tachyarrhythmie und Elektrische Kardioversion), und die Reliabilität des Zufriedenheitsfragebogens lag bei 0,8. Schlussfolgerungen: Das KKS-Modell ist für die Lehre der Studenten/Studentinnen im Bereich Notfalldiagnostik und -behandlung von Tachyarrhythmien anwendbar und wurde gut angenommen. Die interne Reliabilität der Bewertungsinstrumente wurde durch Messung der Zufriedenheit und Leistung in der explorativen Studie als zufriedenstellend betrachtet

Lines Of Curvature On Surfaces Immersed In ℝ4

The differential equation of the lines of curvature for immersions of surfaces into ℝ4 is established. It is shown that, for a class of generic immersions of a surface into ℝ4 in the Cr-topology, r ≥ 4, all of the umbilic points are locally topologically stable. This type of umbilic points is described. © 1997, Sociedade Brasileira de Matemática.282233251Burnside, W.S., Panton, A.W., (1912) The Theory of Equations, , [B-P] Dover Publications, Inc. New YorkGuadalupe, I., Gutiérrez, C., Sotomayor, J., Tribuzy, R., Principal Lines on Surfaces Minimally Immersed in Constantly Curved 4-spaces (1987) Dynamical Systems and Bifurcation Theory, Pitman Research Notes in Mathematics Series, 160, pp. 91-120. , [GGST]Gutierrez, C., Sotomayor, J., Principal Lines on Surfaces Immersed with Constant Mean Curvature (1986) Trans, of the Ame. Math. Soc., 293 (2), pp. 751-766. , [G-S]Jacobowitz, H., The Gauss-Codazzi Equations (1982) Tensor, N., S., 39, pp. 15-22. , [Jac]Little, J.A., On Singularities of Submanifolds of a Higher Dimensional Euclidean Space (1969) Ann. Mat. Pura App., 83, pp. 261-335. , [Lit]Palis, J., De Melo, W., (1982) Geometric Theory of Dynamical Systems, , [M-P] Springer-VerlagRamírez-Galarza, A., Sánchez-Bringas, F., Lines of Curvature near Umbilic Points on Surfaces Immersed in ℝ4 (1995) Annals of Global Analysis and Geometry, 13, pp. 129-140. , [R-S]Spivak, M., (1979) A Comprehensive Introduction to Differential Geometry, 5. , [Spi] Publish or Perish Inc., Berkele

Invasion of a rocky intertidal shore by the tunicate Pyura praeputialis in the Bay of Antofagasta, Chile

Invasion by marine nonindigenous species (NIS) is a spread phenomenon. The tunicate Pyura praeputialis shows pronounced disjoint geographical distribution: along thousands of kilometers in wave-swept headlands on the southeastern coast of Australia, from where it appears to have originated, and exclusively along 60–70 km inside the Bay of Antofagasta, Chile. mtDNA sequences suggested that the species invaded this rocky shore recently. We used field manipulations and juvenile P. praeputialis transplant techniques to test hypotheses regarding the capacity of the tunicate to survive and grow at different sites and tidal heights inside and outside Antofagasta, and its competitive performance for primary space (inside the Bay) against the native mussel Perumytilus purpuratus. We conclude that survival and growth of P. praeputialis showed no significant differences among sites inside and outside the Bay, and suggest that the restrictive distribution of the species in Chile is caused by a specific oceanographic retention mechanism and/or its brief larval dispersal. We demonstrated that, inside the Bay, P. praeputialis outcompetes Perumytilus from the Mid–Low intertidal, constraining Perumytilus to the Upper Mid-Intertidal, modifying the local pattern of intertidal zonation. We show that predation on P. praeputialis juveniles by starfish and snails constitutes a regulatory mechanism for the setting of its low intertidal limit. Major ecological impacts caused by NIS invasions to rocky shores by aggressive primary space users may result in negative aspects, but also may contribute to biodiversity enhancement. We call attention to the need for increment manipulations and testing of ecological hypotheses regarding marine NIS