Identification and functional characterization of LEF-1 associated kinases

0\. Titelblatt und Inhaltsverzeichnis 1. Einleitung 1 2\. Material 17 3\. Methoden 26 4\. Ergebnisse 49 5\. Diskussion 71 6\. Zusammenfassung und Ausblick / Summary 79 7\. Literaturverzeichnis 82 8\. Anhang 94Mit den Ergebnissen meiner Arbeit konnten folgende neue Erkenntnisse gewonnen werden: * Die beiden Serin/Threonin-Proteinkinasen CK1 und CK2 binden an den Transkriptionsfaktor LEF-1 und phosphorylieren diesen. * Die CK1- bzw. CK2-abhängige Phosphorylierung führt zu strukturellen Änderungen innerhalb des LEF-1 Proteins, die wiederum zu strukturellen Änderungen der DNA führen. * Die durch CK1 katalysierte Phosphorylierung von LEF-1 führt zu einer Dissoziation des LEF-1/β-Catenin Komplexes. Übereinstimmend damit zeigte CK1 einen negativen Einfluss auf die Transkriptionsaktivität eines durch den LEF-1/β-Catenin Transkriptionskomplex regulierten Promotors eines Wnt- Zielgens. * Die Phosphorylierung von LEF-1 durch CK2 zeigte keinen Einfluß auf die Integrität des LEF-1/β-Catenin Komplexes, stimulierte aber in Reportergen- Assays die Transkriptionsaktivität. CK1 und CK2 besitzen damit nicht nur auf der Ebene des β-Catenin Degradationskomplexes, sondern auch auf der Ebene des LEF-1/TCF-β-Catenin Transkriptionskomplexes eine regulatorische Funktion im Wnt-Signalweg. Nach den Ergebnissen dieser Arbeit hat CK1 die Rolle eines negativen Regulators und für CK2 wird die Rolle eines positiven Regulators postuliert. In Hinsicht auf die dramatischen Konsequenzen, die durch eine fehlregulierte Aktivierung von Wnt-Zielgenen induziert werden, stellt die koordinierte Regulation auf zwei Ebenen einen wichtigen, zellulären Mechanismus für die Kontrolle des kanonischen Wnt-Signalwegs dar. Zu diesem Zeitpunkt bleibt allerdings unklar, welche Signale die Aktivität von CK1 und CK2 beeinflussen und zu den in dieser Arbeit beschriebenen positiven bzw. negativen Effekten auf der Ebene des LEF-1/TCF-β-Catenin Transkriptionskomplexes führen.With the results of my work the following new findings could be gained: * The serine/threonine proteinkinases CK1 and CK2 directly bind to and phosphorylate LEF-1. * CK1- and CK2-dependent phosphorylation induces a conformational change in the LEF-1 protein that in consequence is tranduced to the DNA. * CK1-dependent phosphorylation of LEF-1 disrupts the LEF-1/β-catenin complex. Consistent with this results CK1 had a negative regulatory effect on the transcriptional activity of a LEF-1/β-catenin regulated Wnt target gene promotor. * Phosphorylation of LEF-1 by CK2 did not significantly affect formation of the LEF-1/β-catenin complex. However, in reporter gene assays the transcriptional activity of the LEF-1/β-catenin transcription complex was enhanced. CK1 and CK2 therefore are involved in Wnt-signalling by regulating β-catenin stability not only at the level of the β-catenin destruction complex but in addition modulate the pathway at the level of the LEF-1/β-catenin transcription complex. The results of this work show that CK1 acts as a negative regulator, whereas CK2 is supposed to function as a positive regulator. With respect to the dramatic consequences induced by misregulated activation of Wnt target genes in cells, a coordinated regulation at two levels appears to be an elegant mechanism to tightly control canonical Wnt- signalling. However, currently it is not understood how the activity of CK1 and CK2 is regulated and lead to the observed positive and negative effects at the level of the LEF-1/β-catenin transcription complex

Community pharmacy-based intervention to improve self-monitoring of blood glucose in type 2 diabetic patients.

Self-monitoring of blood glucose (SMBG) is clearly correlated with increased life expectancy and quality of life in type 2 diabetic patients. Objective: The objective of our study was to record and assess the errors patients make in preparing, performing, and processing self-monitoring of blood glucose (SMBG). Furthermore, the study aimed to determine to what extent a single standardized SMBG instruction session in a community pharmacy might reduce the number of patients making errors or the number of errors per patient. Methods: Between May and October 2005, SMBG of 462 randomly selected patients with type 2 diabetes was monitored in 32 pharmacies specialized in diabetes care. The patients performed blood glucose self-tests using their own blood glucose meters. Self-testing was monitored using a standardized documentation sheet on which any error made during the performance of the test was recorded. If necessary, patients were instructed in the accurate operation of their meter and the use of the necessary equipment. Additionally, patients obtained written instructions. Six weeks later, assessment of the quality of patient¿s SMBG was repeated. Results: During the first observation, 383 patients (83%) made at least one mistake performing SMBG. By the time of the second observation, this frequency had fallen to 189 (41%) (p<0.001). The average number of mistakes fell from 3.1 to 0.8 per patient. Mistakes that may potentially have led to inaccurate readings were initially recorded for 283 (61%) and at study end for 110 (24%) patients (p<0.001). Conclusion: It is important to periodically instruct type 2 diabetic patients in the proper SMBG technique in order to ensure accurate measurements. In this study it was shown that community pharmacies specialized in diabetes care can provide this service effectively