Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan’s presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins

A Framework for the Application of Robust Design Methods and Tools

Robust Design Methods (RDM) have become a powerful concept to design more reliable products. However, even though there are many tools available and have been discussed widely in academia, there is still confusion and doubts in the industry about the use and effectiveness of these methods. Mostly the problems experienced in industry are related to a poor application or knowledge of the methods by the companies (Hasenkamp, et Al, 2007). Based on the authors� experiences in working in development engineering, it has shown that also the phase of application of the specific tools and methods and what those can deliver are not always clear. Expectations to the output are sometimes misleading and imply the incorrect utilization of tools. A categorization of tools, methods and techniques typically associated with robust design methodology in the literature is provided in this paper in terms of purpose and deliverables of the individual tool or method. The majority of tools aims for optimizing an existing design solution or give an indication of how robust a design is, which requires a somewhat settled design. Furthermore, the categorization presented in this paper shows a lack in the methodology for tools in the area of robustness prediction in early design. This categorization clarifies the underlying premises and deliverables of RDM tools for professionals working with design processes and can serve as guidance for an organization how to structure its development process and how to make most efficient use of the existing tools. When to apply, what tool or method, for which purpose can be concluded. The paper also contributes with a framework for researchers to derive a generic landscape or database for RDM build upon the main premises and deliverables of each method

The Translation between Functional Requirements and Design Parameters for Robust Design

AbstractThe specification of and justification for design parameter (DP) tolerances are primarily based on the acceptable variation of the functions’ performance and the functions’ sensitivity to the design parameters. However, why certain tolerances are needed is often not transparent, especially in complex products with multi-disciplinary development teams. In those cases, tolerance synthesis and analysis get complicated which introduces ambiguities and difficulties for system-integrators and lead engineers for the objective decision making in terms of trade-offs but also in terms of an efficient computer aided functional tolerancing. Non-optimal tolerances yield potentials for cost improvements in manufacturing and more consistency of the functional performance of the product. In this contribution a framework is proposed to overcome the observed problems and increase the clarity, transparency and traceability of tolerances by analyzing the translation between the DPs and their influence on the final function

La représentation politique dans la démocratie

La forme politique de la République fédérale d’Allemagne, telle que la définit la Loi fondamentale, est la démocratie représentative. Le peuple souverain n’exerce le pouvoir politique lui-même et directement qu’à l’occasion des élections, et, de manière strictement limitée, lors des référendums, tandis qu’il ne l’exerce le reste du temps que par l’intermédiaire d’ « organes spéciaux ». Dans l’activité législative du Parlement, il est représenté par des députés qui « ne sont liés ni par des ma..

Molekulare Regulation des Steroidstoffwechsels in Comamonas testosteroni

Comamonas testosteroni wird als Modellorganismus für den bakteriellen Steroidabbau gesehen. Bislang wurde kein umfassender Mechanismus der Steroidregulation in C. testosteroni publiziert. Der Fokus der Arbeit liegt auf dem Regulator TeiR von C. testosteroni. TeiR ist in C. testosteroni polar lokalisiert. Im zentralen Proteinteil wurde eine Testosteron bindende, hydrophobe Region beschrieben. An diese schließt sich ein Bereich an, der wie der C-Terminus, für die Funktionalität essentiell. Weiterhin wurde eine GTP-abhängige Phosphatübertragung an einem Histidin in der Signalübertragung gezeigt. Auch weitere, am Testosteronabbau beteiligte Proteine zeigten sich abhängig von TeiR. Die Untersuchung von teiR-überexprimierenden Zellen vervollständigte das Bild des Steroid-Master-Regulators TeiR. Mechanismus und Effekte von TeiR auf unterschiedlichen Ebenen konnten aufgedeckt werden. Somit trägt diese Arbeit maßgeblich zum Verständnis der Regulation des Steroidstoffwechsels in Bakterien bei