Development,standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17β-estradiol (E2), at concentrations from 0.2 to 6.0 μg E2 L⁻¹ in Experiment 1A, and 0.015 to 2.0 μg E2 L⁻¹ in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC₅₀ of 0.12 μg E2 L⁻¹. The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis.     

Thomas de Padova: Alles wird Zahl: Wie sich die Mathematik in der Renaissance neu erfand

Mathematische Semesterberichte -     

Coupling artificial actin cortices to biofunctionalized lipid monolayers

We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.     

Frictional drag and electrical manipulation of recombinant proteins in polymer-supported membranes

We establish a lipid monolayer supported by a polymer interface that offers advantages over conventional solid-supported membranes for determining the frictional drag at the membrane-protein interface as well as for electric field manipulation of membrane-anchored proteins. Polymer-supported monolayers with functional lipid anchors allow for the specific docking of His-tagged green fluorescent protein variants (His-EGFP and His-DsRed tetramer) onto the membrane surface at a defined surface density. In the first part, we measure the lateral diffusion coefficients of lipids and proteins and calculate the frictional drag at the protein-membrane interface. The second part deals with the electric field-induced accumulation of recombinant proteins on a patterned surface. The mean drift velocity of proteins, which can be obtained analytically from the shape of the steady-state concentration gradient, can be controlled by tuning the interplay of electrophoresis and electroosmosis. The results demonstrate the potential of such molecular constructs for the local functionalization of solid substrates with membrane-associated proteins.