Biomedical subjects

Thomas Weidemann

Publications and source records attributed to Thomas Weidemann.

4 recordsLinked to original sources

PubMed2006

Beyond dimerization: a membrane-dependent activation model for interleukin-4 receptor-mediated signalling.

Class I cytokine receptors efficiently transfer activation signals from the extracellular space to the cytoplasm and play a dominant role in growth control and differentiation of human tissues. Although a significant body of literature is devoted to this topic, a consistent mechanistic picture for receptor activation in the membrane environment is still missing. Using the interleukin-4 receptor (IL-4R) as an example, we propose that the membrane-proximal stem-loop of the extracellular domains contains pivotal elements of a rotational switch. Interfacial energies of amino acid side-chains contained in the highly conserved WSXWS at the surface of the lipid bilayer suggest a new functional role for this motif. A generic activation mechanism for this receptor class is presented, which may impact the design of a new generation of biophysical assay systems.

Amino Acid Motifs↗

PubMed2004

The chemical hunt for the identification of drugable targets.

Chemical biology has emerged as a new scientific discipline to change the way scientists approach and study the interface between chemistry, biology, and physics. By integrating the knowledge base of the human genome with the power of diverse and flexible chemical technology platforms, the ultimate goal is to define the 'rules of engagement' for small molecules and their use in basic biology and in drug discovery. Herein, we highlight the current counterpoles of the chemical biology philosophy in the framework between conformational diversity and informational complexity. Expanding the growing molecular recognition information matrix into classification of diseases and immediate mechanistic in-vivo proof of concept models represent the next development phase in a field that, unlike any other due to its multidisciplinary nature, unifies basic scientists and drug discoverers.

Animals↗

PubMed2003

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging.

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.

Flow Cytometry↗

PubMed2003

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Bacterial Proteins↗