Center for Advanced Bioanalysis GmbH

Linz, Austria

Center for Advanced Bioanalysis GmbH

Linz, Austria
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Juhasz K.,Center for Advanced Bioanalysis GmbH | Juhasz K.,Institute of Biochemistry | Thuenauer R.,Center for Advanced Bioanalysis GmbH | Spachinger A.,Center for Advanced Bioanalysis GmbH | And 6 more authors.
Current Pharmaceutical Design | Year: 2013

Tumor specific cell surface localization and release of the stress inducible heat shock protein 70 (Hsp70) stimulate the immune system against cancer cells. A key immune stimulatory function of tumor-derived Hsp70 has been exemplified with the murine melanoma cell model, B16 overexpressing exogenous Hsp70. Despite the therapeutic potential mechanism of Hsp70 transport to the surface and release remained poorly understood. We investigated principles of Hsp70 trafficking in B16 melanoma cells with low and high level of Hsp70. In cells with low level of Hsp70 apparent trafficking of Hsp70 was mediated by endosomes. Excess Hsp70 triggered a series of changes such as a switch of Hsp70 trafficking from endosomes to lysosomes and a concomitant accumulation of Hsp70 in lysosomes. Moreover, lysosomal rerouting resulted in an elevated concentration of surface Hsp70 and enabled active release of Hsp70. In fact, hyperthermia, a clinically applicable approach triggered immediate active lysosomal release of soluble Hsp70 from cells with excess Hsp70. Furthermore, excess Hsp70 enabled targeting of internalized surface Hsp70 to lysosomes, allowing in turn heat-induced secretion of surface Hsp70. Altogether, we show that excess Hsp70 expressed in B16 melanoma cells diverts Hsp70 trafficking from endosomes to lysosomes, thereby supporting its surface localization and lysosomal release. Controlled excess-induced lysosomal rerouting and secretion of Hsp70 is proposed as a promising tool to stimulate anti-tumor immunity targeting melanoma. © 2013 Bentham Science Publishers.


Lanzerstorfer P.,Upper Austria University of Applied Sciences | Stadlbauer V.,Upper Austria University of Applied Sciences | Chtcheglova L.A.,Center for Advanced Bioanalysis GmbH | Haselgrubler R.,Upper Austria University of Applied Sciences | And 8 more authors.
British Journal of Pharmacology | Year: 2014

Background and Purpose Insulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin. Experimental Approach Total internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein. Key Results Using our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR. Conclusions and Implications Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2011.1.1-1 | Award Amount: 5.55M | Year: 2012

We want to further develop our tools and technologies for high-throughput research, with the final goals being (I.) the particle-based combinatorial synthesis of 1 Mio different peptides on a glass slide for chemical costs of ~50 (KIT, CBL, MS, TUV), and (II.) the labelling-free parallel readout of binding affinities by a variant reflectometric interference spectroscopy method for ~10.000 peptide spots per cm(\2) when staining the array with an unlabeled protein (BIA, KIT). These tools provide the basis (III.) for a standardized, fast, and reliable high-throughput procedure that we want to develop in order to find high-affinity peptide binders against any pharmaceutically interesting target protein. Such a procedure might have an important impact in medicine and in the biotechnology industry. In order to achieve this goal, we will use display techniques that in combination with high-throughput sequencing typically will identify ~100.000 putative peptide binders per target protein (ISO). These will be synthesized in array format to validate binding to the target protein by an independent method (PPP, DKFZ). Next, based on binders from initial screens, many variant peptides are synthesized in high-density array format for iterative screens (PPP, DKFZ, KIT), whereby massive parallel labelling-free detection of binders pinpoints higher-affinity binders (BIA). In order to validate our novel high-throughput procedure, (IV.) we want to find high-affinity peptide binders against relevant target proteins (delivered by APO and OXF), and test these binders in biological assays (OXF, APO).


Van Es M.H.,Leiden University | Tang J.,Institute of Biophysics | Preiner J.,Center for Advanced Bioanalysis GmbH | Hinterdorfer P.,Institute of Biophysics | Oosterkamp T.H.,Leiden University
Ultramicroscopy | Year: 2014

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, Kon as determined from single molecule TREC data is in agreement with Kon determined using traditional biochemical methods. © 2014.


Schlapak R.,Center for Advanced Bioanalysis GmbH | Schlapak R.,Hightech Coatings GmbH | Danzberger J.,Johannes Kepler University | Haselgrubler T.,Center for Advanced Bioanalysis GmbH | And 5 more authors.
Nano Letters | Year: 2012

We present a generic and flexible method to nanopattern biomolecules on surfaces. Carbon-containing nanofeatures are written at variable diameter and spacing by a focused electron beam on a poly(ethylene glycol) (PEG)-coated glass substrate. Proteins physisorb to the nanofeatures with remarkably high contrast factors of more than 1000 compared to the surrounding PEG surfaces. The biological activity of model proteins can be retained as shown by decorating avidin spots with biotinylated DNA, thereby underscoring the universality of the nano-biofunctionalized platform for the binding of other biotinylated ligands. In addition, biomolecule densities can be tuned over several orders of magnitude within the same array, as demonstrated by painting a microscale image with nanoscale pixels. We expect that these unique advantages open up entirely new ways to design biophysical experiments, for instance, on cells that respond to the nanoscale densities of activating molecules. © 2012 American Chemical Society.


Zhu R.,Johannes Kepler University | Howorka S.,Center for Advanced Bioanalysis GmbH | Howorka S.,University College London | Proll J.,Elisabethinen Hospital | And 10 more authors.
Nature Nanotechnology | Year: 2010

Atomic force microscopy (AFM) is a powerful tool for analysing the shapes of individual molecules and the forces acting on them. AFM-based force spectroscopy provides insights into the structural and energetic dynamics of biomolecules by probing the interactions within individual molecules, or between a surface-bound molecule and a cantilever that carries a complementary binding partner. Here, we show that an AFM cantilever with an antibody tether can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4 Å, thereby revealing the DNA methylation pattern, which has an important role in the epigenetic control of gene expression. The antibody is able to bind two 5-methylcytidine bases of a surface-immobilized DNA strand, and retracting the cantilever results in a unique rupture signature reflecting the spacing between two tagged bases. This nanomechanical approach might also allow related chemical patterns to be retrieved from biopolymers at the single-molecule level. © 2010 Macmillan Publishers Limited. All rights reserved.


Zhu R.,Johannes Kepler University | Zhu R.,Charité - Medical University of Berlin | Rupprecht A.,University of Veterinary Medicine Vienna | Rupprecht A.,Charité - Medical University of Berlin | And 6 more authors.
Journal of the American Chemical Society | Year: 2013

A tight regulation of proton transport in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis, heat production, or regulation of the reactive oxygen species as proposed for the uncoupling protein family members (UCP). Specific regulation of proton transport is thus becoming increasingly important in the therapy of obesity and inflammatory, neurodegenerative, and ischemic diseases. We and other research groups have shown previously that UCP1- and UCP2-mediated proton transport is inhibited by purine nucleotides. Several hypotheses have been proposed to explain the inhibitory effect of ATP, although structural details are still lacking. Moreover, the unresolved mystery is how UCP operates in vivo despite the permanent presence of high (millimolar) concentrations of ATP in mitochondria. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 reconstituted into lipid bilayers and to analyze the ATP-protein interaction at a single molecule level. The comparison of recognition patterns obtained with anti-UCP1 antibody and ATP led to the conclusion that the ATP binding site can be accessed from both sides of the membrane. Using cantilever tips with different cross-linker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 Å precision. Together with the recently published NMR structure of a UCP family member (Berardi et al. Nature, 2011, 476, 109-113), our data provide a valuable insight into the mechanism of the nucleotide binding and pave the way for new pharmacological approaches against the diseases mentioned above. © 2013 American Chemical Society.


Howorka S.,University College London | Howorka S.,Center for Advanced Bioanalysis GmbH | Hesse J.,Center for Advanced Bioanalysis GmbH
Soft Matter | Year: 2014

Biomolecules positioned at interfaces have spawned many applications in bioanalysis, biophysics, and cell biology. This Highlight describes recent developments in the research areas of protein and DNA arrays, and single-molecule sensing. We cover the ultrasensitive scanning of conventional microarrays as well as the generation of arrays composed of individual molecules. The combination of these tools has improved the detection limits and the dynamic range of microarray analysis, helped develop powerful single-molecule sequencing approaches, and offered biophysical examination with high throughput and molecular detail. The topic of this Highlight integrates several disciplines and is written for interested chemists, biophysicists and nanotechnologists. © 2014 The Royal Society of Chemistry.


Juhasz K.,Hungarian Academy of Sciences | Juhasz K.,Center for Advanced Bioanalysis GmbH | Buzas K.,Hungarian Academy of Sciences | Buzas K.,University of Szeged | And 2 more authors.
Expert Review of Clinical Immunology | Year: 2013

TNF-related ligands (with the exception of lymphotoxin-α) are synthesized as type II transmembrane proteins, though many of them also have soluble forms. An increasing number of publications report that these 'ligands' behave as receptors, activating intracellular signaling pathways when interacting with cognate 'receptors' or agonistic antibodies. Most members of the TNF family and their receptors influence survival, proliferation, differentiation or activation of immune cells. The elicited 'reverse signals' also have significant importance. They proved to be involved in the activation of APCs, T and B cells, differentiation of osteoclasts and apoptosis of activated macrophages. They influence the balance between destructive immune response and tolerance. Several examples show that therapeutic manipulation of the reverse signal can help to treat malignancies as well as autoimmune disorders. © 2013 Expert Reviews Ltd.


Juhasz K.,Center for Advanced Bioanalysis GmbH | Lipp A.-M.,Center for Advanced Bioanalysis GmbH | Nimmervoll B.,Center for Advanced Bioanalysis GmbH | Sonnleitner A.,Center for Advanced Bioanalysis GmbH | And 3 more authors.
Cancers | Year: 2014

Elevated expression of the inducible heat shock protein 70 (Hsp70) is known to correlate with poor prognosis in many cancers. Hsp70 confers survival advantage as well as resistance to chemotherapeutic agents, and promotes tumor cell invasion. At the same time, tumor-derived extracellular Hsp70 has been recognized as a "chaperokine", activating antitumor immunity. In this review we discuss localization dependent functions of Hsp70 in the context of invasive cancer. Understanding the molecular principles of metastasis formation steps, as well as interactions of the tumor cells with the microenvironment and the immune system is essential for fighting metastatic cancer. Although Hsp70 has been implicated in different steps of the metastatic process, the exact mechanisms of its action remain to be explored. Known and potential functions of Hsp70 in controlling or modulating of invasion and metastasis are discussed. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

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