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ABSTRACT: We developed a sequencing assay for genotypic HIV-1 tropism determination. The assay allows examination of HIV RNA from plasma and HIV DNA from peripheral blood mononuclear cells (PBMC), including PBMC samples from patients with undetectable viral loads. Assessment of 100 pairs of plasma and PBMC samples showed a high concordance of 90%. With the limitations of population-based sequencing, the assay was found to be robust and suitable for the routine clinical laboratory.
Journal of clinical microbiology 05/2011; 49(7):2697-9. · 4.16 Impact Factor
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Julian Weghuber,
Mario Brameshuber,
Stefan Sunzenauer,
Manuela Lehner, Christian Paar,
Thomas Haselgrübler,
Michaela Schwarzenbacher,
Martin Kaltenbrunner,
Clemens Hesch,
Wolfgang Paster,
Bettina Heise,
Alois Sonnleitner,
Hannes Stockinger,
Gerhard J Schütz
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ABSTRACT: Our understanding of complex biological systems is based on high-quality proteomics tools for the parallelized detection and quantification of protein interactions. Current screening platforms, however, rely on measuring protein interactions in rather artificial systems, rendering the results difficult to confer on the in vivo situation. We describe here a detailed protocol for the design and the construction of a system to detect and quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput, making it applicable as a screening tool. The proof-of-concept is demonstrated for the interaction between CD4, a major coreceptor in T-cell signaling, and Lck, a protein tyrosine kinase essential for early T-cell signaling.
Methods in enzymology 01/2010; 472:133-51. · 1.90 Impact Factor
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Biophysical Journal 12/2009; 98:22. · 3.65 Impact Factor
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ABSTRACT: The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr(505) upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr(505) to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr(394) to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr(505) and Tyr(394) phosphorylation and conformational alterations.
The Journal of Immunology 03/2009; 182(4):2160-7. · 5.79 Impact Factor
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ABSTRACT: A sequencing assay for detection of mutations conferring resistance to human immunodeficiency virus type 1 (HIV-1) integrase inhibitors raltegravir and elvitegravir was developed using the automated TruGene sequencing system. The assay returned clear sequencing results for samples with >or=500 RNA copies/ml for mutation detection and HIV-1 subtyping across a spectrum of HIV-1 subtypes.
Journal of clinical microbiology 10/2008; 46(12):4087-90. · 4.16 Impact Factor
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ABSTRACT: Monitoring protein function with high throughput at individual cell level is of high interest both for basic research and diagnostic applications. For this, following the changes in fluorescence resonance energy transfer (FRET) between a donor/acceptor pair, genetically encoded in the proteins of interest, is a frequently used tool. As proteins attached to or located in the plasma membrane represent a considerable fraction of total proteins, there is a need for high throughput imaging techniques suited for observation of proteins in the cell membrane only. A system is presented, which allows rapid imaging of large areas via total internal reflection fluorescence microscopy (TIRFM) conditions, using a focus-hold system, multiwavelength excitation and dual color detection. The developed imaging system enables screening of large numbers of cells under TIRFM illumination combined with FRET imaging, thereby providing the means to record, e.g., FRET-efficiency of a membrane-associated protein labeled with a donor-acceptor pair. The capability of the system to perform live-FRET scanning with TIRFM on stoichiometric FRET constructs, reaching throughput of up to 1,000 cells/s at the optical resolution limit is demonstrated. A comparison with confocal microscopy shows that TIRFM offers a 4.2-fold advantage in our conditions over confocal microscopy in detecting contributions from membrane-localized proteins.
Cytometry Part A 06/2008; 73(5):442-50. · 3.73 Impact Factor
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ABSTRACT: Expression of prion protein (PrP) has been reported for a variety of cell types including neuronal cells, haematopoietic stem cells, antigen-presenting cells, as well as lymphocytes. However, besides this widespread occurrence little is known about the physiological roles exhibited by this enigmatic protein. In this study, the contribution of PrP to the classical T-lymphocyte activation process was characterized by clustering the T-cell receptor component CD3epsilon as well as PrP with soluble and surface-immobilized antibodies, respectively. We present evidence that PrP is a component of signaling structures recently described as plasma membrane microclusters established during T-lymphocyte activation. The formation of immunological synapses, however, did not depend on the presence of PrP as proven by siRNA knockdown experiments, indicating very subtle physiological roles of PrP in vivo within the immune system.
European Journal of Cell Biology 06/2007; 86(5):253-64. · 2.81 Impact Factor
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BioTechniques 05/2005; 38(4):527-8, 530. · 2.67 Impact Factor
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ABSTRACT: While long-term effects of temperature treatment in respect of, e.g., gene-expression and cellular function have already been studied in some detail, nothing is known on the physiological responses of lymphocytes during short-term hypothermal shifts. In this report, we characterized the effects of such a stimulation using the human lymphocyte cell line Jurkat E6.1 and present evidence that warming from 4 to 37 degrees C for only 2 min is sufficient to cause co-localization of CD3, prion protein and the lipid-raft ganglioside GM1 paralleling lymphocyte activation as observed by Ca(2+) mobilization and mitogen-activated protein kinase-phosphorylation.
FEBS Letters 06/2004; 566(1-3):121-5. · 3.54 Impact Factor
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Beata Polgar,
Gyula Kispal,
Margit Lachmann, Christian Paar,
Eszter Nagy,
Peter Csere,
Eva Miko,
Laszlo Szereday,
Peter Varga,
Julia Szekeres-Bartho,
Gabriella Paar
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ABSTRACT: Previous studies from our laboratory showed that the immunomodulatory effects of progesterone are mediated by a 34-kDa protein, named the progesterone-induced blocking factor (PIBF). Lymphocytes of women with threatened abortion fail to produce this factor. Via inducing a Th2 biased cytokine production and blocking of NK activity, PIBF prevents induced pregnancy loss in mice, suggesting that substitution therapy with PIBF could be useful as an alternative treatment of certain forms of recurrent spontaneous abortions. Our study was aimed at mapping the sequence and structure of PIBF coding cDNA and characterizing the encoded protein product. Screening of a human liver cDNA library revealed a 2765-bp clone with a 2271-bp open reading frame. The PIBF1 cDNA encodes a protein of 757 amino acid residues with an 89-kDa predicted molecular mass, which shows no significant amino acid sequence homology with any known protein. PIBF produced via recombinant technique is recognized by the Ab specific for the secreted lymphocyte PIBF Ab, and possesses the biological activities of the secreted lymphocyte PIBF. The full-length PIBF is associated with the nucleus, whereas secretion of shorter forms, such a 34-kDa protein is induced by activation of the cell. The 48-kDa N-terminal part of PIBF is biologically active, and the part of the molecule, responsible for modulating NK activity is encoded by exons 2-4. These data provide an initial step for exploiting the possible diagnostic and therapeutic potential of this immunomodulatory molecule.
The Journal of Immunology 01/2004; 171(11):5956-63. · 5.79 Impact Factor
