| 1. |
- Ahlin, Gustav, 1977-, et al.
(författare)
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Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs
- 2009
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 37:12, s. 2275-2283
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate the gene and protein expression profiles of important drug transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters (HeLa, HEK293) and leukaemia cell lines used to study drug resistance by ABC-transporters (HL-60, K562) were investigated, and compared with organotypic cell lines (HepG2, Saos-2, Caco-2 and Caco-2 TC7). For gene expression studies, real-time PCR was used, while monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression and nine for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1; SLC16A1 was investigated using 14C-lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression and the expression patterns were barely affected by transfection. The leukaemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, while the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. Importantly, the monocarboxylic acid transporting protein MCT1 was significantly expressed in all, and functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.
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| 2. |
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| 3. |
- Andersson, Ann-Catrin, et al.
(författare)
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Analysis of protein expression in cell microarrays : A tool for antibody-based proteomics
- 2006
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Ingår i: Journal of Histochemistry and Cytochemistry. - 0022-1554 .- 1551-5044. ; 54:12, s. 1413-1423
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Tidskriftsartikel (refereegranskat)abstract
- Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TIVIA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.
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| 4. |
- Andrade, Jorge, et al.
(författare)
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Using Grid Technology for Computationally Intensive Applied Bioinformatics Analyses
- 2006
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Ingår i: In Silico Biology. - 1386-6338. ; 6:6, s. 495-504
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Tidskriftsartikel (refereegranskat)abstract
- For several applications and algorithms used in applied bioinformatics, a bottle neck in terms of computational time may arise when scaled up to facilitate analyses of large datasets and databases. Re-codification, algorithm modification or sacrifices in sensitivity and accuracy may be necessary to accommodate for limited computational capacity of single work stations. Grid computing offers an alternative model for solving massive computational problems by parallel execution of existing algorithms and software implementations. We present the implementation of a Grid-aware model for solving computationally intensive bioinformatic analyses exemplified by a blastp sliding window algorithm for whole proteome sequence similarity analysis, and evaluate the performance in comparison with a local cluster and a single workstation. Our strategy involves temporary installations of the BLAST executable and databases on remote nodes at submission, accommodating for dynamic Grid environments as it avoids the need of predefined runtime environments (preinstalled software and databases at specific Grid-nodes). Importantly, the implementation is generic where the BLAST executable can be replaced by other software tools to facilitate analyses suitable for parallelisation. This model should be of general interest in applied bioinformatics. Scripts and procedures are freely available from the authors.
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| 5. |
- Asplund, C., et al.
(författare)
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Real-time RT-PCR of protein epitope signature tags
- 2005
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Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 4:8, s. S60-S60
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 6. |
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| 7. |
- Barbe, Laurent, et al.
(författare)
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Toward a confocal subcellular atlas of the human proteome
- 2008
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Ingår i: Molecular and cellular proteomics. - 1535-9476 .- 1535-9484. ; 7:3, s. 499-508
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Tidskriftsartikel (refereegranskat)abstract
- Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
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| 8. |
- Bengtsson, Sofia, et al.
(författare)
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Large-scale proteomics analysis of human ovarian cancer for biomarkers
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:4, s. 1440-1450
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Tidskriftsartikel (refereegranskat)abstract
- Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues.
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| 9. |
- Berglund, Lisa, et al.
(författare)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Forskningsöversikt (refereegranskat)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 10. |
- Berglund, Lisa, et al.
(författare)
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A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 8:14, s. 2832-2839
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Tidskriftsartikel (refereegranskat)abstract
- Here, we present an antigen selection strategy based on a whole-genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in-house generated data. The web-based bioinformatics platform has been designed for selection of multiple, non-overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain- and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome-wide analysis demonstrates that antigens for approximately 80% of the human protein-coding genes can be selected with this strategy.
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