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- Landegren, Ulf, et al.
(författare)
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Molecular tools for a molecular medicine : analyzing genes, transcripts and proteins using padlock and proximity probes
- 2004
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 17:3, s. 194-7
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Tidskriftsartikel (refereegranskat)abstract
- Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.
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- Landegren, Ulf, et al.
(författare)
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Padlock and proximity probes for in situ and array-based analyses : tools for the post genomic era
- 2003
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Ingår i: Comparative and functional genomics. - : Hindawi Limited. - 1531-6912 .- 1532-6268. ; 4:5, s. 525-30
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Tidskriftsartikel (refereegranskat)abstract
- Highly specific high-throughput assays will be required to take full advantage of the accumulating information about the macromolecular composition of cells and tissues, in order to characterize biological systems in health and disease. We discuss the general problem of detection specificity and present the approach our group has taken, involving the reformatting of analogue biological information to digital reporter segments of genetic information via a series of DNA ligation assays. The assays enable extensive, coordinated analyses of the numbers and locations of genes, transcripts and protein.
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- Nilsson, Joacim, et al.
(författare)
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Differentiation-associated redox-regulation in human B cell lines from stem cell/pro-B to plasma cell
- 2004
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Ingår i: Immunology Letters. - : Elsevier BV. - 0165-2478 .- 1879-0542. ; 94:1-2, s. 83-89
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Tidskriftsartikel (refereegranskat)abstract
- Redox-regulation of receptors and transcription factors are important for lymphocyte activation, differentiation and apoptosis. Thioredoxin (Trx) is a key redox-regulating protein and oxidative stress sensor operating in synergy with Trx-reductase and protein disulfide isomerase (PDI). The expression of Trx, PDI, and the Trx-regulated transcription-factor Pax5 were analyzed in a panel of human B cell lines and were compared with that of the Bcl-2 family proteins, also redox-controlled. The panel included representative cells from various stages: FLEB14-4 (pro-B), REH and NALM-6 (pre-B), Rael and Daudi (small mature B), U-698 and NC0467.3 (B-blasts), LP-1, U-1996, and U-266 (plasma cells). We found a significant congruence and co-variation of Trx and Bcl-2 levels in the B-lineage, with high expression levels in early stages (pro-B and pre-B) and in the late stage representing terminally-differentiated plasma cells, whereas mid-stage small resting B cells showed a very low expression. PDI increased significantly in plasma-blasts and plasma cells, indicating its importance in the highly specialized immunoglobulin assembly-machinery, including disulfide-bond isomerization. Pax5 was expressed in early and mid-stages, but was silenced in terminal stages. We conclude that the high Trx and Bcl-2-expression early and late in the B cell maturation pathway reflects a redox-strategy favoring an increased survival potential of the B cells at those stages. © 2004 Elsevier B.V. All rights reserved.
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- Nilsson, J, et al.
(författare)
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Thioredoxin prolongs survival of B-type chronic lymphocytic leukemia cells
- 2000
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Ingår i: Blood. - 0006-4971 .- 1528-0020. ; 95:4, s. 1420-1426
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Tidskriftsartikel (refereegranskat)abstract
- Thioredoxin (Trx) is a ubiquitous protein disulfide oxidoreductase with antioxidant, cytokine, and chemotactic properties. Previously, we showed that Trx, in synergy with interleukin 1 (IL-1), IL-2, IL-4, tumor necrosis factor alpha (TNF-alpha), and CD40-ligation induced S-phase entry and mitosis in normal B cells and B-type chronic lymphocytic leukemia (B-CLL) cells. The viability of B-CLL cells stimulated by these protocols is high, and it has been hypothesized that the overexpression of Bcl-2 found in B-CLL protects the cells from apoptosis in vitro and in vivo. In this study, we have analyzed the response of cells derived from 12 samples of patients with B-CLL to recombinant human Trx in spontaneous apoptosis, with special reference to the Bcl-2 expression. Long-term cultures of B-CLL clones showed significantly higher viability when supplemented with human Trx (P =.031), also exemplified with clones surviving more than 2 months. Short-term cultures of B-CLL cells exposed to 1 microg/mL of Trx for 1, 5, or 12 days maintained expression or delayed down-regulation of Bcl-2 compared with control cultures containing RPMI 1640 medium and 10% fetal calf serum only (P =.032,. 002,.026, respectively). All B-CLL cells expressed constitutive Trx at varying but low levels, in contrast to adult T-cell leukemias, which overexpress Trx, as previously reported. We found that Trx added to B-CLL cells increased in a dose-dependent fashion the release of TNF-alpha, which has been suggested to be an autocrine growth factor for these cells. In conclusion, we have found that human recombinant Trx induced TNF-alpha secretion, maintained Bcl-2, and reduced apoptosis in B-CLL cells.
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- Thunberg, Ulf, et al.
(författare)
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Polymorphism in the P2X7 receptor gene and survival in chronic lymphocytic leukaemia
- 2002
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Ingår i: The Lancet. - 0140-6736 .- 1474-547X. ; 360:9349, s. 1935-1939
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The P2X7 receptor is a ligand-gated cation channel that mediates ATP-induced apoptotic death in haemopoietic and chronic lymphocytic leukaemia (CLL) cells. We aimed to investigate the clinical effect of a single nucleotide polymorphism in the P2X7 receptor gene (1513A-->C) and correlate findings with the immunoglobulin heavy chain variable (V(H)) gene mutation status, a prognostic marker in CLL. METHODS: We investigated tumour DNA in 170 patients with CLL using PCR-RFLP analysis with HhaI restriction enzyme cleavage to screen for the polymorphism in the P2X7 receptor gene. The VH gene mutation status was assessed in 165 patients by PCR amplification and nucleotide sequencing. We correlated the findings of the P2X7 receptor genotype with the V(H) gene mutation data and overall survival and screened for the P2X7 receptor polymorphism in 200 healthy controls. FINDINGS: Of the 170 patients, 35 (21%) were heterozygous and one (1%) homozygous for the 1513C allele; whereas 134 (79%) had the 1513A/A genotype of the P2X7 receptor gene. Overall survival was significantly longer for patients with CLL heterozygous for the 1513C allele than those with the 1513A/A genotype. Median survival for heterozygous patients was 104 months (range 33-467) and 72 months (1-190) for homozygous patients (p=0.009). Of the 165 patients with CLL in whom we assessed the V(H) gene mutation status, the V(H) genes were mutated in 71 (43%) patients and unmutated in 94; 18 (25%) of the 71 patients with mutated genes had 1513C allele compared with 17 (18%) of 94 who had unmutated genes. In patients with mutated VH genes, those with CLL who were 1513C positive had 53 months' longer median survival than did those with the 1513A/A genotype (151 vs 98 months, p=0.011). INTERPRETATION: The P2X7 polymorphism could affect clinical outcome in CLL, especially in patients with mutated V(H) genes. Studies are necessary to elucidate the biological role of the P2X7 polymorphism in CLL in vivo.
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