| 1. |
- Planck, Maria, et al.
(författare)
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Cytogenetic aberrations and heterogeneity of mutations in repeat-containing genes in a colon carcinoma from a patient with hereditary nonpolyposis colorectal cancer.
- 2002
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Ingår i: Cancer Genetics and Cytogenetics. - 0165-4608. ; 134:1, s. 46-54
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Tidskriftsartikel (refereegranskat)abstract
- The majority of tumors from patients affected by hereditary nonpolyposis colorectal cancer (HNPCC) exhibit a mutator phenotype characterized by widespread microsatellite instability (MSI) and somatic mutations in repeated sequences in several cancer-associated genes. An inverse relationship between MSI and chromosomal instability (CIN) has been demonstrated and HNPCC-associated tumors are generally characterized by diploid or near-diploid cells with few or no chromosomal rearrangements. We have studied MSI, somatic mutations in repeat-containing genes, DNA-ploidy, and cytogenetic aberrations in a colon carcinoma from a patient with a germline MLH1 mutation. Mutations in coding repeats were assessed in 10 macroscopically separate areas of the primary tumor and in two lymph nodes. Some of the genes studied (E2F4, MSH3, MSH6, TCF4, and TGFBRII) showed a consistent lack of mutations, whereas others (BAX, Caspase-5 and IGFIIR) displayed alterations in some tumor regions but not in others. The tumor had DNA-index 1.1-1.2 and a stable, aberrant karyotype with extra copies of chromosomes 7 and 12 and the structural aberrations i(1q), der(20)t(8;20), and der(22)t(1;22). The finding of CIN, MSI, and somatic mutations in coding repeats in this tumor suggests that these phenomena may act together in HNPCC tumorigenesis. Furthermore, the observed intratumoral heterogeneity of mutations in coding repeats implies these changes occur late in tumorigenesis and, thus, probably play a role in tumor progression rather than initiation.
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| 2. |
- Gisselsson Nord, David, et al.
(författare)
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Differentially amplified chromosome 12 sequences in low- and high-grade osteosarcoma.
- 2002
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Ingår i: Genes, Chromosomes and Cancer. - : Wiley. - 1045-2257. ; 33:2, s. 133-140
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Tidskriftsartikel (refereegranskat)abstract
- Most osteosarcomas are highly aggressive malignancies characterized by a complex pattern of chromosome abnormalities. However, a subgroup of low-grade, parosteal tumors exhibits a relatively simple aberration pattern dominated by ring chromosomes carrying amplified material from chromosome 12. To assess whether sequences from this chromosome were differentially amplified in low- and high-grade osteosarcomas, copy numbers of the CCND2, ETV6, KRAS2, and D12S85 regions in 12p and the MDM2 region in 12q were evaluated by interphase or metaphase fluorescence in situ hybridization (FISH) in 24 osteosarcomas. Amplification of MDM2 was detected in all five low-grade and four high-grade osteosarcomas, all of which showed ring chromosomes. An overrepresentation of 12p sequences was found in 1/5 low-grade and in 9/19 high-grade tumors. Multicolor single-copy FISH analysis of metaphase cells from six high-grade tumors showed that extra 12p material either occurred together with MDM2 in ring chromosomes or was scattered over the genome as a result of complex structural rearrangements. Most tumors (8/10) not containing amplification of the assessed chromosome 12 loci exhibited a nondiploid pattern at evaluation with probes for centromeric alpha satellite sequences. These findings indicate that gain of sequences from the short arm of chromosome 12 could be a possible genetic pathway in the development of aggressive osteosarcoma.
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| 3. |
- Gisselsson Nord, David, et al.
(författare)
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Locus-specific multifluor FISH analysis allows physical characterization of complex chromosome abnormalities in neoplasia
- 2000
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Ingår i: Genes, Chromosomes and Cancer. - 1045-2257. ; 28:3, s. 347-352
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Tidskriftsartikel (refereegranskat)abstract
- Novel techniques in molecular cytogenetics have radically improved the ability to characterize genetic changes in neoplastic cells. In parallel, a rapid development in high-throughput genomics has resulted in detailed physical maps of the human genome. Combining these two fields, we have developed a method for the simultaneous visualization of several physically defined segments along a chromosome. Seven YAC clones and one subtelomeric cosmid clone from chromosome 12 were labeled with unique combinations of four fluors and hybridized to metaphase chromosomes from neoplastic cells. In a uterine leiomyoma and a myxoid liposarcoma with translocations 12;14 and 12;16, the breakpoints in chromosome 12 could be localized to the HMGIC and CHOP regions, respectively. In the other tumors, more complex aberrations were visualized, including two inversions in 12q with a common breakpoint between MDM2 and D12S332 in a pleomorphic adenoma, amplification of MDM2 and CDK4 in ring chromosomes from a malignant fibrous histiocytoma, and amplification of KRAS2 together with other unbalanced rearrangements in two pancreatic adenocarcinomas. Combinatorially labeled single-copy probes may thus simultaneously provide physical localization of breakpoints and an overview of complex structural rearrangements.
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| 4. |
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| 5. |
- Nandakumar, Madayi P., et al.
(författare)
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Superporous agarose monoliths as mini-reactors in flow injection systems
- 2000
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Ingår i: Bioseparation. - 1573-8272. ; 9:4, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15×5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 μm in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 Å) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min−1). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular β-galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against β-galactosidase).
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| 6. |
- Pålsson, Eva
(författare)
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Fast isolation of proteins from bioreactors using affinity chromatography techniques
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The purification of proteins from raw materials like fermentation broth and other biological sources usually involves several purification steps and is rather time consuming and expensive. This is a major concern in the production of many important protein products e.g. pharmaceuticals such as insulin and blood coagulation factors. One way to speed up the isolation of protein from particle-containing liquids is by the use of expanded bed adsorption. By a selective adsorption of the desired protein, it can be clarified from particles, concentrated and purified from many molecular impurities in one step. A new improved support material for the use in expanded bed adsorption was developed and is described in this thesis. It was shown to have excellent adsorption efficiency at flow rates higher than normally used today. The new, pelicular, matrix was used to capture antibodies from cell-containing fermentation broth. When particle-free samples are to be purified, a packed bed can be used. In an industrially relevant application, the isolation of factor VIII from clarified fermentation broth, the recently developed matrix material “superporous agarose” was employed. Compared to traditional agarose supports it showed considerably higher efficiency at increased flow rates. This was ascribed to the presence of superpores that allow intraparticle flow, which reduces the diffusion distances. In analyses of cell-containing samples small columns permeable to cells and cell debris are valuable tools. A miniaturised expanded bed column and a small superporous monolithic column were designed and used in experimental set-ups in which cell containing samples were analysed for e.g. IgG and glucose. When new support materials are developed it is important to be able to compare their performance with existing supports. The methods used in determining the efficiency of expanded beds were critically examined and found unsuitable when comparing beds of different degrees of expansion. Quantities specially modified for expanded bed evaluation were therefore developed.
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| 7. |
- Pålsson, Eva, et al.
(författare)
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Miniaturised expanded-bed column with low dispersion suitable for fast flow-ELISA analyses
- 2000
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Ingår i: Biotechnology Letters. - 0141-5492. ; 22, s. 245-250
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Tidskriftsartikel (refereegranskat)abstract
- Flow-ELISA measurements of the monoclonal antibody concentration in cultivation broth containing murine hybriboma cells were carried out using a small expanded-bed column (0.5×2.5 cm) charged with protein A. A new specialised pellicular agarose/stainless steel matrix designed for high flow rates with fast mass transport properties was used. Special care was taken to get an efficient flow distribution. The axial dispersion coefficient was very low (2×10−6 m2 s−1 for latex particles at a linear velocity of 10 cm min−1). Breakthrough curves for polyclonal IgG on the protein A-derivatised support (at 2–11 cm min−1) further emphasised its advantageous properties. No significant change in dynamic capacity was found over the entire speed range.
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| 8. |
- Pålsson, Eva, et al.
(författare)
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Pellicular expanded bed matrix suitable for high flow rates
- 2000
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Ingår i: Journal of chromatography. A. - 0021-9673. ; 878:1, s. 17-25
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Tidskriftsartikel (refereegranskat)abstract
- A new type of expanded bed matrix with a heavy core of stainless steel covered with an agarose layer was prepared. Two bead size fractions, the smaller one (32–75 μm ∅) having a single particle core and the larger (75–180 μm ∅) with an agglomerate of stainless steel particles constituting the core, were chosen for further characterisation. The dispersion behaviour was determined both in packed bed and expanded bed modes by the retention time distribution method (RTD) and compared with the Streamline matrix (Amersham Pharmacia Biotech). The comparison turned out in favour of the new matrix. Flow rates as high as 3000 cm/h were used with the larger fraction, giving stable expanded beds with good mass transfer properties. The matrices were mechanically stable without any tendency to crack or peal, even after prolonged use.
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| 9. |
- Surace, Cecilia, et al.
(författare)
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A novel FISH assay for SS18-SSX fusion type in synovial sarcoma
- 2004
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Ingår i: Laboratory Investigation. - : Elsevier BV. - 1530-0307 .- 0023-6837. ; 84:9, s. 1185-1192
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Tidskriftsartikel (refereegranskat)abstract
- Synovial sarcoma is a morphologically, clinically and genetically distinct entity that accounts for 5-10% of all soft tissue sarcomas. The t(X;18)(p11.2;q11.2) is the cytogenetic hallmark of synovial sarcoma and is present in more than 90% of the cases. It produces three types of fusion gene formed in part by SS18 from chromosome 18 and by SSX1, SSX2 or, rarely, SSX4 from the X chromosome. The SS18-SSX fusions do not seem to occur in other tumor types, and it has been shown that in synovial sarcoma a clear correlation exists between the type of fusion gene and histologic subtype and, more importantly, clinical outcome. Previous analyses regarding the type of fusion genes have been based on PCR amplification of the fusion transcript, requiring access to good-quality RNA. In order to obtain an alternative tool to diagnose and follow this malignancy, we developed a fluorescence in situ hybridization (FISH) assay that could distinguish between the two most common fusion genes, that is, SS18-SSX1 and SS18-SSX2. The specificity of the selected bacterial artificial chromosome clones used in the detection of these fusion genes, as well as the sensitivity of the analysis in metaphase and interphase cells, was examined in a series of 28 synovial sarcoma samples with known fusion gene status. In all samples, the type of fusion was correctly identified by FISH. Thus, the assay described here should be useful for clarifying unresolved chromosome markers and for identifying fusion gene status in samples from which RNA of sufficient quality for PCR could not be extracted.
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