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- Koethe, Susanne, et al.
(författare)
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Pivotal Advance: CD45RB glycosylation is specifically regulated during human peripheral B cell differentiation.
- 2011
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Ingår i: Journal of leukocyte biology. - : Oxford University Press (OUP). - 1938-3673 .- 0741-5400. ; 90:1, s. 5-19
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Tidskriftsartikel (refereegranskat)abstract
- A screen of cell surface markers differentially expressed during peripheral B cell differentiation identified that the CD45RB epitope detected by the mAb MEM-55 was highly expressed on CD27(+) memory B cells and absent on CD27(-) naïve B cells. IgG(+)CD27(-) memory and a previously unacknowledged CD27(-) population in blood also expressed high levels of CD45RB(MEM55). Naïve and memory B cells from tonsils followed the pattern observed in blood, and CD38(high) B cells had a bimodal expression pattern when analyzed using flow cytometry. No CD38(high) GC B cells, however, expressed the CD45RB(MEM55) epitope when assayed using immunohistochemistry. Rather, CD38(high)CD45RB(MEM55high) B cells had a distinct cellular phenotype and were localized outside of GCs. CD45RB epitopes, detected by other antibody clones, were expressed at high levels through B cell differentiation, and no changes in splicing of the CD45RB exon were observed during B cell differentiation. Instead, B cells regulated their expression of the CD45RB(MEM55) epitope through site-specific modifications of an O-linked glycochain. CD4(+) T cells differentially spliced CD45 but did not vary the glycosylation of the CD45RB(MEM55) epitope, and CD8(+) cells modified CD45RB(MEM55) expression in a similar manner as B cells. Monocytes expressed the CD45RB exon but not the CD45RB(MEM55) epitope. As CD45 is a highly expressed tyrosine phosphatase that regulates antigen receptor signaling strength in lymphocytes, we conclude that regulated O-linked glycosylation of CD45RB can be used to follow B cell differentiation and that this regulation may be involved in fine-tuning antigen signaling in the cell.
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| 2. |
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| 3. |
- Zander, Linda, 1976, et al.
(författare)
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Identification of genes deregulated in a Burkitt´s lymphoma cell line when adapted for
- 2008
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Ingår i: Cell Proliferation. - : Wiley. - 1365-2184 .- 0960-7722. ; 41:1, s. 136-155
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Serum is usually added to growth media when mammalian cells are cultured in vitro to supply the cells with growth factors, hormones, nutrients and trace elements. Defined proteins and metal ions, such as insulin, growth factors, transferrin and sodium selenite, are sometimes also included and can in some cases substitute serum components. How adaptation to serum free media influences cells has not been studied in detail. Materials and Methods: We have adapted the Burkitt's lymphoma line Ramos to a serum-free medium that supports long-term survival and studied gene expression changes that occurred during the adaptation process. Results and Conclusions: The adaptation process was characterized by initial cell population growth arrest, and after that extensive cell death, followed by proliferation and long-term survival of clonal cultures. Proliferation and cell cycle progression of the serum-free cultures closely mimicked that of serum-dependent cells. Affymetrix micro-array technology was used to identify gene expression alterations that had occurred during the adaptation. Most changes were subtle, but frequently the genes with altered expression were involved in basal cellular functions such as cell division, cell cycle regulation, apoptosis and cell signalling. Some alterations were restored when the cells were transferred back to serum-containing medium, indicating that expression of these genes was controlled by components in serum. Others were not, and may represent changes that were selected during the adaptation process. Among these were, for example, several genes within the Wnt signalling pathway.
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| 4. |
- Zander, Linda, 1976, et al.
(författare)
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Immortalized mouse cell lines that lack a functional Rev3 gene are hypersensitive to UV irradiation and cisplatin treatment.
- 2004
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Ingår i: DNA repair. - : Elsevier BV. - 1568-7864. ; 3:7, s. 743-52
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.
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| 5. |
- Zander, Linda, 1976
(författare)
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The Germinal Centre reaction - Genetic and proteomic analysis of factors important for survival and growth of B lymphocytes
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During an immune response the B lymphocytes main function is to produce antibodies in the specific defence against the pathogen. When naïve B lymphocytes become activated by binding of an antigen, the cells differentiate to become antibody producing plasma cells. During this process, some cells form germinal centres after interaction with T lymphocytes, where the immunoglobuline (Ig) genes are differentiated, to evolve high affinity plasma cells or memory B lymphocytes. We have used the Burkitt lymphoma cell line Ramos to study the germinal centre reaction. Ramos cells are normally cultured in medium containing serum, but we have adapted Ramos cells to long-term survival in a serum-free medium. The serum-free cells are more sensitive to dilution, which indicates a production of autocrine growth factors. We have studied the gene expression changes that occur during adaption to serum-free media by global gene expression analysis, and we found several deregulated genes involved in cell-cycle regulation and apoptosis. We also identified a Ramos cell line deficient in MHC class II expression, resembling the situation during Bare lymphocyte syndrome. The cause of this deficiency is studied by examining the function of transcription factors regulating MHC class II expression. Germinal centre B lymphocytes are highly susceptible to apoptosis unless rescued by survival signals from T lymphocytes and follicular dendritic cells. In an effort to study these interactions we have isolated and identified secreted extracellular proteins produced by serum-free Ramos cells. The expressions of these proteins were also examined in tonsil germinal centre B lymphocytes and the levels were compared with cells in the pre- and post-germinal centre stage of the tonsils. During the germinal centre reaction the antibody gene of B lymphocytes are differentiated through somatic hypermutation and class switch recombination. These events are dependent on the AID mediated cytidine deamination and involve different DNA repair systems, many of which involve error-prone polymerases. We have studied the function of one of these, Polymerase ?, by establishing mouse fibroblast cell lines deficient of the Rev3 subunit of Pol ?. Rev 3 deficient cells are more sensitive to cell cycle arrest caused by UV-radiation or cisplatin treatment than cells with a functional Pol ?, confirming a function of Pol ? in the translesion synthesis over DNA nicks and crosslinking lesions.
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