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- Angerth, Tomas, et al.
(författare)
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Cloning and structural analysis of a gene encoding a mouse mastocytoma proteoglycan core protein : Analysis of its evolutionary relation to three cross hybridizing regions in the mouse genome
- 1990
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 93:2, s. 235-240
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Tidskriftsartikel (refereegranskat)abstract
- Serglycin (SGC) is a Ser-Gly-repeat-containing protein, used as proteoglycan core protein in the parietal yolk sac and in mast cell, where glycosaminoglycan side chains are attached to the serine residues of the repeat region. In this article, the structure of the gene SGC encoding mouse SGC is reported. The gene is divivided into three exons, which are all contained within a region of approximately 13 kb. Nucleotide (nt) sequence analysis was carried out on a region of 1.2 kb upstream from the first exon. The region containing the two promoters (active in parietal yolk sac and in mast cells, respectively) was analyzed for the presence of recognition sites for known DNA-binding proteins. A number of sequence closely related to known recognition sites were found in both promoters, and one consensus octamer-binding site could be identified in the putative yolk-sac promoter. Multiple regions in the mouse genome hybridizing with DNA fragments covering the Ser-Gly repeat region have previously been described, and it has been sugggested that these loci may represent other proteoglycan core proteins. Analysis of nt sequence was carried out on three out of the more than 15 of three regions present in the mouse genome. However, none of the clones analyzed was found to have any open reading frame in the region of cross-hybridization which possibly could code for a SGC protein. Instead, one of the clones was found to contain an exon encoding a highly basic protein, unrelated to SGC protein. Instead, one of the clones was found to contain an exon a highly basic protein, unrelated to SGC. Hence, no evidence ws found for a multigene family of Ser-Gly-repeat-containing proteoglycan-encoding genes.
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| 2. |
- Blom, Thomas, et al.
(författare)
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Phenotypic characterization of KU812, a cell line identified as an immature human basophilic leukocyte
- 1992
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 22:8, s. 2025-2032
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Tidskriftsartikel (refereegranskat)abstract
- The knowledge about the differentiation of basophilic leukocytes is fragmentary. This report discusses a detailed phenotypic characterization of molecular markers for hematopoietic differentiation in a basophilic leukemia cell line, KU812. The expression of markers for lymphoid, erythroid, neutrophil, eosinophil, monocytic, megakaryocytic, mast cell and basophil differentiation was analyzed at the mRNA level by Northern blots in the KU812 cells, and for reference, in a panel of human cell lines representative of the different hematopoietic differentiation lineages. KU812 was found to express a number of mast cell and basophil-related proteins, i.e. mast cell tryptase, mast cell carboxypeptidase A, high-affinity immunoglobulin (IgE) receptor α and γ chains and the core protein for heparin and chondroitin sulphate synthesis. We found no expression of a number of monocyte/-macrophage or neutrophil leukocyte markers except for lysozyme. From earlier studies, it has been shown that lysozyme is not expressed in murine mucosal mast cell lines. This finding, together with the expression of the mast cell carboxypeptidase in KU812 might distinguish the phenotype of this cell line from that typical of mucosal mast cell lines in rodents. We found a low level of expression of the eosinophil and basophil marker, major basic protein, which might indicate a relationship between basophils and eosinophils. No expression is, however, detected with the eosinophil-specific markers, eosinophil cationic protein, eosinophil-derived neurotoxin or eosinophil peroxidase. We also report an extensive screening for inducers of basophilic differentiation of the KU812 cells. The most efficient protocol of induction included serum starvation which led to a dramatic increase in a number of markers specific for mast cells and basophils such as tryptase, carboxypeptidase A and the heparin core protein. Finally, diisopropylfluorophosphate analysis of total protein extracts from KU812 show four labeled protein bands with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that this cell line expresses at least three previously undescribed serine proteases of which one or more could be a potential basophil-specific marker(s).
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| 3. |
- Huang, Ranyang, et al.
(författare)
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Expression of a Mast Cell Tryptase in the Human Monocytic Cell Lines U-937 and Mono Mac 6
- 1993
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 38:4, s. 359-367
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Tidskriftsartikel (refereegranskat)abstract
- Expression of a mast cell tryptase mRNA was detected in two human monocytic cell lines, the U-937 and the Mono Mac 6, and in normal human peripheral blood(PB) monocytes. In the U-937 cell line but not in normal PB monocytes, the tryptase expression was upregulated 3-50 fold following phorbol ester (PMA)-induced differentiation, but no such induction was seen after retinoic acid, interferon-gamma or vitamin D3 exposure. The tryptases expressed in PMA-induced and non-induced U-937 and in Mono Mac 6 were characterized by PCR amplification and nucleotide sequence analysis. The U-937 cell line was found to express a tryptase identical to one of the previously cloned mast-cell beta tryptases (Tryptase I), and the tryptase expressed in Mono Mac 6 was found to be nearly identical to the previously cloned alpha tryptase. By northern blot analysis with oligonucleotide probes specific for the alpha and beta tryptases both cell lines were found to express only one type of tryptase. Densitometric quantifications of tryptase mRNA levels, in the two cell lines, showed approximately 80 times higher mRNA levels in Mono Mac 6 compared to non-induced U-937. Immunohistochemical staining for tryptase showed a marked heterogeneity in the Mono Mac 6 cell line. Only one out of 10 cells were positive for the protein but the levels in these cells were very high, equivalent, or even higher than the levels seen in the human mast cell line HMC-1. This shows that the expression of a single tryptase, in this case the alpha tryptase, is sufficient for the production of a stable protein and probably also a stable proteolytically active tetramer. The family of human mast-cell tryptases has been considered to represent a class of proteases specifically expressed in mast cells and basophilic leucocytes. The expression of tryptases in two monocytic cell lines and in normal PB monocytes indicate that in humans, the lineage specificity of these serine proteases is less restricted than earlier expected. The cloning of a full length cDNA for the murine counterpart to the human mast cell tryptases, the MMCP-6, is presented. No expression of the MMCP-6 was detected in a panel of mouse monocyte or macrophage cell lines indicating a species difference in the lineage specificity of the 'mast cell tryptases'.
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