| 1. |
- Aveskogh, Maria, et al.
(author)
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A single major transcript encodes the membrane-bound form of rat immunoglobulin E
- 1995
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In: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 42:5, s. 535-539
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Journal article (peer-reviewed)abstract
- The primary structure of the membrane-bound form of rat immunoglobulin E was determined by PCR amplification and nucleotide sequence analysis of its mRNA. The sequence was found to correspond to the previously identified membrane exons of the rat epsilon chain gene. The donor splice site in the C4 exon was mapped to a position 35 nt upstream of the stop codon for the secreted form of rat IgE. Therefore, the membrane-bound IgE lacks the 12 C-terminal amino acids present in the secreted form of the protein. Recently, five novel epsilon chain transcripts were isolated from human IgE producing B-cells or B-cell lines. Four of these transcripts encode proteins which differ in their C-terminal ends from the classical membrane or secreted forms of human IgE. To investigate if these transcripts were likely to represent functional mRNAs, their evolutionary conservation was studied by screening a rat IgE producing B-cell line for the expression of similar transcripts. By PCR amplification and cloning of transcripts, containing both the C3 and the M2 exons, approximately 10,000 independent cDNA clones were obtained. These clones were screened with probes directed against regions specific for each of the five novel human epsilon chain mRNAs. However, no evidence was found for the presence of transcripts with a similar structure, indicating that no specific function associated with these transcripts and their corresponding proteins has been conserved between human and rat. The lack of additional M2-containing transcripts in the rat suggest that the novel human IgE transcripts are byproducts of differential splicing and that they most likely encode proteins with no evolutionarily important function.
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| 2. |
- Aveskogh, Maria, et al.
(author)
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Characterization of cDNA clones encoding mouse proteinase 3 (myeloblastine) and cathepsin G
- 1997
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In: Immunogenetics. - : Springer Science and Business Media LLC. - 0093-7711 .- 1432-1211. ; 46:3, s. 181-191
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Journal article (peer-reviewed)abstract
- Serine proteases are the most abundant granule constituents of several major hematopoietic cell lineages. Due to their high abundance and their strict tissue specificity they have become important phenotypic cell markers used for studies of various aspects of hematopietic cell development. Using a polymerase chain reaction (PCR)-based strategy for the isolation of trypsin-related serine proteases, we were able to isolate cDNAs for two of the major neutrophil and monocyte serine proteases in the mouse, cathepsin G and mouse protease 3 (myeloblastin). The internal PCR fragments were used as probes to screen a mouse mast cell cDNA library and a cDNA library originating from a mouse monocytic cell line (WEHI-274.1). Full-length cDNAs for mouse cathepsin G and proteinase 3 were isolated and their complete sequences were determined. Northern blot analysis revealed expression of cathepsin G in immature cells of the monocyte macrophage lineage but also in the connective tissue mast cell line MTC. Proteinase 3 was expressed in several cell lines of myelo-monocytic origin and in one B-cell line, but not in any of the other cell lines tested. The isolation of cDNAs for mouse cathepsin G and mouse proteinase 3, together with the previous characterization of the gene for mouse N-elastase, and the entire or partial amino acid sequences for porcine azurocidine, equine N-elastase and proteinase 3, rat, dog, and rabbit cathepsin Gs in evolutionary relatively distantly related mammalian species, indicates that these four members of the serine protease family have been maintained for more than 100 million years of mammalian evolution. This latter finding indicates a strong evolutionary pressure to maintain specific immune functions associated with these neutrophil and monocyte proteases. All amino acid positions of major importance for the cleavage site selection have also been fully conserved between mouse and human proteinase 3 and a few minor changes have occurred between mouse and human cathepsin G.
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| 3. |
- Aveskogh, Maria, et al.
(author)
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Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica
- 1999
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In: Developmental and Comparative Immunology. - 0145-305X .- 1879-0089. ; 23:7-8, s. 597-606
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Journal article (peer-reviewed)abstract
- To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE (ε chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.
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| 4. |
- Aveskogh, Maria, 1956-, et al.
(author)
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Evidence for an early appearence ofmodern post switch isotypes in mammalian evolution : Cloning of IgE, IgGand IgA from the marsupial Monodelphis domestica
- 1998
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In: European Journal of Immunology. - 0014-2980 .- 1521-4141. ; 28:9, s. 2738-2750
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Journal article (peer-reviewed)abstract
- In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.
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| 5. |
- Lützelschwab, Claudia, et al.
(author)
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Characterization of mouse mast cell protease-8, the first member of a novelsubfamily of mouse mast cell serine proteases, distinct from both theclassical chymases and tryptases
- 1998
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In: European Journal of Immunology. - 0014-2980 .- 1521-4141. ; 28:3, s. 1022-1033
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Journal article (peer-reviewed)abstract
- Using a recently developed PCR-based strategy, a cDNA encoding a novel mouse mast cell (MC) serine protease (MMCP-8) was isolated and characterized. The MMCP-8 mRNA contains an open reading frame of 247 amino acids (aa), divided into a signal sequence of 18 aa followed by a 2-aa activation peptide (Gly-Glu) and a mature protease of 227 aa. The mature protease has an M(r) of 25072, excluding post-translational modifications, a net positive charge of +12 and six potential N-glycosylation sites. MMCP-8 showed a high degree of homology with mouse granzyme B in the critical regions for determining substrate cleavage specificity, indicating that MMCP-8, similar to granzyme B, preferentially cleaves after Asp residues. A comparative analysis of the aa sequence of MMCP-8 with other hematopoietic serine proteases shows that it is more closely related to cathepsin G and T cell granzymes than to the MC chymases. We therefore conclude that MMCP-8 belongs to a novel subfamily of mouse MC proteases distinct from both the classical chymases and tryptases. Southern blot analysis of BALB/c genomic DNA indicated that only one MMCP-8 gene (or MMCP-8 like gene) is present in the mouse genome. Northern blot analysis of rodent hematopoietic cell lines revealed high levels of MMCP-8 mRNA in a mouse connective tissue MC-like tumor line. However, MMCP-8 mRNA could not be detected in mouse liver, intestine, lung or ears, indicating very low expression in normal tissues. Analysis of the expression of different MMCP in the tissues of Schistosoma mansoni-infected BALB/c mice showed a strong increase in MMCP-8 levels in the lungs but not in the intestines of infected animals, suggesting the presence of a novel subpopulation of MC in the lungs that expressed MMCP-8, either alone or in combination with MMCP-5 and carboxypeptidase A. The dramatic increase in MMCP-1 and MMCP-2 levels but not of MMCP-8 in the intestines of parasitized animals also shows that MMCP-8 is not expressed in mucosal MC in the mouse. This latter is in clear contrast to what has been observed in the rat where the MMCP-8 homologues, RMCP-8, -9 and -10, can be considered as true mucosal MC proteases.
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| 6. |
- Lützelschwab, Claudia, et al.
(author)
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Secretory granule proteases in rat mast cells : Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations
- 1997
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In: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 185:1, s. 13-29
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Journal article (peer-reviewed)abstract
- Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP- 1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of monospecific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.
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| 7. |
- Åbrink, Magnus, et al.
(author)
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Isolation of cDNA clones for 42 different Krüppel-related zinc finger proteins expressed in the human monoblast cell line U-937
- 1995
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In: DNA and Cell Biology. - : Mary Ann Liebert Inc. - 1044-5498 .- 1557-7430. ; 14:2, s. 125-136
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Journal article (peer-reviewed)abstract
- To study the complexity and structural characteristics of zinc finger proteins expressed during human hematopoiesis and to isolate novel regulators of blood cell development, a degenerate oligonucleotide probe specific for a consensus zinc finger peptide domain was used to isolate 63 cDNA clones for Krüppel-related zinc finger genes from the human monoblast cell line U-937. By extensive nucleotide sequence and Northern blot analysis, these cDNA clones were found to originate from approximately 42 different genes (HZF1-42) of which only 8 have previously been described. Northern blot analysis showed that a majority of these genes were expressed at comparable levels inU-937 and HeLa cells. The large number of individual genes represented among the 63 clones and their apparent non-cell-type-specific expression suggest that the majority of the Krüppel-related zinc finger genes are likely to be expressed in most human tissues. In contrast, some of the genes displayed are stricted expression pattern,indicating that they represent potential regulators of monocyte differentiation or proliferation. Detailed structural analysis of the first 12 cDNAs (HZF 1-10) and apartial characterization of HZF 11-42 revealed that a common feature of human Krüppel-related zinc finger proteins is the presence of tandem arrays of zinc fingers ranging in number from 3 to over 20 that are preferentially located in the carboxy-terminal regions of the proteins. In addition, several novel KRAB-containing zinc finger genes and a novel conserved sequence element were identified.
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