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W C Raschke

Publications and source records attributed to W C Raschke.

At least 19 recordsLinked to original sources
PubMed1992

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.

Amino Acid Sequence↗
PubMed1991

Analysis of Ly5 chromosome 1 position using allelic differences and recombinant inbred mice.

Differences between the mouse Ly5a and Ly5b alleles can be distinguished on the basis of polymerase chain reaction (PCR)-restriction enzyme analysis and differential monoclonal antibody reactivities. To more precisely map the Ly5 gene on the mouse chromosome 1, analytical DNA and protein tests were performed on recombinant inbred strains of mice prepared from SJL/J (Ly5a) and BALB/cke (Ly5b) progenitor strains. Each recombinant inbred strain was characterized to determine whether it carried the Ly5a or Ly5b allele. Both assays, DNA-PCR and protein-immunofluorescence, yielded identical results for each strain examined. Placement of the Ly5 gene with respect to other characterized markers of mouse chromosome 1 for these recombinant inbred mouse strains shows a gene order of Idh-1:Ity:Pep3:[Ly5, Cfh].

Alleles↗
PubMed1991

Comparison of mouse Ly5a and Ly5b leucocyte common antigen alleles.

The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains. To investigate the divergence between alleles, cDNA clones to the SJA (Ly5a) LCA gene have been isolated and sequenced. A comparison of this information to the Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These base substitutions correspond to five amino-acid changes within the extracellular domain of the LCA molecule. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat LCA sequences. Thus, these two mouse LCA alleles exhibit a pattern of sequence conservation that mimics that found over a much broader scale of evolution. Analysis of antigenicity profiles for each of the allelic sequence changes reveals three molecular domains of altered antigenicity that could account for observed serological differences between the two alleles. Sequence information from the 5' end of the Ly5a LCA gene, generated using polymerase chain-reaction techniques on genomic DNA, reveals eight additional nucleotide differences between the Ly5a and Ly5b alleles.

Alleles↗
PubMed1988

Expression of oncogenes in normal and transformed murine B lymphocytes.

Proliferating, lipopolysaccharide-stimulated murine B lymphoblasts and a number of transformed murine B cell lines representing various differentiation stages of B cell lineage all express the myc, H-ras and K-ras oncogenes. N-ras transcripts are also present in the B cell blasts and some of the cell lines. In addition, abl, fms, fos, myb, src, and yes are transcribed in some or all of the cell lines but not in the normal B cell blasts. Only myb is expressed in a differentiation stage-specific manner; transcripts are present in the pre-B cell lines and a few of the B lymphomas but not in any plasmacytomas tested. The erbB, fes, mos, and sis probes do not hybridize with mRNA from any of the 15 cell lines or normal B cells. ErbA is not detectable in transformed cells but is expressed in the normal B cell blasts at a low level suggesting its possible involvement in normal growth regulation.

Animals↗
PubMed1987

Cloned murine T200 (Ly-5) cDNA reveals multiple transcripts within B- and T-lymphocyte lineages.

The murine T200 family of cell-surface glycoproteins is expressed on hematopoietic lineage cell types in a developmentally regulated manner with different lymphocyte subpopulations expressing cross-reactive but structurally distinct forms. To investigate the differences between these forms and the regulation of these differences, murine T200 cDNA clones were isolated by using a probe obtained by subtractive hybridization. This procedure made use of a T200+ L-cell transfectant and the parent Ltk- cell line. The 1.9-kilobase (kb) cDNA clone was sequenced and found to contain the coding region of the COOH-terminal 331 amino acids and 0.9 kb of 3' untranslated region. Where the sequence overlapped with the rat sequence, 80-90% homology was observed. RNA blot analysis revealed that B-lineage cell lines express either a 5.6-kb or a 6.5-kb mRNA correlating to the size difference of the T200 glycoprotein synthesized. Similarly, in the T-cell lineage a helper T-cell clone and a cytotoxic T-cell clone express T200 transcripts of 5.6 kb and 5.9 kb, respectively, which correlate with the distinct sizes of their T200 glycoproteins. A T200-negative mutant of a T-cell line was found to express full-length T200 mRNA, although at a diminished level.

Amino Acid Sequence↗
PubMed1986

Two types of immunoglobulin-negative Abelson murine leukemia virus-transformed cells: implications for B-lymphocyte differentiation.

Both alleles of immunoglobulin (Ig) heavy-chain joining region (JH) genes in three Ig-negative Abelson murine leukemia virus (Ab-MuLV)-transformed cell lines were characterized by DNA cloning and nucleotide sequence determination. These studies unambiguously identified two distinct types of Ig-negative B-lineage cells. The first type of cell (e.g., R8) is an "immature pre-B cell," and it contains at least one intermediate recombinant structure containing heavy-chain diversity (DH) and JH sequences but no variable region (VH) sequence. This type of cell, which has also been characterized by other investigators, generates mu-positive sublines during subsequent culturing of cells and represents a precursor stage to pre-B cells. The second type of cell (e.g., RAW253) is an "abortive pre-B cell," in that both JH alleles contain nonfunctional VH-DH-JH structures. The nucleotide sequence determinations in this study demonstrated that these nonfunctional V-D-J structures were generated by nonproductive somatic recombinations, involving either out-of-phase joining events, or the formation of termination codons in the DH coding sequences. The identification of abortive pre-B cells suggests that the recombinational joining of Ig VH, DH, and JH segments is not actively regulated by a putative recombinase to preserve the translational reading frame. This in turn implies that a large portion of precursor cells at the early stage of B-cell differentiation are abortive and possibly blocked to further differentiation.

Abelson murine leukemia virus↗
PubMed1985

Stable expression of the mouse lymphocyte T200 antigen in L-cells after transfection with lymphoma DNA.

L-cells, which normally do not express the mouse lymphocyte T200 antigen, were transfected with DNA from the mouse T-cell lymphoma, BW5147, and a T200+ L-cell line isolated. The detection and enrichment of T200+ L-cells from a pool of transfectants was accomplished using monoclonal anti-T200 antibody and fluorescence-activated cell sorting. A subclone has been selected that is stable for expression of T200. The T200 molecules expressed by BW5147 and the T200+ L-cell are similar in size, around 190 Kd, compared to the 220-Kd B-cell form of T200. The BW5147 T200 molecule is 3000 daltons larger than the molecule expressed by the transfected L-cell, a size difference due to glycosylation moieties, since treatment of the molecules with Endoglycosidase F or treatment of cells with tunicamycin yields T200 molecules of the same size from the 2 cell sources. In comparison, the B-cell form of T200 retains a size difference of 25,000 daltons from the T-cell form after these treatments. Monoclonal antibodies specific for the B-cell form of T200 do not recognize the T200+ L-cell, providing further evidence that the T-cell form of T200 is expressed by the transfected L-cell.

Animals↗
PubMed1981

Evidence that the Abelson virus protein functions in vivo as a protein kinase that phosphorylates tyrosine.

Both lymphocytes and fibroblasts that have been transformed by ABelson murine leukemia virus contain 6- to 12-fold increased levels of the rare modified amino acid phosphotyrosine in their proteins. This observation, coupled with the fact that the p120 protein encoded by this virus has been shown to undergo an apparent autophosphorylation to yield phosphotyrosine in vitro, suggests that Abelson virus encodes a protein kinase that phosphorylates tyrosine in transformed cells. These results are similar to those obtained previously with Rous sarcoma virus and suggest, by analogy, that the modification of cellular polypeptides through the phosphorylation of tyrosine may be involved in cellular transformation by Abelson virus. p120 isolated from transformed cells contains phosphoserine, phosphothreonine, and phosphotyrosine. The phosphotyrosine is found at two sites in the protein. p120 therefore may be a protein kinase that undergoes autophosphorylation in vivo.

Abelson murine leukemia virus↗
PubMed1980

Transformation by Abelson murine leukemia virus: properties of the transformed cells.

Ab-MuLV transforms mouse 3T3 fibroblasts, macrophages, and lymphocytes, which have properties of immature cells in the B-lymphocyte differentiation pathway. Differences in expression of intracellular immunoglobulin and surface antigens the lymphoma lines suggest that more than one B-lymphocyte differentiation stage may be transformed, each of which must be before the B lymphocyte acquires cell-surface immunoglobulin receptors for antigen and antigen sensitivity. The plasmacytomas induced by Ab-MuLV plus pristane tested thus far show no evidence of infection by Ab-MuLV. The transformation of these immunoglobulin-secreting cells may be due to causes other than the transforming elements of the Ab-MuLV genome.

Abelson murine leukemia virus↗
PubMed1979

Assembly and secretion of pentameric IgM in a fusion between a nonsecreting B cell lymphoma and an IgG-secreting plasmacytoma.

A new immunoglobulin product has been obtained by hybridization of mouse cell lines arrested at different stages in B lymphocyte development. One line was shown to have the characteristics of an undifferentiated B cell that synthesizes monomeric IgM as a membrane receptor but does not express J chain. The second line was represent of a fully differentiated plasma cell synthesizing large amounts of IgG and J chain, but no IgM. Fusion of the two cell types yielded independent hybrid clones that secreted pentameric IgM, normally the first product of antigen-driven B cell differentiation. Analyses of the hybrid cells indicated that the IgM was expressed as a result of complementation between the synthetic capacities of the parental lines. The hybrid cells synthesized both monomeric IgM and J chain and assembled these components into a pentameric molecule with the expected stoichiometry of one J chain per five monomeric units. These findings provided further evidence that the induction of B cell differentiation includes a signal for de novo synthesis of the J chain. Moreover, the complementation achieved by this hybridization provides a system for identifying other intracellular events in B cell differentiation to IgM secretion.

Animals↗
PubMed1979

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.

Alleles↗
PubMed1975

Oncogenic transformation of murine lymphoid cells by in vitro infection with Abelson leukemia virus.

Spleen cell cultures stimulated to DNA synthesis by antigen or mitogen were infected with Abelson virus, a C-type RNA virus inducing nonthymic lymphomas in mice. After 3 days the cells were transferred to mice and caused 100 percent incidence of lymphomas in as few as 29 days. That a number of the tumors were of donor origin, as shown by female karyotypes in recipient male mice, indicated that cells infected by virus in vitro were transformed. The process depended upon both virus and stimulation of lymphocytes in culture. Lymphoid tumors did not develop in mice receiving cells from virus-infected cultures not exposed to antigen or mitogen.

Animals↗