William C. Raschke

Functional macrophage cell lines transformed by abelson leukemia virus

Abstract Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing and phagocytosis of sheep erythrocyte targets, and secrete high levels of lysozyme. None of these properties was exhibited by the lymphocyte lines. Of the two macrophage cell lines tested, neither was capable of replacing the adherent cell population required for the induction of in vitro immune responses. An agent that activates normal macrophages, bacterial lipopolysaccharide, specifically inhibits the growth of the transformed macrophages in culture. Secretion of infectious Abelson leukemia virus by two of the macrophage lines, RAW 309Cr and WR 19M, provides conclusive evidence that the Abelson virus is capable of productively infecting the macrophage cell type. The other macrophage line, RAW 264, fails to secrete detectable virus particles and is negative in the XC plaque formation assay, as well as the fibroblast transformation assay for Abelson virus, but becomes positive for Abelson virus production after rescue by Moloney leukemia virus.

The immunoglobulin μ chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments

The B lymphocytes synthesizes two forms of IgM molecules during its development from a stem cell to a mature antibody-secreting plasma cell. The monomeric receptor IgM molecule is affixed to the plasma membrane and triggers the later stages of B cell differentiation, whereas the pentameric secreted IgM molecule is an effector of humoral immunity. The structural differences between membrane-bound and secreted IgM molecules are reflected in the differences between their heavy or mu chains. We have previously determined the complete amino acid sequence of a murine secreted mu (microsecond) chain. In this study, we have compared the structures of the secreted and membrane-bound mu (micron) heavy chains by peptide mapping, micro-sequence and carboxypeptidase analyses. These studies demonstrate that the micron and microsecond chains are very similar throughout their VH, C mu 1, C mu 2, C mu 3 and C mu 4 domains. The micron and microsecond chains differ in the amino acid sequence of their C-terminal segments. These studies in conjunction with those carried out on the micron and microsecond mRNAs and the C mu gene suggest that the micron and microsecond chains from a given B cell are identical except for their 41 and 20 residue C-terminal segments, respectively. The amino acid sequence of the 41 residue C membrane terminal segment predicted from the corresponding micron mRNA is in agreement with all the protein studies reported in this paper.

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

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimers amyloid β-protein precursor (AβPP) 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-2AβPP. 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 AβPP (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-2AβPP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2AβPP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2AβPP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2AβPP.

Lymphosarcoma cell growth is selectively inhibited by B lymphocyte mitogens: LPS, dextran sulfate and PPD

Summary A series of murine lymphosarcomas, including those induced by Abelson leukemia virus, were adapted to culture. Their cell growth was inhibited by B lymphocyte mitogens at concentrations which did not affect other hematopoietic cell lines: myelomas, T lymphomas, mastocytoma and erythroleukemia. The pattern of inhibition among different lymphosarcoma lines suggests that they represent tumors of B lymphocytes at different stages of differentiation.

Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid β-protein precursor-like protein-2

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.

Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris.

Pichia pastoris (Pp) and Hansenula polymorpha (Hp) are methylotrophic yeasts commonly used for industrial purposes. Growth of either of these yeasts in the presence of methanol as the carbon source results in high-level induction of alcohol oxidase expression. The respective alcohol oxidase genes, AOX1 in Pp and MOX in Hp, have similar regulatory characteristics. Our studies show that the Pp AOX1 promoter (AOX1p) can be used for methanol-induced expression of a heterologous gene in Hp. Furthermore, the size of an AOX1p-heterologous gene-AOX1 terminator cassette transcript synthesized in Hp is indistinguishable from that synthesized in Pp suggesting that transcription both initiates and terminates at the same sites in both yeast species. Induction of AOX1p in Hp demonstrates that the methanol-inducible regulatory mechanism in Hp is able to recognize and activate the Pp promoter in spite of extensive sequence variations between AOX1p and MOXp.

Growth of mouse plasmacytoma cells in serum-free, hormone-supplemented medium: Procedure for the determination of hormone and growth factor requirements for cell growth

Abstract The procedure of analyzing hormone and growth factor requirements for the growth of MPC-11 cells and of developing a serum-free medium for this cell line has been described. In this medium, MPC-11 cells grow as fast as in serum-supplemented medium, up to 50 generation. MPC-11 cells grown in serum-supplemented medium secrete IgG2b and K light chain into the medium as they do in serum-containing medium.

Monoclonal Antibodies to Human Tumor Antigens

Human tumors are diagnosed and classified by a variety of criteria, one of which is serological analysis of cell surface antigens. In this chapter, a major recent development in serological classification, the production of monoclonal antibodies, will be evaluated.