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M D Scharff

Publications and source records attributed to M D Scharff.

At least 127 records · Page 7Linked to original sources
PubMed1979

Site of binding of mouse IgG2b to the Fc receptor on mouse macrophages.

Three mouse immunoglobulins with altered heavy chains have been used to study the specificity of the mouse IgG2b Fc receptor on mouse macrophages. These immunoglobulins were synthesized by variant clones derived from the MPC 11, IgG2b-producing mouse myeloma cell line. One variant, whose Fc receptor. A second variant, which makes a short heavy chain lacking the CH3 domain, binds specifically to the IgG2b Fc receptor. The third variant makes a hybrid IgG2b-IgG2a heavy chain whose CH3 domain is enterely IgG2a-like and binds to both IgG2a and IgG2b Fc receptors. These data suggest that the binding of mouse IgG2b immunoglobulins to the mouse macrophage Fc receptor involves a site within the CH2 domain and indicate that immunoglobulins with altered heavy chains are a useful tool to probe Fc receptors.

Animals↗
PubMed1979

A mouse myeloma variant with a defect in light chain synthesis.

A spontaneous assembly variant, B 50, has been isolated from the MPC-11 mouse myeloma cell line. This variant synthesizes fewer light chains per cell than the parent resulting the production of a slight molar excess of heavy chains. These changes are associated with a delay and change in the pathway of assembly and a delay in secretion. Spontaneous revertants of B 50 have been obtained, all of which synthesize normal amounts of light chains and assemble and secrete the immunoglobulin molecule through the same pathways and with the same kinetics as the parental cells. A comparison of the tryptic-chymotryptic peptides of the parental, variant and revertant heavy and light chains did not reveal any differences. These studies indicate that variants in mouse myeloma cells can arise with defects in the quantitative expression of the immunoglobulin gene and suggest that the presence of excess light chains facilitates the assembly and secretion of some immunoglobulin molecules.

Animals↗
PubMed1978

IgG2a-producing variants of an IgG2b-producing mouse myeloma cell line.

12 variant cell lines producing an IgG2a (kappa) immunoglobulin derived via different routes from the IgG2b (kappa) synthesizing MPC 11 were studied. These variants all have the parental MPC 11 idiotype as shown by a radioimmunoassay. A comparison of the variants by charge, peptide maps, and assembly patterns has shown that most of them differ from one another, and some can be grouped. One group consists of three primary variants generated with two mutagenic agents: these three have almost indistinguishable peptide maps. Two other primary variants which arose in a similar fashion differ markedly from each other and from this group. A second group is comprised of the four secondary variants which arose from two short heavy chain producing primary variants. Other secondary variants and the one spontaneously arising variant cannot be grouped. Possible genetic mechanisms such as translocation, expression of previously silent genes and recombination are discussed.

Cell Line↗
PubMed1978

Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: identification of tumor origin by idiotypic analysis.

Multiple lymphoblastoid cell lines have been derived from two patients with chronic lymphocytic leukemia with an associated monoclonal immunoglobulin (Ig) band. Idiotypic antisera raised against the monoclonal serum Ig bands were shown to be specific for the membrane Ig of the patients' leukemic cells. The idiotypic determinants in these patients thereby constitute tumor-specific antigens. Surface and intracellular immunofluorescence studies utilizing these idiotypic antisera were used to identify the cell lines of leukemic origin. These studies showed that certain cell lines from each patient were derived from the leukemic cells while other cell lines were derived from residual normal B lymphocytes. The leukemic cell lines were variable and contained different percentages of lymphoid cells with the idiotype-specific membrane Ig and, in addition, different percentages of plasma cells with intracellular Ig of the same specificity. Specific Ig synthesis was also demonstrated by hemagglutination-inhibition analysis of cell line supernatants. Aside from Ig specificity, no differences have been found between the leukemic cell lines and those derived from normal cells. One of the leukemic cell lines was cloned in soft agarose. All the clones were shown to be of leukemic origin.

Aged↗
PubMed1977

Abnormalities in the glycosylation of immunoglobulin heavy chain and an h-2 transplantation antigen in a mouse myeloma mutant.

Two mutant cell lines derived from the MPC-11 mouse myeloma synthesize immunoglobulin with abnormal heavy chains and normal light chains. The defective heavy chains have molecular weights of 38,000-42,000 (M3.11) and 50,000 daltons (ICR 11.19) as compared to 55,000 daltons of the wild-type. The glycosylation of the defective heavy chains demostrated several unusual features: first, 30-50% of the M3.11 heavy chain contained no carbonydrate, while 100% of the wildtype and ICR 11.19 heavy chains were glycosylated; second, the glycopeptides of the M3.11 heavy chains revealed an altered gel filtration pattern when compared with the wild-type; and third, digestion with an endoglycosidase indicated that the heterogeneity of the wild-type and M3.11 glycopeptides involved structural changes in the core region of the oligosaccharide. Examination of two other glycoproteins (the major histocompatibility complex antigens) in these cell lines showed that in M3.11, the H-2D but not the H-2K product was abnormally glycosylated and contained a smaller glycopeptide. However, in a subclone of M3.11 that had lost the ability to produce immunoglobulin heavy chains, the H-2D glycopeptide had returned to wild-type size. We concluded from these studies that the defective M3.11 immunoglobulin heavy chain interfered both with its own glycosylation and the glycosylation of another protein, H-2D.

Animals↗
PubMed1977

Antigen-binding mutants of mouse myeloma cells.

A cultured mouse myeloma cell line, S107, that secretes an IgA phosphocholine-binding immunoglobulin has been cloned in soft agar and overlaid with phosphocholine-hemocyanin. Spontaneous mutants that secrete immunoglobulin with a decreased ability to precipitate antigen were detected with this plate assay and occur at a very high frequency. From one such mutant, phenotypic revertants arise spontaneously with a frequency of 0.28-2.8%. This mutant and one of its revertants were studied, and they were found to differ from the parent and from each other serologically and in antigen binding. While it is not yet clear whether these findings bear any relationship to the normal generation of antibody diversity, they do indicate that it is possible to generate antigen binding diversity in somatic cells.

Antibody Specificity↗
PubMed1976

Synthesis of immunoglobulin by substrate attached mouse myeloma cells.

Cultured mouse myeloma cells grow in suspension and synthesize and secrete large amounts of immunoglobulin. Mouse myeloma cells which attach to a plastic substratum have been obtained by mutagenesis and subsequent selection. Normal mouse myeloma cells will also attach to plastic tissue culture dishes pre-treated with poly-L-lysine. The attached cells synthesize and secrete the same large amounts of immunoglobulin as the suspended cells.

Acridines↗
PubMed1976

Somatic cell hybridization of mouse myeloma cells.

Somatic cell hybrides between different mouse myeloma cell lines have been readili isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antifen expression. Three hybrid cell lines are described here in detail: an IgG2B, K X LgG2a, k; an IgG1, k X IgG2b, k; and an IgG1, k X IgM, Lambda. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.

Cell Fusion↗
PubMed1976

Regulatory variants for the expression of H-2 antigens. I. Isolation and characterization.

By subjecting "mutagenized" mouse myeloma cells repeatedly to selection with antiserum against H-2Kd, we isolated variants that expressed little or no H-2Kd alloantigens. The phenotype was unstable, with the culture accumulating revertants in the absence of selective pressure. The variants not only failed to express the selected antigen, but also the antigen coded by the closely liked H-2Dd gene. The cells reexpressed this antigen with the same kinetics as for the selected antigen. Several physiologic parameters (karyotype, cell morphology, size, and growth curves) were not altered by the loss of H-2 antigens.However,when injected iv, the H-2-deficient cells did not lodge in the spleen as readily as did the wild-type cells.

Animals↗
PubMed1976

Mutations in mouse myeloma cells: implications for human multiple myeloma and the production of immunoglobulins.

Multiple myeloma raises a number of puzzling questions about the production of immunoglobulins and the malignant transformation of lymphoid cells. Some of these questions can be approached by studying mouse plasmacytomas and by genetic and biochemical studies of mouse myeloma cells in culture. The synthesis, assembly, glycosylation, and secretion of immunoglobulin has been analyzed in detail using the mouse myeloma system. The development of a technique that detects variants in clones of cultured mouse myeloma cells has led to the demonstration of a unique genetic instability in these cells. Based on these results a model is presented to explain the high frequency of ligh-chain producing (Bence Jones) myelomas in patients. Finally, mutant cell lines have been recovered which produce defective immunoglobulins similar to those found in heavy-chain disease and some other lymphoproliferative disorders.

Amino Acid Sequence↗
PubMed1976

Immunoglobulin biosynthesis by the MOPC 173 mouse myeloma tumor and a variant spleen clone.

The IgG2a producing MOPC 173 tumor synthesizes heavy (H) and light (L) chains and assembles them into H2L2 utilizing heavy chain dimers (H2) and H2L as the major precursors. Although the tumor cells secrete only H2L2, they synthesize excess L chains which appear to be degraded and are not secreted. A L chain-producing variant arising spontaneously from the MOPC 173 tumor also failed to secrete any L chains. MOPC 173 tumor cells were cloned in the spleens of normal BALB/c mice and 1 of 23 spleen clones appeared to differ from the parent tumor in demonstrating a) a transient block in the assembly of H2L2 at the H2 level, leading to delayed formation and secretion of H2L2, b) appreciable amounts of non-covalently bonded H2L, c) an abnormal intracellular immunoglobulin species, H4, d) production of equimolar amounts of H and L chains. The patterns of immunoglobulin synthesis and assembly demonstrable in the above studies were also observed when tumor cells were studied in situ. Tryptic peptide mapping of the H and L chains produced by the MOPC 173 tumor and the variant spleen clone failed to demonstrate any differences in the H chains, but there were definite chemical differences in the L chains. These studies indicate that variant myeloma cells producing structurally altered immunoglobulins may continually be arising in myeloma tumors.

Animals↗
PubMed1975

Characterization of light chain and light chain constant region fragment mRNAs in MPC 11 mouse myeloma cells and variants.

Cultured MPC 11 mouse myeloma cells synthesize not only gamma2b heavy and kappa light chains but also a carboxyl terminal (constant region) fragment of kappa light chain. In vitro translational analysis of total cytoplasmic and microsomal RNA indicates that these cells contain RNA which directs synthesis of both a light chain precursor and a light chain fragment precursor. Variant clones which do not synthesize either heavy or light chains continue to synthesize the light chain fragment. One such "nonproducing" variant was studied in detail. It does not contain translatable mRNA for the intact light chain but does contain RNA which is translated into the light chain fragment precursor. Nucleic acid hybridization analysis with a cDNA probe specific for the constant region of kappa light chains revealed that microsomal RNA from the wild-type cell contains both 14S and a 10S species of kappa specific RNA, whereas the variant contains only the 10S species. Translational analysis of these same RNAs indicates that the 14S species codes for the light chain precursor, while the 10S RNA codes for the light chain fragment precursor.

Animals↗