Complexes of nuclear matrix DNA with proteins tightly bound to DNA contain a specific small-size RNA of a novel type.

Analysis of DNA-protein structures composed of nuclear matrix attached DNA and the most tightly bound proteins was performed. Although the previously described non-histone proteins (1) were present the buoyant density of the complex was the same as that of pure DNA. RNA inaccessible to RNase in 0.4 M NaCl but digestible in low ionic strength buffer was detected. This RNA is not a nascent one. It turned out to be homogeneous and represent a novel type of small nuclear RNA. Partial sequence of this RNA is presented.

Extramedullary plasmacytoma of skeletal muscle. A case report with immunocytochemistry and ultrastructural study.

An uncommon origin of a rare tumor is described in a 60-year-old white man. Extramedullary plasmacytoma was localized in the left biceps muscle. Tumor immunostaining corresponded with immunocharacterization of the atypical midgamma band on electrophoresis or the serum monoclonal M protein of IgG kappa. The latter disappeared 6 months after the operation. The diagnosis was confirmed ultrastructurally.

Testicular plasmacytoma.

The case of a sixty-five-year-old man with multiple myeloma and a testicular plasmacytoma is described. This represents the thirty fourth reported case of testicular plasmacytoma and the first in which immunoperoxidase histochemistry has been used to demonstrate that the testicular plasma cells contain immunoglobulin of the same isotype as the patient's paraprotein. The clinical and morphologic features of previously reported testicular plasmacytoma are reviewed.

Cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study.

Forty-nine cutaneous plasmacytomas in 46 dogs were studied. Tumors occurred at solitary sites in middle-aged to old dogs (mean age, 9.7 years) and most commonly involved the skin of the digits, lips, and ears. Initial diagnosis was made on the basis of light microscopic morphologic findings. Tumors were graded according to the extent of cellular differentiation and immunoreactivity to a panel of immunohistochemical markers (cytokeratins, canine IgG F[ab]2, neurofilament, neuron-specific enolase, S-100 protein, and vimentin). Immunoreactivity was limited to antibodies directed at canine IgG F(ab)2 and vimentin. Vimentin immunoreactivity was usually greater than that of canine IgG F(ab)2, but there was no correlation between immunoreactivity and histologic grade of the tumors. Thirty-six of 39 dogs (92.3%) followed (mean follow-up, 13 months) were cured by surgical excision. The results of this study indicate that canine cutaneous plasmacytomas are benign neoplasms that should be included in the differential diagnosis of cutaneous round cell tumors in dogs.

Induction of vimentin synthesis in a murine myeloma cell line by TPA is strongly dependent on the composition of the cell culture medium.

MPC-11 mouse plasmacytoma cells virtually lacking intermediate filament (IF) proteins can be induced to synthesize and accumulate the IF protein vimentin by treatment with the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Like MPC-11 cells, X63-Ag8.6.5.3 mouse myeloma cells (Ag8) proved to be vimentin-negative, as assayed by immunoblotting of whole cellular protein using goat antiserum to vimentin and [125I]protein A. Vimentin synthesis could be elicited by a TPA concentration as low as 10(-9) M in cells grown in HB-102 serum-free medium. Transfer of these cells to medium containing 15% fetal calf cerum (FCS) greatly reduced the ability of these cells to synthesize vimentin upon TPA treatment. After 50 generations of culture in the presence of FCS, induction of vimentin synthesis was barely detectable even at a TPA concentration of 10(-6) M. Addition of FCS to cells grown in serum-free medium partially suppressed vimentin induction by TPA. This suppression seems to be due, at least in part, to nondialyzable, heat-sensitive components of FCS, since the dialyzable fraction even enhanced vimentin induction by TPA. When cells grown in the presence of FCS were transferred back to serum-free medium, their ability to synthesize vimentin in response to TPA treatment was readily restored. The individual components of serum-free medium which proved to support vimentin induction by TPA were insulin and the unsaturated fatty acids oleic acid and linoleic acid. An even stronger TPA response could be elicited by a combination of these components.(ABSTRACT TRUNCATED AT 250 WORDS)

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors.

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.

Identification of a novel 9-kDa polypeptide from nuclear extracts. DNA binding properties, primary structure, and in vitro expression.

Using a modified DNA mobility shift assay, we have identified and purified a novel 9-kDa polypeptide (designated p9) from plasmacytoma nuclear extracts which forms salt-stable DNA-protein complexes without apparent sequence specificity. In competition studies, p9 bound exclusively with naturally occurring DNA relative to RNA and preferentially with polydeoxypyrimidines among 10 homopolynucleotides tested. Nuclear extracts prepared from various murine and human cell lines contained a common factor which, when cross-linked with photoreactive [32P]DNA, co-migrated with covalent [32P]DNA-p9 complexes on polyacrylamide gels. An oligonucleotide, constructed on the basis of the N-terminal 29 amino acid residues of p9, was employed to isolate a 700-base pair cDNA clone encoding the complete polypeptide. On Northern blots, p9 cDNA hybridized with a mRNA species of comparable size from both mouse and human cell lines, suggesting a significant degree of interspecies sequence conservation. Amino acid and cDNA sequence analyses demonstrated that p9 derives from the 77-residue C-terminal domain of a 14-kDa polypeptide comprised of 127 amino acids. DNA binding activity was exhibited by peptides synthesized in vitro from run-off RNA transcripts corresponding to the truncated 9-kDa C-terminal domain, but not to the 14-kDa precursor, implicating proteolysis as a post-translational mechanism required for functional activation of p9.

Complex protein binding within the mouse immunoglobulin heavy-chain enhancer.

We have begun to purify and characterize several proteins which bind to the mouse immunoglobulin heavy-chain enhancer to understand the molecular interactions important for enhancer activity. Three proteins which bind to different sites on the immunoglobulin heavy-chain enhancer have been chromatographically separated and partially purified. One protein binds a site which has not been reported previously and does not bind to other reported protein-binding sites on the immunoglobulin heavy-chain enhancer. Binding-site boundaries for the three partially purified proteins have been precisely mapped by methylation interference, DNase I footprinting, and orthophenanthroline/copper chemical nuclease footprinting. We have also characterized these three proteins with respect to dissociation rate constants.

Parental legacy determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting.

We have created a transgenic mouse strain in which an autosomal transgene bearing elements of the RSV LTR and a translocated c-myc gene obeys very unusual rules. If the transgene is inherited from the male parent, it is expressed in the heart and no other tissue. If it is inherited from the female parent, it is not expressed at all. This pattern of expression correlates precisely with a parentally imprinted methylation state evident in all tissues. Methylation of the transgene is acquired by its passage through the female parent and eliminated during gametogenesis in the male. These observations provide direct molecular evidence that autosomal gene expression can depend upon the sex of the parent from which the gene is inherited. They also provide a plausible mechanism for understanding parental imprinting that may be relevant to the failure of parthenogenesis in mammals, the apparent non-Mendelian behavior of some autosomal genes, and the role of methylation in gene regulation.

Effect of a zinc-deficient diet on mitochondrial and microsomal lipid composition in TEPC-183 plasmacytoma.

A zinc-deficient diet caused an increase in microsomal membrane phospholipid levels compared to ad libitum controls. Cholesterol levels were found to be decreased 50% compared to either pair-fed or ad libitum controls, resulting in a sharp decline in the cholesterol/phospholipid ratio. No differences were observed in the distribution of phospholipid classes among all three groups, either in mitochondrial or microsomal membrane fractions. Fatty acid analysis of PC and PE revealed a rise in the 18:2 fraction from zinc-deficient mitochondrial and microsomal membrane fractions. Mitochondrial PE and PC from zinc-deficient animals revealed a rise in the 22:6 fatty acid fraction while microsomal PC also revealed a corresponding decrease in 20:4. None of the zinc-deficient preparations differed significantly from either ad libitum or pair-fed controls in the content of long-chain alk-l-enyl ethers. The results of this study point to an effect of a zinc-deficient diet on lipid metabolism in tumor subcellular membranes which may account for the decreased rate of tumor growth observed in zinc-deficient animals.

Immunohistochemical studies with a monoclonal antibody on the distribution of phosphophoryn in predentin and dentin.

A monoclonal antibody was raised against phosphophoryn, a unique noncollagenous phosphoprotein in dentin. Mouse myeloma NS-I cells were fused with spleen cells obtained from BALB/c mice immunized with phosphophoryn from fetal calf tooth germs. Mice inoculated with the hybridoma produced ascites fluid containing the antibody and this reacted only with a band of phosphophoryn transblotted from polyacrylamide gel. Immunohistochemical studies with the antibody showed that phosphophoryn was present in odontoblasts, odontoblastic processes and dentin, but not in the matrix of predentin, and that the phosphophoryn content of the dentin layer was high at and around the predentin-dentin junction and gradually decreased toward the enamel layer. The area corresponding to mantle dentin was not stained with the antibody.

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins.

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.

Isolation, purification, and characterization of a mouse plasmacytoma cell surface glycoprotein involved in the resistance of the tumor cells to immune destruction.

Cells of a subline of the mouse plasmacytoma LPC-1 are resistant to lysis by cytotoxic T-lymphocytes, probably as a result of the blocking of the major histocompatibility gene complex-encoded cell surface antigens by a trypsin-sensitive glycoprotein of approximately 160 kilodaltons. This glycoprotein (gp 160) was extracted from LPC-1 cells with 1.5 M urea and was further purified by ammonium sulfate precipitation and Sephacryl S-300 gel filtration. The gp 160 consists of a single peptide chain rich in sialic acid residues (10% of total molecular weight) and has an acidic isoelectric point. The amino acid composition of gp 160 is compatible with the linkage of carbohydrates (galactose, glucosamine, and sialic acid) to the protein portion. The apparent weak attachment of gp 160 to the cell membrane could explain the finding that LPC-1 cells easily revert from the resistant to the sensitive to the immune lysis phenotype.

Ig RNA expression in normal B cells stimulated with anti-IgM antibody and T cell-derived growth and differentiation factors.

An increase in mRNA encoding the secretory form of the mu heavy (H) chain (mus) and in the ratio of mus mRNA to the mRNA encoding the membrane form of the microH chain (micron) occurs in normal B cells stimulated with anti-IgM and BSF-p1 together with the two B cell differentiation factors B15-TRF and EL-TRF. Stimulation of cells with anti-IgM and BSF-p1 with either B15- or EL-TRF causes no change in mus levels or mus/micron ratios. The requirements for induction of high rate IgM synthesis in normal B cells stimulated with anti-IgM were precisely the same as those required for elevation of mus, mRNA levels and for increase in mus/micron mRNA ratios. A very close correlation also exists between induction of mRNA for J chain and increase in mus mRNA levels. Similarly, the increase in J chain protein concentration and percent of cells with cytoplasmic IgM were correlated to each other and to levels of mus and J chain mRNA. These results indicate that elevation of mus mRNA and mus/micron mRNA ratios occur in normal B cells only upon commitment to IgM synthesis, and reaffirm the close relation between IgM synthesis and the presence of J chain.

[An autopsy case of extramedullary plasmacytoma with systemic dissemination 2 years after the onset of a localized tumor in the nasal cavity].

A 73-year-old male was admitted because of a localized tumor in the nasal cavity, which was suspected to be malignant lymphoma by biopsy specimens. Laboratory findings were within normal limits except for small amounts of M protein (IgG-lambda) in the serum. The patient was successfully treated by radiotherapy, with the disappearance of both the tumor and M protein. Two years after the onset, however, a solid tumor was observed in the soft tissue, followed by rapid systemic dissemination. An increase in M protein was obvious, and the biopsy specimens disclosed plasmacytoma. The patient died three months after admission, and post-mortem examinations revealed a diffuse infiltration of plasmacytes predominantly in the soft tissues.

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion.

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.

Nuclear proteins associated with gene expression in mouse plasmacytoma cell lines.

Nucleoplasmic and non-histone chromatin proteins from two unrelated and four related mouse plasmacytoma cell lines have been analysed by biosynthetic labelling with [35S]-methionine followed by one- and two-dimensional polyacrylamide gel electrophoresis. We have attempted to find a relationship between the patterns of nuclear proteins and gene expression in mutant plasmacytoma cell lines. The majority of nuclear proteins are common to all of the cell lines studied as would be expected if the majority of nuclear proteins are concerned with functions common to all plasma cells. There are, however, both qualitative and quantitative differences in the nuclear protein patterns of mutant and parent cell lines which appear to correlate with differences in gene expression. The turnover of nuclear proteins in two of the cell lines, MOPC 315.40 (IgA producer) and MOPC 315.32 (lambda 2 chain producer) was studied using pulse-chase techniques.