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J C Roder

Publications and source records attributed to J C Roder.

At least 73 records · Page 4Linked to original sources
PubMed1989

Murine lymphocytes with natural killer activity express CTL-derived serine protease genes.

Serine protease genes (C11, B10 and HF) derived from activated cytolytic T lymphocytes have been shown to be important in CTL-mediated cytotoxicity. In this study, we examined the expression of these genes in fresh natural killer (NK) cells from severe combined immunodeficiency (SCID) and athymic nude mice, as well as in T-cell lines with NK activity. All of these serine protease genes were expressed in NK cells freshly isolated from SCID and athymic nude mice. In addition, all lines showed similar strong levels of expression of C11 and B10 genes, but not the HF gene. However, levels of expression in the T-cell lines did not correlate with levels of NK-like cytotoxicity. These results suggest that C11, B10 and HF serine protease genes are necessary but not sufficient for NK-like cytotoxicity.

Animals↗
PubMed1988

Differential splicing of MAG transcripts during CNS and PNS development.

Myelin-associated glycoprotein (MAG) is expressed on the surface of glial cells and is thought to act as a glial-neuronal adhesion molecule during early stages in the myelination process. Sequencing of several cDNA clones predicted the existence of two classes of MAG mRNAs which differ in the presence or absence of a 45 nucleotide insert near the 3' end. These two mRNAs are sufficient to encode the two MAG proteins previously described (p67MAG and p72MAG) and show that they differ only in their carboxyl terminal regions. The results of RNAse protection experiments reported here confirm the existence of two mRNAs for MAG which arise by alternative splicing of exon 12, as shown by Lai et al. Our results show that the p72MAG mRNA is expressed during the time of active myelin formation in the CNS, reaching a peak by post-natal day 22 and thereafter declining to adult levels by day 62. Conversely, p67MAG mRNA is produced as the minor species during myelin formation, but becomes the predominant form in adult brain. Cultures of oligodendrocytes express both forms of MAG. In the PNS, mRNA coding for p67MAG is predominant throughout development, reaching peak levels at day 6-10, whereas p72MAG mRNA is a very minor species. Alternative splicing also occurs at the 5' terminus. One form of mRNA lacking exon 2 from the 5' non-coding region is predominant in PNS, whereas mRNA containing exon 2 predominates in the CNS. Therefore, at least two and possibly four different mRNA species encode MAG. These results confirm the hypothesis that the two forms of MAG (p67 and p72) are generated by alternative splicing and show that each form is differentially regulated during development in the CNS and PNS.

Aging↗
PubMed1988

Myelin-associated glycoprotein, a cell adhesion molecule of oligodendrocytes, is phosphorylated in brain.

Myelin-associated glycoprotein (MAG) has been implicated in the mediation of interactions between oligodendrocytes and neurons during the development of the myelin sheath. Here we show that MAG is phosphorylated in intact myelinating mouse brain primarily at serine residues and to a lesser extent at threonine and tyrosine residues. In vivo, only the larger of the two developmentally regulated MAG isoforms is phosphorylated. MAG can be phosphorylated at tyrosine by the v-fps and v-src protein-tyrosine kinases in vitro and by a kinase endogenous to myelin membrane preparations. MAG phosphorylated in myelin membranes in vitro also contains phosphoserine and phosphothreonine. These observations suggest that phosphorylation of MAG is physiologically significant in regulating oligodendrocyte-neuron interactions.

Animals↗
PubMed1988

Neuronal proteoglycans: biosynthesis and functional interaction with neurons in vitro.

Glycosaminoglycans (GAGs) with electrophoretic mobilities on cellulose acetate similar to heparin (H), heparan sulfate (HS), and chondroitin sulfate (CS) were detected in cell extracts and in conditioned medium of high-density, neuron-enriched cultures labeled with 35SO4. Heparitinase digestion revealed that heparan sulfate proteoglycans (HSPGs) were heterogeneous in charge density and were responsible for neurite outgrowth activity for sensory neurons in conditioned medium. In the presence of beta-D-xyloside, an inhibitor of proteoglycan assembly, there was an increase of released GAGs with the mobility of CS and heavily sulfated HS but a decrease in neurite outgrowth activity on a laminin substrate at times greater than 14 hr. In the presence of beta-D-xyloside or the monoclonal antibody HNK-1 (Leu 7), which recognizes a neuronal cell surface epitope, there was a time-dependent inhibition of process formation that was half-maximal at 7-8 hr and independent of laminin concentration or cell adhesion to the laminin substrate. The kinetics and magnitude of the inhibitory effects of beta-D-xyloside and HNK-1 (Leu 7) were similar, and the influence of HNK-1 (Leu 7) could no longer be observed in the presence of beta-D-xyloside. Pretreatment of the laminin substrate with conditioned medium from high-density neuron cultures resulted in an increased rate of neurite formation compared with untreated laminin. Where the laminin substrate had been pretreated with conditioned medium, maximal inhibition by HNK-1 (Leu 7) was apparent from the earliest times. However, if the conditioned medium had been digested with heparitinase, neither enhanced neurite outgrowth nor the inhibitory influence of HNK-1 (Leu 7) were observed. The present biosynthetic and functional studies suggest that neurons are one source of HSPGs. These data complement and extend earlier studies suggesting a role for HSPGs in neurite formation. The experiments also provide evidence for modulation of laminin by HSPGs which interact with laminin and promote neurite outgrowth that is mediated by a cell surface receptor at or in juxtaposition to the HNK-1 (Leu 7) epitope.

Animals↗
PubMed1987

DNA amplification and metastasis of the human melanoma cell line MeWo.

A positive correlation was found between the presence of amplified DNA in the form of homogeneously staining regions (HSRs), and the formation of spontaneous metastases by the human melanoma cell line MeWo. HSR+ and HSR- MeWo sublines and clones were injected s.c. into BALB/c nude mice. All MeWo lines produced primary tumors that were allowed to grow to a similar size before the animals were sacrificed and examined for the presence of metastatic nodules in the lungs and abdominal cavity. HSR+ lines gave extensive metastases (greater than 100 nodules) in the lungs and/or liver and abdomen in 18 of 19 animals. The HSR- MeWo lines were effectively nonmetastatic, producing metastases in 3 of 20 animals, two of which had only a single metastatic lung nodule each. Evidence for the presence of HSRs in the primary tumors and metastatic nodules was obtained by DNA dot-blot hybridization to a sequence (D15Z1) amplified in the HSRs, flow cytofluorometry for cellular DNA content, and quinacrine staining of metaphase spreads. The HSR+ clones also colonized the lung to a much greater extent than HSR- clones following i.v. injection. In addition, the HSR+ clone had a selective advantage in lung colonization, since i.v. injection of a 50:50 mixture of HSR+ and HSR- clones resulted in extensive metastases populated exclusively by HSR+ cells. The results suggest that DNA sequences amplified in the HSRs of human melanoma MeWo cells may confer enhanced metastatic properties to these cells.

Animals↗
PubMed1987

Enhanced lytic susceptibility of Ha-ras transformants after oncogene induction is specific to activated NK cells.

C3H 10T1/2 mouse fibroblasts were transfected with a plasmid vector composed of EJ, the mutated c-Ha-ras, and a metallothionein promotor that induced amplified ras expression when activated by culture in the presence of zinc. Experiments were conducted to compare the effect of induction on killing by activated natural killer (NK) cells, cytotoxic T lymphocytes, activated macrophages, and antibody plus complement. The only effector that recognized increased ras expression and exhibited high-inducible cytolysis was an activated NK cell. The effectors from spleen were poly I.C. boostable, Lyt-1.1 negative, NK 1.2 positive, and asialo GM1 positive. Spleen cells from T cell-deficient nude mice, but not NK-deficient beige mice, exhibited high levels of killing activity, and experiments with NK cell clones demonstrated that these lines were also highly cytolytic and killed Ha-ras transfectants in parallel to YAC. Transfection of the same fibroblast line with c-myc did not alter the level of activated NK sensitivity. Cold target competition experiments revealed that Ha-ras-transfected and non-transfected 10T1/2 fibroblasts competed equally for lysis of either YAC or Ha-ras transfectants. Rat-1 fibroblasts did not compete, but gained this capacity when transformed with the v-Ki-ras oncogene but not v-fps. These data suggest that Ha-ras acts in target cells at a post-binding step, whereas Ki-ras may affect expression of target-effector binding structures. The findings that activated NK cell lysis may be specifically influenced by ras expression support a role for NK cells in host surveillance against early neoplastic changes.

Animals↗
PubMed1987

Double restriction in NK cell recognition is linked to transmethylation and can be triggered by asparagine-linked oligosaccharides on tumor cells.

The determinants of tumor cell susceptibility to NK cell-mediated cytolysis were analyzed in a two stage model. The binding of tumor cells to NK effectors was measured by target-effector conjugation and cold target competition in 51Cr-release assays, whereas triggering was measured by assaying phospholipid methylation in NK cells stimulated by intact targets. Representative targets could be grouped into three phenotypes based on the data. Those such as YAC 1.2 could bind and trigger NK cells whereas the mutagenized variant, YAC 6.28.8, could bind but was unable to trigger NK cells and therefore resisted lysis. The third phenotype was represented by HL-60 which could neither bind nor trigger NK cells and was therefore completely NK resistant. The oligosaccharide nature of the triggering molecules was demonstrated by showing that purified, high mannose containing, asparagine-linked oligosaccharides from tumor cell targets were potent stimulators of NK transmethylation at submicromolar levels. Tunicamycin pretreatment of target cells inhibited their triggering capacity but not their NK binding function. These results suggest a double restriction in NK specificity involving two independent but sequential stages in recognition represented in binding and triggering by asn-linked oligosaccharides on the tumor cell surface.

Animals↗
PubMed1986

An expanded population of natural killer cells in mice with severe combined immunodeficiency (SCID) lack rearrangement and expression of T cell receptor genes.

Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, whereas myelopoiesis remains unaffected. We report here that spleens from SCID mice have undergone phenotypic expansion of cells bearing the NK-2 and asialo GM1 markers (70-80%) characteristic of NK cells and this expansion is accompanied by a 3-4-fold enrichment in NK cytolytic activity over their normal C.B-17 littermates. Furthermore, the NK cells from SCID mice do not rearrange or express T cell receptor alpha or beta genes, or a third T cell rearranging gene, gamma. These findings suggest that (a) T cell receptors are not necessary for NK-mediated cytolysis, and (b) either NK cells constitute an entirely distinct lineage or NK cell function is acquired in pre-T cells prior to the expression of T cell receptor genes.

Animals↗
PubMed1986

Establishment of a human large cell lung tumor line (QU-DB) with metastatic properties in athymic mice.

A continuous human cell line was established from a patient with large cell anaplastic lung carcinoma. This cell line, designated QU-DB, has been in culture for over 36 months and grows as an adherent monolayer with a doubling time of 10-12 hours. Its morphology, ultrastructure, karyotype, ability to grow in soft agar and heterotransplantability, indicate it is a large-cell lung tumor cell line of human origin. Three cell lines were established from metastatic tumors in nude mice receiving subcutaneous injections of QU-DB cells. The morphology and growth characteristics exhibited by these cell lines were similar to the primary cell line. Karyotypic analysis of cell lines derived from the primary tumor and a metastasis to the diaphragm were similar, but cells from a liver metastasis culture showed additional karyotypic changes. This large cell lung tumor cell line may prove useful as a model system for studies of human tumor progression and metastasis.

Agar↗
PubMed1986

Rearrangements of chromosomal regions containing ribosomal RNA genes and centromeric heterochromatin in the human melanoma cell line MeWo.

A chromosomal examination of cells from the earliest available passage of the human melanoma cell line MeWo revealed the presence of seven hypodiploid cell types that shared common complex marker chromosomes. Two of the cell types had long homogeneously staining regions (HSR) by Q-banding on three different chromosomes. Distamycin A/DAPI staining and silver staining for active nucleolar organizing regions (NOR) confirmed that the HSR were derived from chromosome #15. All HSR-containing cells had 4-9 pairs of large NOR distributed along the length of each HSR, with all acrocentric chromosomes being negative. The HSR-lacking cells differed primarily with respect to the morphology of the short arm of one #13 chromosome and NOR activity. One cell type had four chromosomes with active NOR, whereas all other cell types had a single active NOR on one #13. One of these cell types had a satellited #8 with NOR. Cells from three other MeWo cultures at higher passages were examined. Two of these contained both hypodiploid and hypotetraploid cells, some of which had satellited X chromosomes or satellited #3 chromosomes with active NOR. The majority of the new chromosomal rearrangements in cells from the later cultures involved the NOR-containing regions, many of which were associated with the distamycin A/DAPI-positive centromeric heterochromatin from chromosome #15. These results indicate that the chromosomal instability in the MeWo cultures is mainly limited to sequences containing active NOR and centromeric heterochromatin from chromosomes #13 and #15. This may be due to a selective pressure to increase the number of active NOR in the MeWo cells. If this is so, it would appear that amplification of active NOR occurs more readily than the activation of the many silent NOR present in these cells.

Cell Line↗
PubMed1986

Adhesion properties of a neuronal epitope recognized by the monoclonal antibody HNK-1.

A carbohydrate epitope on adhesion proteins of the developing nervous system, and on myelin-associated glycoprotein, is recognized by the monoclonal antibody HNK-1. The HNK-1 epitope bearing proteins and the monoclonal antibody alter, in a dose-dependent manner, the interaction between neurons and neurite-promoting substrate-attached materials released from cultured neural cells.

Animals↗
PubMed1986

T cell receptor gene rearrangements in cells with natural killer activity in the mouse.

Cell-mediated recognition can operate at different levels of complexity and specificity based largely on the time of appearance of effector mechanisms during the course of evolution. Antigen-specific cytotoxic T lymphocytes require both T cell receptor genes and lectin-like cell adhesion molecules (LFA-1, LFA-2, lymphocyte function-associated) to initiate and maintain stable effector target cell conjugates. Natural killer (NK) cells, on the other hand, do not require expression of T cell receptor genes in the recognition and killing of tumor cells and virally infected cells. Adhesion is mediated by a family of glycoprotein molecules, of which the LFA-1 and LFA-2 molecules appear as the most likely candidates. NK-mediated cytolysis proceeds in the absence of MHC restriction, but nevertheless appears to be triggered by depressed levels of self MHC products on the cell surface of target cells. Finally, interleukin 2-dependent, cloned cell lines with NK-like cytotoxic activity should no longer be considered as bona-fide NK cells but rather reclassified as a subset of T cells which displays NK function.

Animals↗
PubMed1985

Transfection of a rat cell line with the v-Ki-ras oncogene is associated with enhanced susceptibility to natural killer cell lysis.

Transfection of the v-Ki-ras oncogene into rat-1 fibroblasts resulted in the establishment of cell lines that were transformed, tumorigenic, and sensitive to lysis by natural killer (NK) cells. Characterization of effectors indicated that the killing was not related to Lyt-1+ or Lyt-2+ cells (T cells) but was associated with cells bearing NK markers (asialo GM1, NK-1.2+, and NK-2.1+). Transfected targets were also killed by cloned NK lines. The transformation determinants on rat-1 transfectants cross-competed with YAC 1.2 lymphoma cells, suggesting a common target structure on these two diverse cell types. The results indicate that the NK surveillance system can recognize and kill cells newly transformed by a member of the ras oncogene family.

Animals↗
PubMed1985

Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas.

Human X human hybridomas constructed with the B6 lymphoblastoid clone, which produces antitetanus toxoid (TT) antibody, and the lymphoblastoid cell line KR-4 or human hybrid myeloma KR-12, were adapted to growth as ascites in pristane-treated BALB/c nude mice by a single prior passage as a solid subcutaneous (s.c.) tumor in irradiated nude mice followed by in vitro culture. Both B6 X KR-4 and B6 X KR-12 hybrids produced anti-TT antibody and phenotypically resembled the lymphoblastoid KR-4, or the hybrid myeloma KR-12 parent, respectively. Growth as ascites increased the tumorigenicity of both hybrids in nude mice as measured by tumor incidence and rate of tumor growth. The observed increase in tumorigenicity of these hybrid cells after ascites growth was associated with a substantial loss of chromosomes. Passage of the B6 X KR-4 lymphoblastoid hybrid resulted in several reversible morphological changes characteristic of myeloma cells. These changes correlated with increased human Ig production. These observations provide a system for greatly amplifying human monoclonal antibody production.

Animals↗
PubMed1985

Accelerated regenerative neurite formation by a neuronal surface epitope reactive with the monoclonal antibody, Leu 7.

A family of glycoproteins sharing an epitope with myelin associated glycoprotein as recognized by the monoclonal antibody Leu 7 (HNK-1) has been found to be present on neurons grown in culture from embryonic chicks and rats. Immunofluorescent staining demonstrates that, in vitro, 100% of the neurons from dorsal root ganglia and spinal cord from 7-8 day chick embryos react with Leu 7. Analysis of in vitro regenerative neurite formation by neurons on substrates enriched with Leu 7 showed accelerated regenerative process formation under limiting conditions. These results indicate that the Leu 7 epitope on neurons is appropriate for substrate adhesion and promotes rapid process extension.

Animals↗
PubMed1985

Antibody production by human X human hybridomas in serum-free medium.

Four human X human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human X human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies.

Antibodies, Monoclonal↗