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S Rudikoff

Publications and source records attributed to S Rudikoff.

At least 37 records · Page 2Linked to original sources
PubMed1993

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion.

Animals↗
PubMed1993

Long-term thymic reconstitution by peripheral CD4 and CD8 single-positive lymphocytes.

Significant immigration of peripheral T cells into SCID thymus was observed following reconstitution with normal Peyer's patch, mesenteric lymph node or peripheral lymph node cells. Immunohistologic and flow cytometric analyses reveal that T cells from these tissues are found in the thymus for as long as 177 days and can account for up to 67% of intrathymic cells. The returning cells express the CD3/T cell receptor alpha/beta complex, indicative of mature cells, and are equally divided among helper (CD4+CD8-) and cytotoxic (CD4-/CD8+) phenotypes. The immigration of peripheral T cells is not accompanied by the appearance of immature, double-positive (CD4+CD8+) thymocytes as seen in similar reconstitutions using bone marrow. Taken together, these results suggest that peripheral T cells from a variety of lymphoid organs may regularly re-enter the thymus and, thus, possibly play a role in normal thymic development.

Animals↗
PubMed1993

Characterization of a V kappa family in Mus musculus castaneus: expansion at the subset level.

We have examined the same kappa chain variable (V kappa) region family in several mouse species in order to observe short-term, incremental change at immunoglobulin (Ig) multigene loci. In the present study, the Igk-V24 family has been characterized in a Mus m. castaneus colony derived from individuals originating in Thailand and compared to the same family in Mus m. domesticus (BALB/c) and Mus pahari, representing about 1-2 and about 5-9 million years of evolution, respectively. Southern hybridization of genomic DNA with a probe encoding the prototype Igk-V24 coding region reveals restriction fragment patterns indicative of two distinct M. m. castaneus haplotypes. These haplotypes appear to result from an unequal recombination between similar gene arrays, as their restriction patterns are unique but contain many common fragments. The complexity of these patterns indicates a marked expansion in the Igk-V24 family of M. m. castaneus relative to BALB/c and M. pahari. Additional analyses using probes specific for individual subsets demonstrate that the expansion is not general throughout the entire family, but is restricted to particular subsets and therefore to relatively short chromosomal segments. One subset alone accounts for most of the expansion and comprises over 40% of the entire M. m. castaneus family. The wide range of Igk-V24 family complexity seen among M. m. castaneus, M. m. domesticus, and M. pahari, as well as among the different M. m. castaneus family subsets, suggests a model of random evolution in V kappa family copy number rather than one which is selective.

Animals↗
PubMed1993

Characterization of a V kappa family in Mus musculus castaneus: sequence analysis.

To examine genetic variation at immunoglobulin (Ig) multigene loci over short spans of evolutionary time, we have compared members of an Ig kappa chain variable (V kappa) region family from several mouse species. In this study, seven unique Igk-V24 family members have been isolated from Mus m. castaneus and characterized by nucleotide sequence determination for comparison to their counterparts in Mus m. domesticus (BALB/c), and Mus pahari, representing 1-2 million years of evolution in the former case and 5-8 million years in the latter. Parsimony, together with evolutionary distances calculated for various pairs of Igk-V24 family coding regions, relate all family members to a common progenitor existing roughly 24 million years ago (Mya). A significant portion of the M. m. castaneus family consists of pseudogene segments in various degrees of progressive degeneration. The substitution patterns and divergence rates for all gene segments are characteristic of their respective subsets, especially in the areas flanking the coding regions. Complex and variable patterns of diversity are seen in potentially expressed coding regions, which appear to reflect quite different selective pressures on various subregions within the V kappa protein domain. These results indicate that evolutionary pressures are operating at the level of family subsets, their individual members, and subregions within similar molecules.

Animals↗
PubMed1993

Susceptibility and resistance to J3V1 retrovirus-induced murine plasmacytomagenesis in reconstituted severe combined immunodeficient mice.

To date much is known about the genetics of susceptibility and resistance to plasmacytoma induction in mice, however little is known about the cellular aspects of these phenotypes. The complexity of plasmacytomagenesis allows for susceptibility and resistance to reflect differences in B cells, T cells, accessory cells and/or stromal elements contributing to the disease process. Alternatively, these phenotypes may result from differential abilities to affect events critical to plasmacytomagenesis, such as myc deregulation. To address these possibilities, the v-myc-raf-containing retrovirus, J3V1, was used to induce plasmacytomas (PCTs) in severe combined immunodeficient (SCID) mice reconstituted with susceptible (Balb/c) and/or resistant (DBA/2) cells. The results demonstrate that Balb/c bone marrow (BM)-reconstituted SCID mice yielded PCTs of donor origin, while DBA/2 BM-reconstituted mice did not. Mice reconstituted with both DBA/2 BM and Balb/c peripheral lymphocytes, as well as those reconstituted with Balb/c peripheral lymphocytes alone, also yielded only Balb/c PCTs. These results indicate that: (1) a microenvironment supportive of plasmacytomagenesis is insufficient to allow PCT development among resistant cells; (2) DBA/2 BM-derived cells do not suppress plasmacytomagenesis by target cell elimination or microenvironment destruction; (3) resistance is not solely attributable to the inability of DBA/2 B cells to deregulate myc; and (4) potential PCT targets reside in a number of lymphoid tissues. Taken together, these results demonstrate that a major aspect of resistance/susceptibility to plasmacytomagenesis is dictated by the genotype of the target B cell.

Animals↗
PubMed1992

Exon-specific gene correction (conversion) during short evolutionary periods: homogenization in a two-gene family encoding the beta-chain constant region of the T-lymphocyte antigen receptor.

The two genes encoding the beta-chain constant region of the T-lymphocyte antigen receptor appear to have undergone gene conversion in a number of species, including wild and laboratory mice. To examine the frequency of such events during short evolutionary periods, we have characterized the corresponding genes from an African pygmy mouse, Mus minutoides. Sequence analysis indicates that exon 1 regions from these genes have undergone conversion events independent of those observed in other mouse species. Furthermore, the conversion events in all murine species are limited to exon 1 sequences. One such event involves the insertion and subsequent transfer of an entire codon between the two genes. Comparisons with other murine C beta sequences suggest that gene conversion has occurred on the order of every 0.3 Myr during the evolution of a family consisting of only two genes.

Animals↗
PubMed1991

Instability of immunoglobulin genes in S107 cell line.

Somatic mutation occurs frequently in rearranged and expressed immunoglobulin variable region genes in vivo. In contrast, V region hypermutation seldom occurs in antibody-forming cells in culture. The S107 mouse myeloma cell line is one of the few cell lines that has been observed to generate V region mutations frequently and spontaneously in vitro. Detailed examination reveals that both the S107 tumor and the cell line derived from it contain and express a duplicated heavy-chain gene. In culture, only one of the two heavy-chain genes undergoes both V and C region mutation, and variants with complex phenotypes and genotypes arise as a result of mutation and segregation of these duplicated genes.

Animals↗
PubMed1990

Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli.

The ATP-dependent Clp protease of Escherichia coli contains two dissimilar components: the Clp A regulatory polypeptide, with two ATP binding sites and intrinsic ATPase activity, and the Clp P subunit, which contains the proteolytic active site. The DNA sequence of the clpP gene predicts a protein of 207 amino acids (Mr 21,679), which is in close agreement with the size determined by sodium dodecyl sulfate-gel electrophoresis of purified Clp P. Clp P has a native Mr of approximately 240,000, and electron micrographs of the protein show superimposed disk-like structures with a central cavity, similar in appearance to purified proteasomes from eukaryotic cells. Clp P is synthesized with a 14-amino acid leader which is rapidly cleaved in vivo to yield the 193-amino acid protein which has activity in vitro. The clpP gene is at 10 min on the E. coli map, close to that for the ATP-dependent Lon protease of E. coli and far from the gene for clpA. Primer extension experiments indicate that transcription initiates immediately upstream of the coding region for Clp P, with a major transcription start at 120 bases in front of the start of translation. Insertion mutations in clpP have been isolated and transferred to the chromosome; strains devoid of Clp P are viable in the presence or absence of Lon protease. Mutations in clpP stabilize the same Clp A-beta-galactosidase fusion protein specifically stabilized by clpA mutations, providing the first genetic evidence that Clp A and Clp P act together in vivo.

ATP-Dependent Proteases↗
PubMed1990

Expression of a class I MHC transgene: regulation by a tissue-specific negative regulatory DNA sequence element.

In vivo patterns of expression of a miniature swine class I major histocompatibility gene, PD7, were analyzed both in situ in the pig, and in transgenic mice. Structural analysis of PD7 DNA sequences revealed that PD7 is highly homologous to the pig gene PD1, which encodes a classical transplantation antigen. Despite the extensive homology, PD7 is expressed in situ at markedly lower levels than PD1 in nearly all tissues. Introduction of PD7 into mice results in a pattern of PD7 expression in the transgenic animals that parallels that observed in situ in the pig. Comparison of two lines of PD7 transgenic mice, which differ only in the extent of 5' flanking sequence, reveals the presence of a silencer element. The silencer activity is tissue specific: differences in PD7 expression are observed only in lymphoid tissues and skin. Skin from both lines of transgenics mediates graft rejection, but the rate of rejection correlates with the level of PD7 expression.

Amino Acid Sequence↗
PubMed1989

T cell derived IL-6 is differentially required for antigen-specific antibody secretion by primary and secondary B cells.

IL-6 (formerly PCTGF, HP-1, BSF-2, HGF, IFN-beta 2, 26 kDa) is a recently defined lymphokine demonstrating activity on multiple cell types, including hepatocytes, thymocytes, T cells, plasmacytomas, and B cells. The biologic effects of IL-6 on lymphocytes, particularly B cells, suggest this factor may be involved in the regulation of normal immune responses. Accordingly, we have investigated the role of IL-6 in Ag-specific responses of B cells from both naive and Ag-primed mice. When Ag-primed splenic T cells were used as a source of help, naive (primary) B cell responses specific for the hemagglutinin molecule of the influenza A virus (PR8) were fully inhibited by the addition of an anti-IL-6 antiserum, and are thus IL-6 dependent. In contrast, secondary B cell responses were essentially IL-6 independent, being unaffected by this antiserum even at concentrations 10-fold higher than required to completely inhibit primary responses. This differential IL-6 requirement was further investigated by using a panel of hemagglutinin molecule-specific Th clones. Consistent with the above findings, a Th1 clone secreting biologically active IL-6 enables antibody secretion by both primary and secondary B cells, whereas Th1 clones that do not produce IL-6 support secondary responses, but fail to help primary B cell responses unless exogenous IL-6 is added. These results provide the first instance of differential lymphokine requirements among primary vs secondary B cell responses, and suggest T cell-derived IL-6 plays a critical role during the regulation of humoral immune responses. Moreover, functionally distinct Th1 clones were identified that differed in IL-6 secretion and their corresponding ability to induce Ig secretion by primary and secondary B cells.

Animals↗
PubMed1989

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice.

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.

Adenocarcinoma↗
PubMed1989

The murine Il-6 gene maps to the proximal region of chromosome 5.

Murine Il-6 cDNAs were isolated by cross-hybridization with a human IL-6 cDNA from an IL-1 activated bone marrow stromal cell line (W20). Mouse-hamster somatic cell hybrids were utilized to localize murine Il-6 to chromosome 5. Genetic mapping with respect to En-2, AlbH, and Gus in backcross progeny from an interspecific mating between C57BL/6J and Mus spretus positioned Il-6 3 cM distal to En-2. The syntenic relationships of Il-6 and En-2 in mouse and man, as well as the potential role of IL-6 in tumorigenesis, are discussed.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of a newly identified growth factor specific for epithelial cells.

A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinocyte growth factor (KGF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C4 reversed-phase HPLC column. KGF was both acid and heat labile and consisted of a single polypeptide chain of approximately 28 kDa. Purified KGF was a potent mitogen for epithelial cells, capable of stimulating DNA synthesis in quiescent BALB/MK epidermal keratinocytes by greater than 500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lack of mitogenic activity on either fibroblasts or endothelial cells indicated that KGF possessed a target cell specificity distinct from any previously characterized growth factor. Microsequencing revealed an amino-terminal sequence containing no significant homology to any known protein. The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimulation of normal epithelial cell proliferation.

Animals↗
PubMed1989

The role of plasmacytoma growth factor in the in vitro responses of murine plasmacytoma cells.

Many plasmacytomas arising in BALB/c mice are dependent upon a specific, macrophage-derived plasmacytoma growth factor for survival and proliferation in vitro. Adherent cells taken from the peritoneal oil granuloma in which the early plasmacytomas arise and proliferate produce 50 times more PCT-GF activity in vitro than do normal peritoneal cells, thus suggesting a possible in vivo role for PCT-GF. Purification and amino acid sequencing of PCT-GF derived from the murine macrophage cell line, P388D1, have identified a 23 kDa protein with a unique NH2-terminal sequence. This molecule is now known as murine IL6. As part of the characterization of murine Il-6, genomic sequences have been localized to the proximal end of mouse chromosome 5 via Southern analysis of restriction fragment length polymorphisms. The removal of IL6 from IL6-dependent PCT cell lines results in a growth arrest in early G1. This is accompanied by a rapid and specific loss of transferrin receptor expression and results in eventual cell death. It appears that the response to IL6 is at least partially dependent on Ca++ because functional Ca++ channels are necessary for the PCT cells to pass through G1 and to maintain transferrin receptor expression.

Animals↗
PubMed1988

The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component.

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.

ATP-Dependent Proteases↗
PubMed1988

Interaction and sequence diversity among T15 VH genes in CBA/J mice.

Nucleotide sequences of the four genes composing the T15 heavy chain variable region (VH) family of the CBA/J mouse have been determined. Comparison of these sequences with their published BALB/c and C57BL/10 homologues reveals that nucleotide differences found between given alleles of two strains, i.e., CBA/J and BALB/c, are observed in other family members of the same strain. We suggest that these patterns of sequence variation are most readily explained by gene interaction (conversion). Additionally, the sequence of a CBA/J hybridoma, 6G6, proposed to have been generated by gene conversion, is directly encoded by the CBA/J V11 gene indicating that the putative conversion has occurred meiotically in the germline. These results are consistent with the premise that gene correction is occurring frequently among members of this family and that such processes may contribute significantly to the evolution of Ig variable region genes even in the relatively short time frame of inbred strain derivation.

Alleles↗
PubMed1988

Relationships between B cell and myeloid differentiation. Studies with a B lymphocyte progenitor line, HAFTL-1.

A cell line, HAFTL-1, derived by in vitro transformation of fetal liver cells with v-Ha-ras, was found to have molecular and phenotypic characteristics of pro-B cells recently committed to the Ly-1+ B cell differentiation pathway. Stimulation of these cells with LPS resulted in their differentiation within either the B or myelomonocytic lineages. Thus, lines derived from LPS-stimulated HAFTL-1 cells were shown to be clonally related, as evidenced by common v-ras integrations, but to exhibit characteristics of pre-B cells (ThB expression, continuing DJ heavy chain rearrangements) or mature macrophages (expression of Mac-1 and Mac-2, lysozyme and nonspecific esterase production, phagocytosis) while maintaining their Ly-1+ phenotype. These results suggest that events resulting in the irrevocable commitment to a single lineage occur late in differentiation, at least within the pathway yielding Ly-1+ B cells and a proposed subpopulation of Ly-1+ monocytes and macrophages. Final commitment to these lineages is carefully orchestrated, as evidenced by restricted expression of Ly-5 isoforms and production of IgH transcripts.

Animals↗