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E Shacter

Publications and source records attributed to E Shacter.

At least 37 records · Page 2Linked to original sources
PubMed1995

Disposition of the plasmacytomagenic alkane pristane (2,6,10,14-tetramethylpentadecane) in mice.

The intraperitoneal administration of pristane (2,6,10,14-tetramethylpentadecane) induces peritoneal plasmacytomas in genetically susceptible BALB/c mice. The purpose of this study was to estimate the disposition of an amount of intraperitoneally injected pristane that would conventionally be used in a tumor induction protocol. The distribution of 3H-labeled pristane in various tissues was monitored by liquid scintillation counting at different times after injection. The data show that pristane is present in the blood and detectable in all tested tissues during an observation period of one to 64 days. The levels of pristane fluctuate in some tissues such as lymph node and bone marrow but show a clear tendency to accumulate in others such as liver, spleen and kidney. Evidence is also presented for the in vivo metabolism of pristane based on the observed urinary excretion of tritium and on the high levels of radioactivity in the gall bladder fluid. It is concluded that intraperitoneally administered pristane is distributed throughout the mouse and is stored in tissues in sufficient amounts to allow interactions with the cells residing there.

Animals↗
PubMed1994

Differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay.

Plasma proteins are exposed to oxidants in a variety of circumstances in vivo, such as during tissue injury and inflammation. In this report, the relative susceptibility of each of the major plasma proteins to oxidative modification was assessed by exposing whole plasma to a metal-catalyzed radical generating system and detecting oxidation (protein carbonyl groups) using a novel Western blot immunoassay. Proteins were derivitized with dinitrophenylhydrazine, separated by SDS-gel electrophoresis, and screened with antibodies against dinitrophenyl groups. As little as 1 pmol of protein-associated carbonyls could be detected (100 ng of a 50 kD protein containing 0.5 mol carbonyl/mol protein). Individual plasma proteins were identified by their comigration with standards, crossreactivity with specific antibodies, and by comparison of plasma to serum. Using this approach, we found that plasma fibrinogen was much more susceptible to oxidative modification compared to the other major plasma proteins, albumin, immunoglobulins, and transferrin. The results emphasize the utility of this method for studying oxidation of proteins in cell extracts and tissues and indicate that experiments on the effects of oxidation on fibrinogen function are merited.

Animals↗
PubMed1994

Fusion between enveloped viruses and erythrocyte membranes is induced by the isoprenoid alkane pristane (2,6,10,14-tetramethylpentadecane).

Pristane (2,6,10,14-tetramethylpentadecane) is an isoprenoid alkane that induces plasma cell tumors in mice. Infection with certain retroviruses accelerates tumorigenesis but the nature of the cooperation between pristane and viruses is unknown. The purpose of this study was to investigate the potential influence of pristane on the fusion between enveloped viruses and mammalian plasma membranes. Using a fluorescence dequenching assay, we found that micromolar amounts of pristane induced fusion between erythrocyte membranes and both vesicular stomatitis virus and influenza virus. Induction of fusion occurred with as little as 5 microM pristane and reached saturation at roughly 50 microM alkane. Control experiments revealed that induction of fluorescence dequenching was not due to extraneous phenomena such as lipid transfer or non-specific interactions with the carrier for pristane (beta-cyclodextrin). Fusion was also induced by standard protocols which involve lowering the pH of the incubation medium. In the presence of pristane, low pH-triggered fusion was enhanced. The extent to which pristane induced fusion was dependent upon the orientation of the lipids in the target membrane. That is, fusion was most effective with erythrocyte ghosts which had a symmetric lipid distribution and was less effective with ghosts in which the native lipid asymmetry was maintained. Intact erythrocytes, which have an asymmetric lipid distribution, were the least effective targets. This result exactly parallels the pattern observed with acid-induced fusion. Similar patterns were also observed in the temperature dependence of fusion induced by these two protocols. The novel fusogenic activity of pristane is discussed with regard to current models of virus/membrane fusion.

Biophysical Phenomena↗
PubMed1993

Stimulation of interleukin-6 and prostaglandin E2 secretion from peritoneal macrophages by polymers of albumin.

Interleukin-6 (IL-6) is a multifunctional cytokine that is elevated in vivo during acute infection, chronic inflammation, and some hematopoietic malignancies. To understand how IL-6 becomes elevated in vivo, it is important to identify factors that can stimulate its secretion from effector cells. We found that commercial preparations of bovine serum albumin (BSA) stimulated murine macrophages to secrete high levels of IL-6. In fact, BSA was at least as potent as bacterial lipopolysaccharide (LPS) in stimulating IL-6 production. Stimulation was clearly visible at concentrations as low as 20 micrograms/mL and reached saturation at 0.5 to 1 mg/mL albumin, at which concentration 1.1 x 10(6) oil-elicited macrophages produced 6,000 +/- 700 B9 units of IL-6 in an overnight incubation. Prostaglandin E2 production was induced by the same concentrations of BSA. Both resident and oil-elicited peritoneal cells were responsive to the albumin. The stimulatory activity did not derive from contamination of the protein with Escherichia coli LPS; when compared directly with LPS, the response to BSA was more rapid, had a higher amplitude, and was not inhibitable by polymyxin B. In addition, macrophages isolated from C3H/HeJ mice, which have an inherited defect in their ability to respond to LPS, secreted IL-6 in response to BSA but not to LPS. The stimulatory activity was stable to heat, mild acid, and reduction/alkylation and copurified with albumin on Cibachron Blue agarose (Sigma, St Louis, MO) and anti-albumin immunoaffinity chromatography. Comparison of different sources and preparations of albumin showed differences in the levels of IL-6-inducing activity; three different lots of commercial fatty acid-free BSA and one lot of polymer-enhanced BSA stimulated IL-6 secretion by more than 100-fold over basal levels whereas other preparations showed more limited activity. A sample of BSA that was active in vitro caused a marked elevation of IL-6 when injected into BALB/c mice, thus demonstrating inflammatory activity in vivo. When the albumin preparations were fractionated by ion exchange and gel filtration chromatography and then analyzed by sodium dodecyl sulfate-gel electrophoresis and Western blot immunoassay, it was found that the IL-6-inducing activity resided in high molecular weight polymers of albumin. The ability of albumin polymers to stimulate IL-6 production represents a novel mechanism for modulation of this cytokine.

Animals↗
PubMed1993

Activated murine neutrophils induce unscheduled DNA synthesis in B lymphocytes.

Activated neutrophils induce DNA damage in neighboring cells by secreting reactive oxygen compounds into the extracellular milieu. Repair of this damage is required to prevent mutagenesis and neoplastic transformation. Conditions were established to detect the activation of excision-repair pathways (unscheduled DNA synthesis) by measuring stimulated thymidine uptake in target B lymphocytes exposed to activated neutrophils. Murine neutrophils were cocultured in serum-free medium with splenic B cells or with murine plasmacytoma cells for 2 h. Unscheduled DNA synthesis in the B cells was detected at neutrophil:target cell ratios of 1:1 to 4:1 when the neutrophils were activated with phorbol myristate acetate. Reagent H2O2 alone (> or = 6 microM) also induced UDS whereas HOCl (up to 4 mM) did not. No repair synthesis was observed within the neutrophils themselves. Control experiments indicated that the induction of UDS by neutrophils and H2O2 was not due to formation of a stable genotoxic compound from HU. On the contrary, scavenging of free H2O2 by HU probably lowered the levels of UDS that could be detected by these agents. Induction of unscheduled DNA synthesis by neutrophils and H2O2 occurred under conditions of less cytostasis than was found with other DNA-damaging agents such as 4-nitroquinoline 1-oxide or gamma-irradiation. This may reflect a heightened responsiveness of the cells to repair of damage from physiological oxidants. The results demonstrate that DNA damage induced by reactive oxygen intermediates can be repaired by nucleotide excision-repair pathways.

Animals↗
PubMed1992

Elevation of interleukin-6 in response to a chronic inflammatory stimulus in mice: inhibition by indomethacin.

Intraperitoneal (i.p.) injection of a mineral oil such as pristane induces a chronic inflammatory response in mice. This is characterized by a large influx of macrophages and other inflammatory cells into the peritoneal cavity for months after injection of the oil. By using the B9 cell bioassay, it was found that injection of pristane caused a marked and prolonged elevation of interleukin-6 (IL-6) levels in the peritoneal cavities of the mice. IL-6 was undetectable (less than 15 U/mL) in the peritoneal fluids of unprimed mice and during the first week after injecting pristane. From 4 to 20 weeks, the concentration of IL-6 increased to an apparent plateau with concentrations ranging from 200 to 2,000 U/mL. Increasing the dose of pristane did not substantially increase the peritoneal levels of IL-6 established at 20 weeks after pristane treatment. At later times (by day 250), the level decreased to 263 +/- 217 U/mL. However, mice that developed plasma cell tumors around day 300 showed high levels of IL-6 in the ascites fluid (650 to 2,400 U/mL). Serum levels of IL-6 were also elevated in pristane-primed mice but were substantially lower than those found in the peritoneal cavity. Chronic administration of the nonsteroidal anti-inflammatory drug indomethacin decreased the levels of IL-6 by 75% to 80%. Experiments performed in vitro showed that pristane-elicited macrophages secreted low levels of IL-6 constitutively and high levels of IL-6 in the presence of lipopolysaccharide. Both IL-6 and prostaglandin E2 production were inhibited by addition of indomethacin to macrophage cultures in vitro. Treatment of mice with pristane may provide a model system for studying the inflammatory pathways that control IL-6 levels in vivo. The relevance of these results to elucidation of the role of IL-6 in plasma cell tumorigenesis is discussed.

Animals↗
PubMed1991

A new method for delivering alkanes to mammalian cells: preparation and preliminary characterization of an inclusion complex between beta-cyclodextrin and pristane (2,6,10,14-tetramethylpentadecane).

Pristane (2,6,10,14-tetramethylpentadecane) is an isoalkane which induces plasma cell tumorigenesis in genetically susceptible strains of mice. Attempts to study the biological activity of pristane on cells in vitro have been hindered by the extreme hydrophobicity and hence complete immiscibility of the compound in aqueous cell culture media. In comparing different solubilization protocols such as using organic solvents, liposomes, and molecular encapsulation into beta-cyclodextrin (beta-CyD), it was found that beta-CyD/pristane inclusion complexes were optimal for delivery of the hydrocarbon to cells. After solubilization in beta-CyD, pristane was cytotoxic (51Cr release assay) to murine B lymphocyte lines in culture (P388, NSF-1, and SJL-4) and inhibited the lipopolysaccharide-induced stimulation of splenic B lymphocyte proliferation and blast formation (Coulter counter analysis) when added in the micromolar concentration range. Moreover, sub-toxic concentrations of beta-CyD/pristane inclusion complexes were found to have a small but reproducible mitogenic effect [( 3H]thymidine incorporation) on SJL-4 and 308 (murine initiated keratinocyte) cells in culture but not on P388 cells. The results indicate that molecular encapsulation of pristane into beta-CyD provides a new and effective method for delivering low concentrations of alkanes to mammalian cells in vitro.

Alkanes↗
PubMed1991

DNA repair in the c-myc proto-oncogene locus: possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice.

This report describes an unexpected difference in the efficiency of removal of UV-induced DNA damage in the c-myc locus in splenic B lymphoblasts from two inbred strains of mice. In cells from plasmacytoma-resistant DBA/2N mice, 35% of UV-induced damage in the regulatory and 5' flank of c-myc is removed by 12 h. However, in cells from plasmacytoma-susceptible BALB/cAn mice, damage is not removed from this region. In the protein-encoding region and 3' flank of c-myc as well as in two dihydrofolate reductase gene fragments, UV damage is repaired with similar efficiency in B lymphoblasts from both strains of mice. Furthermore, in the protein-encoding portion and 3' flank of c-myc, damage is selectively removed from only the transcribed strand. No repair is detected in the nontranscribed strand. In contrast, DNA repair in the 5' flank of c-myc is not strand specific; in DNA from DBA/2N cells, UV damage is rapidly removed from both the transcribed and nontranscribed strands. In BALB/cAn cells no repair was detected in either strand in the 5'flank, consistent with the results with double-stranded, nick-translated probes to this region of c-myc. In addition to the repair studies, we have detected post-UV-damage formation: in most of the genes studied, we find that additional T4 endonuclease-sensitive sites are formed in the DNA 2 h after irradiation. Our findings provide new insights into the details of gene-specific and strand-specific DNA repair and suggest that there may be close links between DNA repair and B-cell neoplastic development.

Animals↗
PubMed1990

DNA damage induced by phorbol ester-stimulated neutrophils is augmented by extracellular cofactors. Role of histidine and metals.

Activated neutrophils cause extensive DNA damage in neighboring nonphagocytic cells. To determine whether compounds in the extracellular milieu participate in the DNA damage process, murine neutrophils were cocultivated with target tumor cells in media of varying composition. Using the alkaline elution assay, it was found that the level of strand breaks induced was significantly higher (2.8-fold) in complex cell culture media than in minimal phosphate-buffered saline. Addition of amino acids in general and of histidine in particular increased the level of damage nearly to that observed in complete media (2.7- and 2.1-fold, respectively). The histidine stimulation was concentration-dependent and reached a maximum at 100-400 microM. The mechanism whereby this occurred is not proven but probably derived from chelation of metals and participation in a site-specific Fenton reaction. Addition of the cell-impermeable chelator EDTA dramatically inhibited induction of strand breaks by neutrophils in complete media and prevented the enhancement of damage induced by histidine in phosphate-buffered saline. None of the effects on neutrophil-induced damage could be attributed to modulation of the oxidative burst activity of the cells (O2- and H2O2 production). Histidine also enhanced induction of strand breaks by reagent H2O2. However, EDTA had no effect or actually increased the level of damage induced by both a bolus of H2O2 and a flux of H2O2 generated by glucose oxidase. The cell-permeable chelator o-phenanthroline inhibited both neutrophil- and H2O2-induced damage. The results indicate that secondary reactions involving extracellular amino acids and metals contribute significantly to neutrophil-induced DNA damage to neighboring cells. Moreover, the data show that the mechanism whereby neutrophils induce this damage cannot be attributed solely to secretion of H2O2.

Amino Acids↗
PubMed1990

DNA repair in the c-myc locus.

We have studied DNA repair after UV damage in the murine c-myc locus. It appears that a region in B cells upstream of the murine c-myc gene is repaired with a different efficiency in plasmacytoma-resistant DBA/2N mice than in plasmacytoma-susceptible BALB/cAn mice. The region just upstream of c-myc is inefficiently repaired in B lymphoblasts derived from BALB/cAn mice. In contrast, this same region of c-myc is efficiently repaired in B lymphoblasts derived from DBA/2N mice. DNA fragments located in the coding region of c-myc and in another gene, dihydrofolate reductase (DHFR), are repaired with equal efficiency in cells from these two strains of mice. It is possible that repair efficiency of the 5' flank of c-myc may be involved in tumor susceptibility of the mouse strain.

Animals↗
PubMed1988

Activated neutrophils induce prolonged DNA damage in neighboring cells.

We have measured the capacity of highly-purified, paraffin oil-elicited neutrophils to induce DNA single-strand breaks in a newly established plasmacytoma cell line, RIMPC 2304, which was induced by a retrovirus containing the c-myc and V-Ha-ras oncogenes. This cell line effectively repairs DNA damage induced by gamma-irradiation. DNA damage induced by neutrophils was correlated with the oxidative burst of the neutrophils. The levels of superoxide anion, H2O2, and HOCl produced after stimulation of the neutrophils (6 X 10(5)/cm3) with the tumor promoter phorbol myristate acetate (100 nM) were 33.8 microM, 12.8 microM and 1.7 microM respectively in 15 min, and 98 microM, 20 microM and 8.7 microM respectively in 90 min. The results of alkaline elution experiments revealed that when the same concentration of neutrophils was co-incubated for 15 min in serum-free medium with an equal number of radioactively labeled RIMPC 2304 cells, the latter incurred a level of damage that approximated that caused by 300 rad equivalents of gamma-irradiation or by a 1-min treatment with 20 microM H2O2 at 37 degrees C. Damage from neutrophils was coincident with the oxidative burst; it was induced rapidly (within 5 min) but remained high for more than 90 min. The level of damage achieved was dependent upon the ratio of neutrophils: target cells and was clearly detectable at ratios as low as 0.25:1. Induction of single-strand breaks was completely inhibited by catalase and partially inhibited by superoxide dismutase, mannitol, and reduced glutathione but not by Na azide. Addition of the non-steroidal anti-inflammatory drug indomethacin either enhanced (at 50 microM) or had no effect (at 2 microM) on the damage detected. Finally, repair of strand breaks induced by neutrophils was significantly slower (half-time approximately 10 min) than that observed for repair of similar levels of damage induced by H2O2 or gamma-irradiation (half-times approximately 3 min, each). The results indicate that neutrophils cause prolonged DNA damage in neighboring cells. Moreover, they indicate that although H2O2 produced in the oxidative burst is an essential mediator of the damage observed, additional reactive oxygen intermediates including the superoxide anion are also implicated. The data are discussed in relation to the possible role of neutrophils in chronic inflammation and in pristane-induced plasmacytoma formation in mice.

Animals↗
PubMed1987

Serum-free medium for growth factor-dependent and -independent plasmacytomas and hybridomas.

A serum-free medium was developed for culture of plasmacytomas and hybridomas that are dependent upon or independent of the presence of specific P388D1-derived polypeptide growth factors. Possibly due to the stringency of requirements for culturing such plasmacytomas, a highly advantageous combination of components was developed. The medium, SFM, contains RPMI 1640, beta-mercaptoethanol, Hepes buffer, reduced glutathione (GSH), selenite (Se), pyruvate, transferrin, albumin, soybean lipids, and low density lipoproteins. A cooperative effect of GSH and Se is observed. SFM supports the continuous, prodigious growth of every B cell line tested, including a variety of the plasmacytomas, hybridomas, myeloma fusion partners and lymphoma cell lines, as well as the macrophage-like cell line P388D1. All of the B cell lines grow equally as well in SFM as in serum-containing medium. Hybridoma lines from mouse-mouse and rat-mouse fusions continue to produce monoclonal antibodies which can then be purified in a single-step procedure. The cells can be cloned out and frozen down in SFM. In addition, the medium is highly effective for establishment of plasmacytoma cell lines from some transplantable plasmacytomas, including early generation tumors.

Animals↗
PubMed1986

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice.

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.

Amino Acid Sequence↗
PubMed1985

Immunological characterization of phosphoprotein phosphatases.

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.

Animals↗