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D H Pluznik

Publications and source records attributed to D H Pluznik.

At least 55 records · Page 3Linked to original sources
PubMed1985

Structural requirements in hydroxyeicosanoids for the activation of the 5-lipoxygenase in PT-18 mast/basophil cells.

We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE) stimulated the 5-lipoxygenase in the murine PT-18 mast/basophil cell line to produce leukotriene B4 and 5-HETE from exogenously added arachidonic acid. In order to determine the structural requirements in the HETE molecule that are necessary for the activation of this 5-lipoxygenase, various isomeric HETEs, derivatives and analogs were prepared, purified and tested. The order of stimulatory potencies was: 15-HETE acetate greater than 15-HETE = 15-hydroperoxyeicosatetraenoic acid (15-HPETE) greater than 5-HPETE = 12-HPETE greater than 5-HETE. 15-HETE methyl ester, 12-HETE and prostaglandin E2 were ineffective over the concentration range tested. Several diHETEs were also tested. 5S,15S-DiHETE was somewhat less potent than 15-HETE, whereas both 8S,15S-diHETE and leukotriene B4 were inactive. The calcium ionophore A23187 was much less effective than 15-HETE. These structure-activity studies indicate the importance of the nature, position and location of the various functional groups in the HETE molecule and suggest that a specific recognition site is involved in the activation of the 5-lipoxygenase in PT-18 cells.

Animals↗
PubMed1985

Susceptibility of mice reconstituted with trisomy 19 hematopoietic cells to infection with Rauscher leukemia virus.

Radiation chimeras with trisomy 19 hematopoietic cells were constructed to test the sensitivity of the trisomic hematopoietic system to infection with Rauscher leukemia virus: Hematopoietic cells from livers of trisomic fetuses were rescued by transplantation into lethally irradiated adult mice. These Ts 19 radiation chimeras show a stable and sufficiently long-lived trisomic hematopoiesis to allow experimental induction of Rauscher leukemia. Rauscher leukemia virus (RLV) induced a marked proliferation of erythroblasts in the spleens of Ts 19 mice and control chimeras within 3 weeks. The onset of erythroblast proliferation was significantly delayed in the Ts 19 mice, suggesting a smaller number of target cells for the RLV and/or reduced susceptibility of the target cells to RLV. Both Ts 19 and control chimeras developed nonlymphocytic leukemia 2-4 months after RLV injection. The course of leukemogenesis was similar in the two experimental groups. No numerical chromosome abnormalities associated with leukemogenesis were detectable in Ts 19 or control cells. The numbers of chromosomal sister chromatid exchanges 2 weeks after RLV injection were elevated to the same degree in both Ts 19 and control cells. Thus, cells with constitutional trisomy do not show increased chromosomal instability due to leukemogenesis.

Animals↗
PubMed1985

Antigen-induced increase in protein kinase C activity in plasma membrane of mast cells.

Bridging of cell-bound IgE antibody molecules on colony-stimulating factor-dependent mouse mast cell line (PT-18) cells by multivalent antigen induces phospholipid methylation, a transient rise in intracellular cAMP, intracellular mobilization and uptake of Ca2+, and formation of diacylglycerol followed by histamine release. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C activity in the plasma membrane, which is accompanied by a slight decrease in the enzyme in cytosol. Protein kinase C activity in the membrane fraction reached maximum within 30 sec after antigen challenge and then gradually declined. The increase of the enzyme activity in the membrane could not be explained by a shift of the enzyme from cytosol, and it suggested that bridging of IgE-receptor may induce a modulation of existing enzyme to a state of higher catalytic activity. Phorbol 12-myristate 13-acetate also induced a rapid but persistent increase in protein kinase C activity in the membrane fraction of mast cells. However, the increase in the enzyme activity in the membrane was accompanied by a marked decrease in the enzyme in cytosol.

Animals↗
PubMed1985

Possible role of gangliosides in the interaction of colony-stimulating factor with granulocyte-macrophage progenitor cells.

Our previous studies have shown that preincubation of murine bone marrow (BM) cells with cholera toxin (CT) or with its B subunit inhibited their responsiveness to colony-stimulating factor (CSF). Because ganglioside GM1 is a component of the CT receptor, the present study was undertaken to determine whether gangliosides interact with CSF and therefore might play a role in the binding sites for CSF. Preincubation of CSF with increasing concentrations of bovine-brain mixed gangliosides resulted in decreased numbers of colonies of BM-derived granulocyte-macrophage progenitor cells (CFU-C) in soft agar. The inhibitory effect of the gangliosides could be reduced by increasing the concentrations of CSF. Evidence for direct binding of CSF to gangliosides was obtained by affinity chromatography of CSF on gangliosides-sepharose beads. CSF activity was retained on the beads and could be eluted with 6 M guanidine HCl. Four different individual gangliosides (GM1, GM2, GD1a, GT1b) were tested for their inhibitory effect on CSF-induced clonal growth of CFU-C. GM1 was the most effective with a 50% inhibition (I50) of clonal growth at a concentration of 15 microM. while the other three gangliosides had slight inhibitory activity (I50 at a concentration greater than 100 microM). In addition, preincubation of BM cells with rabbit anti-GM1 antibodies before addition of CSF reduced the clonal growth of CFU-C to 45%. These data indicate that GM1 interacts with CSF and suggest that gangliosides may play a role in the interaction of CSF with CFU-C and that the binding site for CSF on the surface of these cells might either consist of or contain this ganglioside.

Animals↗
PubMed1984

Histamine release from the cultured mouse mast cell line PT18 in response to immunologic and non-immunologic stimuli.

Cultured mouse mast cells that are dependent on spleen-derived factors for their proliferation and maintenance have been shown rapidly to release histamine in response to immunologic stimuli, concanavalin A, and ionophores A23187 and Br-x537A. These cells did not, however, release histamine when treated with compound 48/80 or adenosine 5'-triphosphate. Immunologically mediated histamine release was inhibited by theophylline and quercetin, but not by disodium cromoglycate. The results presented here are discussed in terms of the possible relationship of this cell line to mucosal mast cells.

Adenosine Triphosphate↗
PubMed1984

Inhibition of interleukin-2-dependent cytotoxic T-lymphocyte growth by gangliosides.

Previous work has shown that exogenous gangliosides suppress lectin-induced T-lymphocyte mitogenesis in mixed populations of immune cells. As one potential cellular site of this inhibition, the influence of gangliosides on interleukin-2-dependent T-cell proliferation was tested, using cultures of cytotoxic T cells (strain CT-6). Incubation of CT-6 cells with mixed bovine brain gangliosides resulted in a dose-dependent inhibition of cell proliferation. Cell viability was unaffected by gangliosides, and the inhibition was totally reversed when the gangliosides were removed. Individual purified gangliosides were tested and GM2 was most inhibitory (I50 = 15 microM). GD1a and GT1b were somewhat less potent, whereas GM1 and GM3 were only weakly inhibitory. Various nonpolar lipids, sulfatides, and sialic acid did not inhibit CT-6 cell growth. The results suggest that a primary mechanism whereby gangliosides inhibit lectin-induced lymphocyte mitogenesis is by inhibition of the interleukin-2-stimulated proliferation of T cells in these cultures.

Animals↗
PubMed1984

Colony-stimulating factor (CSF) controls proliferation of CSF-dependent cells by acting during the G1 phase of the cell cycle.

Proliferation of granulocyte/macrophage (GM) progenitor cells in soft agar cultures is dependent on the continuous presence of colony-stimulating factors (CSF). To elucidate this dependency we studied the effect of deprivation and readdition of CSF on the cell cycle kinetics of a GM-CSF-dependent murine mast/basophil cell line (PT-18). Flow cytometry and [3H]thymidine incorporation have been used to analyze the complete cell cycle. Removal of CSF from the culture medium resulted in accumulation of the cells in the G1 phase. Eighteen hours after removal of CSF, 85% of the cells were arrested in G1 phase. Readdition of GM-CSF to such quiescent cells was followed by progression of the cells from G1 into S phase with a lag period of 10 hr. A similar lag period was observed when cells released from G2+M arrest progressed, in the presence of CSF, to S phase via the G1 phase. These findings indicate that deprivation of GM-CSF does not move PT-18 cells out of the cycle to a G0 phase but rather arrests them at early G1 phase. Finally, we demonstrate that, for the cells to progress through the cell cycle, the requirement for the presence of GM-CSF is limited to the first 6 hr of the 10-hr duration of the G1 phase.

Animals↗
PubMed1984

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers.

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.

Animals↗
PubMed1984

Chromosomal assignment of two murine genes controlling susceptibility to spleen focus formation by Rauscher leukemia virus.

Two murine genes affecting susceptibility to spleen focus formation by RLV were assigned to chromosomes. The gene Rv-2, which controls susceptibility to spleen focus formation, was assigned to chromosome 9, and closely linked to the Fv-2 gene, by using the two congenic strains B10.C(47N)/Sn and B6.C-H-7b/By. These congenic strains, which were previously shown to carry the BALB/cJ allele for susceptibility to FLV on chromosome 9, were also susceptible to RLV, as determined by number of foci on the spleens of infected mice. A second gene which contributes to the susceptibility to spleen focus formation by RLV was assigned to the X chromosome because hemizygous male offspring of crosses between resistant-strain females and susceptible-strain males (C57BL/6J X CBA/Lac) showed significantly lower numbers of foci/spleen than the female littermates and than the offsprings, males and females, of the reciprocal cross (CBA/Lac X C57BL/6J). We suggest this gene be called Rv-3.

Animals↗
PubMed1983

Rearrangement of Con A and WGA receptors on murine mastocytoma cell membranes: relationship to cell cycle.

Incubation of interphase murine mastocytoma cells with fluorescein-labeled wheat germ agglutinin (FITC-WGA) or with rhodamine-labeled concanavalin A (Rh-Con A) shows binding of the lectins to all the cells in a ring form distribution. Treatment of the cells with trypsin before addition of FITC-WGA, or with colchicine before addition of Rh-Con A, promoted rearrangement of the lectin binding sites into cap formation in 27-29% of the cells. Trypsin had no effect on cap formation by Rh-Con A, and colchicine had no effect on cap formation by FITC-WGA. When the same cells were pretreated consecutively with trypsin and colchicine followed by incubation with FITC-WGA and Rh-Con A, no increase in the percentage of cells bearing caps was observed. Moreover, the same cells showed co-capping with both lectins. Experiments with synchronous cultures of mastocytoma cells have indicated that capping induced by either lectin is maximal in the G2 phase and minimal in the G1 and S phases of the cell cycle. At the G2 phase 60% of the cells show cap formation either after trypsin-WGA treatment or after colchicine-Con A treatment. These data indicate that rearrangement of membrane receptors for Con-A and WGA in murine mastocytoma cells can occur only at certain phases of the cell cycle.

Animals↗
PubMed1983

Use of mice tolerant to lipopolysaccharide to demonstrate requirement of cooperation between macrophages and lymphocytes to generate lipopolysaccharide-induced colony-stimulating factor in vivo.

Injection of lipopolysaccharide (LPS) into mice was followed by a rapid elevation of colony-stimulating factor (CSF) in the serum. A second, challenging injection of LPS given 3 to 4 days later failed to induce elevated levels of CSF in the serum. Such mice tolerant to LPS were used as an experimental tool to identify the CSF-producing cells which respond to LPS. We observed that generation of LPS-induced CSF in mice tolerant to LPS could be restored by an intraperitoneal injection of spleen cells 24 h before the challenging injection of LPS. Depletion of the adherent cells from the spleen cells reduced the ability of the splenic lymphocytes to restore the capacity of the mice tolerant to LPS to generate serum CSF. Reconstitution of the splenic lymphocytes with 5% thioglycolate-elicited peritoneal macrophages, however, reestablished the restorative capacity of these cells, whereas almost no restoration was observed after direct injection of elicited peritoneal macrophages. These data suggest that the spleen cells are active in generating CSF, provided that macrophages are present and can interact with the splenic lymphocytes to generate LPS-induced CSF in the serum.

Animals↗
PubMed1983

Studies on the optimal conditions of CSF generation by endotoxic LPS and its PS derivative in mice.

Not only the endotoxic LPS preparations, but a non-toxic, lipid-free, non-mitogenic hydrolytic breakdown product of it (called PS) is also capable of inducing colony stimulating factor (CSF) release (1). Due to difficulties to reproduce above findings it became necessary to study the optimal conditions to obtain CSF active PS preparations. It was found that the CSF generating component of the highly heterogeneous PS mixture is sensitive to acidic hydrolyses, but it is less sensitive than the toxic site in the lipid moiety of the LPS. Carefully controlled optimal hydrolytic conditions give PS preparations which have less than one percent residual endotoxicity but maintained 40 to 80% of the original CSF generating capacity. Prolonged hydrolysis will destroy this activity too. Optimal dose of LPS and PS for CSF induction in mice differed widely. For LPS the optimal dose is 25 micrograms, injecting more gave a much reduced or non-detectable CSF level. Optimal dose for PS was 160 micrograms, and this generated a significantly higher CSF level than 25 micrograms LPS. At concentrations below 25 micrograms, LPS was clearly more active than PS. The CSF level reached its peak at 3-4 hours after other LPS or PS injection. Intravenous route was sometimes but not always more effective than intraperitoneal.

Animals↗
PubMed1983

Hemin stimulating effect on colony formation of leukemic and bone marrow cells.

The hemin enhancing activity on colony formation of leukemic and normal bone-marrow (BM) cells is described. The colony growth of Friend erythroleukemic cells (FL) and mastocytoma cells (M) was markedly enhanced. On the other hand, myeloid leukemic cells (P) and normal bone-marrow cells (BM) were only slightly affected. Inhibition of colony formation was observed with lymphoid leukemic cells (L). For M and BM cells, the horse serum could be replaced by BSA with preservation of hemin enhancing activity.

Animals↗
PubMed1982

New role for 15-hydroxyeicosatetraenoic acid. Activator of leukotriene biosynthesis in PT-18 mast/basophil cells.

Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line (PT-18) was used to investigate this problem. Exogenously supplied [14C]arachidonic acid is not appreciably converted to leukotrienes by untreated PT-18 cells. However, when the cells were preincubated with the lymphocyte product 15-hydroxyeicosatetraenoic acid (15-HETE), addition of [14C]arachidonic acid consistently resulted in a dose-dependent synthesis of large amounts of both [14C]leukotriene B4 and [14C]5-HETE. These metabolites were isolated by high pressure liquid chromatography, converted to the methyl ester trimethylsilyl ether derivatives, and the structures confirmed by gas chromatography-mass spectrometry. These findings indicate that 15-HETE induces a direct activation of a cryptic 5-lipoxygenase in these cells. The closely related 12-HETE was ineffective. The activation phenomenon occurs rapidly and is reversible. Furthermore, the activation appears to be highly cell- and enzyme-specific, since lipoxygenases in three primary cell types including one that contains a 5-lipoxygenase and six other cell lines did not show this specific induction of leukotriene biosynthesis by 15-HETE. This report is the first evidence that 15-HETE, a major arachidonate metabolite in lymphocytes, can act as a signal to activate leukotriene production by susceptible mast cells.

Animals↗
PubMed1982

Correlation between LPS-induced mitogenic and virogenic activities in mouse spleen cell cultures.

The present study provides direct evidence for a correlation between mitogenicity and induction of type C particles (virogenic activity) in cultures stimulated by LPS. This evidence is based on the following observations: 1) The complex of the antibiotic polymyxin B with LPS (LPS-PB) inhibits mitogenicity and virogenic activity in spleen cell cultures from BALB/c mice, but does not inhibit the generation of colony stimulating factor (CSF) from such cultures; 2) CBA/N, a genetically-defective mouse strain in its mitogenic response to LPS, was also defective in virus induction by LPS, but generated high levels of LPS-induced CSF.

Animals↗
PubMed1982

Inhibition by cholera toxin of clonal growth of murine granulocyte/macrophage progenitor cells in soft agar cultures.

Proliferation in vitro of the committed granulocyte-macrophage progenitor cell (CFUC) is inhibited by cholera toxin (CT) in a dose-dependent manner. This inhibition is reduced and counteracted by higher doses of colony stimulating factor (CSF), an obligatory growth stimulator of CFUC. Mixing of CT with its specific receptor, the monosialoganglioside, GM1, before exposure to bone marrow (BM) cells, blocks the toxin's effect, and a restoration of colony formation is achieved. A dose-dependent inhibition of CSF-induced colony formation is also observed in the presence of choleragenoid, the B subunit of the toxin which binds to the specific receptor on the cell surface, but is biologically inactive. Incubation of BM cells with CT prior to cloning in soft agar cultures supplemented with CSF inhibits clonal proliferation of CFUC, whereas a brief in vitro exposure of BM cells to CSF prior to CT protects the cells against subsequent CT inhibition. The ability of CT to inhibit the CSF-induced clonal proliferation of CFUC and the effectiveness of CSF in reducing and even counteracting CT inhibition suggests that CT, by binding to BM cells through the B subunit, might either directly or indirectly interfere with the stimulatory activity of CSF upon its target cells.

Animals↗
PubMed1981

Trisomic hemopoietic stem cells of fetal origin restore hemopoiesis in lethally irradiated mice.

Autosomal trisomy in the mouse is invariably associated with fetal or early postnatal death. Hemopoietic stem cells from fetuses trisomic for chromosome 12 or 19 can be rescued by transplantation into lethally irradiated mice. These trisomic cells restore hemopoiesis, including lymphopoiesis, in the irradiated mice and establish a permanent and almost complete engraftment. There is no evidence that hemopoietic cells with trisomy 12 or 19 are cytogenetically unstable.

Animals↗