摘要:

AbstractVectors that generate antisense RNA targeted to granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA sequences were constructed using a strong viral promoter and a T cell-specific control element from the human CD2 gene. Stably transfected lymphoid clones expressing antisense RNA were tested for their ability to synthesize GM-CSF in response to stimulation with phorbol 12-myristate 13-acetate (TPA) and ionomycin. At early time points (4 and 8 hr) following stimulation, mean GM-CSF production by clones expressing antisense RNA was 10% the mean of control clones (p<0.001). Analysis of mean log data for 15 antisense clones demonstrated that GM-CSF production remained depressed at 12 and 24 hr time points, averaging 37% of that of the control clones (p<0.01). We conclude that antisense inhibition of growth factor production may be an effective strategy to investigate the role of specific growth factors in hematopoiesis in vivo in transgenic mice.

ISSN:0897-7194    

DOI:10.3109/08977199109000281

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractMouse macrophage BAM3 cells produced colony-stimulating factors (CSFs) after stimulation with bacterial lipopolysaccharide (LPS). By assaying the CSF using various interleukin 3-dependent cell lines, it was shown that most of the CSFs produced by BAM3 cells were granulocyte CSF (G-CSF). The granulocyte-macrophage CSF (GM-CSF) gene was also expressed in BAM3 cells after stimulation with LPS. When BAM3 cells were fused with the mouse renal adenocarcinoma cell line RAG which does not produce G-CSF, two of four hybrid cell lines constitutively produced large quantities of G-CSF. About 300 bp of the promoter region of mouse G-CSF chromosomal gene was inserted upstream of theEscherichia colichloramphenicol acetyltransferase gene, and introduced into BAM3, RAG and hybrid cells. The G-CSF promoter was activated by stimulation with LPS, in BAM3 cells, but was inert in RAG cells. On the other hand, there was significant constitutive CAT activity in the hybrid cells.

ISSN:0897-7194    

DOI:10.3109/08977199109000282

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractThe effect of recombinant interleukin-2 (IL-2) on the proliferation of T-cell depleted leukemic blasts was evaluated in 23 patients with acute myelogenous leukemia (AML). For this purpose, the effect of IL-2 on cell growth, [3H]-thymidine incorporation into the blasts and the expression of IL-2 receptors on cell surface using T-cell depleted blasts were studied. The results showed that IL-2 stimulated [3H]-thymidine incorporation significantly in blasts of 8 out of 23 cases of AML. An IL-2 induced increase in cell number was directly demonstrated in seven out of eight IL-2 responsive patients studied.IL-2 stimulated the proliferation of blasts in monocytic lineage (M4 and M5), but not all M4/M5 leukemics responded to rIL-2. Stimulation of the growth of leukemic cells was not correlated with the expression of Tac antigen on the cell surface, but it was significantly correlated with the expression of IL-2 receptor (IL-2R)βchain on the cell surface. These results indicate that IL-2 is an active growth factor in certain myeloid leukemia cells, especially of monocytic type.

ISSN:0897-7194    

DOI:10.3109/08977199109000283

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractWe have demonstrated that both recombinant and purified IL-2 exert a direct effect on quiescent human microvascular endothelial cells in vitro, causing the cells to enter the cell cycle and proliferate (Hicks et al., 1989). In this study we have identified IL-2 receptors (R) on both human umbilical vein (HUVEC) and neonatal foreskin (HCEC) endothelial cells. The techniques used to identify the receptors included proliferation studies, flow cytometry and immunofluorescence. Results indicate that both HUVEC and HCEC possess low numbers of receptors since both cell types proliferate in response to IL-2. The number of receptors on the cell surface vary according to passage number and culture conditions. Immunofluorescent studies show discrete areas of staining on the cell membrane. These combined results suggest that human vascular endothelial cells possess IL-2R.

ISSN:0897-7194    

DOI:10.3109/08977199109000284

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractUsing mild conditions of SDS-PAGE, i.e. no heating of the sample, and the PhastSystem (Pharmacia), we found that bFGF, either natural bovine or recombinant human migrated at a 27 kD position in addition to the classical 18 kD one. By the cell-blot technique, we found that the biological activity toward rat astroblasts and 3T3 mouse fibroblasts was always restricted to the 27 kD band. Partial heat denaturation experiments revealed a close correlation between the remaining biological activity of bFGF in solution and the ratio of the 27 kD band versus the 18 kD band seen on SDS gels. These observations suggest that the bFGF which is biologically active in solution migrates at an apparentMrof 27 kD in our conditions of electrophoresis, keeping its biological activity after electrophoresis, and the molecules which are inactive (denatured) in solution migrate at 18 kD and remain inactive. These experimental conditions, in which the biological activity appears to be preserved, could be referred to as“non-denaturing SDS-polyacrylamide gel electrophoresis”and could be useful, associated to cell-blot, for the search and characterization of new growth factors active on cells in culture.

ISSN:0897-7194    

DOI:10.3109/08977199109000285

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractWe have previously shown that only adult brain contained a detectable amount of high affinity receptors for basic Fibroblast growth factor (bFGF) whereas adult liver, kidney, lung, intestine or stomach showed only low affinity binding sites. We now have studied and compared the distribution of the receptors for acidic Fibroblast growth factor (aFGF) with that of bFGF receptors in the same tissues. Membrane binding of15I-aFGF was time dependent, reversible and displaced by an excess of unlabeled aFGF. Scatchard analyses of binding data obtained with all tissue membrane preparations revealed the presence of at least one class of low affinity/high capacity interaction sites characterized by apparentKdvalues ranging from 3.9 to 6.9×10−8M.Interestingly and as for bFGF, high affinity receptors for aFGF could be detected only in adult brain membranes. Cross-linking and Scatchard analyses indicate that this family of interaction was characterized by four molecular species of 175, 125, 95 and 70 kDa and by an apparentKdvalue of 1.8×10−10M. Moreover, cross-competition binding assay revealed that these brain high affinity receptors were common for both acidic and basic FGF. These results suggest that these growth factors may share identical functions mediated by the same receptors highly expressed in the brain. Using a cDNA probe for theBekform of FGF receptors, we were able to show that all the tissues studied expressed this mRNA (4.5 kb transcript) but probably not in sufficient amounts to account for the number of high affinity receptors that we detected only in the brain.

ISSN:0897-7194    

DOI:10.3109/08977199109000286

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractIncubation of Swiss 3T3 murine fibroblasts at low temperatures induces phosphorylation on tyrosine of a transmembrane protein of 175 kDa. This phenomenon is time and temperature dependent and reaches a maximum after 2 hr at 4°C. The 175 kDa protein phosphorylatedin vivoat low temperatures can be immunoprecipitated by phosphotyrosine antibodies and displays auto-kinase activityin vitroin the presence of radiolabelled ATP. This molecule was found to react with anti-peptide antibodies directed against the product of the HER2/neuproto-oncogene only when immunoprecipitated with phosphotyrosine antibodies from cold-stimulated cells. Activation of protein kinase-C by treatment of the cells with phorbol esters, bombesin or PDGF inhibits the effect of the exposure to low temperatures. Phosphorylation of p1 75 is not induced by treatment of the cells with the phosphatases inhibitor sodium ortho-vanadate. These results suggest, that, at low temperatures, the tyrosine kinase associated with the putative receptor encoded byc-neuis activated by physicochemical modifications of the plasma membrane.

ISSN:0897-7194    

DOI:10.3109/08977199109000287

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

AbstractTransforming growth factor-β1 (TGF-β1) is synthesized and secreted as a biologically latent complex. It has been proposed that one role of the latent complex is to prevent premature interaction of ligand and receptor intracellularly during biosynthesis (Wakefield et al,J. Cell Biol.(1987)105, 965-9751. To test this hypothesis, the endoplasmic reticulum retention sequence Lys-Asp-Glu-Leu (KDEL) was added to the C-terminus of the wildtype TGF-β1 coding sequence, and to a construct in which mutagenesis of two cysteine residues in the precursor pro region results in the synthesis and secretion of active, as opposed to latent, TGF-β. Addition of either SEKDEL, or the control sequence SEKDVS to the TGF-β1 protein abolished biological activity. Western blot analysis indicated that the extended gene products are synthesized, but that the extension sequence partially interferes with the normal dimerization of the protein product, and totally inhibits the normal proteolytic processing and glycosylation of the precursor protein. The data suggest that correct folding of the highly conserved C terminus of TGF-PI is critical for subsequent proteolytic cleavage and glycosylation at sites that are quite distant in the primary sequence. Thus molecular strategies for the generation of TGF-βantagonists or superagonists should avoid extensive modification of this region of the molecule. Since synthesis of the endogenous TGF-β1 is unaffected by the presence of the mutated analog, the data further indicate that transfection with the KDEL-extended TGF-β1 sequence cannot be used as a dominant negative mutation to prevent secretion of the endogenous TGF-Pβprotein.

ISSN:0897-7194    

DOI:10.3109/08977199109000288

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor