Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, several human bone marrow stromal cell lines have established and produced hematopoietic growth factors. One of these factors, a burst-promoting activity (BPA), was purified from 6 liters of serum-free conditioned medium cultured from stromal cell line KM-102, which was stimulated by phorbol myristate acetate (PMA) and calcium ionophore A23187. This stimulation induced 60 times more production of BPA than the unstimulated control culture. BPA was purified 4000-fold by sequential fractionation using ammonium sulfate precipitation, anion-exchange and lentil lectin affinity chromatographies, high performance gel filtration chromatography, and reversed phase high performance liquid chromatography. Purified BPA gave a single broad band of protein with a molecular weight of approximately 18 kd, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The concentration required for half maximal growth of early erythroid colonies was estimated as 10 pg/ml or 0.6 pM. At a higher concentration (125 pg/ml) this factor also stimulates the growth of granulocyte, macrophage, and eosinophil colonies in agar culture. The profile of amino acid composition is very similar to that of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) deduced from its complementary DNA sequence. The result of amino-terminal sequence analysis strongly suggests that the purified material consists of GM-CSF and tetrapeptide-deleted GM-CSF. Moreover, antibody against GM-CSF completely neutralized the biological activities of this factor. These results indicate that the human bone marrow stromal cell line secretes GM-CSF as a burst-promoting activity and GM-CSF may play a significant role in the interaction between stem cells and stromal cells in the hematopoietic microenvironment.
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PMID:A burst-promoting activity derived from the human bone marrow stromal cell line KM-102 is identical to the granulocyte-macrophage colony-stimulating factor. 313 50

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was purified from 3 liters of serum-free conditioned medium of the Hodgkin's tumor cell line L428 KSA. The conditioned medium contained a high specific activity of 2.5 X 10(5) units of total colony-stimulating factor per mg protein. Colony-stimulating factor activity was determined by colony formation by human fetal liver cells or mouse bone marrow cells. The latter bioassay discriminated colony-stimulating factor 1, a subclass specific for monocyte/macrophage production, and G-CSF, specific for granulopoiesis, from GM-CSF. The starting material contained predominantly GM-CSF with CSF-1 and G-CSF constituting 10% and 12%, respectively, of the total activity. A seven-stage purification scheme was employed. The first stage involved concentration by batch chromatography on calcium phosphate gel. Subsequent stages involved gel filtration on Ultrogel AcA44, affinity chromatography on concanavalin A-Sepharose, batch chromatography on calcium phosphate gel and high-performance liquid chromatography on C1 reversed-phase (TSK TMS-250), gel permeation and C8 reversed-phase columns. The purified material showed a single disperse band, having an Mr of 30,000, by silver staining on sodium dodecyl sulfate polyacrylamide gel electrophoresis. An amino-terminal sequence of 20 amino acids was determined in a gas-phase sequencer with 500 ng of purified material. The sequence was identical to that predicted from the cDNA sequence. It was active on human fetal liver cells with half-maximum colony formation at 1 X 10(-12) M, but was not active on mouse bone narrow cells.
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PMID:Human granulocyte-macrophage colony-stimulating factor purified from a Hodgkin's tumor cell line. 353 1

Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band of Mr 42,000-50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 degrees C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, and N-ethylmaleimide did not affect the biological activity at the concentration of 1 mM. However, CSF-1 activity was inhibited totally by the combination of 10 mM DTT and 1 mM SDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band of Mr 21,000-25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-beta-D-N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000-40,000, and to 18,000-20,000 in the presence of mercaptoethanol. Because of the specificity of endo-beta-D-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked "complex-type" units.
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PMID:Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells. 354 83

Radioiodinated granulocyte-macrophage colony-stimulating factor (125I-GM-CSF) binds to specific receptors (molecular weight approximately 50,000 daltons) on the murine myelomonocytic leukemia, WEHI-3BD+. At 4 degrees C 125I-GM-CSF remains on the surface of the cells and can be eluted by washing the cells with acidified isotonic buffer. When the cells are warmed to 37 degrees C, the 125I-GM-CSF is internalized rapidly (t 1/2: 7 min). The internalisation appears to be entirely receptor mediated and is independent of energy sources inhibited by sodium azide. This GM-CSF-mediated internalisation is not due to a general increase in the turnover of cell surface molecules as the specific binding of 125I-transferrin is not affected by incubation of WEHI-3BD+ cells with GM-CSF. The initial 125I released when the cells are warmed to 37 degrees C appears to be intact 125I-GM-CSF; however, after 2 h 80% of the 125I released was not precipitable with trichloroacetic acid and presumably represented degraded 125I-GM-CSF. Ammonium chloride or monensin reduced the release of 125I-GM-CSF from the cells, suggesting that the receptor-bound ligand was processed through the lysosomes. A considerable proportion of the internalised GM-CSF receptors were recycled to the surface and were available for ligand binding. Synthesis of new GM-CSF receptors contributed to the re-expression of GM-CSF receptors after down-regulation and it is possible that the GM-CSF enhances the synthesis of its own receptors.
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PMID:Internalisation and recycling of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on a murine myelomonocytic leukemia. 354 40

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.
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PMID:Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium. 403 29

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.
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PMID:Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor. 619 Aug 15

Colony-stimulating factor (CSF), a protein required for the in vitro formation of colonies composed of granulocytes and/or macrophages, was isolated from the urine of anemic patients by using a seven-step procedure. The purified, homogeneous CSF had a specific activity of 1.9 X 10(8) U/absorbance unit at 280 nm (AU). This represents an overall purification of 25,330-fold and a total recovery of 3.8%. Upon iodination of the protein, the radioactivity migrated on sodium dodecyl sulfate (SDS) gel electrophoresis as a single peak with an apparent molecular weight of 46,000; reduction with mercaptoethanol caused dissociation to a single component of molecular weight 23,000. Only the dimer is active in stimulating colony formation. Urinary CSF stimulates formation of colonies comprising only macrophages in the mouse bone marrow cell culture assay. A neutralizing antibody raised against mouse L-cell CSF did not neutralize the activity of the urinary CSF but did bind it. This may indicate that the relative positions of antibody binding sites and the active sites are different in these two glycoproteins.
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PMID:Purification of a human urinary colony-stimulating factor. 660 42

Interleukin 2 (IL-2), produced with and without co-stimulation by the Burkitt's lymphoma line Daudi, was purified 37,000-fold to apparent homogeneity from lymphocyte conditioned medium by (NH4)2SO4 precipitation, DEAE-cellulose ion-exchange chromatography, gel filtration, and chromatography on blue agarose and on Procion-red agarose. The purified IL-2 showed a 10(6) U/mg protein sp act. IL-2 produced in the absence of Daudi cells had a mol wt of 26,000 as measured by gel filtration and an isoelectric point of 6.7. This IL-2 showed a 16,000 and 17,000 mol wt in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IL-2, produced in the presence of Daudi cells (10(6)/ml), showed a mol wt of approximately 14,000, as measured by both gel filtration and SDS-PAGE, and an isoelectric point of 8.1. The purified IL-2 lacked detectable interferon (alpha and gamma), granulocyte-macrophage colony-stimulating factor, B cell growth factor, T cell-replacing factor, and thymocyte-differentiating activity and was free of any contaminating proteins as judged by silver staining in SDS-PAGE. All three molecular forms of IL-2 were biologically active at concentrations of 10(-11) - 10(-10) M, supporting the growth of human and murine cytotoxic T cell lines.
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PMID:Purification of human interleukin 2 to apparent homogeneity and its molecular heterogeneity. 698 Feb 56

To analyze the activity of the CD11c promoter during myeloid differentiation without the limitations of transient expression systems, we have stably transfected the myeloid U937 cell line with the pCD11C361-Luc plasmid, in which the expression of the firefly luciferase cDNA is driven by the CD11c promoter region -361/+43, previously shown to confer myeloid specificity to reporter genes. The stable transfectants (U937-C361) retained the ability to differentiate in response to phorbol-ester (PMA), sodium butyrate (SB), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other differentiating agents. U937-C361 differentiation correlated with increased cellular luciferase levels, showing the inducibility of the CD11c promoter during myeloid differentiation and establishing the U937-C361 cells as a suitable system for studying the myeloid differentiation-inducing capacity of cytokines, growth, factors, and other biological response modifiers. Unexpectedly, the inducibility of the CD11c gene promoter showed distinct kinetics and magnitude on the PMA-, SB-, GM-CSF-triggered differentiation. Moreover, SB synergized with either PMA or GM-CSF in enhancing both the CD11c promoter activity and the cell surface expression of p150,95 on differentiating U937 cells. Furthermore, we showed the existence of a c-Myb-binding site at -85, the importance of the -99/-61 region in the CD11c promoter inducibility during PMA- or SB-triggered differentiation, and the dependency of the GM-CSF and PMA responsiveness of the CD11c promoter on an intact AP-1-binding site located at -60. These results, together with the lack of functional effect of mutations disrupting the Sp1-and Myb-binding sites within the proximal region of the CD11c promoter, indicate that the myeloid differentiation pathways indicated by SB and phorbol esters (or GM-CSF) activate a distinct set of transcription factors and show that the myeloid differentiation-inducibility of the CD11c gene maps to the -99/-53 proximal region of the promoter.
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PMID:Granulocyte-macrophage colony-stimulating factor, phorbol ester, and sodium butyrate induce the CD11c integrin gene promoter activity during myeloid cell differentiation. 757 38

We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c-jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)-induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c-jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF-alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Arachidonic acid induces c-jun gene expression in stromal cells stimulated by interleukin-1 and tumor necrosis factor-alpha: evidence for a tyrosine-kinase-dependent process. 757 89


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