scholarly journals Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1924-1933 ◽  
Author(s):  
John P. de Koning ◽  
Amrita A. Soede-Bobok ◽  
Anita M. Schelen ◽  
Louise Smith ◽  
Daphne van Leeuwen ◽  
...  
Keyword(s):  
Distal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Signaling Mechanisms ◽  
Fold Reduction ◽  
Multiple Signaling ◽  
Stimulating Factor

Abstract The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

scholarly journals Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1924-1933 ◽  
Author(s):  
John P. de Koning ◽  
Amrita A. Soede-Bobok ◽  
Anita M. Schelen ◽  
Louise Smith ◽  
Daphne van Leeuwen ◽  
...  
Keyword(s):  
Distal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Signaling Mechanisms ◽  
Fold Reduction ◽  
Multiple Signaling ◽  
Stimulating Factor

The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

scholarly journals Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4759-4766 ◽  
Author(s):  
Keiko Okuda ◽  
Lorie Smith ◽  
James D. Griffin ◽  
Rosemary Foster
Keyword(s):  
Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Stat Pathway

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.

scholarly journals Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2148-2153 ◽  
Author(s):  
RM Barge ◽  
JP de Koning ◽  
K Pouwels ◽  
F Dong ◽  
B Lowenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Distal Region ◽  
Proximal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Tyrosine Residues ◽  
Membrane Proximal Region ◽  
Stimulating Factor ◽  
Proliferative Signaling ◽  
Factor G

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.

scholarly journals Differential role of segments of α-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Sakshi Aggarwal ◽  
Saroj Mishra
Keyword(s):  
Amino Acids ◽  
Pichia Pastoris ◽  
Fine Tuning ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Secretion Signal ◽  
Cargo Protein ◽  
Stimulating Factor

Abstract Background The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. Results Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57–70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30–43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47–49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30–43 in the WT-GCSF was partially restored (by 60%) when the Δ57–70 was added. The effect of Δ47–49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57–70 indicating the importance of the 47–49 region. A stimulatory effect of Δ57–70 was confirmed in the double deletion (Matα:Δ57–70;47–49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57–70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. Conclusions Among the different truncations (Matα:Δ57–70, Matα:Δ47–49, Matα:Δ30–43, Matα:Δ57–70;30–43, Matα:Δ57–70;47–49) in pro-peptide of α-MAT secretion signal, Matα:Δ57–70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30–43 and Matα:Δ47–49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

scholarly journals The Expression and Role of Multiple Forms of Granulocyte Colony-Stimulating Factor in Medaka Fish

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-36
Author(s):  
Ayame Ogawa ◽  
Ryo Yamagishi ◽  
Takeru Matsumoto ◽  
Satoshi Ansai ◽  
Kiyoshi Naruse ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Severe Neutropenia ◽  
Granulocyte Colony Stimulating Factor ◽  
Medaka Fish ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Hematopoietic Growth Factors ◽  
Gene Expressions ◽  
Stimulating Factor

Diversity in the repertoires of granulocytes, such as neutrophils, basophils, and eosinophils, has been known among more than seventy thousand vertebrate species. Granulocyte colony-stimulating factor (G-CSF; csf3) affects the differentiation, survival, and proliferation of neutrophils. However, the function of G-CSF in many animal species whose repertoire of granulocytes differs from that of mammals is not clear. We, therefore, attempt to investigate G-CSF function in adult d-rR strain of Medaka fish (Oryzias latipes). Of the three types of granulocytes, Medaka fish possess only neutrophils. First of all, we assessed the distribution of neutrophils by detecting myeloperoxidase (MPO) -positive cells in the various organs, including the kidney, spleen, heart, liver, and intestine. Among those tissues, the cell proportion of neutrophils was the highest in the kidney (approx. 15%). We then identified the genome sequences of Medaka G-CSFa1x1 and G-CSFa1x2, as orthologs of the mammalian genes encoding G-CSF. They are transcriptional variants and located on chromosome 19. Phylogenetic analysis and locus analysis revealed that these sequences are classified as G-CSFa of teleost fish. The synteny and functional domains of the Medaka G-CSF receptor (G-CSFR; csf3r) is also conserved with those of other species of G-CSFR. They are different from typical mammalian G-CSFs because they have N-linked glycans. We compared the expression of Olcsf3a1 and Olcsf3r in each tissue and found higher expression in the spleen, kidney, and bone of the Medaka fish. The Olcsf3a1x2 expressed much higher than that of Olcsf3a1x1 in the kidney and spleen. In the Medaka exposed to breeding water containing lipopolysaccharide (LPS; Escherichia coli 055: B5), the number of peripheral neutrophils at 3 hours significantly elevated; while that of splenic neutrophils at 3 hours reduced, suggesting that splenic neutrophils were released to the circulation. The expression of Olcsf3a1 mRNA increased at 12 hours after LPS exposure in the bone The number of neutrophils in the kidney increased at 24 hours after LPS exposure, demonstrating that Olcsf3a1 stimulated myelopoiesis shortly after splenic neutrophil release. To elucidate the role of OlG-CSFa1 in a steady-state, we generated the Olcsf3a1 deficient Medaka (G-CSFa1-deficient) by CRISPR/Cas9 genome editing using short guide RNA for the shared sequence of both G-CSFa1x1 and G-CSFa1x2. As we expected, the heterozygous G-CSFa1-deficient Medaka exhibited severe neutropenia. The number of peripheral neutrophils significantly decreased to 1.80×109 cells/L, whereas that of wild-type Medaka was 14.2×109 cells/L. Remarkably, the number of peripheral erythrocytes displayed 2.91×1012 cells/L in the G-CSFa1-deficient Medaka, whereas that of the wild-type Medaka was 5.85×1012 cells/L. We, therefore, tested the gene expressions of hematopoietic growth factors and found that the relative gene expression of erythropoietin decreased in the kidney of G-CSFa1-deficient Medaka. The results show that G-CSF-deficient Medaka should be a valuable model to examine specific functions of G-CSF in producing granulocytes and its synergistic effect on erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Definition of the Role of Tyrosine Residues of the Common β Subunit Regulating Multiple Signaling Pathways of Granulocyte-Macrophage Colony-Stimulating Factor Receptor

1998 ◽  
Vol 18 (2) ◽  
pp. 742-752 ◽  
Author(s):  
Tohru Itoh ◽  
Rui Liu ◽  
Takashi Yokota ◽  
Ken-ichi Arai ◽  
Sumiko Watanabe
Keyword(s):  
Colony Stimulating Factor ◽  
Tyrosine Residues ◽  
Gm Csf ◽  
Mapk Cascades ◽  
Macrophage Colony Stimulating Factor ◽  
The Common ◽  
Macrophage Colony ◽  
Multiple Signaling ◽  
Stimulating Factor

ABSTRACT Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces various functions, including the proliferation and differentiation of a broad range of hematopoietic cells. We previously reported that at least two distinct pathways are involved in human GM-CSF receptor signaling; both require the box 1 region of the common β subunit (βc). This region is essential for the activation of JAK2, which is necessary for all the biological functions of GM-CSF. The activation of JAK2 by GM-CSF leads to rapid tyrosine phosphorylation of cellular proteins, including the βc. However, the significance of βc phosphorylation with regard to the regulation of signaling molecules and the expression of GM-CSF functions is less well understood. Here we investigated the role of the cytoplasmic tyrosine residues of the βc by using a series of βc mutants expressed in murine BA/F3 cells. A mutant βc with all eight cytoplasmic tyrosines converted to phenylalanine (Fall) activated JAK2 but not SHP-2, MAPK cascades, STAT5, or the c-fos promoter in BA/F3 cells, and it did not effectively induce proliferation. Adding back each tyrosine to Fall revealed that Tyr577, Tyr612, and Tyr695 are involved in the activation of SHP-2, MAPK cascades, and c-fos transcription, while every tyrosine, particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired growth was also restored, at least partly, by any of the tyrosines. These results provide evidence that βc tyrosines possess distinct yet overlapping functions in activating multiple signaling pathways induced by GM-CSF.

scholarly journals Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4759-4766 ◽  
Author(s):  
Keiko Okuda ◽  
Lorie Smith ◽  
James D. Griffin ◽  
Rosemary Foster
Keyword(s):  
Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Stat Pathway

Abstract The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.

scholarly journals Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4435-4444 ◽  
Author(s):  
SS Tian ◽  
P Tapley ◽  
C Sincich ◽  
RB Stein ◽  
J Rosen ◽  
...  
Keyword(s):  
Immediate Early Genes ◽  
Signaling Molecules ◽  
Immediate Early ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Multiple Signaling ◽  
Stimulating Factor ◽  
Dependent Pathway

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of proliferation and differentiation of neutrophilic granulocyte precursor cells. G-CSF activates multiple signaling molecules, including the JAK1 and JAK2 kinases and the STAT transcription factors. To investigate G-CSF signaling events regulated by the JAK-STAT pathway, we have generated UT7-epo cells stably expressing either wild-type (wt) G-CSF receptor or a series of C-terminal deletion mutants. Gel mobility shift and immunoprecipitation/Western analysis showed that STAT5 is rapidly activated by G-CSF in cells expressing the wt G-CSF receptor, in addition to the previously reported STAT3 and STAT1. Mutants lacking any tyrosine residues in the cytoplasmic domain maintain their ability to activate STAT5 and STAT1 but cannot activate STAT3, implying that STAT5 and STAT1 activation does not require receptor tyrosine phosphorylation. We also observed significant changes in the ratio of STAT1:STAT3:STAT5 activated by various G-CSF receptor C-terminal deletion mutants. These mutant receptors were further used to investigate the role of JAKs and STATs in G-CSF-mediated responses in these cells. We found that JAK activation correlates with G-CSF-induced cell proliferation, whereas STAT activation is not required. We have also identified three classes of G-CSF immediate early genes, whose activation correlates with the activation of distinct JAK-STAT pathways. Our data show that, whereas c-fos is regulated through a pathway independent of STAT activation, oncostatin M, IRF-1, and egr-1 are regulated by an STAT5-dependent pathway and fibrinogen is regulated by an STAT3-dependent pathway. In conclusion, our results suggest that G-CSF regulates its complex biologic activities by selectively activating distinct early response genes through different JAK-STAT signaling molecules.

scholarly journals Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF)

1988 ◽  
Vol 256 (1) ◽  
pp. 213-218 ◽  
Author(s):  
P Wingfield ◽  
R Benedict ◽  
G Turcatti ◽  
B Allet ◽  
J J Mermod ◽  
...  
Keyword(s):  
Coli Strain ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Disulphide Bonds ◽  
Disulphide Bond Formation ◽  
Stimulating Factor ◽  
Factor G

Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

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