摘要:

AbstractThe fluorescence emission of a single tryptophan residue present in both FGF-1 and FGF-2 was used as a structural probe to directly assess the interaction of the growth factors with heparin orβ-cyclodextran tetradecasulfate. About 20–25% of the fluorescence of either FGF-1 or FGF-2 is quenchable, and is dependent on sulfation of the ligands. The quenchable fluorescence is associated with about 20% of total FGF, suggesting the presence of two fluorospectrometric forms of the protein. The equilibrium dissociation constants, determined by this method, for heparin orβ-cyclodextrin tetradecasulfate binding to FGF-1 are about 1 nM, whereas the values for FGF-2 are 1 and 23 nM, respectively. The method provides a direct tool to evaluate FGF-ligand interaction and assess the structural integrity of the proteins.

ISSN:0897-7194    

DOI:10.3109/08977199409015046

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

AbstractThree preparations of recombinant basic fibroblast growth factor (bFGF, FGF-2) were evaluated, using a variety ofin vitrobioassays and immunoassays, by 11 laboratories in six countries for their suitability to serve as international standards (IS) for bFGF. Two preparations of human sequence bFGF showed broadly similar behavior in different assays, but the relative activity of the third preparation, a modification of the human sequence, showed more variability. All three preparations were predicted to be sufficiently stable to serve as an IS. On the basis of the results reported here, the World Health Organization (WHO) established one of the preparations (coded 90/712) as the international standard for basic fibroblast growth factor, and assigned it a content of 1600 International Units (IU) per ampoule. The IS may be obtained for use in the calibration of local standards by writing to NIBSC, PO Box 1193, Potters Bar, EN6 3QH, UK.

ISSN:0897-7194    

DOI:10.3109/08977199409015047

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

AbstractThe thrombin receptor has been shown to be a novel member of the family of G-protein coupled receptors (Vu, T.-K. H., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. (1991)Cell64, 1057–1068). This receptor appears to be activated through a thrombin-mediated proteolytic mechanism which exposes a“tethered ligand”responsible for receptor activation. In order to investigate the initial interactions of thrombin with this receptor, we have constructed cell lines which express high levels of the human thrombin receptor and studied the binding of various forms of thrombin to the cell surface. Analysis of transfected cells with thrombin receptor monoclonal antibodies identified a particular cell line (clone #5–18) which displayed>150,000 thrombin receptors per cell. Clone #5–18 appeared to express functional receptors since treatment with thrombin resulted in both a 15–20 fold increase of cytoplasmic phosphoinositide levels and a comparable shift in the EC50of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells.Binding of125I-α-thrombin to clone #5–18 did not reach equilibrium at 37°C. However, direct binding studies of125I-α-,125I-diisopropylphospho (DIP)-α-, and125I-β-thrombin to clone #5–18 demonstrated that binding at 4°C was saturable and reversible for each ligand. Analysis of the binding data revealed Kd's of 0.8 nM, 0.7 nM and 9.7 nM for125I-α-,125I-DIP-α- and125I-β-thrombin respectively. Association of125I-α-, DIP-α, andβ-thrombin could be competed by unlabelledα- and DIP-α-thrombin. Unlabelledβ-thrombin, which has a modified anion-binding exosite, was a poor competitor for125I-α-and125I-DIP-α-thrombin, but did compete for125I-β-thrombin. In addition, the hirudin54–65peptide competed at submicromolar concentrations for the binding ofα- and DIP-α-thrombin, but not forβ-thrombin. This peptide binds specifically at the anion-binding exosite ofα-thrombin and has been shown to have a lower affinity forβ-thrombin. These results demonstrate directly a high affinity interaction between thrombin and its receptor, and suggest that an important component is the high affinity association of the thrombin receptor with the anion-binding exosite of thrombin.

ISSN:0897-7194    

DOI:10.3109/08977199409015048

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

AbstractTransforming growth factor-beta (TGF-β) is produced by a number of tumor cell types including human malignant mesothelioma (MM), but its role as a direct or indirect factor in tumorigenesis is incompletely understood. We have investigated the expression of TGF-βisoforms by human and murine MM cells and have analysed the effects of inducible antisense RNA-mediated inhibition of TGF-βexpression on murine MM in vitro and in vivo. The results showed that (a) TGF-βand -β2 were produced by both human and mouse MM cells, (b) antisense RNA against either TGF-β1 or -β2 cross-inhibited both TGF-β1 andβ2 expression, (c) inhibition of TGF-βexpression reduced the anchorage-independent growth of MM cellsin vitroand the tumorigenicity of MM cellsin vivo, and (d) inhibition of TGF-βexpression led to increased T lymphocyte infiltration into tumors. The data suggest that TGF-βhas multiple tumor-enhancing effects in MM.

ISSN:0897-7194    

DOI:10.3109/08977199409015049

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

AbstractBone Morphogenetic Proteins (BMPs) induce the differentiation of Serum-free Mouse Embryo (SFME) cells into astrocytes (D'Alessandro et al., 1994) as demonstrated by change in morphology, increase in Glial Fibrillary Acidic Protein (GFAP) content and classification as both type 1 and 2 astrocytes. Further analyses showed that in the presence of BMP, cells which had differentiated into astrocytes were inhibited from proliferation. Moreover, removal of BMP resulted in a resumption of proliferation accompanied by a loss of GFAP expression over time, indicating that under thesein vitroconditions the differentiation was reversible. Since EGF is absolutely required for the survival of SFME cells, we examined the effect of its removal in the presence of BMP. Cell survival was>80% in the presence of BMP-2, 7 or 2/7 and<10% in the presence of TGF-β1. These data demonstrate that BMPs have effects on the proliferation, differentiation and survival of cells in the astrocyte lineage.

ISSN:0897-7194    

DOI:10.3109/08977199409015050

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

AbstractSerum-free mouse embryo (SFME) cells express Glial Fibrillary Acidic Protein (GFAP), a specific marker of the astrocyte lineage, when treated with either Transforming Growth Factor Beta (TGF-β) or calf serum. We examined the effects of the related Bone Morphogenetic Proteins (BMPs) which are expressed in the developing murine nervous system. Treatment with the heterodimers BMP-2/6 and 2/7 followed by the homodimers BMP-2, 4, 5, 6, and 7 induced higher levels of GFAP in these cells than either TGF-β1 or activin when tested at the same concentration. The BMP-induced cells resembled classically described astrocytes and were characterized by antibody markers as type 1 and type 2. In addition, these astrocytes also showed increased levels of the cell adhesion molecules CD44 and neural cell adhesion molecule (N-CAM), both known to be expressed by this cell type. These data clearly demonstrate that the BMPs function as differentiation factors as well as regulators of adhesion molecule expression for cells of the astrocyte lineage and suggest a key role in glial development in the nervous system.

ISSN:0897-7194    

DOI:10.3109/08977199409015051

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor

摘要:

AbstractInterleukin 3 (IL-3) induces proliferation and differentiation of mast cell progenitorsin vitro, whereas it induces granulocytosisin vivo. In this paper, a positive feedback mechanism of granulopoiesis was studied in order to elucidate the granulocytosis induced by IL-3 in mouse. IL-3 induced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in total bone marrow cells and a marrow adherent cell population. In fractionated marrow cell populations, a different expression pattern of induction by IL-3 stimulation was observed; GM-CSF was expressed in macrophages and fraction 1 (P<1.061) and 2(1.061

ISSN:0897-7194    

DOI:10.3109/08977199409015052

出版商:Taylor&Francis    

年代:1994

数据来源: Taylor