Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.
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PMID:Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg). 844 92

To characterize angiogenic factors produced by ovine corpora lutea (CL) during early pregnancy, two experiments were performed. In Experiment 1, luteal explants from days 12, 18, 24, and 30 (n=4 ewes/day) after mating were incubated in serum-free medium for 6 h. Luteal-conditioned media (LCM) were evaluated for their ability to stimulate proliferation of endothelial and 3T3 cells, as well as migration of endothelial cells. Pools of the LCM (one pool/day) then were characterized biochemically. In Experiment 2, two pools of LCM from days 24 of pregnancy were evaluated for their effects on endothelial cell, 3T3 cell, and ovine luteal cell proliferation. These pools of LCM then were concentrated by ultrafiltration and subjected to heparin-agarose affinity chromatography with salt gradient (0-4 M NaCl in buffer) elution, and fractions were evaluated for mitogenic activity for endothelial and 3T3 cells. The resulting five peaks of mitogenic activity from heparin-agarose chromatography were characterized biochemically. The five peaks of mitogenic activity were further purified by using chromatography, then were concentrated and subjected to SDS-PAGE and Western analysis for FGF-2. Ovine CL from each day of early pregnancy secreted mitogens (P<0.05) for endothelial (285 +/- 8% of unconditioned media controls) and 3T3 (142 +/- 7%) cells as well as factors which stimulated migration of endothelial cell (153 +/- 8% of controls). LCM pool from day 24 of pregnancy also stimulated (P<0.05) proliferation of ovine luteal cells in a dose-dependent manner. In Experiment 1, mitogenic activity for endothelial cells was greater than 100 kDa, heat-labile, trypsin-sensitive and bound to DEAE-Sephacel and heparin-agarose columns, but not to a CM-Sepharose column. Antibody against FGF-1 did not affect mitogenic activity of LCM for endothelial and 3T3 cells, whereas treatment with FGF-2 antibody decreased (P<0.05) mitogenic activity of LCM for both endothelial and 3T3 cells. In Experiment 2, heparin-agarose affinity chromatography resolved five peaks of mitogenic activity: a non-heparin-binding peak that was specific for 3T3 cells, three heparin-binding peaks that were specific for endothelial cells, and one heparin-binding peak that was specific for 3T3 cells. In Experiment 2, heparin-, heat-, or trypsin-treatment and immunoneutralization with FGF-1 or FGF-2 antibodies influenced mitogenic activity of all of the peaks. Whereas SDS-PAGE demonstrated several bands of protein within each peak, Western analysis was unable to detect the presence of FGF-2 in any of the heparin-binding peaks. These data demonstrate that ovine CL from early pregnancy produce mitogenic factors that can be resolved into 5 separate peaks of activity with differing affinities for heparin. These data also indicate that the endothelial mitogens produced by CL of early pregnancy are immunologically related to, but biochemically distinct from FGF-2. Mitogens for endothelial and other cells likely play a role in regulation of luteal function during early pregnancy in sheep.
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PMID:Initial characterization of mitogenic activity of ovine corpora lutea from early pregnancy. 867 47

Proliferation of vascular smooth muscle cells with the accumulation of proteoglycans in the extracellular matrix is one of the significant changes found in atherosclerotic lesions. In order to clarify the relationship between pericellular proteoglycan and cell growth, we established a simple method for quantitatively estimating the amount of pericellular proteoglycans and investigated the effects of various growth factors on the synthesis of pericellular proteoglycans by cultured A10 rat smooth muscle cells. Analysis of trypsin accessible [35SO4]-labeled material in the pericellular area of the A10 cell culture by Q-sepharose anion-exchange chromatography showed two peaks. One peak, eluted at 0.55 M NaCl, disappeared after treatment with 2 mU/ml of heparitinase, indicating that heparan sulfates (HS) were present. The other peak, which eluted at 0.65 M NaCl, disappeared with 20 mU/ml of chondroitinase ABC, indicating the presence of chondroitin sulfates and dermatan sulfates (CS/DS). We estimated the effects of several growth factors on the synthesis of the pericellular proteoglycans by measuring heparitinase- and chondroitinase-ABC-sensitive radioactivities. Although PDGF-AB significantly stimulated cell proliferation and the synthesis of pericellular CS/DS, its dose-dependent effect on the cell growth did not coincide with that on the proteoglycan synthesis. IGF-I (1 nM) increased pericellular CS/DS but not the cell number, while basic FGF (1 nM) and EGF (1 nM) increased the cell number but not pericellular CS/DS. All the growth factors we examined had no effect on the synthesis of pericellular HS. These results indicate that growth factors increase pericellular proteoglycans independently of their mitogenic effects.
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PMID:Growth factors increase pericellular proteoglycans independently of their mitogenic effects on A10 rat vascular smooth muscle cells. 959 53

To characterize mitogenic factors produced by ovine endometrium during early pregnancy, endometrial explant-conditioned media (ECM) were obtained from ewes on day 12, 18, 24, or 30 after mating. These ECM contained mitogenic activity for both endothelial and 3T3 cells across all days. The endothelial mitogenic activity was greatest on day 24, whereas mitogenic activity for 3T3 cells did not differ across days. By ultrafiltration, ion exchange, and heparin-affinity chromatography, the endothelial mitogenic activity was found to have a molecular mass greater than 100 kDa, to be anionic, and to be heparin binding, respectively. Three peaks of endothelial mitogenic activity were recovered from heparin-affinity chromatography. The major peak, H3, was mitogenic for endothelial but not for 3T3 cells. H3 was further purified, and the single peak of heparin-binding activity, designated H3b, represented a 681-fold purification of endothelial mitogenic activity from endometrial ECM. H3 and H3b were heat labile and trypsin sensitive, and their biological activity was heparin enhanced. The majority of the endothelial mitogenic activity was immunoneutralized by antibodies against acidic and basic FGF. Nevertheless, we were unable to detect bFGF in H3 or H3b by Western immunoblot analysis. Thus, in this study we have extended our previous observations and demonstrated that (i) during early pregnancy the ovine endometrium produces mitogenic activity for both endothelial and 3T3 cells, (ii) the endothelial mitogenic activity is greatest on day 24 after mating. which corresponds with the onset of endometrial vascular growth, and (iii) the major endothelial mitogen has a high affinity for heparin, and although it is immunologically related to FGF, it differs from known FGF in its apparent molecular size and biological activities.
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PMID:Characterization of heparin-binding endothelial mitogen(s) produced by the ovine endometrium during early pregnancy. 966 10

Basic fibroblast growth factor (bFGF) is a polypeptide that is mitogenic for a wide variety of cell types. We used Northern blot analysis and immunohistochemistry to determine if bFGF is expressed in the nasal polyp tissue; bFGF messenger RNA was detectable in the polyps examined by Northern blot analysis. Strong immunostaining for bFGF was found in blood vessels and along the basement membrane of the epithelial cell layers. Basal epithelial cells and some infiltrating mononuclear cells also stained for bFGF. Proliferating cell nuclear antigen colocalized with bFGF to basal epithelial cells, endothelial cells, and areas of focal epithelial metaplasia. The polyp tissue was double-labeled with a mouse monoclonal antitryptase, a specific mast cell marker, and anti-bFGF. A significant number (65% +/- 19%) of the bFGF-positive mononuclear cells in the polyp tissues were positive for tryptase. These findings suggest that bFGF may contribute to the endothelial and epithelial proliferation in nasal polyp tissues and that mast cells are one source of this growth factor.
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PMID:Expression of basic fibroblast growth factor in nasal polyps. 979 21

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), FGF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.
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PMID:Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures. 995 Jun 79

Basic fibroblast growth factor (bFGF) is a potent mitogenic and chemotactic factor for endothelial cells and fibroblasts. To investigate the pathological role of bFGF in hypertrophic scar, we performed an immunohistochemical study on bFGF and bFGF receptor (bFGF-R) in hypertrophic scar (HS) including keloid, in comparison with normal scar (non-HS) and normal skin. To identify bFGF and bFGF-R positive cells, double immunostaining with antibody to mast cell (MC, tryptase) or tissue macrophage (CD68) was carried out. The expression of bFGF and bFGF-R in cultured fibroblasts from scars was also examined. In HS, many positive cells for bFGF or bFGF-R were observed between collagen bundles in addition to the positive area in normal skin. Although most of the positive cells for bFGF or bFGF-R were fibroblasts, the positive rates of bFGF in macrophages was also increased (p < 0.005). The positive rate of bFGF in MCs and the positive rates of bFGF-R in macrophages and MCs were not changed. No obvious difference was observed between non-HS and normal skin in the expression of bFGF and bFGF-R. Cultured fibroblasts from HS showed a strong nuclear staining of bFGF, but not from non-HS and normal skin. bFGF-R was equally expressed with a diffuse cytoplasmic pattern by fibroblasts from all sources. bFGF may play an important role in the pathological fibrotic process of HS in which fibroblasts are persistently activated. Cellular source of the abnormal bFGF in HS may be both fibroblasts themselves and macrophages.
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PMID:Expression of basic fibroblast growth factor and its receptor by fibroblast, macrophages and mast cells in hypertrophic scar. 1041 37

To test the hypothesis that basic fibroblast growth factor and mast cells play a key role in the phenotypic differences between human dystrophinopathies and hypertrophic feline muscular dystrophy, serial sections of dystrophin-deficient, carrier and normal cat muscle biopsy specimens were examined. They were stained immunohistochemically for dystrophin and different markers of differentiation such as desmin, vimentin and utrophin. Basic fibroblast growth factor was increased in the myofibers of dystrophic cats compared to normal controls and carriers. An association of basic fibroblast growth factor with fiber regeneration and necrosis was shown. The amount of mast cells was markedly increased in muscle tissue of dystrophic cats with a clear predominance of tryptase-positive cells present in large amounts in the endomysium. Mast cells, like basic fibroblast growth factor, were concentrated in areas of muscle fiber regeneration and necrosis. Our data concerning basic fibroblast growth factor and mast cells are consistent with a highly abnormal cellular environment in feline dystrophic muscle with very high levels of basic fibroblast growth factor which is likely modulated by mast cells.
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PMID:Mast cell proliferation and alterations in bFGF amount and localization are involved in the response of muscle to dystrophin deficiency in hypertrophic feline dystrophy. 1116 67

Elastic cartilage responds mitogenically in vitro to transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (basic FGF). We studied the effects of these growth factors separately or in a combination on porcine auricular chondrocytes in vitro and on the autologous elastic cartilage produced. Cells were harvested from the elastic auricular cartilage of 16- to 18-kg Yorkshire swine. Viability and quantification of the cells was determined. Cells were plated at equal concentration and studied in vitro in one of four identical media environments except for the growth factors: Group I contained Ham's F-12 with supplements but no growth factors, Group II also contained basic-FGF, Group III also contained TGF-beta, and Group IV also contained a combination of both growth factors. After 3 weeks in vitro, the cells were chemically dissociated with 0.25% trypsin. Cell suspensions composed of 3 x 10(7) cells/cc in 30% Pluronic F-127/Ham's F-12 were injected subcutaneously. Implants were harvested at 6, 8, 10, and 12 weeks of in vivo culture and then were examined with histologic stains. After 3 weeks of in vitro culture the total number of cells was as follows: Group I, 1.8 x 10(8); Group II, 3.5 x 10(8); Group III, 1.3 x 10(8); Group IV, 2.5 x 10(8). After 8 weeks of in vivo autologous implantation, the average weight (g) and volume (cm3) of each group was as follows: Group I, 0.7 g/0.15 cm3; Group II, 1.5 g/0.8 cm3; Group III, 0.6 g/0.1 cm3; Group IV, 1.2 g/0.3 cm3. Histologically, Groups I, II, and IV generated cartilage similar to native elastic cartilage, but Group III specimens demonstrated fibrous tissue ingrowth. Basic FGF produced the most positive enhancement on the quantity and quality of autologous tissue engineered elastic cartilage produced in this porcine model both in vitro and in vivo.
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PMID:The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage. 1122 26

Human acidic fibroblast growth factor (hFGF-1) is a potent mitogen and is involved in the regulation of key cellular process such as angiogenesis, differentiation, and morphogenesis. hFGF-1 is a signal peptide-less protein that is released into the extracellular compartment as a multiprotein complex consisting of S100A13, synaptotagmin (Syt1), and a hFGF-1 homodimer. Cu(2+) is known to play an important role in the formation of the multiprotein release complex. The source of Cu(2+) required for the formation of the multiprotein release complex is not clear. In this study, we show that the cytoplasmic C2A domain of synaptotagmin binds to Cu(2+) ions with high affinity. Results from the isothermal calorimetry (ITC), near-UV circular dichroism (CD), and absorption spectroscopy experiments suggest that four Cu(2+) ions bind per molecule of C2A domain. Far-UV CD and limited trypsin digestion analysis reveal that the C2A domain undergoes a mild conformational change upon binding to Cu(2+). Competition experiments monitored by ITC and fluorescence resonance energy transfer indicate that Cu(2+) and Ca(2+) ions share common binding sites on the C2A domain. Cu(2+) ions compete with and replace Ca(2+) ions bound to the C2A domain. Two-dimensional nuclear magnetic resonance spectroscopy data clearly show that Cu(2+) ions bind to the Ca(2+) binding sites in the loops (loops 1-3) located at the apex of the structure of the C2A domain. In addition, there is a unique Cu(2+) binding site located in the loop connecting beta-strands 7 and 8. It appears that the C2A domain provides the Cu(2+) ions required for the formation of the multiprotein FGF release complex.
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PMID:The C2A domain of synaptotagmin exhibits a high binding affinity for copper: implications in the formation of the multiprotein FGF release complex. 1626 43


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