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D Barritault

Publications and source records attributed to D Barritault.

At least 91 records · Page 5Linked to original sources
PubMed1991

Mitogenic properties of a new endothelial cell growth factor related to pleiotrophin.

A growth factor was isolated from a neutral pH extract of adult bovine brain. Purification of this polypeptide was achieved by a three step procedure including cationic exchange, heparin-Sepharose affinity and Mono S chromatography. This heparin binding protein had a molecular weight of 18,000 as assessed by silver-stained SDS-PAGE and was not immunologically and structurally related to acidic or basic FGF. Freshly purified protein had a maximal mitogenic effect on bovine brain capillary cells at a concentration of 100 pM. Microsequencing revealed an unique amino-terminal sequence homologous to heparin-binding growth-associated molecule (HB-GAM), a neuronal maturation protein, to pleiotrophin (PTN), a fibroblast cell growth factor and to one form of the putative protein product of the MK gene, a retinoic acid induced-gene.

Amino Acid Sequence↗
PubMed1991

Enhanced MYCN oncogene expression in human neuroblastoma cells is associated with altered FGF receptor expression and cellular growth response to basic FGF.

We have examined the effect of human basic fibroblast growth factor (bFGF) on the proliferation of human neuroblastoma cells with normal and enhanced MYCN oncogene expression. bFGF stimulated the proliferation of the neuroblastoma cells with enhanced, but not normal, MYCN expression. Both cell species express FGFR-1, but not FGFR-2, receptors and both harbor FGF receptor species of Mr 145.000, but they differ in their pattern of lower and higher-molecular weight FGF receptor species. Our results demonstrate that enhanced MYCN expression confers to neuroblastoma cells the ability to respond to bFGF, possibly by inducing functional FGF receptors. This mechanism may contribute to the advanced malignant phenotype of human neuroblastomas with enhanced MYCN expression.

Blotting, Northern↗
PubMed1991

Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain.

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured, aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

Animals↗
PubMed1991

Biotinylated basic fibroblast growth factor is biologically active.

Basic fibroblast growth factor (bFGF) was modified by biotinylation via amino group substitution, using biotin-N-hydroxysuccinimide ester at molar reaction ratios of 20, 200, and 2000 per bFGF molecule (respectively named bio-bFGF.20, bio-bFGF.200, and bio-bFGF.2000). The biotinylated bFGF derivatives, bio-bFGF.20 and bio-bFGF.200, conserved the same affinity for heparin as native bFGF, in contrast to bio-FGF.2000 which lost this property. Bio-bFGF.20 and bio-bFGF.200 were as effective as native bFGF in their capacity to compete with 125I-bFGF for binding to bFGF receptor on bovine brain membranes. The biological activity of these bFGF derivatives was tested on CCL39 cells; bio-bFGF.20 and bio-bFGF.200 were as able as native bFGF to promote growth of CCL39.

Biotin↗
PubMed1991

MCF7 mammary cancer cells respond to bFGF and internalize it following its release from extracellular matrix: a permissive role of cathepsin D.

High and low affinity receptors for basic fibroblast growth factor (bFGF) were detected by binding experiments on MCF7 breast cancer cells. These cells were stimulated for growth by physiological concentrations of bFGF. However, in contrast to endothelial cells, these MCF7 cells did not produce detectable amounts of biologically active bFGF or related heparin-binding growth factor(s) of the FGF family. In vitro, the cathepsin D (cath-D) secreted by MCF7 cells was able to digest extracellular matrix (ECM) and to release ECM-bound 125I-bFGF. When MCF7 cells were cultured on ECM containing bound bFGF, they internalized bFGF, which was slowly and partially proteolyzed in the cells. Processing occurred in acidic compartments and was inhibited by leupeptin. Pepstatin A, an inhibitor of aspartyl proteases, had no effect on the processing but reduced internalization of matrix-bound bFGF by MCF7 cells. Taken together, these results suggest a cooperation between cath-D and bFGF, by which the protease could facilitate the release of bFGF from ECM and its subsequent use by breast cancer cells and/or adjacent cells involved in angiogenesis.

Breast Neoplasms↗
PubMed1991

Internalization of basic fibroblast growth factor by Chinese hamster lung fibroblast cells: involvement of several pathways.

Subconfluent Chinese hamster lung fibroblast cells (CCL39) which express high- and low-affinity binding sites for basic fibroblast growth factor (bFGF) were used to study bFGF internalization. Kinetics at 37 degrees C indicated that this process was complex and involved various pathways with regard to the ligand concentration used. Internalization with 6 to 45 pM of 125I-r-bFGF led to a steady state that lasted up to 3 h without any appearance of 125I-labeled degradation products in the cell-culture medium, suggesting that the endocytosis reached equilibrium. Furthermore, binding data at steady state, at 37 degrees C, revealed a two-phase Scatchard curve suggesting the involvement of two families of interaction sites in the process of internalization. Apparent dissociation constants were estimated to be 20 pM and 58 nM, respectively, and the number of bFGF molecules involved per cell, 4300 and 1.3 x 10(6), respectively. These data were in good agreement with those obtained from binding experiments at equilibrium at 4 degrees C. Besides, higher concentrations of 125I-r-bFGF (greater than 47 pM) induced an internalization process which did not reach steady state and was not saturable. These results suggest that CCL39 cells could internalize bFGF by various pathways involving high- and low-affinity binding sites.

Animals↗
PubMed1991

High affinity receptors to acidic and basic fibroblast growth factor (FGF) are detected mainly in adult brain membrane preparations but not in liver, kidney, intestine, lung or stomach.

We 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 of 125I-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 apparent Kd values ranging from 3.9 to 6.9 x 10(-8) M. 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 apparent Kd value of 1.8 x 10(-10) M. 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 the Bek form 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.

Animals↗
PubMed1990

High-performance affinity chromatography of basic fibroblast growth factor on polystyrene sulphonate resins modified with serine.

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor, so it was usually purified by affinity chromatography on a heparin-grafted support. It was previously observed that, among several modified polystyrene that exhibit anticoagulant heparin-like properties, insoluble polystyrenes modified by chlorosulphonation substituted with serine (PSSer) develop specific interactions with bFGF in solution. These PSSer resins were used as stationary phases in high-performance affinity chromatography in order to separate the radiolabelled growth factor from a bovine brain crude extract. The growth factor is strongly bound to the solid phase, as demonstrated by the adsorption isotherms. It can be adsorbed on the resin at low ionic strength and be desorbed by raising the salt concentration in the eluent. The effects of flow-rate, of the initial buffer and of the slope of the salt gradient on the adsorption and on the desorption of bFGF were investigated in order to study the thermodynamic and the kinetic parameters of the interactions and to define the optimum conditions for a fast and efficient separation of the growth factor.

Animals↗
PubMed1990

Evidence of high and low affinity binding sites for basic fibroblast growth factor in mouse placenta.

The placenta has been shown to contain bFGF, but the presence of specific binding sites for this growth factor in this tissue remained to be established. In order to study the role of bFGF in the placenta growth, we looked for specific binding sites on mouse placental cell membranes at days 12, 14, 16, and 18 of pregnancy. At day 12, Scatchard analyses indicated that two classes of specific interaction sites for bFGF were detected. One class of high affinity binding sites was characterized by an apparent Kd of 10 pM and a binding capacity of 10 fmoles per mg of membrane protein. A second class of low affinity binding sites was detected with an apparent Kd of 60 nM and a binding capacity of 26 pmoles per mg of membrane protein. At days 14, 16 or 18, Scatchard analyses only showed low affinity binding sites with an apparent Kd of 24 nM and a binding capacity of 230 pmoles per mg of membrane protein. The characterization of these binding sites was performed by cross linking experiments that revealed two forms of specific complexes. This result suggested that the high affinity binding sites correspond to putative receptors with relative molecular masses equal to 65,000 and 85,000. The dramatic decrease of the high affinity receptor number after the 12th day of pregnancy, which is synchronous with the 9-fold increase of the low affinity binding site number, suggests that the biological activity of bFGF could be regulated by a balance between both the numbers of high and low affinity binding sites on placenta cell membranes. Thus, as it was shown for other growth factors, bFGF could only be involved at specific pregnancy stages.

Animals↗
PubMed1990

Affinity chromatography of fibroblast growth factors on substituted polystyrene.

The heparin-binding growth factors aFGF and bFGF (acidic and basic fibroblast growth factor) from crude bovine brain extract were co-eluted with purified [125I]aFGF and/or [125I]bFGF as tracers from heparin-Sepharose and from several insoluble substituted polystyrenes used as stationary phases in low-pressure affinity chromatography. The ability of the resins to isolate FGFs was determined by measuring the eluted radioactivity. It was demonstrated that the various substituted polystyrene resins retain [125I]aFGF and [125I]bFGF with different specificities according to the chemical nature of the substituted groups bound to the polystyrene support. Bifunctional resins substituted with sulphonate and phenylalanine sulphamide groups adsorbed both [125I]aFGF and [125I]bFGF whereas bifunctional resins substituted with sulphonate and sulphamide serine adsorbed only [125I]bFGF. These stationary phases could be adapted to high-performance affinity chromatography and used to isolate growth factors of the FGF family.

Animals↗
PubMed1990

Proliferating satellite cells express acidic fibroblast growth factor during in vitro myogenesis.

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.

Antibodies↗
PubMed1990

Acidic fibroblast growth factor is a modulator of epithelial plasticity in a rat bladder carcinoma cell line.

During normal embryogenesis and neoplastic transformation epithelia change their state of differentiation and degree of cohesiveness. It is thus essential to identify the signals modulating these transitions. We report here that acidic fibroblast growth factor (FGF) induces cells derived from a rat bladder carcinoma to lose their epithelial character and to acquire some properties typical of mesenchymal cells. The structurally related basic FGF did not have such an effect; both factors, however, had a mitogenic activity for these cells. Two distinct populations of receptors for acidic FGF and basic FGF were distinguished by their ligand-binding characteristics. The observations that both acidic and basic FGFs had a mitogenic effect on NBT-II cells and that only acidic FGF caused cell dissociation and dispersion strongly suggest that these two biological activities could be medicated through distinct signaling pathways.

Animals↗