Search PubMed⌕ Search

Biomedical subjects

Y Courtois

Publications and source records attributed to Y Courtois.

At least 127 records · Page 7Linked to original sources
PubMed1991

A new heparin binding protein regulated by retinoic acid from chick embryo.

A 19 KDa heparin binding protein was previously purified from chicken embryos. Essentially localized within basement membranes in early embryonic tissues, this protein is very rich in basic and cystein residues. Its N-terminal fragment is similar to corresponding fragment of two other proteins expressed during embryogenesis and postnatal period. Its synthesis and secretion are induced by retinoic acid in chicken myoblasts and fibroblasts. This new retinoic acid induced heparin binding protein (RI-HB) does stimulate neurite outgrowth and proliferation on PC12 cells. These results suggest that retinoic acid could regulate some aspect of differentiation and development by inducing the synthesis of a new family of growth and neurotrophic factors.

Adrenal Gland Neoplasms↗
PubMed1991

In vitro kinetics of basic fibroblast growth factor diffusion across a reconstituted corneal endothelium.

We have studied the ability of bFGF to traverse and be trapped within basement membranes. An extract of EHS tumor (matrigel) coated on culture chamber filters was used as an in vitro model of basement membranes. Our results showed a slow diffusion of bFGF dependent on the amount of low affinity binding sites present within the matrigel. High amounts of bFGF and heparin increased the initial rate of diffusion by displacement of bFGF bound to matrigel. An in vitro corneal endothelium model (endothelial cells overlying matrigel) was also developed. This monolayer, with a weak permeability, decreased the kinetic rate of bFGF diffusion compared with matrigel alone. These results indicate that modulation of bFGF distribution in a tissue by a basement membrane is dependent on bFGF concentration, basement membrane composition, permeability of associated cells, and local presence of heparin. This selective control may be a regulating step in bFGF action.

Animals↗
PubMed1991

Differential localization by in situ hybridization of specific crystallin transcripts during mouse lens development.

The embryonic development of the mammalian lens is well known at the biochemical and histological level. However few data are available at the molecular level concerning gene expression during the continuous differentiation of the lens. In the present study, we have investigated by in situ hybridization the changes in the distribution of mouse crystallin mRNA as a marker of differentiated lens cells, during development of the lens primordium, when tissue interactions are known to be essential. The transcripts of alpha and beta crystallins are first detected at the early elongation stage of primary fibres; gamma-crystallin-transcripts do not appear until the late elongation phase. All areas of the lenses exhibited crystallin mRNA until the beginning of secondary fiber formation at 18 days of development. Hybridization for alpha and beta crystallin is confined at that time to the equatorial part of the lens. The gamma crystallin transcripts are no longer found in the equatorial region after 1 post-natal day, but remain in the lens core, decreasing gradually. A possible mechanism is discussed.

Animals↗
PubMed1991

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved.

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.

Animals↗
PubMed1991

Localization of acidic fibroblast growth factor in proliferative vitreoretinopathy membranes.

The pathogenesis of proliferative vitreoretinopathy (PVR) membranes remains poorly understood. We have studied the presence of acidic fibroblast growth factor (aFGF), a potent mitogen for many cells, within these membranes. We have used affinity purified monospecific anti-aFGF polyclonal antibodies, in conjunction with highly sensitive immunofluorescence techniques. The labelling was exclusively localized to cell bodies and was absent from the extracellular matrix. Double labelling techniques revealed that all cytokeratin positive cells (probably pigmented epithelial cells) and macrophages contained aFGF-like immunoreactivity, whilst glial cells were unlabelled. Appropriate controls indicated the specificity of the antibodies. Hence, the presence of this mitogenic molecule within certain cell types constituting PVR membranes may contribute to the pathogenesis.

Animals↗
PubMed1991

Effect of FGFs on adult bovine Muller cells: proliferation, binding and internalization.

A new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS), alpha cristallin (alpha C) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC50 = 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of 125I-bFGF bound to the Muller cell receptors did not occur at 4 degrees C. At 37 degrees C, however, there was a rapid decrease in receptor-bound 125I-bFGF due to the downregulation of bFGF receptors. Concomitantly 125I-bFGF appeared inside the Muller cells. After 2 h, 125I-bFGF began to be degraded and after 6 h three fragments of 16 kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these factors may be the mediators involved in the communication between glial cells and neurons.

Animals↗
PubMed1991

Acidic fibroblast growth factor overexpression in corneal epithelial wound healing.

aFGF expression was studied in normal and regenerating cornea of adult rats. aFGF mRNA and proteins were expressed mainly in corneal epithelium but not in stroma. After burning of the epithelium by iodine vapours, the intact epithelial cells migrated to cover the wounded area during the first 4 days and then divided to reconstitute a normal multilayered epithelium 6 days after injury. aFGF mRNA localized by in situ hybridization on regenerating epithelium showed a peak between 6 hr and 2 days after denudation, decreasing to basal levels 6 days later. This induction of aFGF mRNA preceded the increased amount of aFGF peptides, as assessed by indirect immunofluorescence staining. Thus aFGF overexpression is clearly correlated with active migration in epithelial wound healing.

Animals↗
PubMed1991

[FGF and physiopathological implications].

The role of FGF in retinal pathologies can be considered in two respects: 1-as a causative agent (for instance, stimulating aberrant growth or abnormally affecting some aspects of cellular behaviour), or 2-as a possible therapeutic drug (for instance, its ability to rescue neuronal cells from programmed or surgically-induced death), there is evidence for both: the retina, particularly the photoreceptors (PR), seems to be highly succeptible to a host of genetic defects, for instance, in the rat mutant RCS a defect in the pigmented epithelial cells (RPE) leads to the destruction of the PR. Transocular injection of FGF into RCS rats reduces the rate of PR cell loss. On the other hand, we have recently identify a difference in FGF-Receptor cell biology in the RCS mutant which may underline this particular dystrophy. Cultures of purified RPE cells were established from the dystrophic RCS rat and its congenic normal sighted control, and the number and affinity of cell surface FGF-R determined by incubation with 125I-bFGF. Scatchard analysis of the bining data indicated that whereas affinites were in both strains around 60pM, there was a large reduction in receptor number, about 70% in the mutant. Analysis of other FGF/FGF-R criteria, such as mitogenic activity, receptor molecular weight and the profile of FGF degradatoin showed no significant differences between the strains. Thus, defects in the FGF/FGF-R regulating system may actually led to retinal degeneration (RCS rat). But, once again, the underlying principles of FGF mechanisms of action in vivo are at present too incompletely understood to permit rationalization of these various observations.

Animals↗
PubMed1990

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.

Animals↗
PubMed1990

Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells.

We postulated that Fibroblast Growth Factor (FGF) involved in fetal or regenerative morphogenesis of skeletal muscle originated from this tissue. Using a bovine retina cDNA probe encoding acidic FGF, we showed that growing muscles from bovine fetuses express this mRNA, but that this expression is reduced in neonate muscles. Cultures of proliferating satellite cells isolated from adult rat muscles expressed aFGF mRNA strongly but bFGF mRNA weakly; these mRNAs disappeared in cells differentiated into myotubes. 10(-7)M 12-O-tetradecanoyl phorbol -13-acetate (TPA) increased aFGF mRNA expression in both proliferating and differentiated satellite cells. Contrastingly, proliferating L6 myogenic cells only expressed aFGF mRNA significantly under TPA treatment. Therefore, the satellite cells did seem to be a possible source for FGF, especially aFGF, which might regulate the myogenic process.

Animals↗
PubMed1990

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.

Biological Assay↗
PubMed1990

Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues.

Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.

Animals↗
PubMed1990

The growth and behaviour of rat retinal Müller cells in vitro. 1. An improved method for isolation and culture.

Eyeballs were enucleated from young (postnatal day 8-12) pigmented rats and the retinas were dissected free after soaking the globes overnight in growth medium. The retinas were digested with enzymes, dissociated and maintained in stationary culture in 10% serum supplemented growth medium. Cultures displayed extensive cellular outgrowth after 1-5 days, with abundant fusiform and epithelioid cells. Removal of aggregates and cellular debris after 6-7 days yielded a purified flat cell preparation, which could be maintained either as a primary culture for several weeks or passaged repeatedly as rapidly proliferating epithelioid cells. Staining with monoclonal antibodies RET-G1, G3 and G7, and polyclonal S-100, glutamine synthetase and carbonic acid anhydrase antisera, all markers for Müller cells, showed positive labelling of all cells present in these purified cultures, both primary and passaged cells. This contrasted with the use of RET-G2, anti Factor VIII and anti glial fibrillary acidic protein (GFAP) antibodies. RET-G2, another Müller cell marker, failed to recognize passaged cells. Anti Factor VIII also did not label any cells, and anti GFAP stained very few cells: these remained associated with aggregated material so that vigorous washing to remove loosely adherent tissue from primary cultures resulted in the total absence of GFAP positive cells. In addition, no GFAP positive cells were detected in passaged cells or in cells regrown following freezing and storage. The Müller cell nature of these flat cells in soaked retinal cultures was further supported by the specific uptake of 5-bromo-deoxyuridine by nuclei located within the inner nuclear layer of retinal fragments in vitro. Hence the soaking treatment greatly reduces the number of surviving astrocytes whilst stimulating the rapid growth of cells expressing many properties of mature retinal Müller cells.

Animals↗
PubMed1990

Analysis of lens protein synthesis in a cataractous mutant mouse: the Cat Fraser.

The cataract produced by the dominant Cat Fraser gene in mouse is associated with quantitative changes in lens proteins (crystallin) and with capsule abnormalities. We have analyzed and compared the protein synthesis in control and mutant lenses using [3H]leucine and [3H]proline incorporation. The specific activities of free [3H]leucine in the intracellular pools of the two mouse strains were identical, while the incorporation of both labelled amino acids in proteins was largely increased in Cat Fraser lens. These data indicate that the higher labelling of Cat Fraser lens proteins reflects a true change in the cellular synthesis activity by Cat Fraser lens cells. Despite the enhanced type IV collagen synthesis by Cat Fraser epithelial cells, the amount of type IV collagen in Cat Fraser capsule is lower than in control. This altered type-IV collagen metabolism may disturb the structure of Cat Fraser capsule which becomes thicker.

Animals↗
PubMed1990

Related fibroblast growth factor receptor genes exist in the human genome.

We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors. This has been demonstrated by specific binding assays and chemical cross-linking experiments using 125I-labeled growth factors. It appears, therefore, that the human genome contains at least two distinct genes, for TK14 and FLG, that code for related fibroblast growth factor receptors.

Amino Acid Sequence↗
PubMed1990

Localization of basic fibroblast growth factor binding sites in the chick embryonic neural retina.

We have investigated the localization of basic fibroblast growth factor (bFGF) binding sites during the development of the neural retina in the chick embryo. The specificity of the affinity of bFGF for its receptors was assessed by competition experiments with unlabelled growth factor or with heparin, as well as by heparitinase treatment of the samples. Two different types of binding sites were observed in the neural retina by light-microscopic autoradiography. The first type, localized mainly to basement membranes, was highly sensitive to heparitinase digestion and to competition with heparin. It was not developmentally regulated. The second type of binding site, resistant to heparin competition, appeared to be associated with retinal cells from the earliest stages studied (3-day-old embryo, stages 21-22 of Hamburger and Hamilton). Its distribution was found to vary during embryonic development, paralleling layering of the neural retina. Binding of bFGF to the latter sites was observed throughout the retinal neuroepithelium at early stages but displayed a distinct pattern at the time when the inner and outer plexiform layers were formed. During the development of the inner plexiform layer, a banded pattern of bFGF binding was observed. These bands, lying parallel to the vitreal surface, seemed to codistribute with the synaptic bands existing in the inner plexiform layer. The presence of intra-retinal bFGF binding sites whose distribution varies with embryonic development suggests a regulatory mechanism involving differential actions of bFGF on neural retinal cells.

Animals↗