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Biomedical subjects

Y Courtois

Publications and source records attributed to Y Courtois.

At least 145 records · Page 8Linked to original sources
PubMed1990

Phosphorylation and lipocortin-like activity of a 34-kD surface protein in lens epithelial cells: relation to mitogenesis induced by basic fibroblast growth factor.

In order to elucidate the molecular mechanism whereby basic fibroblast growth factor (bFGF) exerts its action on cell proliferation, we investigated the possible relationship between the mitogenic response to bFGF of bovine lens epithelial cells and the phosphorylation and phospholipase A2 (PLA2) activity of a 34-kD protein (p34) present in the basal plasma membrane of these cells. When p34 was obtained from the peripheral epithelial cells which were not capable of being stimulated with bFGF, pretreatment with bFGF led to decreased phosphorylation and PLA2 activity of p34. In contrast, both an increased phosphorylation of p34 and a strong activation of PLA2 occurred when p34 was derived from the central epithelium which was induced to proliferate in the presence of bFGF. It is hypothesized that p34 might play a key role as part of signal transduction of bFGF in controlling DNA synthesis of bovine lens epithelial cells.

Animals↗
PubMed1990

Binding of basic fibroblast growth factor to normal and neovascularized rabbit cornea.

The labeling pattern of frozen sections of rabbit cornea incubated with radioiodinated basic fibroblast growth factor (bFGF) was investigated in normal corneas and prostaglandin-induced neovascularized corneas by autoradiography followed by image analysis. 125I-bFGF binds to Bowman's, Descemet's, and vascular basement membranes in a dose-dependent manner. The specificity of the binding of bFGF to basement membrane was demonstrated by the following experiments: 1) an excess of unlabeled growth factor displaced the labeling; 2) histones did not modify the labeling; and 3) 2 M NaCl washing and enzymatic treatment with heparitinase prevented binding of labeled growth factor without apparent destruction of the overall structure of the basement membrane. Our results suggest that bFGF binds to the heparan sulfate proteoglycan of basement membranes. Both normal limbal vessels and the newly formed corneal vessels exhibited the same type of labeling but with different intensities, according to the degree of maturation of the new vessels. bFGF binding also is located clearly on the endothelial cells in both types of vessels. This second binding site could correspond to the high affinity receptors on the cell surface and suggests a direct interaction of bFGF with endothelial cells during new vessel formation.

Alprostadil↗
PubMed1990

Biomarkers of aging.

This paper summarises a conceptual discussion of some aspects of biomarkers of aging and the logical strategy for developing them. A biomarker of aging is a biological parameter of an organism that either alone or in some multivariate composite will, in the absence of disease, better predict functional capability at some late age than with chronological age. The criteria for putative biomarker measurement and assessment of putative biomarkers are discussed.

Aged↗
PubMed1989

Identification of a new heparin-binding protein localized within chick basement membranes.

A protein with a molecular mass of 19 kDa has been purified to homogeneity from 11-day-old chick embryos using a procedure involving chromatography on heparin - Sepharose, immunoaffinity resin and C4 reversed-phase. Indirect immunofluorescence studies, using polyclonal and monoclonal antibodies raised against this protein, indicate that it is essentially localized within the basement membranes in early embryonic tissues. After the 18th day of embryonic life and in post-hatched chicken, this protein could only be detected in some eye basement membranes. It appears to be bound to heparan sulfate chains of the proteoglycan present in these structures. Thus, the protein exhibits similar properties to those previously described for fibroblast growth factors (FGF), such as heparin affinity, molecular mass and localization in the basement membranes. In contrast, this protein is present in much larger amounts than FGFs, at least in 11-day-old embryos. Furthermore, the first 17 amino acid residues of the N-terminal sequence show that it does not strictly correspond to any previously described protein.

Amino Acid Sequence↗
PubMed1989

Identification and isolation from bovine epithelial lens cells of two basic fibroblast growth factor receptors that possess bFGF-enhanced phosphorylation activities.

Cross-linking of bound 125I-basic fibroblast growth factor (bFGF) to bovine epithelial lens cells identified two labelled species whose apparent molecular weights were identical with those of two phosphorylated proteins. The bFGF-stimulated phosphorylation of these proteins was shown to be rapid, suggesting an autophosphorylation process. To demonstrate that the phosphorylated proteins were indeed the bFGF-binding molecules, the two components were purified to homogeneity and their bFGF-binding activity was examined. We conclude that bFGF stimulates the phosphorylation of two receptors of 130 and 160 kDa in bovine epithelial lens cells.

Adenosine Triphosphate↗
PubMed1989

Fibroblast growth factor phosphorylation and receptors in rod outer segments.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been isolated and purified from rod outer segments (ROS). aFGF is tightly bound to ROS membranes and can be specifically released by ATP. We show that this mechanism is dependent on the phosphorylation of aFGF itself. Phorbol 12-myristate 13-acetate (PMA) enhances this phenomenon independently of rhodopsin phosphorylation. This demonstrates that aFGF release from ROS membranes is dependent on its phosphorylation by endogenous kinase C. In addition specific binding sites for exogenous FGFs have been identified on ROS and disc membranes. A single high affinity site with a Kd of 40 pM was present in intact ROS while an additional low affinity site with a Kd of 300-600 pM was present in leaky ROS or in disc membranes. Light or ATP modified neither these Kd nor the apparent number of sites. The presence of specific receptors for FGFs and the kinase C dependent release of endogenous membrane bound aFGF suggest an autocrine mechanism which may be involved in photoreceptor cell biology.

Animals↗
PubMed1989

Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells.

The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells (KD of 42.0 +/- 3.8 pM and 70,526 +/- 6121 binding sites/cell for the high-affinity sites, KD of 0.933 +/- 0.27 nM and 630,252 +/- 172,459 sites/cell for low-affinity binding sites). The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely 125I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound 125I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At 37 degrees C, 30% of the cell-associated 125I-bFGF became resistant to the acidic wash after 90 min, suggesting that this fraction of bound 125I-bFGF was internalized. At this temperature, degradation of the internalized ligand was followed after 1 h by the appearance of three major bands of 15,000, 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

Binding Sites↗
PubMed1989

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers.

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.

Animals↗
PubMed1989

Lens fiber differentiation correlated with activation of two different DNAases in lens embryonic cells.

In order to identify the different DNAases present in the lens differentiating tissue, we have used an assay which reveals their activity directly on DNA-containing gels after SDS polyacrylamide gel electrophoresis. DNAase renaturation from nuclear embryonic lens extracts does not occur after separation in 0.1% SDS polyacrylamide gel electrophoresis in contrast to that observed with purified micrococcal nuclease. When the SDS concentration in the running buffer and separating gel is decreased to 0.075%, renaturation of lens DNAase and enzyme activities are observed. Isoelectrofocusing was carried out in a polyacrylamide gel which was overlaid with an agarose gel containing DNA, permitting the visualization of the pI of DNAase activity. The presence of several DNAase isoenzymes was demonstrated in 11-day embryonic lenses. In epithelial lens nuclei, high molecular weight (MW) isoenzymes with basic pI were predominant. In post-mitotic fiber lens nuclei, two lower MW isoenzymes with acidic pI were detected as well as high MW activity with a basic pI.

Animals↗
PubMed1989

Filipin-induced deformations in plasma membranes of cultured bovine corneal endothelial cells with incomplete belts of tight junctions.

Complete belts of intact tight junctions are thought to act as barriers to the movement of cholesterol in the plane of the plasma membrane. As cholesterol can be revealed by filipin-induced membrane deformations on freeze-fracture images, we studied the distribution of these deformation in cultured bovine corneal endothelial cells with incomplete belts of tight junctions. While the extent of filipin-induced deformations differed between individual cells, there is a homogeneous distribution of filipin-induced deformations on both sides of the incomplete junctional belt of endothelial cells. Our results suggest that cultured endothelial cells do not polarize cholesterol, possibly because of the incomplete tight-junctional barrier.

Animals↗
PubMed1989

The effect of eye-derived-growth-factor (EDGFs) on methionine incorporation in the different cell populations of bovine adult lens in organ culture.

When adult bovine lenses were cultured in vitro, the purified retina-derived growth factors EDGF I or EDGF II, as well as the soluble fraction of the retina RE, increased the rate of incorporation of [35S]methionine into protein in cells belonging to different populations in the anterior epithelium as well as in fibers from the most superficial region of the cortex. These fiber cells were the most sensitive to stimulation by the retinal factors as they exhibit a significant increase of total protein synthesis 24 hr after addition of the factors to the culture medium. The epithelial cells studied--central epithelial cells and germinative cells--appeared stimulated only 1 day later. The stimulation of incorporation was not directed towards a particular subset of proteins but to all major polypeptides constituting the electrophoretic pattern of each cell population. It is suggested that this type of ocular signal, which stimulates the expression of a definite program, may act as a permissive signal.

Animals↗
PubMed1989

Differential assemblage of the basal membrane-cytoskeleton complex in bovine epithelial lens cells.

We have investigated the membrane-cytoskeleton complex involved in interactions between the epithelial cells and the capsule of the bovine eye lens. The organization of the molecular complex was determined from cell extraction, immunoprecipitation and immunoblotting experiments and from ultrastructural studies by scanning electron microscopy. We show that marked differences exist in the organization of this basal complex between the central epithelium (mitotic quiescent) and the peripheral epithelium which initiates lenticular differentiation into fibres. Our results support the view that: (a) the organization of several major membrane components in the peripheral epithelium differs from that of the central epithelium; (b) microfilaments and vimentin filaments are independent of each other in the peripheral epithelium, whereas microfilaments are involved in an inter-relationship with vimentin filaments in the central epithelium; (c) two surface proteins of 24 and 27 kD and two surface glycoproteins of 46 and 220 kD appear to be bound to vimentin filaments in the peripheral region, whereas the intermediate filaments appear to be solely in close association with the 46 and 220 kD glycoproteins in the central zone.

Actin Cytoskeleton↗
PubMed1989

Evidence for a relationship between longevity of mammalian species and a lens growth parameter.

The lens growth was studied in a number of curves representing 16 mammalian species. Emphasis was placed on determining the length of the lens development stage. All the lens curves studied went through a growth crisis: It is a period of apparent growth slowing down, lasting linear during the resting lifespan (adult stage). The growth crisis occurred at a length that was characteristic for the species and was shown to be directly correlated with the species maximum lifespan potential. These results may indicate the prevalence of a common functional basis regulating the lens growth and thus operating in the longevity of mammalian species.

Animals↗
PubMed1989

Colocalization of fibroblast growth factor binding sites with extracellular matrix components in normal and keratoconus corneas.

Basic and acidic fibroblast growth factor (bFGF, aFGF) binding sites were determined in frozen sections of normal and keratoconus corneas. After incubation with I-125 radiolabelled growth factors, corneal binding sites were revealed by autoradiography. The growth factors were localized mainly to Descemet's membrane and to the epithelial basement membrane. FGF binding sites were generally similar in normal and keratoconus corneas. The binding specificity was demonstrated by competitive inhibition experiments with an excess of unlabelled growth factors. The binding sites were sensitive to pretreatment of the corneal sections with heparitinase. We have attributed FGF's basement membrane affinity to one of its constituents, proteoheparan sulfate. Proteoheparan sulfate, laminin, collagen type IV, and fibronectin were all revealed by immunofluorescent techniques. While keratoconus cornea stroma had less laminin but more fibronectin than normal corneas the main difference lied in type IV collagen which was overexpressed in keratoconus epithelium.

Adult↗
PubMed1989

In vivo binding of topically applied human bFGF on rabbit corneal epithelial wound.

We present the results of the first evaluation of human placenta extracted basic fibroblast growth factor (bFGF) in a rabbit corneal epithelium wound-healing model. Healing dose-response experiments after selective epithelial wounding with iodine vapors demonstrated that bFGF accelerated the repair process in a saturable manner. Corneal binding of topically applied 125I-labeled bFGF was investigated using radioassay and autoradiographic techniques. Basic FGF was shown to bind specifically to denuded epithelial basement membrane in a very stable fashion and not to the intact epithelium. No transfer of the topical bFGF to the aqueous humor or any intraocular structure could be observed. The stability of this interaction was further demonstrated by reextracting and characterizing the labeled factor from treated corneas. The specificity of the fixation was documented by in vivo topical competition with unlabeled bFGF or heparin. We propose that bFGF-basement membrane interactions play a role in corneal wound healing.

Administration, Topical↗
PubMed1988

Characterization of a bovine acidic FGF cDNA clone and its expression in brain and retina.

A cDNA encoding the acidic eye-derived growth factor (EDGF II) similar to the acidic fibroblast growth factor (aFGF), a potent cell mitogen, has been isolated from a bovine retinal cDNA library. The cDNA, 4.1 kb in size, has a sequence coding for the 155 amino acids of bovine aFGF, and shows similarity with human aFGF (87% identity). The coding sequence is flanked by a 5'-untranslated region of 0.8 kb and a 3'-untranslated end of 3.0 kb. Northern blot analysis of bovine brain and retina poly(A+) RNAs showed the existence of four aFGF mRNA species. Two of these species are 9.9 and 6.0 kb in size, not abundant and could represent premessengers. The other two species, 4.2 and 2.5 kb, are abundant.

Amino Acid Sequence↗