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J F Tournier

Publications and source records attributed to J F Tournier.

14 recordsLinked to original sources
PubMed2000

Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells.

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.

Animals↗
PubMed1997

12-O-tetradecanoylphorbol-13-acetate inhibits aminophospholipid translocase activity and modifies the lateral motions of fluorescent phospholipid analogs in the plasma membrane of bovine aortic endothelial cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent mitogenic factor which can replace the growth promoting activity of basic fibroblast growth factor (bFGF) on bovine aortic endothelial cells. However, TPA-treated cells lose their strict contact inhibition at confluence, which is a characteristic of cells grown in the presence of bFGF. We have examined whether these changes could be related to modifications of the transbilayer and lateral motions of fluorescent lipids, namely 1-acyl-2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-p hosphatidylcholine (C6-NBD-PC), -phosphatidylserine (C6-NBD-PS), and -phosphatidylethanolamine (C6-NBD-PE) inserted in the outer leaflet of the cell plasma membrane. In TPA-treated cells, the three fluorescent phospholipids remained located in the outer leaflet for at least 1 h at 20 degrees C after their insertion, indicating a blockade of the aminophospholipid translocase activity which is normally present in the plasma membrane of bFGF-treated cells. TPA also induced a large increase in the percentage of C6-NBD-PC and C6-NBD-PE probes which were free to diffuse laterally. The mobile fractions M reached values of approximately 100% for the two lipids, while for bFGF-treated cells they were found around 85 and 75%, respectively. For the C6-NBD-PS probe, M remained unchanged in bFGF and TPA-treated cells, at around 85%. TPA treatment also induced a twofold increase in the lateral diffusion coefficients of C6-NBD-PC and C6-NBD-PE, while that of C6-NBD-PS remained nearly unchanged. These effects of TPA may be related to the observed loss of differentiated properties of vascular endothelial cells and not to its mitogenic properties.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Basic fibroblast growth factor modulates the aminophospholipid translocase activity present in the plasma membrane of bovine aortic endothelial cells.

Vascular endothelial cells form the inner nonthrombogenic lining of the large blood vessel. Through back-exchange and fluorescence recovery after photobleaching experiments and using the two fluorescent lipids 1-acyl-2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl]glycerophosphocholine and 1-acyl-2[6-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]hexanoyl]glycerophosphoethanolamine, we have recently shown that an energy-dependent and protein-dependent aminophospholipid translocase activity is present in the plasma membrane of cultured bovine aortic endothelial cells, which specifically transports phosphatidylethanolamine from the outer leaflet toward the inner leaflet of the membrane lipid bilayer. In the present study, using the same approach and 1-acyl-2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl] glycerophosphoserine as the probe, it is shown that this conclusion is also valid for phosphatidylserine. Furthermore, evidence is presented indicating that this aminophospholipid translocase activity can be maintained, suppressed, and restored at will, depending on the conditions of cell incubation. Thus, the translocase activity is detected for cells maintained in their normal culture medium or in a serum-free incubation medium [Dulbecco's modified Eagle's medium (DMEM)] supplemented with the basic fibroblast growth factor, whereas inhibition is observed for cells exposed for at least 2 h to DMEM. The translocase activity is restored when these pretreated cells are further incubated at least for 1 h in the presence of serum or of basic fibroblast growth factor. In view of the importance of basic fibroblast growth factor as a mitogenic and differentiating agent for vascular endothelial cells, various growth factors were tested (acidic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, transforming growth factors alpha and beta, vascular endothelial growth factor, interferon gamma, tumor-necrosis factor, insulin, and interleukin 4). Only basic fibroblast growth factor was active in the maintenance and restoration of the translocase activity. With respect to the effects of serum, evidence is presented showing that high-density lipoproteins might play a role in the control of the translocase activity. However, the positive effects of basic fibroblast growth factor, serum and high-density lipoproteins on the translocase activity were suppressed when experiments were carried out in the presence of an anti-(basic fibroblast growth factor) IgG, thus indicating that in all cases, basic fibroblast growth factor was directly involved in the modulation of the aminophospholipid translocase activity present in the plasma membrane of bovine aortic endothelial cells.

Adenosine Triphosphate↗
PubMed1994

Lipid domains and lipid/protein interactions in biological membranes.

In the fluid mosaic model of membranes, lipids are organized in the form of a bilayer supporting peripheral and integral proteins. This model considers the lipid bilayer as a two-dimensional fluid in which lipids and proteins are free to diffuse. As a direct consequence, both types of molecules would be expected to be randomly distributed within the membrane. In fact, evidences are accumulating to indicate the occurrence of both a transverse and lateral regionalization of membranes which can be described in terms of micro- and macrodomains, including the two leaflets of the lipid bilayer. The nature of the interactions responsible for the formation of domains, the way they develop and the time- and space-scale over which they exist represent today as many challenging problems in membranology. In this report, we will first consider some of the basic observations which point to the role of proteins in the transverse and lateral regionalization of membranes. Then, we will discuss some of the possible mechanisms which, in particular in terms of lipid/protein interactions, can explain lateral heterogenities in membranes and which have the merit of providing a thermodynamic support to the existence of lipid domains in membranes.

Animals↗
PubMed1993

Differences in the transbilayer and lateral motions of fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine in the apical plasma membrane of bovine aortic endothelial cells.

In the plasma membrane of various eucaryotic cell types, in particular blood platelets and erythrocytes, it is known that phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer and that this transverse asymmetry is controlled by an aminophospholipid translocase activity. In this respect, it was of interest to check whether there are differential transbilayer movements between amino- and neutral phospholipids in the apical plasma membrane of vascular endothelial cells which form the inner nonthrombogenic lining of the large blood vessel. In the first step we compared the transbilayer localization and also the rate of lateral motion of two fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine, namely C6-NBD-PC and C6-NBD-PE, inserted into the apical plasma membrane of bovine aortic endothelial cells, in vitro. By the use of back-exchange experiments we have found that C6-NBD-PC could be removed from the cell membrane toward the culture medium regardless of the incubation conditions used, i.e., just after cell labeling at 0 degrees C or even after further cell incubation for 1 h at 0 or 20 degrees C. In contrast, C6-NBD-PE could be removed only when the cells were maintained at 0 degrees C. After incubation for 1 h at 20 degrees C, 85% of the probe molecules remained nonexchangeable, indicating probe translocation from the outer to the inner leaflet of the lipid bilayer. This "flip" process, which occurred at 20 degrees C, was abolished when the endothelial cells were preincubated with N-ethylmaleimide, diamide, vanadate (VO4(3-)) and vanadyl (VO2+) ions, a set of substances which inhibit aminophospholipid translocase activity in various systems, and with a combination of sodium azide and 2-deoxyglucose which led to nearly complete ATP depletion in the cells. Fluorescence recovery after photobleaching experiments were also carried out to specify more precisely the localization and dynamics of the probes in the two leaflets of the plasma membrane lipid bilayer. They produced lateral diffusion coefficients D of 1.2 +/- 0.05 x 10(-9) cm2/s for C6-NBD-PC and 2.8 +/- 0.3 x 10(-9) cm2/s for C6-NBD-PE, when the two probes were located in the outer leaflet of the plasma membrane, just after cell labeling at 0 degree C.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1990

The respective 27 kDa and 28 kDa protein kinase C substrates in vascular endothelial and MCF-7 cells are most probably heat shock proteins.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the phosphorylation of two distinct 27 kDa and 28 kDa proteins, respectively, in bovine vascular endothelial cells and in MCF-7 human breast cancer cells. These protein phosphorylation events were correlated to striking opposite cell growth responses to TPA, i.e., stimulation of vascular endothelial cell proliferation and inhibition of MCF-7 cell growth. Exposure of both vascular endothelial and MCF-7 cells to heat shock induced synthesis of the respective 27 kDa and 28 kDa proteins among a set of common and distinct other proteins as well as an increase in the degree of phosphorylation of the two 27 kDa and 28 kDa proteins. These results suggest that the two protein kinase C substrates very likely belong to the family of low molecular mass stress proteins.

Breast Neoplasms↗
PubMed1989

Lipid lateral diffusion and membrane organization.

It is shown that investigating the lateral motion of lipids in biological membranes can provide useful information on membrane lateral organization. After labeling membranes with extrinsic or intrinsic lipophilic fluorescent probes, fluorescence recovery after photobleaching experiments strongly suggests that specialized cells like spermatozoa, eggs and epithelia exhibit surface membrane regionalization or macrocompartmentation and that lateral microheterogeneities or lipid microdomains exist in the plasma membrane of many cellular systems.

Animals↗
PubMed1989

Proliferative effect of high density lipoprotein (HDL) and HDL fractions (HDL1,2, HDL3) on virus transformed lymphoblastoid cells.

The growth-promoting activities of plasma lipoproteins (LDL, HDL, HDL1,2, HDL3) and total HDL apolipoproteins on a virus transformed lymphoblastoid cell line in vitro, has been compared. When maintained in lipoprotein-deficient serum-supplemented medium, these cells do not proliferate optimally. The addition of either HDL, HDL1,2 or HDL3 induced optimal cell proliferation as compared to the result observed in fetal calf serum-supplemented medium. The HDL1,2 subfraction was found to be more potent than the HDL3 subfraction in supporting cell growth. Total HDL apolipoproteins were able to support significant cell proliferation. In contrast, LDL did not promote cell growth. In serum-free conditions and in the presence of transferrin, only HDL and HDL subfractions induced cell proliferation. These results suggest that HDL and HDL subfractions could initiate B lymphoblastoid cell growth and that total HDL apolipoproteins could support a part of cell proliferation.

B-Lymphocytes↗
PubMed1989

The lateral motion of lipid molecules in the apical plasma membrane of endothelial cells is reversibly affected by the presence of cell junctions.

As with other epithelia, the question arises of whether the endothelial cell junctions participate in cell polarization, restrict the localization of lipid molecules, and lead to differences in their lateral motion between the apical and basolateral plasma membranes. We found that in bovine aortic endothelium in culture, the localization of the lipophilic probe 5N-(hexadecanoyl)-aminofluorescein (HEDAF) was markedly affected by the presence of cell junctions. At confluence, the probe was observed to be restricted to the exoplasmic leaflet of the apical plasmalemma. It was detected in the basal membrane only after disruption of the cell junctions, whereas the cells remained firmly bound to the underlying extracellular matrix. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the endothelial confluent cell monolayer forms a mosaic of closed diffusion areas in which the probe molecules are free to diffuse. For the first time, and using a new mathematical approach, it was possible to estimate the diameter of these diffusion areas. Values in the range 14-33 microns were found which correlate well with the actual measured diameters of 14 to 26 microns for the apical pole of individual cells. Moreover, cell junctions were found to affect the dynamics of the probe. When the cell junctions were disrupted, the lateral diffusion coefficient D of HEDAF was found to be the same in both apical and basal membranes. It regained its initial higher value in the apical poles when cell contacts were restored. This strongly suggests that in vascular endothelium, cell junctions affect overall plasma membrane organization in a reversible manner.

Animals↗
PubMed1989

Effect of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells.

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.

Animals↗
PubMed1986

Possible role of protein phosphorylation in the mitogenic effect of high density lipoproteins on cultured vascular endothelial cells.

The implication of protein phosphorylation in the mitogenic action of high density lipoproteins (HDL) on bovine vascular endothelial cells was investigated by incubating endothelial cell cultures in the presence of 32P-labeled phosphoric acid. The incorporation of 32P into proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and autoradiography of the gel. In endothelial cells seeded at low density and made quiescent by serum starvation, HDL markedly and consistently enhanced the degree of phosphorylation of a Mr 27,000 protein in a time- and dose-dependent manner. Using 500 micrograms/ml HDL, 32P labeling of the 27-kDa protein was already measurable after 10 min of incubation and reached a maximum at 20-30 min. Minimal effective dose of HDL during a 30-min incubation period was in the range of 5-10 micrograms/ml. While the apolipoprotein moiety of HDL was able to mimic the effect of total HDL, the lipid part of HDL was not. Furthermore, fibroblast growth factor appeared to potentiate the effect of HDL on 27-kDa protein phosphorylation, in agreement with the synergism observed between fibroblast growth factor and HDL on endothelial cell proliferation. Two activators of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetylglycerol also induced the phosphorylation of the 27-kDa protein. These results suggest that the 27-kDa protein may be a physiological substrate for protein kinase C and that HDL could exert their mitogenic effect on endothelial cells through activation of protein kinase C and subsequent protein phosphorylation.

Amino Acids↗
PubMed1985

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions.

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures ("confluent-log" cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that "confluent-log" cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.

Breast Neoplasms↗
PubMed1984

Rapid purification and activity of apolipoprotein C1 on the proliferation of bovine vascular endothelial cells in vitro.

The growth-promoting activity of human high-density lipoproteins (HDL) and of their apolipoprotein components on bovine vascular endothelial cells in vitro has been compared. When maintained on plastic culture dishes and exposed to medium containing lipoprotein-deficient serum and fibroblast growth factor, these cells do not proliferate. Addition of either HDL or the total HDL apolipoproteins induces significant cell proliferation. Apolipoprotein C1, purified by chromatography on the ion-exchanger resin Polybuffer exchanger 94, has an effect on the cell growth similar to that of the total apolipoproteins of HDL.

Animals↗