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International audience; The syncytiotrophoblast layer plays a major role throughout pregnancy, since it is the site of numerous placental functions, including ion and nutrient exchange and the synthesis of steroid and peptide hormones required for fetal growth and development. Inadequate formation and regeneration of this tissue contributes to several pathologies of pregnancy such as intrauterine growth restriction and preeclampsia, which may lead to iatrogenic preterm delivery in order to prevent fetal death and maternal complications. Syncytiotrophoblast formation can be reproduced in vitro using different models. For the last ten years we have routinely purified villous cytotrophoblastic cells (CT) from normal first, second and third trimester placentas and from gestational age-matched Trisomy 21 placentas. We cultured villous CT on plastic dishes to follow the molecular and biochemical aspects of their morphological and functional differentiation. Taking advantage of this unique collection of samples, we here discuss the concept that trophoblast fusion and functional differentiation may be two differentially regulated processes, which are linked but quite distinct. We highlight the major role of mesenchymal-trophoblast cross talk in regulating trophoblast cell fusion. We suggest that the oxidative status of the trophoblast may regulate glycosylation of proteins, including hCG, and thereby modulate major trophoblast cell functions.

High and low affinity receptors for basic flbroblast 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.

Objectives Placental ribonuclease inhibitor (PRI) is a 50-kDa leucine-rich repeat protein characterised by its property to inhibit ribonucleases (RNase) of the pancreatic type. PRI is a tight-binding inhibitor of angiogenin (RNase 5), a 14-kDa secreted protein with ribonucleolytic and angiogenic activities that promotes cell growth and survival. PRI inhibits angiogenesis and blocks tumour growth and metastasis. PRI was first purified to homogeneity from human placenta but is largely expressed [1]. Angiogenin is associated with tumour development but is also present in normal biological fluids and tissues [2]. Its expression has been previously mapped in human placentas and shown associated with blood vessel formation [3-5]. To address the physiological role of PRI, we studied its expression in situ and in vitro, during human placental development.

Lipid-linked oligosaccharides containing at least five mannose residues (0.7 nmol/g of tissue) were isolated from calf pancreas by chloroform-methanol-water extraction and purification on DEAE-cellulose and Sephadex LH-20, and affinity chromatography on concanavalin A-Sepharose in the presence of nonionic detergent. The addition, prior to the chromatographic steps, of 14C-labeled lipid-linked oligosaccharides (synthesized in vitro by calf pancreas microsomes in the presence of GDP-D-[14C]mannose and UDP-D-[14C]glucose, respectively) as internal standards, indicated a final yield ranging from 38 to 50%. Analysis of the oligosaccharide residues by liquid chromatography of the lipid-free preparation, monitored by u.v. absorbance and radioactivity measurement of the tritiated compounds, indicated a heterogeneous mixture of oligosaccharides. Its components, ranging from Man5(GlcNAc)2 to Glc3Man9(GlcNAc)2, cochromatographed with the 14C-labeled derivatives from in vitro synthesis. Calf pancreas contains lipid intermediates bearing at least six mannose residues, such as Man9(GlcNAc)2-P-P-lipid, in almost equal or even higher amounts than Glc3Man9(GlcNAc)2-P-P-dolichol.

Summary Eight patients (4 suffering from acute myeloid leukemia) exhibiting a loss of ABO red cell antigens, as seen by a mixed-field reaction pattern in agglutination tests, were selected and examined for the level of the A, — B, — H blood group glycosyltransferases within membranes prepared from erythrocyte subpopulations (A or B positive and A or B negative red cells). A or B enzyme activities were largely decreased in membranes which had lost A or B antigens (A or B negative subpopulations) but were within normal level in membrane from cells which had not lost A or B antigens (A or B positive subpopulations). The H enzyme level which was frequently low in the serum was within normal limits in the membrane preparations examined. Since A or B negative subpopulations were normally glycosylated in vitro into A or B reactive structures, the results demonstrate that loss of A or B antigens is related to some alteration of the blood group gene products rather than to significant abnormalities of the membrane precursors.

International audience; We described a protocol for purification of bovine brain membranes suitable to study the binding of iodinated basic fibroblast growth factor (FGF) to bovine brain membrane preparation. The binding of 125I basic FGF to brain membranes reached equilibrium within 30 min at 20 degrees C, was reversible, and displaced by an excess of unlabeled basic FGF. Scatchard analysis of the data revealed that two classes of binding sites could be detected with an apparent Kd of 30 pM and a capacity of 0.24 pmol/mg of membrane proteins for the high affinity binding site and Kd of 3 nM with a capacity of 51 pmol/mg of membrane proteins for the low affinity binding site. Cross-linking experiments of labeled basic FGF to brain membrane receptor yield the formation of a single major complex with an apparent molecular mass of 170 kDa which is similar to the value obtained for the high affinity binding site for basic FGF on target cells in tissue culture. Hence these data present the first biochemical evidence suggesting that membrane purified from bovine brain contain two classes of specific binding sites for basic FGF and confirm results described with cells grown in vitro.