Biomedical subjects

D Rainteau

Publications and source records attributed to D Rainteau.

17 recordsLinked to original sources

PubMed2005

Building up of the liquid-ordered phase formed by sphingomyelin and cholesterol.

The long-range and molecular orders and dynamics in codispersions of egg sphingomyelin-cholesterol have been investigated by synchrotron x-ray diffraction and electron spin resonance using phosphatidylcholine spin-labeled at several positions on the sn-2 chain. Mixtures containing 0, 17, 33, 41, 50 mol% cholesterol exhibited a single phase by x-ray diffraction methods. The temperature dependence of the d-spacing between 20 and 50 degrees C is attenuated with increasing proportions of cholesterol, becoming invariant for cholesterol contents of 41 and 50 mol% on completion of the liquid-ordered phase. Electron spin resonance revealed two sites for 17 and 33 mol% cholesterol. One site is highly ordered and the other is less ordered than the fluid phase of pure sphingomyelin as shown by the molecular and the intramolecular order parameters reflecting the segmental motions of the probe. The two-sites exchange rate indicates a mean lifetime of the sites of approximately 0.1 micros during which the lipid displacement is approximately 1 nm. The short lifetime of the sites probed by ESR and the single phase detected by x-ray diffraction support in this binary mixture, the building up of the Lo phase by a progressive accumulation of randomly distributed sphingomyelin-cholesterol condensed complexes rather than by diffusional exchange between extended domains.

Biophysical Phenomena↗

PubMed2000

Establishment of plasma membrane polarity in mammary epithelial cells correlates with changes in prolactin trafficking and in annexin VI recruitment to membranes.

Mammary epithelial cells (MEC) of lactating animals ferry large amounts of milk constituents in vesicular structures which have mostly been characterized by morphological approaches (Ollivier-Bousquet, 1998). Recently, we have shown that under conditions of lipid deprivation, perturbed prolactin traffic paralleled changes in the membrane phospholipid composition and in the cytosol versus membrane distribution of annexin VI (Ollivier-Bousquet et al., 1997). To obtain additional information on the membrane events involved in the vesicular transport of the hormone to the apical pole of the cell, we conducted a biochemical study on prolactin-containing vesicles in MEC at two different stages of differentiation. We first showed that MEC of pregnant and lactating rabbits exhibited membrane characteristics of non-polarized and polarized cells respectively, using annexin IV and the alpha-6 subunit of integrin as membrane markers. Incubation of both cell types with biotinylated prolactin for 1 h at 15 degrees C, followed by a 10-min chase at 37 degrees C revealed that prolactin transport was activated upon MEC membrane polarization. This was confirmed by subcellular fractionation of prolactin-containing vesicles on discontinuous density gradients. In non-polarized MEC, (125)I-prolactin was mainly recovered in gradient fractions enriched with endocytotic vesicles either after incubation at 15 degrees C or after a 10-min chase at 37 degrees C. In contrast, in polarized MEC, the hormone switched from endocytotic compartments to a fraction enriched in exocytotic clathrin-coated vesicles during the 10-min chase at 37 degrees C. Association of annexin VI to prolactin carriers was next studied in both non-polarized and polarized cells. Membrane compartments collected at each gradient interface were solubilized under mild conditions by Triton X-100 (TX100) and the distribution of annexin VI in TX100-insoluble and TX100-soluble fractions was analyzed by Western blotting. Upon MEC polarization, the amount of annexin VI recovered in TX100-insoluble fractions changed. Quite interestingly, it increased in a membrane fraction enriched with endocytotic clathrin-coated vesicles, suggesting that annexin VI may act as a sorting signal in prolactin transport.

Animals↗

PubMed1997

Lipid-depleted diet perturbs membrane composition and intracellular transport in lactating mammary cells.

When rats were fed a control or a lipid-depleted diet for five generations, reproduction was not disturbed but pup growth was affected. The membrane organization and the secretory activity of mammary epithelial cells from these lactating rats were investigated. This diet induced a large decrease in the level of polyunsaturated fatty acids of membrane phospholipids (26.6% versus 44.0%). The level of 20:4 (n-6) was strongly decreased, mainly in phosphatidylethanolamine. Annexin VI, which interacts preferentially with this phospholipid, accumulated at the periphery of the cell and was largely associated to the hydrophobic region of the bilayer as compared to control membranes. Casein synthesis and casein secretion measured in incubated explants, after pulse-chase metabolic labeling, were both reduced by about 60% in lipid-deprived cells. The secretory ratio (radioactive secreted caseins in %) was not modified, suggesting that the mechanism of basal secretion was not mainly affected. On the contrary, the secretagogue effect of prolactin disappeared. The intracellular transport of the hormone was considerably slowed down by the diet and prolactin did not reach the lumen of the acini after 1 h of chase, in contrast to what occurred in control cells. Addition of 20:4 (n-6), in vitro, to mammary fragments from lipid-deprived rats restored the localization of annexin VI, increased synthesis and secretion of caseins as well as intracellular transport of PRI. Together, these data underline the importance of the level of 20:4 (n-6) in membrane phospholipids for exocytic and endocytic transport in lactating mammary epithelial cells.

Animals↗

PubMed1995

Characterization and ultrastructural localization of annexin VI from mitochondria.

Annexin VI, a member of a family of related intracellular proteins that associate reversibly with membrane phospholipids in a Ca(2+)-dependent manner, has been purified from bovine liver mitochondria and characterized. Moreover, biochemical and immunocytochemical lines of evidence are presented which strongly suggest that annexin VI is closely associated with the cristae in the inner membrane of mitochondria. These findings are consistent with a calcium channel activity of annexin VI in mitochondria.

Amino Acid Sequence↗

PubMed1993

Annexins from bovine adrenal cortex exhibit specific cytosol/membrane solvation.

Cytosol/membrane localization of annexins I to VI was analyzed in tissue extracts from bovine adrenal cortex. Based on their solubility in either aqueous or detergents solutions, they were subfractionated in three groups named cytosolic (C), membrane-bound (MB) and membrane-inserted (MI). Less than 1% of the total annexins present in the tissue were recovered in the C fraction when as much as 76.5 and 22.5% were obtained respectively in the MB and the MI fractions. By immunoblotting after SDS-PAGE, it was shown that the various members of the annexin family were not equally recovered in the different fractions. A-V and A-VI were found present in the three fractions whereas the distribution of A-I, A-II, A-III and A-IV was distinct, suggesting different cellular functions.

Adrenal Cortex↗

PubMed1991

Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: a comparison of six mammalian species.

The distribution of Calmodulin was examined during spermiogenesis and sperm epididymal maturation in rabbit, hamster, mouse, rat, monkey, and human. An affinity-purified antibody to Calmodulin was used to characterize this protein in sperm extracts by immunoblot analysis. Post-embedding immunogold procedures were used to localize Calmodulin at the ultrastructural level. The pattern of Calmodulin distribution was similar in the six species studied. A diffuse labeling was observed in round spermatids. Gold particles accumulated first in the subacrosomal layer of elongating spermatids. The perinuclear ring was also labeled. During the maturation phase of spermatids, Calmodulin labeling extended to the postacrosomal sheath. Dramatic changes occurred at spermiation so that in testicular sperm Calmodulin immunostaining was predominant in the postacrosomal sheath. Some labeling was still detected in restricted areas of the subacrosomal layer. This feature varied from species to species. Calmodulin location did not change during sperm epididymal maturation. A role for Calmodulin in the control of manchette development and regulation of subacrosomal actin aggregation state during spermiogenesis is proposed. The unique location of Calmodulin in the postacrosomal sheath of all species that have been studied in this work, together with the known presence of calcium in this area suggest a pivotal role for Calmodulin in sperm-egg fusion process.

Acrosome↗

PubMed1991

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor.

Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cells, the plasmalemmal undercoat was labeled. Anti-actin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca2(+)-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.

Actins↗

PubMed1990

Annexins I-VI in secretory ameloblasts and odontoblasts of rat incisor.

Immunoblot analyses and ultrastructural immunogold studies have been conducted on annexins in the secretory ameloblasts and odontoblasts of the rat incisor. Annexins I and II were seen in the soluble and particulate fractions of the enamel-related portion but not in the dentin-related portion. These proteins were visualized in the cytosol, near to the plasma membrane of Tomes' processes and in secretory vesicles in the ameloblasts. The forming enamel was also labeled. Annexins III, VI an V were detected in both the soluble and particulate fractions of the enamel-and dentin-related portions. Annexin IV was mainly localized in the proximal and distal areas of the secretory ameloblasts and virtually absent from in the supranuclear area. Annexin V was mainly detected in the cytosol of the cells and to a lesser extent near the plasma membrane. Annexin VI was mainly detected in the particulate fraction of enamel- and dentin-related portions. It was seen in the mitochondria and in the subplasmalemmal undercoat. All these proteins may play a role in exocytosis and endocytosis. They are implied in the regulation of cell calcium, but not in the transfer of calcium through the cells in the direction of the forming enamel and dentin, except annexins I and II since they are both present in the secretory vesicles and in the forming enamel.

Ameloblasts↗

PubMed1989

Effects of calcium and calcium analogs on calmodulin: a Fourier transform infrared and electron spin resonance investigation.

Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopies have been used to monitor changes in the conformation of calmodulin induced by Ca2+ and Ca2+ analogs. Using FTIR spectroscopy we observe that Ca2+: (i) favors the alpha-helical conformation and decreases the flexibility of the molecule; (ii) multiplies the intramolecular hydrogen bonds (the ratio of freely vibrating NH/hydrogen bound NH groups decreases); (iii) induces changes in the C-terminal tyrosine environment; and (iv) increases compactness of the molecule (less NH groups in the peptide bonds can be deuterated). As proved by ESR, Ca2+ binding induces exposure of hydrophobic domains allowing binding of a spin-labelled phenothiazine on calmodulin. When the experiments are performed in the presence of increasing amounts of Ca2+, both ESR and FTIR provide evidence that major conformational changes result after the filling of only two Ca2+-binding sites. But achievement of the spectroscopical changes is only observed when the four binding sites are filled (Ca2+/calmodulin = 4). The effects of analogs are monitored with the same spectroscopical parameters. Zn+ does not induce structural modifications of calmodulin but all other analogs studied mimic the calcium effects to some extent. As regards the amplitude of the spectroscopical effects, analogs rank in the following order: Ca2+ greater than Cd2+ greater than Tb3+ = Eu3+ greater than Gd3+ greater than La3+ greater than Zn2+ = cation depleted. Except for Zn2+, ranking for their activating potency of MLCK, the analogs can be arranged in a similar order.

Animals↗

PubMed1989

Differential localization of calmodulin, the 67kDa calcimedin and calpactin II in secretory ameloblasts.

By using specific antibodies, we have shown by Western blots that dental tissues contain calmodulin, the 67 kDa calcimedin and calpactin II. Moreover, by immunogold electron microscopy, we were able to compare the intracellular distribution of these three calcium-binding proteins in secretory ameloblasts. They were all found in the cytosol of these cells but only calcimedin was detected in the mitochondria. Calpactin II was the only one present in secretory vesicles. Twice as much calmodulin and calpactin II were detected in cell bodies as in Tomes' processes, but calcimedin was more abundant in the latter. The presence of these calcium mediators in well defined areas of ameloblasts may indicate differential ways for these cells to regulate different calcium-dependent processes during enamel formation.

Ameloblasts↗

PubMed1987

Calmodulin in lymphocyte mitogenic stimulation and in lymphoid cell line growth.

Calmodulin levels are elevated three- to fourfold in the dividing cells, resulting from the lectin-induced stimulation of fresh human lymphocytes. This increase in calmodulin appears to be related mainly to progression into S phase and supports the hypothesis that calmodulin might be crucial in regulating the progression of lymphoblasts through their division cycle. Calmodulin levels are higher in a lymphoid cell line derived from human acute lymphoblastic leukemia blood cells than in a lymphoid cell line derived from normal human blood cells, suggesting that calmodulin could be an important mediator of the leukemogenetic process.

Calmodulin↗

PubMed1986

Interaction between calmodulin and five different spin-labelled chlorophenothiazines.

The binding to purified calmodulin of five spin-labelled derivatives of chlorophenothiazine was investigated by e.s.r. spectrometry and by the antagonizing potency on the calmodulin-dependent activation of myosin light chain kinase. The results of a comparative study and the influence of pH and ionic strength on the binding support the occurrence of an electrostatic binding involving the terminal amino group of the side-chain of the chlorophenothiazine. These results are discussed in relation to the specificity of the interaction that holds the antipsychotic drug-calmodulin complex together.

Calcium↗

PubMed1986

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

Animals↗

PubMed1984

Effects of AY 9944 on low density lipoprotein metabolism in cultured human fibroblasts.

Treatment of cultured human fibroblasts with the hypocholesterolemic drug AY 9944 resulted in a marked increase in low density lipoprotein internalization and degradation for concentrations up to 5 X 10(-6)M. Low density lipoprotein binding was less affected. Concentrations above 5 X 10(-6)M resulted in a relative decrease in low density lipoprotein degradation, whereas binding and internalization plateaued. The stimulation of low density lipoprotein internalization took place within the first hours of incubation of cells with the drug, which suggests a direct effect on the cell membrane. Such phenomenon could account at least partially for the hypocholesterolemic effect of the drug, besides its inhibitory effect on 7-dehydrocholesterol reductase.

Cell Line↗

PubMed1984

Binding of a spin-labelled chlorpromazine analogue to calmodulin.

The binding of a spin-labelled derivative of chlorpromazine to calmodulin was investigated by e.s.r. spectrometry. The completion of the spectroscopic changes requires the presence of 4 Ca2+ ions per calmodulin molecule. The influences of various physicochemical factors (pH, ionic strength) are discussed in relation to the nature (hydrophobic and polar) of the interactions that hold the drug-calmodulin complex together.

Calcium↗

PubMed1984

Changes in calmodulin level after fertilization and during first cleavage in the egg of the urodelan amphibian Pleurodeles waltlii.

We report three significant calmodulin rises related to Pleurodeles waltlii egg fertilization and following developmental events. These elevations are correlated to the major obvious Ca2+-dependent events: Na+-H+ exchange, activation of NAD kinase, triggering of cortical reaction, resumption of meiotic division II, initiation of DNA synthesis and regulation of cell division. Therefore, it is suggested that alterations in calmodulin level in fertilized egg may be part of the Ca2+-dependent regulatory mechanisms which turn on metabolisms, initiate development and govern cell cleavages.

Animals↗

PubMed1983

Effects of phenothiazines on low density lipoprotein metabolism in cultured human fibroblasts.

Treatment of cultured human fibroblasts with trifluoperazine or chlorpromazine resulted in a biphasic effect on low density lipoprotein (LDL) catabolism, depending upon the dose. At up to 10(-5) M, a marked increase in LDL binding, internalization and degradation was observed. This phenomenon took place within the first hours of incubation with the drugs, suggesting a direct effect on cell membrane physical characteristics, probably related to the lipophilic properties of phenothiazines. Concentrations above 2 X 10(-5) M resulted in a relative decrease in LDL binding and internalization, and in a dramatic decrease in LDL degradation, which may be related to an inhibition of calmodulin-dependent processes.

Calmodulin↗