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J Gabrion

Publications and source records attributed to J Gabrion.

At least 55 records · Page 3Linked to original sources
PubMed1986

Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study.

Striatal neurons were cultured from the fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). Pretreatment of the culture dishes successively with a polycation followed by fetal calf serum resulted in rapid neuron attachment and neurite proliferation. After 9-10 DIV, electron microscope observations revealed the presence of vesicles in axon terminals forming mature synapses with axons and perikarya of adjacent neurons and in varicosities along extended axons. Synapsin I, a synaptic vesicle-specific protein, was present only in neuronal perikarya after 3 DIV, in perikarya and in varicosities along extended axons after 6 DIV, and in varicosities and contact points between axon terminals and adjacent axons or perikarya after 11-14 DIV. Neurotransmitter-stimulated intracellular formation of cAMP decreased markedly during neuronal differentiation. Inositol phosphate formation in response to neurotransmitters, however, increased significantly throughout the period of striatal neuronal development. K+ (56 mM) depolarization resulted in a 2-fold increase in endogenous gamma-aminobutyric acid (GABA) release from striatal neurons, 50% of which was Ca2+-dependent, between 3 and 11 DIV. Between 11 and 14 DIV, subsequent to synapse formation (as revealed by electron microscope observations), GABA release evoked by 56 mM K+ increased up to 5-fold, 75% of which was Ca2+-dependent. It appears that the complete differentiation of striatal neurons in serum-free medium may provide a suitable model for the study of the physiological and regulatory mechanisms involved in nerve cell development.

Animals↗
PubMed1986

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: an ultrastructural study.

Inside-out porcine thyroid follicles in culture undergo polarity reversal after being embedded in collagen gel. The newly-formed follicles reexpress some specific thyroid functions lost in inside-out follicles (Chambard et al., 1984. We present here an ultrastructural study of the inversion of polarity in this model system. This process takes place within 24 to 48 hr, without any opening of the original tight junctions, as shown by fixation in the presence of ruthenium red. A general shrinkage of cellular aggregates was noted soon after embedding. At the apical pole, three different modifications were observed: structural changes appeared in the kinocilium, microvilli and underlying cytoskeleton as early as 10 min after embedding, mainly when the apical pole of the cells was in close contact with the collagen fibers; large cytoplasmic lamellipod- or pseudopod-like extensions, covering the adjacent apical domain, protruded from outer apical regions; some other apical areas invaginated and formed channels inside the aggregates. The last two processes prevented close contact between apical cell surfaces and collagen fibers and allowed a persistence of the initial polarity in some of the cells. Newly-formed lumens were closed 24 hr after embedding in gel and the outer surface of the cellular aggregates in close contact with collagen fibers looked like a basal membrane. These mechanisms proceeded at different rates and involved different numbers of cells, but they all appeared to be related to the transformation of inside-out follicles into follicular structures.

Animals↗
PubMed1986

Primary culture of striatal neurons: a model of choice for pharmacological and biochemical studies of neurotransmitter receptors.

Striatal neurons were cultured from fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). A double coating of culture dishes with polyornithine and fetal calf serum was needed in order to obtain synaptic differentiation. Synaptic vesicles were present in axon terminals as well as in varicosities along extended axons. The presence of differentiated synapses was confirmed by the immunostaining of the preparation with synapsin I antibody. After 13 days in vitro synapsin I was present in axonal varicosities and particularly concentrated at contact points between axonal terminals and postsynaptic sites on adjacent axons or perikarya. On a surface of 429 mm2 on which 2211 cells were observed under phase contrast microscopy only 7% were stained with an antibody against GFAP (glial fibrillary acidic protein). One or two days after the formation of differentiated synapses (11 DIV), a Ca2+-dependent liberation of GABA was observed. These cultures are an excellent model for studying the coupling of some neurotransmitter receptors with an adenylate cyclase. In particular using this preparation we were able to demonstrate that dopamine (D2) and serotonin-(5-HT1) receptors are negatively coupled with an adenylate cyclase. These cultures are also an excellent model to study the coupling of some neurotransmitter receptors with inositol phosphate producing enzymes. We demonstrated for the first time that the quisqualate subtype of glutamate receptors is able to increase inositol phosphate production in striatal neurons.

Animals↗
PubMed1985

Carbohydrates detected by lectins in the vestibular organ.

In the vestibular organ the presence of carbohydrates in the cupula and otoconia of young (6-day-old) and adult rats was investigated using fluorescent lectins. The following sugars have been identified in both young and adult rats: N-acetyl-glucosamine, galactose, mannose and fucose. In contrast, N-acetyl-galactosamine was not detected. In order to demonstrate the specificity of the reaction, control experiments were performed after preincubation of the lectin with its specific inhibitory sugar. The same sugars were identified in calcified (i.e. otoconia) and non-calcified (i.e. cupula) structures. The role of these sugars in the mineralization and fusion process of otoconia is discussed.

Animals↗
PubMed1985

Lectins demonstrate the presence of carbohydrates in the tectorial membrane of mammalian cochlea.

Histological sections of rat and guinea pig cochleas were exposed to lectins to identify the carbohydrates present in the tectorial membranes. N-Acetylglucosamine, galactose, mannose and fucose were found to be present in both rats and guinea pigs, but N-acetylgalactosamine was not detected. In addition, two control experiments were performed. In the first, each lectin was preincubated with its specific inhibitory sugar. In the second, the unfixed tectorial membranes were exposed to lectins. Radioautographic studies confirmed the presence of glucosamine and fucose in the tectorial membrane of 1-day-old rats.

Acetylglucosamine↗
PubMed1985

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells.

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.

Amiloride↗
PubMed1984

Muscle gelsolin: isolation from heart tissue and characterization as an integral myofibrillar protein.

A 92-kDa polypeptide present in rabbit and dog cardiac muscle was purified to homogeneity and some of its properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar, if not identical to macrophage gelsolin; it cross-reacts immunologically with anti-rabbit macrophage gelsolin antibody, has a Ca2+-sensitive shortening effect on the actin filaments as judged by the high shear viscometry and sedimentation experiments, and has a similar amino acid composition. In addition, immunoblot and SDS polyacrylamide gel analysis of cardiac muscle extracts obtained at high and low ionic strength showed that this protein is tightly bound to myofibrils, both in the absence and presence of Ca2+, in ventricular as well as in atrial muscle cells. Indirect immunofluorescence microscopy revealed a striated gelsolin staining pattern analogous to that previously observed for the skeletal muscle gelsolin, suggesting that in the muscle cell this protein is sharing the same localisation as actin. Because of its severing and nucleating properties the gelsolin may play a major role in the organization, assembly and turnover of the thin filaments within the muscle cells.

Amino Acids↗
PubMed1984

Microtubules in the cochlea of the hypothyroid developing rat.

In order to study the effects of hypothyroidism on the development of microtubules in the cochlea, rat pups were rendered hypothyroid by daily administration of propylthiouracil. Microtubules were studied by immunofluorescence and electron microscopy. The absence of immunostaining of pillar cells with antimicrotubule or antitubulin antibodies was correlated with a retarded morphological development of microtubules within these same structures. The above alterations induced an abnormal development of pillar cells, non-appearance of the tunnel of Corti, and stunted epithelial growth. In contrast, a distinct immunoreaction was observed under the outer hair cells. This was attributed to abnormal persistence of afferent dendrites containing microtubules. The results suggest that, while the effect of thyroid hormone on microtubules in afferent cochlear dendrites could not be demonstrated, thyroid hormone is necessary for the normal development of microtubules in epithelial structures.

Animals↗
PubMed1984

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions.

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.

Animals↗
PubMed1983

Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroid-stimulating hormone receptor-adenyl cyclase complex.

When cultured in collagen gel-coated dishes, thyroid cells organized into polarized monolayers. The basal poles of the cells were in contact with the collagen gel, whereas the apical surfaces were facing the culture medium. Under these culture conditions, thyroid cells do not concentrate iodide nor respond to acute stimulation by thyroid-stimulating hormone (TSH). To allow the free access of medium components to the basal poles, the gel was detached from the plastic dish and allowed to float in the culture medium. After release of the gel, the iodide concentration and acute response to TSH stimulation were restored. Increased cAMP levels, iodide efflux, and formation of apical pseudopods were observed. When the thyroid cells are cultured on collagen-coated Millipore filters glued to glass rings, the cell layer separates the medium in contact with the apical domain of the plasma membrane (inside the ring) from that bathing the basolateral domain (outside the ring). Iodide present in the basal medium was concentrated in the cells, whereas no transport was observed when iodide was added to the luminal side. Similarly, an acute effect of TSH was observed only when the hormone was added to the basal medium. These results show that the iodide concentration mechanism and the TSH receptor-adenylate cyclase complex are present only on the basolateral domain of thyroid cell plasma membranes.

Adenylyl Cyclases↗
PubMed1982

Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles.

Antibodies were elicited against turkey gizzard myosin light chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase--in rabbit fast skeletal, slow skeletal, cardiac, and smooth muscles--by indirect immunofluorescence. When studied on nitrocellulose replicas of NaDodSO4/polyacrylamide gel electrophoretograms, antibodies were specific for the Mr 140,000 MLCK of gizzard smooth muscle. By using the same technique, they were shown to recognize the Mr 140,000 MLCK and a Mr 75,000 polypeptide--presumably derived from the former by proteolysis--in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a Mr approximately equal to 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with approximately equal to 11 mole of antibody needed for 50% inhibition of 1 mole of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. In contrast, sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. This shows that (i) antibodies are not species- or tissue-specific, (ii) they recognize kinases that differ in their molecular weight and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains, and (iii) sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. This raises the possibility that myosin phosphorylation may be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.

Animals↗
PubMed1982

Isolation from thyroid cells or purified plasma membranes with associated actin microfilaments. Proteins bound to actin.

Plasma membranes of thyroid cells were purified from hog thyroid glands following two procedures. Their homogeneity was tested by electron microscopy and by measurements of the activity of membrane-bound enzyme markers. According to the procedure used the membrane fractions obtained present some differences in their morphological features as well as in the repartition of the activities of the membrane-bound enzyme markers. However, whatever the composition of the membrane fraction examined (membrane vesicles, single membrane sheets with junctional complexes), decoration with heavy meromyosin clearly shows the presence of actin filaments attached to these fragments. Analysis of proteins by polyacrylamide gel electrophoresis indicates the presence of about twelve major components with actin. Treatment of membranes with Triton X-100 results in an insoluble core which contains all the actin and most of the major proteins. The selective extraction of these components by buffers differing in their ionic strength, pH, or the presence or absence of ATP X Mg has been used to characterize some of the proteins associated to actin; among them are filamin, myosin, alpha-actinin, tropomyosin.

Actins↗
PubMed1981

Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum. Isolation and characterization of sperm calmodulin.

Upon fluorescent staining with a goat antibody anti-ram testis calmodulin, washed bull sperm appears to contain calmodulin in the acrosome, in the post acrosomal region, in the neck region probably associated with the implantation plates and thin laminated fibers, and in a sheath around the upper part of the flagellum. Heads and midpieces + tails were separated by elutriation of sonicated sperm. Immunofluorescent labeling of fragments confirms the presence of calmodulin in implantation plates, where sonication disrupted heads from midpieces, and in a sheath around the midpiece and the upper part of the principal piece. These results were confirmed by electrophoretic and radioenzymatic assays of calmodulin in the fragments, using calmodulin-deficient Ca2+/calmodulin-dependent myosin light chain kinase. Small but significant amounts (approx. 3 micrograms per 10 (10) sperm) are found in midpieces + tails vs. approx. 280 micrograms in the same number of heads. These results are in agreement with a recent report from Jones et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2772-2776. Sperm calmodulin was purified from a whole sperm 1 M KCl extract and found to exhibit the same characteristics as other mammalian calmodulins isolated so far in terms of ultraviolet absorption spectrum and amino acid composition, including one residue of epsilon-N-trimethyllysine. Its behavior upon SDS-polyacrylamide gel electrophoresis was dependent on the presence or absence of Ca2+. The high performance liquid chromatography tryptic peptide maps were similar, if not identical, to mammalian calmodulin maps (Autric et al. (1980) Biochim. Biophys. Acta 631, 139-147). Sperm calmodulin is therefore probably identical to the somatic cell protein.

Amino Acids↗
PubMed1981

Ultrastructural organization of actin filaments in neurosecretory axons of the rat.

The ultrastructural organization of actin filaments was studied in the neurohypophysial system of the rat after heavy meromyosin (HMM) labeling. This structural pattern is characterized by (1) a straight arrangement of the filaments parallel to the axonal axis in the proximal nondilated parts of axons, (2) a central location within axonal dilatations, and (3) a higher concentration within axonal endings where the filaments form a complex three-dimensional network. The relationships of the filaments to other axonal structures and organelles was further studied by use of electron microscopic stereoscopy. The actin filaments frequently appear anchored to the axolemma with either polar arrangements of the arrowhead decoration (i) at structurally undifferentiated sites, and (ii) more particularly within perivascular endings, at sites with electron-dense thickenings. In all axonal divisions actin filaments are also found to bind to filamentous material surrounding the microtubules and to various organelles. Within the terminal portions of the axons actin filaments exhibit close relationships to neurosecretory granules and to the numerous smooth microvesicles found in this region. Such preferential relationships are particularly observed both in axon ;terminals and in pituicytes, with coated vesicles frequently binding actin filaments. In water-deprived rats, the concentration of actin filaments is conspicuously increased along the axons and more clearly in the axonal swellings and endings, where they form a more complex and interconnected network. These data are discussed in the light of a possible involvement of contractile proteins in the mechanisms of axonal transport and terminal release of neurosecretory products.

Actins↗