Biomedical subjects

J Gabrion

Publications and source records attributed to J Gabrion.

At least 37 records · Page 2Linked to original sources

PubMed1990

Establishment of a long-term primary culture of striatal neurons.

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.

Animals↗

PubMed1990

Cellular distribution and biochemical characterization of G proteins in skeletal muscle: comparative location with voltage-dependent calcium channels.

GTP binding proteins have been proposed to play a role in excitation--contraction coupling. In a precedent study [Toutant et al., (1988), Biochem. J., 405-409], we determined that Bordetella pertussis toxin is able to catalyse ADP-ribosylation of two substrates in the detergent soluble fraction of total muscle extracts. Purified fractions of transverse tubule membranes (T-tubule membranes), a key element of the excitation--contraction coupling, were shown to exhibit a major ADP-ribosylated substrate at 40 kd and an immunoreactivity with antisera raised against purified bovine brain Go alpha or G beta. In the present study, we have investigated the cellular distribution of G protein subunits in comparison with that of the voltage-dependent Ca2+ channels by immunofluorescence on transverse and longitudinal sections of fast and slow muscles. With affinity-purified antibodies against G beta subunits, a fluorescent labelling underlined the myofibrils and sarcolemma, whereas a strong immunoreaction in a dotted pattern evoked the presence of the subunit in repetitive triadic structures. With anti-Go alpha antibodies, the immunofluorescence was more clearly focussed on a dotted pattern and the co-location with the voltage-dependent Ca2+ channel immunoreactivity indicates that both proteins were located in very close subcellular structures. Immunoblot analysis and PTX ADP-ribosylation of the purified light sarcoplasmic reticulum (LSR), heavy sarcoplasmic reticulum (HSR) and T-tubule subcellular fractions indicate the discrete presence of G proteins in LSR, an unambiguous labelling of the HSR fraction, while T-tubule membranes clearly appear very rich in a Go-like protein, confirming the observed preferential immunocytochemical distribution of G protein subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Maturation of the endocrine pancreas in the sea bass, Dicentrarchus labrax L. (Teleostei): an immunocytochemical and ultrastructural study. I. Glucagon-producing cells.

The structure of the endocrine pancreas in the sea bass (Dicentrarchus labrax L.) was studied with special reference to glucagon-immunoreactive cells. As described in most of the teleosts, the sea bass was found to have a diffuse pancreas. In the adult, endocrine cells were clustered in a principal islet and numerous accessory islets where the glucagon A cells were localized peripherally. Under electron microscopy, the A cells displayed a clear hyaloplasm with granules having typical spherical or polyhedral cores, as in other vertebrates. The maturation of the endocrine pancreas was monitored under rearing conditions. The endocrine pancreas appeared during the prelarval stage, 3 days after hatching, and consisted of a single cluster of morphologically similar cells, containing very small cytoplasmic granules. During the larval stage, cytodifferentiation resulted in modifications of cell shape and increased granule size. Typical granules appeared in 8-mm-long larvae. Cells immunoreactive with mammalian glucagon antibodies appeared only at the beginning of the juvenile stage (3 months/20 mm). Electron microscope observations revealed that the storage of hormone in numerous cytoplasmic granules began at this stage.

Animals↗

PubMed1990

Selective distribution of sugars on the tegumental surface of adult Bothriocephalus gregarius (Cestoda: Pseudophyllidea).

Whole specimens and histological and semi-thin sections of Bothriocephalus gregarius adults were exposed to lectins to identify carbohydrates present in the tegument and parenchyma. The sugars N-acetyl glucosamine, N-acetyl galactosamine, galactose, glucose (or mannose) and fucose were detected in the cestode using eight lectins: WGA (Wheat germ agglutinin), HPA (Helix pomatia agglutinin), SBA (Soy bean agglutinin), PHA (Phaseolus vulgaris agglutinin), RCA60 and RCA120 (Ricinus communis toxin and agglutinin), ConA (Concanavalin agglutinin) and UEA-I (Ulex europaeus agglutinin). Combined use of these methodological approaches (whole specimens, paraffin and semi-thin sections) revealed the presence of a gradient in the distribution of most of the sugars over the tegument, with the highest concentrations on the strobila (as shown by most of the lectins). Other sugars were specific for the scolex or strobila (as shown by UEA-I or HPA, respectively). The ultrastructural study showed that the distribution of glycoconjugates was associated with the presence of specific tegumental coats. The significance of this selective distribution and its relevance to cestode physiology and host-parasite relationships are discussed.

Animals↗

PubMed1990

Choroidal ependymocytes in culture: expression of markers of polarity and function.

Long-term primary cultures derived from fetal mouse or rat choroid plexus were obtained in serum-supplemented media. Monoclonal and polyclonal antibodies to basolateral and apical membrane components were used to observe the expression of specific markers of polarity and function. Choroid plexus cultures and thin frozen sections of adult tissues were compared by immunocytochemistry. Two polyclonal antibodies directed against laminin and fibronectin were used on cultured choroid plexus and sectioned tissues, showing that fibronectin and laminin are located on the basolateral membrane domain in ependymocytes in vitro, as well as in vivo. Na(+)-K(+) ATPase was apically detected by light and electron microscopy with a monoclonal antibody (Mab H30) in both cultured cells and sectioned tissues. Double immunofluorescent staining with Mab H30 and affinity-purified polyclonal antibodies to the alpha subunit of G0 protein (G0 alpha) demonstrated the relatively similar distribution of the two antigens on the apical face of the choroidal tissue, both in vivo and in vitro. The distribution of these markers shows a typical differentiation with maintenance of polarized features in choroidal ependymocytes in culture, testifying that this cell culture system constitutes an interesting model for studying the functional characteristics of ependymal cells of the choroid plexus.

Animals↗

PubMed1989

Isolation, characterization and immunocytochemical localization of caldesmon-like protein from molluscan striated muscle.

A 140-kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat-stable protein. It cross-reacts immunologically with anti-(gizzard caldesmon) antibody, binds to calmodulin-Sepharose in a Ca2+-dependent manner, cosediments with F-actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+-ATPase. The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon-like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.

Animals↗

PubMed1989

A thyroid hormone-vasopressin interaction promotes survival and maturation of hippocampal neurons dissociated postnatally.

Hippocampal cells dissociated from 5-day-old rat pups were grown in a suitable chemically defined basal medium, supplemented or not with 3,3',5-triiodo-L-thyronine (T3), [Arg8]-vasopressin (AVP), or both, at different concentrations. Four days after plating, neuron-like cells began to degenerate drastically in the basal medium. Although AVP alone had no effect, T3, and to a greater extent T3 and AVP together, prevented their death. Moreover, T3 and AVP also acted synergically in promoting the maturation of surviving cells, especially AchE-positive neurons, either directly or through glial cells.

Animals↗

PubMed1989

Characteristics and specific localization of receptors for atrial natriuretic peptides at non-neuronal cells in cultured mouse spinal cord cells.

Characteristics of atrial natriuretic peptide receptors were determined in cultured mouse spinal cord cells. Saturation and competition experiments demonstrated the presence of a single class of atrial natriuretic peptide binding sites with high affinity (KD = 0.054 nM) and a density of 1.92 fmoles/10(6) cells. A similar affinity (KD = 0.070 nM) was observed in rat spinal cord membrane preparations. These atrial natriuretic peptide binding sites were functional receptors since the treatment of cells with atrial natriuretic peptide increased cyclic guanosine monophosphate levels within these cells in a classical time-dependent manner. When atrial natriuretic peptide was applied onto the cell body of intracellularly recorded spinal cord neurons, this peptide did not evoke a change of the input resistance or of the resting membrane potential value. Light-microscopic autoradiography studies showed that no atrial natriuretic peptide binding could be detected on typical birefringent neurons but it could be located on astroglial and epithelial cells as identified by immunocytochemical markers. These results show that functional atrial natriuretic peptide receptors with high affinity exist in cultured mouse spinal cord cells and are not located on neurons. The presence of atrial natriuretic peptide receptors on astrocytes suggests that this neuropeptide might be a good candidate for neuron-glial communication. As the atrial natriuretic peptide binding sites previously shown in epithelia responsible for maintaining fluid and electrolyte gradients, the atrial natriuretic peptide receptors on epithelial cells in these spinal cord cultures may be involved in vivo in the control of water balance in the central nervous system.

Animals↗

PubMed1989

Ultrastructural localization of the GTP-binding protein Go in neurons.

The ultrastructural localization of Go, a GTP-binding protein (G protein) highly expressed in nervous tissues, was performed in cultured fetal and adult murine neurons, using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha). These antibodies recognized denatured Go alpha and both the native Go alpha-subunit and the Go alpha beta gamma heterotrimer. At the ultrastructural level, the positive immunoreactivity detected in cultured cells as well as in thin frozen sections, showed that Go was largely distributed in cell bodies and neuritic cytoplasm. Labelling was principally noted on the cytoplasmic face of the plasma membrane lining the cell body and the neurites, especially in 'cell-cell' contacts, but also in the cytoplasmic matrix, between endoplasmic reticulum and Golgi cisternae. No immunoreactivity was observed on the inner face of the pre- or postsynaptic membranes in both adult brain and in cultured neurons. This last finding strongly suggests that the Go protein is not involved in transducing chemical signals at the level of synapses, but more probably modulates the synaptic functions by controlling the activity of effectors localized outside of the synaptic densities.

Animals↗

PubMed1989

Endogenous amino acid release from cultured cerebellar neuronal cells: effect of tetanus toxin on glutamate release.

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.

Amino Acids↗

PubMed1989

Apical localization of the alpha subunit of GTP-binding protein Go in choroidal and ciliated ependymocytes.

The presence of GTP-binding proteins (G proteins) has been studied in murine adult choroid plexuses and cultured fetal choroidal or hypothalamic ependymal cells by ADP-ribosylation catalyzed by Bordetella pertussis toxin (PTX) and by immunodetection using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha), the major brain G protein. ADP-ribosylation with 32P-NAD and PTX of choroid plexus revealed an intense labeling at the 40 kDa level in addition to the known PTX-substrates at 41 kDa (Gi alpha) and 39 kDa (Go alpha). This 40 kDa substrate was also predominant in cultured ependymal cells. However, a positive immunoreactivity with the anti-Go alpha antibodies was detected at the level of the 39 kDa faster component, indicating the presence of Go alpha in both choroid plexuses and cultured ependymal cells. In thin frozen sections as well as in cultured cells, Go alpha was mainly immunolocalized at the apical pole of choroidal ependymocytes and in the kinocilia of ciliated ependymal cells. At the ultrastructural level, using gold immunoprobes, the immunoreactivity of a Go alpha-like protein was detected on the cytoplasmic face of the apical plasma membrane, coated pits and vesicles, and in the apical cytoplasmic matrix. In ciliated ependymal cells, the positive immunostaining displayed a dotted pattern at the surface of demembranated axonema of apical kinocilia. These findings strongly suggest that G proteins, especially Go, are involved in transducing chemical signals that modulate traffic and exchanges between cerebrospinal fluid and ependyma through the apical membrane of ependymocytes.

Animals↗

PubMed1988

The presence of non-neuronal cells influences somatostatin release from cultured cerebral cortical cells.

We examined the effect of non-neuronal cells on somatostatin release from cultured cerebral cortical cells. Three culture models were used: (1) neuron-enriched cultures obtained from cortex of 17-day-old rat embryos and exposed to 10 microM cytosine arabinoside (Ara C) for 48 h between days 3 and 5 after plating; (2) whole cell cultures obtained by using the same protocol but untreated with Ara C; (3) glial primary cultures obtained from newborn rats. We studied: (i) the cellular composition of the cultures by using two astroglial markers: vimentin and glial fibrillary acidic protein (GFAP); (ii) the spontaneous and forskolin-stimulated somatostatin release. In 8-day-old cultures morphological data revealed that Ara C treatment reduced glial cells to 6%. At 7 and 10 days of culture somatostatin spontaneously released from Ara C-treated cells was higher than that measured from untreated cells. On the 17th day of culture, neuron-enriched cultures contained a lower amount of somatostatin than whole cell cultures. Forskolin elicited a dose-dependent release of somatostatin from whole cell cultures, but had no effect on neuron-enriched cultures. Astroglial released media (ARM) from glial primary cultures exposed to forskolin for 20 min induced somatostatin release from neuron-enriched cultures. HPLC analysis of endogenous amino acids of ARM showed that glutamate, glutamine, glycine and alanine were significantly increased after forskolin stimulation. Our results suggest a functional interaction between glial cells and neurons secreting somatostatin.

Amino Acids↗

PubMed1988

Characterization of ependymal cells in hypothalamic and choroidal primary cultures.

Long-term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus were obtained in serum-supplemented and chemically defined media. In order to identify and characterize cell types growing in our cultures, we used morphological features provided by phase-contrast, scanning and transmission electron microscopy. Immunological criteria were recognized, using antibodies against intermediate filament proteins (vimentin, gliofibrillar acid protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin and fibronectin in single or double immunostaining, and monoclonal antibodies known to detect epitopes of ependymal or endothelial cells. Minor cell types such as astrocytes, fibroblasts and endothelial cells were distinguished. Ependymal cells, which exceeded 75% of the cultured cells, were identified by their cell shape and epithelial organization revealed by phase-contrast and transmission electron microscopy, by their apical differentiation evidenced by scanning and transmission electron microscopy, and by certain molecular markers (e.g. gliofibrillar acid or ciliary rootlet proteins) detected by immunofluorescence. Four ependymal cell types were recognized: choroidal ependymocytes, ciliated and unciliated ependymal cells, and tanycytes. All these cultured ependymal cell types showed a remarkable resemblance to in vivo ependymocytes, in terms of marker expression and ultrastructural features.

Animals↗

PubMed1988

Cholesterol-rich microdomains in rat and porcine thyroid membranes involved in TSH-induced endocytotic processes.

Filipin, a polyene antibiotic, was used to detect cholesterol in thyroid membranes in vivo and in culture during TSH stimulation. We found that apical and basolateral plasma membranes were heterogeneously modified by filipin which induced abundant lesions in apical membranes, whereas Golgi apparatus, endoplasmic reticulum, nuclear membranes were unmodified. Small apical vesicles and colloid droplets were generally highly enriched in these complexes, suggesting a high cholesterol concentration in their membranes. Pseudopod membranes, known to be highly specialized domains in the apical plasma membrane, appeared enriched in cholesterol. Consequently, we suggest that an increased cholesterol content may be involved in the stabilization of thyroid membranes during endocytotic processes.

Animals↗

PubMed1988

Duodenal toxicity of dietary Phaseolus vulgaris lectins in the rat: an integrative assay.

It is now generally admitted that phytohemagglutinin (PHA) constitutes the main factor responsible for the dietary toxicity of raw kidney beans. In the growing rat, an impairment of growth is the unique expression of a malnutrition syndrome. The aim of this work was to precise to what extent the intestinal injuries may account for this malnutrition. PHA was administered for 9 days to growing rats at levels ranging from 0.0025 to 0.25% of food dry matter. One group of controls was fed ad libitum and other groups were restrained. In such conditions, PHA reduced the food intake when offered at a level higher than 0.04% as a linear function of the logarithm of lectin rate. Intestinal injuries were also dose-dependent: blebbing of microvilli and loss of alkaline phosphatase occurred at the smallest dose of PHA, cell loss occurred at higher doses. A compensatory hyperplasia was observed as a consequence of both intestinal injury and reduced food intake. Our main results are that, whatever may be the damages caused to the duodenal mucosa, the observed growth impairment was quasi-totally imputable to the reduction of food intake.

Alkaline Phosphatase↗

PubMed1988

[Characterization of surface and intracellular glycoconjugates in larval forms of Echinococcus multilocularis using lectins].

Carbohydrate moieties have been detected with nine fluorescent lectins in Echinococcus multilocularis cysts, developed in liver and lung of infested jirds. Tegument and glycocalyx, and two types of medullary cells were found to selectively and strongly bind lectins which are specifically adsorbed by N acetyl glucosamine, N acetyl galactosamine, galactose or mannose. The significance of these features in the host-parasite interaction are discussed.

Animals↗

PubMed1988

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: structure of the junctions assessed by freeze-fracture and lanthanum permeability.

The organization of tight junctional complexes (TJs) was studied in cultured porcine thyroid cells during the inversion of polarity induced by collagen-embedding of inside-out follicles, using freeze-fracture replicas and lanthanum penetration. During the early steps of polarity reversal, freeze-fractures showed that TJs generally persisted. They increased in width and progressively branched out into the basolateral surfaces, towards the basal pole. Later, the number of TJ strands decreased and gap junctions inserted within TJ networks were found between cells in reversed follicles, in the same manner as in typically polarized follicles, embedded in collagen or in suspension. The de novo formation of TJ complexes was rarely found in the reversing structures. Despite the heterogeneity of TJs assessed by freeze-fracture, impermeability to lanthanum tracer was noted in inside-out structures. During the reversal process, some TJs remained unstained, whereas others displayed permeability to lanthanum. This heterogeneity might be due to the "opening" of a small number of junctions (perhaps only one by aggregate). When the process was achieved after 48 hr in collagen, the tightness of the junctions was complete, confirmed by the absence of lanthanum in luminal cavities of newly formed follicles.

Animals↗

PubMed1987

Epithelial cell polarization in culture: orientation of cell polarity and expression of specific functions, studied with cultured thyroid cells.

Isolated porcine thyroid cells reorganize in culture into various types of multicellular structure, which differ in the orientation of cell polarity and in the surface of the cell layer accessible to molecules present in the culture medium. The types of structure are: (1) follicles: the basal pole is oriented toward the medium; (2) inside-out follicles or monolayers: the apical pole is facing the culture medium; (3) monolayers on a permeable substratum: both sides of the cell layer are accessible to the medium. Follicles can be transformed into inside-out follicles or monolayers and vice versa by manipulation of the external cell environment and without dissociating the cells. Cells concentrate iodide and respond to acute stimulation by thyroid-stimulating hormone (TSH) when the basal pole is accessible, and organification occurs only when cells form a closed follicular lumen. In porous-bottomed culture chambers monolayers are formed with the basal surface accessible to the medium and the apical compartment separated from the medium. Under these conditions 85-95% of the thyroglobulin produced is secreted apically and 5-15% basally. Thyrotropin stimulates (X3) apical accumulation without modifying secretion in the basal compartment. Sodium transport across the cell layer has been characterized. An amiloride-sensitive influx occurs at the apical pole whereas the Na+/K+-ATPase, localized in the basolateral membrane, mediates ouabain-sensitive efflux at the basal pole. The thyroid epithelium in culture appears therefore as a Na+-absorbing epithelium. The role of this transport in the stabilization of cell polarity is discussed.

Animals↗