Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages.

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.

Distribution of an endogenous 16-kd S-lac lectin in the chicken retina.

PURPOSE: To examine by indirect immunofluorescence the distribution of an endogenous 16-kd S-lac lectin (soluble lactose binding lectin) during development of the chicken retina. METHODS: Cryosections of retinal tissue at different developmental stages and cultured retinal cells (either not permeabilized or permeabilized with acetone) were incubated with a rabbit antiserum that specifically reacts with the retinal 16-kd S-lac lectin. After incubation with a fluorescent-labeled secondary antibody, tissue sections and cultured cells were analyzed by fluorescence microscopy. RESULTS: Retina was weakly stained with the antiserum on early embryonic day 7, whereas on embryonic days 13 and 18 it showed a restricted "granular" staining in the outer retina. At embryonic day 18, in addition, there was widespread staining in all retinal layers. This pattern was maintained by postnatal day 5 and in the adult retina, although the intensity of the staining of the outer retina was weaker. In retinal cell cultures, glial-like flat cells and monopolar, bipolar, and multipolar neurons were stained with the antiserum, but only if they had been previously permeabilized with acetone. CONCLUSION: The results suggest that the distribution of a 16-kd S-lac lectin changes during retinal development. Cell culture experiments indicate that most often the lectin is localized intracellularly in the different retinal cell types.

Immunocytochemical study of the distribution of a 16-kDa galectin in the chicken retina.

PURPOSE: To compare the distribution of a developmentally regulated 16-kDa galectin in the chicken retina at two different developmental stages: embryonic day 13 (ED13) and postnatal day 10 (PD10) retinas, by immunocytochemical analysis using light and transmission electron microscopy. METHODS: Semi-thin and thin sections from ED13 and PD10 retinas were incubated with the IgG fraction purified from a rabbit antiserum raised against the 16-kDa chicken galectin. After incubation with colloidal gold particle-labeled goat anti-rabbit IgGs, tissue sections were analyzed by light and transmission electron microscopy. To improve the observation by light microscopy, semi-thin immunostained sections were intensified by silver enhancement. RESULTS: In ED13 retinas a specific galectin labeling was detected in the region corresponding to the outer limiting membrane by light microscopy. This labeling seemed to be associated with the apical villi of Muller glial cells and their specialized junctions, as judged by transmission electron microscopy. In PD10 retinas, the more relevant finding revealed by light microscopy was the detection of a widespread immunostaining at the level of all retinal layers. The ultrastructural analysis indicated that the galectin labeling was detected at the cytoplasmic and nuclear compartments of Muller cells throughout the different retinal layers. Moreover, the labeling was detected in the inner limiting membrane in structures that resemble the end feet of Muller cells. The apical villi, and the specialized junctions of these glial cells, appeared more strongly stained in PD10 retinas than in ED13 retinas. Finally, highly intense labeling in a group of mitochondria localized in the inner segments of cone cells was observed. CONCLUSIONS: The present study clearly supports the idea that the subcellular distribution of the 16-kDa galectin changes during the development of the chicken retina. Morphologic changes associated with developmentally regulated expression and subcellular compartmentalization of the retinal galectin suggest that this lectin may be involved in the modulation of several processes in the visual system. Its presence in the apical villi of Miller cells may be related by modulatory functions between retina and pigment epithelium, but its presence in the cytoplasm and nucleus of these glial cells suggests a potential immunomodulatory role and its involvement in different metabolic processes between Muller and the other retinal cells. Finally, although the presence of galectins inside mitochondria has not been described before, this localization gives rise to the idea that this lectin may be involved in the modulation of mitochondrial processes.

Specific inhibition of lymphocyte proliferation and induction of apoptosis by CLL-I, a beta-galactoside-binding lectin.

Beta-galactoside-binding lectins or galectins are a family of closely related carbohydrate-binding proteins which functions still remain to be elucidated. Several evidence suggest they could play a role in different biological processes, such as cell growth regulation and immunomodulation. In the present study we report that affinity-purified CLL-I (chicken lactose lectin-I), an acidic 16-kDa galectin exhibits specific growth regulatory properties. Con A-stimulated rat spleen mononuclear cells showed a marked dose-dependent growth inhibition upon incubation with the galectin protein. Cell growth arrest was highly prevented by galectin-specific sugars. In addition, biochemical, cytofluorometrical, and morphological evidence are also provided to show that these inhibitory properties are related to a positive control in the apoptotic threshold of spleen mononuclear cells. Flow cytometric analysis showed a dose- and time-dependent increase of cells with hypodiploid DNA content upon exposure to CLL-I. Moreover, cells treated with CLL-I displayed the typical ultrastructural changes compatible with apoptosis, mainly chromatin condensation and margination along the inner surface of the nuclear envelope. Finally, the highly characteristic "ladder" pattern of DNA fragmentation into oligonucleosome-length fragments of approximately 180-200 bp could be found within 6 h of cell culture with CLL-I, mainly in the T cell-enriched population. Induction of apoptosis by a beta-galactoside-binding protein highlights a potentially novel mechanism for regulating the immune response and points to a rational basis for the postulated immunomodulatory properties of this protein family.

The ganglíosides of the chicken retina and optic tectum. The influence of light on their labelling after an injection of labelled precursors.

Cultures of retinas from 8-day-old embryonic chicken in the presence of (3)H -glucosamine showed that GD3 and GM3 are the gangliosides with highest labelling. From this age onward, and apparently responding to the appearance of the required enzymes, the labelling of GM1, GD1a and GT increases and at hatching day GD1a becomes the highest labelled ganglioside. The main site of synthesis of neuronal gangliosides was found in the neuronal perikarya, in a Golgi membrane enriched fraction. In this fraction the nascent gangliosides are protected from the neuraminidase attack. Experiments in the visual system of chicks indicated that the gangliosides synthesized in the soma of retinal ganglion cells are transported axonally to their endings in the contralateral optic tectum. After 3, 5 and 8 h of injection of N (3)H acetylmannosamine into one eye of the chick, the gangliosides in the contralateral optic tectum of animals exposed to 1000 lux were more labelled than in their controls in dark. The glycoproteins followed a pattern of labelling similar to that of gangliosides. Most of the difference in labelling appears in the same subcellular fraction in which normally is found the maximal deposition of gangliosides. The increase of labelling was not due to a greater turnover of gangliosides.

Gangliosides in postmitotic retina of chick embryo: changes in vivo and in cell cultures.

Developmental changes in ganglioside composition of postmitotic neural retina of chick embryo were analyzed by thin-layer chromatography. Gangliosides were identified by comparing their chromatographic mobilities with reference standards. The outstanding changes are decrease in the concentration of GD3L and increase in GD1a and GM1 concentrations. By depleting Müller glia cells from retina tissue of 13- and 16-day embryos (R13, R16) we determined that the bulk of the major gangliosides is associated with the neurons. Analysis of gangliosides in monolayer cultures of R13 and R16 cells highly enriched for Müller cell-derived gliocytes indicated that these cells express the same types of gangliosides as neurons, but in somewhat different concentrations and relative proportions; however, after time in culture these cells showed ganglioside types and changes in ganglioside profile that are not characteristic of normal retina. The latter observation is consistent with other evidence that the phenotype of Müller glia cells becomes altered in monolayer culture. In contrast to cultures of early embryonic retina, in organ cultures of later postmitotic retina, ganglioside composition did not continue to change as in normal development. This suggests that in postmitotic retina, normal developmental progression of ganglioside changes requires systemic and/or other conditions which are missing or altered when this tissue is isolated and cultured in vitro.

Biosynthesis of gangliosides in cultured retina from chick embryos.

Retina tissue from 7-day chick embryos was maintained in culture for up to 10 days. After 5 days in culture the incorporation of [3H]leucine into proteins and of [3H]glucosamine into gangliosides was similar to that found in retinas from 12-day embryos. The incorporation of [3H]thymidine into DNA decreased steadily with time in culture; after 5 days it was about 20% of the initial value and approximately twice that determined in retinas from 12-day embryos. The radioactivity pattern of gangliosides labeled with [3H]glucosamine showed a predominance of the label in disialosyllactosylceramide (GD3); up to the 3rd day of culture. From then on, there was a progressive increase in the labeling of disialosylgangliotetraosylceramide (GD1a); by day 7 of culture, labeling of GD1a predominated and the labeling pattern was indistinguishable from that found in retinas from 12-day-old embryos. The specific activities of the CMP-NeuAc:GM3 sialosyl- and UDP-GalNAc:GM3 N-acetylgalactosaminyl-transferases decreased to 15% and increased to 400%, respectively, of the values determined in the retinas of 7-day embryos. The cultured retinas progressed in their organization into layers with culture time. The labeling transition from GD3 to GD1a was also detected after inhibition of the histotypic organization by addition of 5-bromo 2-deoxyuridine to the culture medium. Results suggest that high activity of GM3:sialosyl transferase and high labeling of GD3 are associated with the proliferative state of retina cells, while high activity of GM3:N-acetylgalactosaminyltransferase and high labeling of GD1a are associated with the non-proliferative, differentiated state of these cells.

Changes in ganglioside profile in chick embryo retina: Studies on tissue and cell cultures.

The developmental profile of gangliosides in the neural retina of the chick embryo is characterized by a progressive decrease in the concentration of GD3 complex from a high level on day 6; by a continuous increase in GD1a concentration; and by less striking increases in GD1b and GT1b concentrations during the growth phase; GM1 increases in the post-mitotic retina. Gangliosides were analyzed by thin layer chromatography and by densitometry of the TLC plates. (Ganglioside nomenclature is according to Svennerholm.(37)) We have examined comparatively ganglioside changes in organ cultures of retina tissue from 6 day embryos (R(36)), in cell aggregates and in primary monolayer cultures of R(26) cells, all maintained for 6 days in vitro. In all cases, the pattern of ganglioside changes was qualitatively similar to that in the retina in vivo. These results suggest that, unlike some other aspects of retina differentiation, the progression of ganglioside changes in the 6-12 day embryonic retina is not critically dependent on histotypic cell organization or on specific contact-dependent cell interactions; these changes appear to be largely preprogrammed in the cells at some earlier phase of development.

Galectins: a key intersection between glycobiology and immunology.

Galectins are a family of evolutionarily conserved animal lectins, widely distributed from lower invertebrates to mammals. They share sequence and structure similarities in the carbohydrate recognition domain and specificity for polylactosamine-enriched glycoconjugates. In the last few years significant experimental data have been accumulated concerning their participation in different biological processes requiring carbohydrate recognition such as cell adhesion, cell growth regulation, inflammation, immunomodulation, apoptosis and metastasis. In the present review we will discuss some exciting questions and advances in galectin research, highlighting the significance of these proteins in immunological processes and their implications in biomedical research, disease diagnosis and clinical intervention. Designing novel therapeutic strategies based on carbohydrate recognition will provide answers for the treatment of autoimmune disorders, inflammatory processes, allergic reactions and tumor spreading.

Isolation and characterization of a soluble lactose-binding lectin from postnatal chicken retina.

We investigated the presence of endogenous lectins in postnatal chicken retinal tissue assaying the hemagglutinating activity of crude soluble extracts of the tissue that was homogenized in a buffer supplemented with different sugars. Lactose was the most effective sugar to extract an hemagglutinating activity. Using similar extraction conditions, other sugars, such as glucose, N-acetylglucosamine, mannose, fucose, glucuronic and sialic acid, were ineffective to extract any significant hemagglutinating activity. The lectin was purified by affinity chromatography on lactosyl-Sepharose. SDS-PAGE and isoelectric focusing analyses showed that it has a subunit molecular weight of 16 kDa and a pI about 4.5. The retinal lectin cross-reacted immunologically with a rabbit antiserum raised against a lectin purified from adult chicken liver, which is a CLL-I (Beyer et al.: J Biol Chem 255:4236-4239, 1980) or C-16 (Sakakura et al.: J Biol Chem 265:21573-21579, 1990) form of chicken endogenous soluble lactose-binding lectins. Gel filtration studies showed that the oligomeric structure of the retinal lectin is dependent on the ionic strength of the elution buffer. The lectin hemagglutinating activity and the amount of lectin protein reached their highest levels at late developmental stages of the retinal tissue, suggesting that retinal lectin might have a functional role during terminal differentiation of retinal cells.

Peanut agglutinin binding glycoproteins in the chick retina: their presence in Müller glia cells.

The histological and cellular distribution and some biochemical characteristics of components that bind peanut agglutinin (PNA), a lectin that recognizes preferentially terminal galactose-beta (1-3) N-acetyl galactosamine disaccharide residues of glycoconjugates, were studied in chick retinal tissue and in dissociated retinal cells after their differentiation in culture. In sections of retinal tissue from animals 7 days after hatching (Rp7), in addition to the inner and outer segments of the photoreceptor layer, the plexiform and optic fiber layers were stained with rhodamine-labeled PNA, indicating that, besides photoreceptor cells, other cellular types contribute to the PNA staining. We present evidence indicating that at least part of this staining is provided by Müller glia cells. In cultures of dissociated cells from retinas at embryonic day 7 (R7), photoreceptor-like cells and flat Müller glia-derived cells but not neurons were stained with rhodamine-labeled PNA. Furthermore, Müller glia cells isolated from Rp7 were also brightly stained with PNA. Western blot assays of extracts from R7 showed the presence of PNA binding glycoproteins of 31-33 kDa and a component that migrates at the dye front. In addition to the components detected in R7, extracts from R14 and Rp7 showed the presence of a major PNA binding glycoprotein of 175 kDa and a minor glycoprotein of 220 kDa. Extracts from the photoreceptor layer contain the 175 and 220 kDa glycoproteins, indicating their association with photoreceptor cells. The 31-33 kDa components were detected in extracts from the remnant inner retina, suggesting their association with the Müller glia cells. Supporting this view, these components and not those of 175 and 220 kDa were detected in cell cultures enriched in flat Müller glia-derived cells. Only the 31-33 kDa components and the component that migrates at the dye front were detected in extracts from cell cultures enriched in photoreceptor-like cells, suggesting the need of some environmental element for the expression of the 175 and 220 kDa components in the differentiated photoreceptor cells.

Regulation of ganglioside composition and synthesis is different in developing chick retinal pigment epithelium and neural retina.

We examined the immunocytochemical expression of GM3 and GD3 in 3-day-old chick embryo retinal pigment epithelium (RPE) and neural retina (NR). We also compared the composition of gangliosides and the activities of key ganglioside glycosyltransferases of the RPE and NR of 8-, 12-, and 15-day old embryos. The immunocytochemical studies in 3-day-old embryos showed heavy expression of GM3 and GD3 at the inner and outer layers of the optic vesicle that are the precursors of the RPE and NR, respectively. The compositional and enzymatic studies showed pronounced differences between RPE and NR of 8-day and older embryos. HPTLC showed that at 8 days the major species were GM3 and GD3 in RPE and GD3 and GT3 in NR. As development proceeded, GD3 decreased in both tissues, GM3 became the major ganglioside in RPE, and ganglio-series gangliosides (mainly GD1a) became the major species in NR. At 15 days the major species were GD1a in NR and GM3 in RPE. Enzyme determinations showed that whereas in RPE from 12-day-old embryos GM2 synthase was under the limit of detection and GD3 synthase activity was about sixfold lower than GM3 synthase, in NR the activities of GM3 and GD3 synthases were similar and both six- to ninefold lower than GM2 synthase. These results evidence a markedly different modulation of the ganglioside glycosylating system in cells of a common origin that through distinct differentiation pathways originate two closely related tissues of the optic system.(ABSTRACT TRUNCATED AT 250 WORDS)

GD3 ganglioside is prevalent in fully differentiated neurons from rat retina.

Adult mammalian retinas contain unusually high amounts of GD3, a ganglioside of the lactosylceramide series. In this respect, they differ from adult avian retina and other regions of the adult avian and mammalian brain, where GD3 is a minor ganglioside and gangliosides of the gangliotetraosylceramide series (GM1, GD1a, GD1b, GT1b) are the predominant ones. We compare here the ganglioside patterns of rat, human, horse, and guinea pig retinas, which are known to differ in the degree of vascularization and astrocytic cell content. All these retinas showed a prevalence of pathway "b" gangliosides over pathway "a" gangliosides but showed no correlation between GD3 content and the degree of vascularization and astrocytic cell content. Immunostaining of rat retina sections showed the presence of GD3 in the inner and outer plexiform layers and also in the ganglion cell and inner nuclear layers. About 60% of the cells dissociated from rat retina showed immuno-colocalization of GD3 and the neuronal marker class III beta tubulin isotype or cholera toxin binding. All morphologically identifiable glial Muller cells coexpress GD3 and gangliotetraosylgangliosides. GD3 was a minor ganglioside among these axonally transported by ganglion cells in rats and guinea pigs, suggesting that it is either not synthesized by ganglion cells or, if so, it is restricted to the cell soma and/or dendritic tree. Our results demonstrate that, unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosylceramide series but also keep a prevalence of gangliosides of the lactosylceramide series (GD3) when they are fully differentiated.

Coexpression of lactosyl and gangliotetraosyl gangliosides in rat cerebellar radial glial cells in culture.

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. To distinguish the major neural cell types present in the cultures the expression of the following cell type-specific markers was investigated: neuron-specific enolase and microtubule-associated protein-2 (MAP-2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells. More than 80% of cells dissociated from cerebellar tissue of 15-day-old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl-GTC, but most are negative for the expression of neuron-specific enolase, MAP-2, and GFAP. After culturing for 4 days (E15 + 4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl-GTC and "inactivate" the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl-GTC, but unlike neuron-like cells, they do not "inactivate" the expression of GD3.

In vivo and in vitro expression of gangliosides in chick retina Müeller cells.

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult rat retina interneurons synthesize GD3: GD3 expression by these cells is regulated by cell-cell interactions.

GD3, a ganglioside of the lactosyl series, is prevalent in rat retina neuronal cells. We studied here whether rat retina neurons synthesize their own surface GD3 or if they acquire it from Müller glia cells. We analyzed the activity of GD3 synthase and the in vivo labeling of gangliosides from N-[3H]acetylmannosamine in adult rat retinas after selective destruction of Müller glia cells with the gliotoxic alpha-D,L-aminoadipate (AAA). Immunostaining of rat retina sections and western blot analysis with an antivimentin antibody confirmed the gliotoxic effect of AAA. Neither GD3 synthase activity nor the in vivo labeling of GD3 and other gangliosides was significantly affected by AAA, indicating that neuronal cells synthesize their own GD3. We next analyzed the regulation of the expression of GD3 by these neurons in culture. About 80% of freshly dissociated cells from retina of 4-day-old rats (R4) immunoexpress surface GD3. After 3 days in dispersed cell culture conditions, GD3 expression was under the limit of detection in 80% of neuronal cells, indicating a failure of these cells to maintain the expression of surface GD3 in these experimental conditions. Most flat Müller glia-derived cells present in these cultures were GD3 positive. Surface GD3 was detected in approximately 60% of neuronal cells dissociated from R4 tissue that was developed in vitro as an organ culture for 3 days. Likewise, approximately 50% of neurites that had grown out from R4 retinal explants within 3 days in culture and whose neuronal character was indicated by immunoexpression of growth-associated protein GAP-43 were GD3 positive. These findings suggest that the tissue organization and/or specific interactions modulate GD3 expression in neuronal cells. Under dispersed-cell culture conditions, c-pathway gangliosides (GQ1c and GT1c), which are built up from the sialylation of GD3 and later completion of the oligosaccharide backbone, were detected in approximately 60% of neuronal cells, suggesting a maintenance of production of GD3 as an intermediate for gangliotetraosyl gangliosides.

GD3 prevalence in adult rat retina correlates with the maintenance of a high GD3-/GM2-synthase activity ratio throughout development.

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a ganglioside of the lactosylceramide series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N-acetylneuraminic acid (NeuNAc)] to adult (30% of total gangliosidic NeuNAc). GD3-synthase specific activity increased about 1.5-fold from birth to day 7 and essentially plateaued thereafter. The GD3-/GM2-synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GD1a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other "b" pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3-/GM2-synthase activity ratio throughout development of the tissue.

The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides.

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.

Disposition of gangliosides and sialosylglycoproteins in neuronal membranes.

Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP-[3H]N-acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [3H]sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 M-KCl, 125 mM-EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of th neuraminidase-labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase-labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labelled in vivo by an intracerebral injection of N-[3H]acetylmannosamine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with behaviour of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.