Fucose Incorporation Research Articles

Myogenesis of normal and dystrophic chick embryonic skeletal muscle cells in vitro: biosynthesis of plasma membrane proteins.

Plasma membranes were prepared from primary cell cultures of normal and genetically dystrophic chick embryonic pectoral muscle. These membranes were analyzed both by one-dimensional sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis and by two-dimensional electrophoresis using isoelectric focusing in the first dimension. No marked and reproducible abnormalities could be detected in the synthesis, or accumulation, of plasma membrane proteins of dystrophic muscle cells maintained in culture for periods of up to 6 days. Analysis of the relative rates of protein turnover, analysis of fucose incorporation into plasma membrane proteins, and comparison of iodinated cell surface proteins also failed to reveal distinct abnormalities in plasma membranes derived from cultured dystrophic muscle cells. Although the results obtained do not rule out an early defect in plasma membrane protein biosynthesis during the development of dystrophic skeletal muscle in vivo, they do demonstrate that the synthesis and assembly of at least the major plasma membrane proteins occur normally during the initial phases of terminal differentiation of isolated dystrophic skeletal muscle cells in tissue culture.

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.

ChemInform Abstract: HALOGENATED L‐FUCOSE AND D‐GALACTOSE ANALOGS: SYNTHESIS AND METABOLIC EFFECTS

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 m m) initially suppresses [ 3H]glucosamine or [ 3H]fucose incorporation into cellular macromolecular material; however, after 3 1 2 h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μ m) produced a similar increase in accumulation of [ 3H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [ 3H]glucosamine-containing immunoprecipitable material was not suppressed during the first 3 1 2 h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [ 3H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [ 3H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.

Synthesis and secretion of glycoproteins by mucosa of rat gastric antrum in organ culture

Measurement of gastric mucus production in vivo poses several problems. We therefore used organ culture of rat antrum to measure mucosal synthesis and secretion of glycoprotein. Tissue maintained good viability for 20 hr. Glycoprotein synthesis rate, assessed by incorporation of [3H]fucose and [3H]glucosamine, was constant during 20 hr. [3H]Fucose incorporation increased in proportion to increasing [fucose] in medium until the latter reached 10(-3) M, then the slope decreased. Cycloheximide abolished [3H]fucose incorporation into glycoprotein. Two methods for measuring secretion gave parallel results: prelabeled glycoprotein was secreted linearly, with 60% of pulse-labeled tissue glycoprotein appearing in medium in 20 hr. The data suggest that (1) antral mucous cells contain a high-concentration pool of endogenous fucose, but possibly only a small-capacity pool of preformed apoglycoprotein, (2) synthesis and secretion of glycoprotein can be successfully examined with organ culture, and (3) secretion in vitro occurs at a steady, slow rate.

3H-glucosamine and 3H-fucose incorporation by murine peritoneal macrophages “non-divided” during differentiation in vitro

Normal murine peritoneal macrophages were maintained in culture for 24 hours and 6 days. These cells continuously incorporated 3H-glucosamine and 3H-fucose in their membrane components. For both precursors culture ages, these components may be considered to undergo turnover according to a given rhythm with a 24 hours doubling time. Treatment with neuraminidase did not modify the doubling time, although a slight increase of incorporation of radioactivity was observed. Variations of 3H-fucose and 3H-glucosamine incorporations were observed between 24 hours old and 6 days old cultures: 6 days old cultures incorporated more glucosamine than 24 hours old cultures whereas no difference in fucose incorporation was observed between the two cultures.

Halogenated L-fucose and D-galactose analogues: synthesis and metabolic effects.

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

A comparison of [3H]galactose and [3H]fucose uptake with the morphological and histochemical changes observed in mucous secretion in chemically induced rat colonic carcinoma.

The alterations in carbohydrate metabolism which occur in the distal colon of rats during carcinogenesis induced by dimethylhydrazine were investigated using [3H]galactose and [3H]fucose as glycoprotein precursors. A statistically significant decrease in [3H]galactose uptake was observed in dysplastic epithelia. These findings are consistent with the alterations in mucin composition with predominance of sialomucins shown in these areas by histochemical methods. Furthermore, changes in the gradient of [3H]galactose incorporation along the crypt epithelium were also found in the histological and histochemically non-involved colonic mucosa of dimethylhydrazine-treated rats, as compared with controls. No significant variations were seen in [3H]fucose incorporation. These results correlate well with our previous histochemical observations and are further evidence of the profound alterations in glycoprotein synthesis affecting the whole colonic mucosa during carcinogenesis.

Incorporation of intrastriatally injected [ 3H]fucose into electrophoretically separated synaptosomal glycoproteins. I. Turnover and molecular weight estimations

The radioactivity profiles of electrophoresed neostriatal P2 fraction glycoproteins were examined at a series of times (2.5, 3 and 4 h; 1, 5 and 10 days) following intracranial injections of [3H]fucose into the neostriatum. Ten major fucosylglycoprotein peaks were discerned in these profiles and certain aspects of their metabolism were characterized. The half-life of fucosylglycoproteins in the P2 fraction was estimated to be 9.7 days. The half-lives of the individual glycoprotein peaks ranged from 4.9 to 17.9 days. The apparent molecular weights of the glycoprotein peaks obtained by our procedures ranged from 32,000 to 180,000 daltons. One peak (peak VIII) incorporated radioactivity primarily at short intervals following the injection. The time course of [3H] fucose incorporation into this peak suggests involvement in the transport, activation and/or incorporation of fucose in brain. Since intracranial injections of [3H]fucose are incorporated into proteins in the cell body, synaptosomal fractions from caudate neurons alone are labeled by this technique. This may be useful in separating pre- and postsynaptic glycoprotein biochemistry. Finally, we tentatively propose that the glycoprotein peaks observed in neostriatum may be identical to previously isolated glycoproteins of known function or subcellular location.

The incorporation of intrastriatally injected [ 3H]fucose into electrophoretically separated synaptosomal glycoproteins. II. The influence of passive avoidance training

Following intrastriatal injections of [3H]L-fucose, male albino rats served as (a) trained subjects in a step-down passive avoidance task, (b) stress controls receiving inescapable shock or (c) handled controls. At a series of time points after treatment the animals were sacrificed and the P2 fraction of the injected neostriatum was isolated. This tissue was electrophoresed on SDS-polyacrylamide gels and radioactivity profiles were constructed from 1 mm gel slices. The profiles of trained subjects were compared to shocked and handled control subjects from the same time point group. No differences in total [3H]fucose incorporation into neostriatal glycoproteins were detected as a result of the behavioral treatment used, nor was an incorporation into the majority of electrophoresed peaks altered. Three radioactive gel peaks were significantly altered as a function of experience. At the one day time point, trained subjects exhibited a significant increase in the tritium content of a 70,000 dalton fucosyl-glycoprotein peak. At the 5 day time point, increased label was detected in a 180,000 dalton peak in both trained and shocked subjects, while a significant increase in a 140,000 dalton peak was observed only in trained animals. The relation of the present findings to perviously reported training related differences in glycoprotein metabolism are discussed.

Dopamine and macromolecule synthesis in rat hippocampus

In hippocampus slices both dopamine and apomorphine lead to an increased incorporation of ( 3H)-fucose into total proteins, whereas the incorporation of ( 14C)-leucine was unchanged or decreased, respectively. Noradrenaline did not alter the incorporation of both precursors, whereas haloperidol partially reduced the dopamine induced increased in incorporation of fucose. Thus, an induction process of the observed macromolecular changes involving dopaminoceptive structures of hippocampus can be assumed.

Polyprenyl phosphate sugars synthesized during slime-polysaccharide production by membranes of the root-cap cells of maize (Zea mays)

Two types of experiments were carried out; either maize roots were incubated in L-[1-3H]fucose or membranes were prepared from root tips and these were incubated with GDP-L-[U-14C]fucose or UDP-D-[U-4C]glucose. The radioactively labelled lipids that were synthesized in vivo and in vitro were extracted and separated into polar and neutral components. The polar lipids had the characteristics of polyprenyl phosphate and diphosphate fucose or glucose derivatives, and the neutral lipids of sterol glycosides (fucose or glucose). A partial separation of the glycolipid synthetase reactions was achieved. Membranes were fractionated into material that sedimented at 20,000g and 100,000g. Most of the polar glycolipid synthetase activity (for the incorporation of both fucose and glucose) was located in the 100,000 g pellet, and this activity was probably located in the endoplasmic reticulum. The neutral lipid, which contained fucose, was synthesized mainly by membranes of the 20,000g pellet, and the activity was probably associated with the dictyosomes, whereas the neutral glucolipids were synthesized by all the membrane fractions. It is suggested that the polar (polyprenyl) lipids labelled with fucose could act as possible intermediates during the synthesis of the glycoproteins and slime in the root tip.

Biosynthesis of membrane glycoproteins in rat hepatoma tissue culture cells.

The early steps in the biosynthesis of glycoproteins associated with the plasma membranes of rat hepatoma tissue culture cells has been analyzed. By measuring the effect of tunicamycin on the incorporation of [3H] mannose and [3H] fucose into cell glycoproteins, it was determined that an interval of about 1 h was required to transfer the glycoprotein from site of mannosylation to the site of fucosylation. This result was corroborated by an analysis of the time required for the appearance of either mannose or fucose-labeled glycoproteins at the cell surface. The separation of membrane glycoproteins by a two-dimensional gel system allowed the visualization of the modifications leading to both size and charge heterogeneity of these proteins. By following the changes in electrophoretic mobility introduced into membrane glycoproteins during a chase period after a pulse labeling, the time course of these molecular alterations could be estimated. Several glycoproteins have apparently higher rates of synthesis than the bulk of membrane-associated glycoproteins. Most of these glycoproteins were released within 2 h after biosynthesis from the intracellular membrane fraction and appear after 3 h in the medium. In addition to the glycoproteins that contain both mannose and fucose and that show a high degree of charge heterogeneity, there are other membrane-bound species that are not noticeably modified by the incorporation of fucose or sialic acids. These glycoproteins could represent constituents limited to the internal membrane system of the HTC cell.

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.

NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells

Rat PC12 pheochromocytoma cells respond to Nerve Growth Factor (NGF) by ceasing to undergo mitosis and acquiring neuronal characteristics, including outgrowth of neurites and electrical excitability. We have found that NGF treatment results in no consistent qualitative and only a few minor quantitative changes in the two-dimensional electrophoresis pattern of 14C-amino acid-labeled proteins synthesized by the cells. On the other hand, NGF stimulates the incorporation of radiolabeled fucose or glucosamine into several components, including one of apparent molecular weight 230,000 daltons on one- and two-dimensional SDS-polyacrylamide gels. This component is removed by mild trypsinization of intact cells and therefore appears to be a glycoprotein (named here NILE) at least partially exposed on the cell surface. Stimulation of NILE glycoprotein labeling can first be detected after 2 days of exposure to NGF and increases progressively with time of treatment. This change is not solely a consequence of the cessation of cell division caused by NGF, since it does not occur in nondividing PC12 populations prepared by cytosine arabinoside treatment. NGF stimulates labeling of NILE glycoprotein even when attachment and process outgrowth are prevented by growing the cells in spinner suspension cultures. The relative rate of labeling attained under these conditions is less, however, than in NGF-treated, substrate-attached cells. Stimulation of NILE glycoprotein labeling by NGF is selectively blocked (as is neurite outgrowth) by camptothecin, an RNA synthesis inhibitor, and thus may require transcription. Despite their similarities in apparent size and exposure on cell surfaces, the NILE and LETS glycoproteins are shown to be immunologically distinct.

Glycoprotein transport in the rat cerebellum.

The fate of tritiated fucose in rat cerebellum has been followed using autoradiographic techniques for a period up to one week after intraventricular injection. At intervals of up to three hours after injection, Purkinje and granule cell bodies were found to be most heavily labelled. Subsequently this labelling declined and the highest grain density was observed over the molecular layer. The data is interpreted in terms of fucose incorporation into glycoproteins in nerve cell bodies followed by rapid dendritic transport in the Purkinje cell and fast transport in granule cells along ascending axons and parallel fibres.

Loss of cell-population-density-dependent incorporation of fucose into the lipid fraction of cultured human tumour cells.

The incorporation of radioactively labelled fucose into the lipid fraction of cultured normal human cells and several human tumour-cell lines was examined as a function of the cell population density. Normal cells exhibited a density-dependent pattern of incorporation, whereas in tumour cells the radioactivity incorporated was independent of the cell population density. An exception was found among the tumour cells, which suggests a possible correlation between the loss of this marker and the ability to produce tumours.

Patterns of cell differentiation revealed by l-[ 3H]fucose incorporation in Dictyostelium

A study of the incorporation of l-[6- 3H]fucose and d-[6- 3H]glucosamine hydrochloride was conducted during the development of the cellular slime mold Dictyostelium discoideum 1-H. Autoradiographs revealed that pulse-labeled vegetative amoebae incorporated [ 3H]fucose intracytoplasmically within 15 min. The majority of the cells had randomly scattered silver grains but the remainder were distinguished by a dense localized labeling which suggested that oligo or polysaccharide synthesis was occurring. The localized pattern of labeling attributed to active synthesis declines at aggregation and early conus formation. As the pseudoplasmodium makes the developmental transition from the conus to the culmination stages the localized pattern of [ 3H]fucose labeling was restricted to the prespore cells while the prestalk cells were devoid of label. Prespore vacuoles were not present at the onset of this transition and consequently [ 3H]fucose incorporation occurred in the cells prior to their differentiation into prespore cells. In contrast to cells composing earlier stages, mature spores exhibited [ 3H]fucose-containing substances at the cell surface. At appropriate stages certain cells actively synthesize slime and stalk sheath which were labeled with either [ 3H]fucose or [ 3H]glucosamine. Prestalk isolates were obtained by transecting migrating slugs. [ 3H]Fucose was incorporated within 10 min among the basal cells of the isolate in the localized pattern typically found in prespore cells. The incorporation of [ 3H]fucose occurred prior to prespore differentiation as certain preparations were devoid of prespore vacuoles. Prespore isolates differentiate prestalk cells which have lost the capacity to incorporate [ 3H]fucose. This investigation suggests that cell contacts and interactions may affect the incorporation of [ 3H]fucose.

Biochemical expression of the galactosemic defect in lymphocytes and the effects on glycoprotein synthesis

Incorporation of radioactive galactose, fucose, mannose, glucosamine, and N-acetylmannosamine into acid-precipitable glycoproteins of purified peripheral blood lymphocytes from three patients with congenital galactosemia, their healthy parents, and control subjects have been measured. In the galactosemic lymphocytes, the incorporation of galactose into acid-precipitable glycoproteins was less than 7% of values obtained with controls and the presumed heterozygotes. The incorporation of other sugars into the glycoprotein fraction did not differ significantly from normal. Attempts to demonstrate a deficiency in cell-surface galactose groups proved negative. Galactosemic lymphocytes responded well to galactose-binding mitogens and were fully sensitive to a galactose-binding toxic lectin. We conclude that a defect in the galactose-1-phosphate uridyl transferase pathway to uridine diphosphate-galactose does not affect appreciably normal carbohydrate chain assembly in galactosemic lymphocytes. However, we cannot rule out minor changes which might account for the decreased responsiveness to serotonin in an experimental animal model of galactosemia. The approaches used in this paper may be useful in detecting other inborn errors of carbohydrate metabolism.

Carbohydrate incorporation in plasma membranes of mouse thymocytes stimulated by concanavalin A.

Thymocytes from CBA/J mice were stimulated with concanavalin A. Incorporation of various carbohydrates, leucine and thymidine into trichloroacetic-acid-precipitable material was compared in stimulated and control cells. Incorporation of all carbohydrates, in particular of galactose, was enhanced in concanavalin-A-treated cells. Leucine and thymidine incorporation was increased 2 and 50-fold respectively in stimulated cultures. The kinetics of fucose, galactose, glucosamine, leucine and thymidine incorporation were studied using 10-h pulses at various times during the cultivation. The incorporation of fucose, galactose and thymidine showed two maxima 5 and 30 h after the beginning of the cultivation. In contrast, there were no pronounced maxima seen with glucosamine and leucine. Plasma membranes from stimulated cells were prepared by a modified procedure of McCollester (Cancer Res. 30, 2832–2840, 1970). The purity of the membrane fraction was examined by electron microscopy, by chemical analysis and by assay of marker enzymes. When prepared from cells radioactively labelled with fucose or galactose the plasma membrane fraction was enriched in carbohydrate label. Gel filtration experiments with papain treated, radioactively labelled membranes, solubilised in sodium dodecylsulphate buffer, showed that fucose was only attached to glycoprotein, while other carbohydrate label was distributed between glycoprotein and a fraction containing glycolipids. Analysis of hydrolysed, carbohydrate-labelled membranes by paper chromatography and paper electrophoresis revealed that the bulk of the fucose and galactose label had not been converted into other substances, although some (10%) galactose is converted into glucose. Mannose label had been partially (14%) converted into fucose. Glucosamine label was found to be distributed in sialic acid (25%), glucosamine (40%), galactosamine (30%) and neutral compounds (5%). The data are discussed in relation to the sequence of biochemical events occurring at the cell surface during stimulation of thymocytes with concanavalin A.