Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts. Evidence for differences related to the age of embryos

Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8-day chick embryos and to stimulate this process in those derived from 16-day embryos. Con-A effects depended on the duration of contact with cells and lectin and were inhibited by alpha-methylmannopyrannoside. Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. Con A only modified the Vmax of the uptake system and did not alter the Km. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.     

Identification of components of phospholipids metabolism in automated phosphate ester chromatography.

Transport accross the cell membrane of 3-O-methylglucose, a non-phosphorylatable glucose analogue, was measured in primary cultures of fibroblasts from 8-, 12- and 16-day chick embryos. Transport of this hexose was found to be 3.5 times and 2 times faster in fibroblasts from 16-day embryos than in fibroblasts from 8- and 12-day embryos, respectively. Compared with 8- and 12-day embryos, the rate of efflux in fibroblasts from 16-day embryos was found to be increased. 3-O-methylglucose transport in these cells did not result in an accumulation of the hexose against a concentration gradient. It was concluded that in fibroblasts from older embryos a facilitated diffusion system for hexose transport was stimulated. Embryo differentiation could be associated with a change in the plasma membrane by increasing either the number or the mobility of the glucose carriers, since the Vmax of the transport system for 3-O-methylglucose increased in fibroblasts from older embryos, while the affinity or Km of the system remained unchanged.     

Embryo Cell Surfaces—Lectin Binding and Cell Proliferation

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Conca navalin A decrease cell number and proportion of cells incorporating [³H] thymidine in case of 8and 10day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-dayold embryo cells. No effect was noticed in 12-day cells.
These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8^th to the 12^th day of development, and slowly increases on the 16–day cells, the affinity constant remaining rather constant.
The results indicate that the age–dependent effect of lectin on embryo cells could not be directly related to the number of lectin–binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of Concanavalin A.
Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.     

Increase in the number of glucose carriers in chick fibroblasts during embryo development

Cytochalasin B (CB) has been used as a tool to ascertain whether the increase in the rate of 3-O-methylglucose (3-O-MeG) uptake between the 8th and the 16th day of development in chick embryo fibroblasts could be attributed to an increase in the number of hexose transport carriers. There was a 2—3-fold difference in glucose-specific CB binding between the 8- and the 16-day cells, a difference which is comparable to the previously reported differences in rates of 3-O-MeG uptake. We therefore suggest that glucose-specific CB binding represents binding to the 3-O-MeG carrier and that the increase in the rate of 3-O-MeG uptake from the 8th and the 16th day of development is probably due to an increase in the number of hexose carriers.     

Inaccessibility of certain Ricinus lectin binding sites due to the increase in hyaluronic acid during chick embryo development

Chick embryo fibroblasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin; however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2-5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells. These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation.     

The hexose transporters at the plasma membrane and the tonoplast of higher plants

The uptake of hexoses in higher plant cells is thought to be catalyzed by an H⁺/hexose contrasporter in the plasma membrane. Transport studies with isolated plant vacuoles indicate that, at the tonoplast, a second hexose transporter is located with properties different from the plasma membrane transporter. Recently membrane vesicles of high purity and defined orientation have been used for a more rigorous individual characterization of these two carriers. Concomitantly, a cDNA for the inducible H⁺/hexose cotransporter of the green alga Chlorella has been sequenced and shown to exhibit homology to a group of hexose transporters (for facilitated diffusion) of other eukaryotic and prokaryotic organisms. With a probe derived from the Chlorella sequence, the first plant gene for an H⁺/hexose contransporter ( Arabidopsis thaliana ) has been isolated, opening the route to molecular studies of structure, function and evolution of the hexose transporters of higher plants. The present review discusses recent work on the kinetic characterization and identification of the higher plant plasma membrane and tonoplast hexose transporters as well as their respective cellular functions. Furthermore, perspectives for future research on the plant hexose transporters are outlined.     

Inaccessibility of certain Ricinus lectin binding sites due to the increase in hyaluronic acid during chick embryo development

Abstract. Chick embryo fibre-blasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin;.however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2–5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells.
These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation.     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development.

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26 plus or minus 0.03)-10-6 sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites fo Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

Effect of fusicoccin and gibberellic acid on primary dormant Avena fatua caryopses

Fusicoccin induced germination in dormant and partially afterripened dormant caryopses of Avena fatua L. The rate of caryopsis germination was slower and final percentage germination lower in the highly dormant inbred line M73 at a given concentration of fusicoccin than in the dormant caryopses of line AN265. Gibberellic acid was more effective than fusicoccin in breaking dormancy in both lines. Promotion of germination of dormant caryopses by fusicoccin was inhibited by a 6-day pretreatment with (2-chloroethyl)trimethylammonium chloride.
The basal rate of proton efflux from embryos isolated from dormant and fully afterripened line AN265 caryopses was similar. Addition of fusicoccin increased the rate of proton efflux from the isolated embryos of dormant and afterripened caryopses by nearly 400%. Gibberellic acid had no effect on the rate of proton extrusion. The uptake of ⁸⁶Rb⁺ in dormant and afterripened A. fatua embryos was similar after a 2 h uptake period. The addition of fusicoccin to the medium doubled the uptake of ⁸⁶Rb⁴ by dormant and afterripened embryos. Gibberelleic acid had no effect on the uptake of ⁸⁶Rb⁺ by isolated embryos from either dormant or afterripened caryopses. The experimental results indicate that gibberellic acid is more versatile in its action than fusicoccin, and gibberellic acid may facilitate dormant A. fatua caryopsis germination by stimulating mechanisms other than the direct H⁺ efflux and K⁺ uptake at the membrane level.     

Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development

Concanavalin A and Robinia pseudoacacia lectin decreased [(3)H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6-10-day chick embryos. In contrast, these lectins stimulated [(3)H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [(3)H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with alpha-methyl mannopyranoside and with anti-(Robinia lectin) serum.     

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.     

Cell growth and thymidine incorporation changes induced by Dolichos lectin in embryo fibroblasts at various stages of differentiation.

We report here the effect of Dolichos lectin on chick embryo fibroblasts from embryos between 6th and 16th day of development. There is evidence that Dolichos lectin decreases cell number and proportion of cells incorporating tritium labelled thymidine in case of chick embryo fibroblasts of 6th, 8th and 10th day of development. Dolichos lectin stimulated the proliferation of 16-day old embryo cells. No effect was noticed on 12-day embryo cells at different concentrations of Dolichos lectin used. This lectin is specifically inhibited by N-acetyl-D-galactosamine and anti-Dolichos lectin serum. The difference in response by cells during different stages of embryonic development could perhaps be explained as some regulatory changes occurring on the cell surface.     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Studies on Polyadenylic Acid-Containing RNA from Developing Nervous System of the Chicken the

Abstract: To investigate certain biochemical aspects of myelination, a study was undertaken of the messenger-like RNA in the nervous system of pre- myelinating 14-day embryos and of myelinating 17-day embryos and 3-day chicks. The central and peripheral nervous systems of the chick were found to contain and to actively synthesize poly(A)⁺ RNA. RNA species binding to oligo(dT)-cellulose contained a relatively high proportion of adenylate residues and were resistant to the actions of pancreatic and T₁ ribonucleases. Preparations labeled by incubation with adenosine in vitro showed a decrease in the proportion of poly(A)⁺ RNA as the age of the animal increased, while preparations labeled in vivo exhibited the opposite trend. Polyacrylamide gel electrophoretograms of both in vivo and in vitro labeled pqeparations showed that the poly(A)⁺ fractions contained mainly heterodisperse RNA species. The average molecular size of poly(A)⁺ RNAs of purified polysomal fractions of nerve RNA from 3-day chicks was smaller than 18S, whereas that of total poly(A)⁻ RNA was larger than 18s. The proportion of poly(A)⁺ molecules larger than 18s was lower in the rapidly myelinating nerve tissues of 17-day embryos and post-hatching chicks than in those of premyelinating 14-day embryos. Similar results were obtained for crude nuclear RNA fractions or RNA preparations fractionated under denaturing conditions. These results are consistent with previous work showing that the embryonic peripheral nerve contains a larger proportion of high-molecular-weight, messenger-like RNA molecules than does nerve tissue from young chicks or adults.     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

Concanavalin A-induced impairment of fibroblast spreading on laminin but not on fibronectin

The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Inhibition of the glucose and amino-acid carriers of Lemna gibba by pretreatment with HgCl2

The electrical resting potential across the plasmalemma of Lemna gibba L. (G 1) cells is −230 to −250 mV and the diffusion potential in the presence of 1 mol m⁻³ KCN + 1 mol m⁻³ salicylhydroxamic acid is about −100 mV. A concentration of 0.01 mol m⁻³ HgCl₂ depolarises the transmembrane electrical potential in a largely reversible way. When the cells after 16 min of HgCl₂-application are returned to Hg-free solution, the transmembrane electrical potential is only depolarised by 24 × 13 mV (SD, n = 13) compared with the potential prior to HgCl₂ treatment. In contrast, a 16 min pretreatment with HgCl₂ followed by a wash with mercury-free solution reduces the transient depolarisations of transmembrane potential observed after addition of 5 mol m⁻³ D-glncose or 1 mol m⁻³ L-alaoine to about 60% of controls. These transient depolarisations are due to the onset of solute uptake. Accordingly, HgCl₂-pretreatment inhibits uptake of ¹⁴C-3-O-methyl- d -glucose by more than 50% and uptake of ¹⁴C- l -alanine by more than 70%. Washing with 1 mol m⁻³ 1,4-dithiothreitol does not reverse this inhibition. It is, therefore, concluded that Hg²⁺ irreversibly binds to essential SH-groups of the H⁺-hexose and the H⁺-amino-acid cotransport carriers of Lemna gibba and inhibits these carriers without appreciably affecting the electrogenic proton-extrusion pump.     

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.     

Changes in ion content and transport during development of embryonic rainbow trout

Wet mass and water content of four lots of whole eggs did not change throughout embryonic development of rainbow trout Oncorhynchus mykiss. Eggs in all four lots accumulated Na⁺. Eggs in lots 2 and 4 also accumulated Ca²⁺ and Cl^-, whereas eggs in lot 1 showed no significant change in Ca²⁺ or Cl^- and eggs in lot 3 showed no change in Cl^-and a small loss of Ca²⁺. Although the Na⁺ content of embryonic tissues increases in the later stages of development, the yolk sac content remained constant, indicating uptake of Na⁺ from the environment. Na+ uptake by whole eggs was non-saturable, consistent with diffusion of Na⁺ across the chorion into the perivitelline fluid. Na⁺ uptake in dechorionated embryos was saturable, as was Ca²⁺ uptake by both whole eggs and dechorionated embryos, consistent with active uptake or facilitated diffusion mechanisms at the surface of embryos. Very low Ca²⁺ uptake rates in dechorionated embryos suggest that the Ca²⁺ uptake mechanism is not fully developed until after hatching.