Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Modulation by all-trans retinoic acid of glycoprotein glycosylation in murine melanoma cells: enhancement of fucosyl- and galactosyltransferase activities

beta-All-trans retinoic acid (RA) treatment of murine S91-C2 melanoma cells decreases in vitro growth and modulates the glycosylation of specific cellular and cell-surface glycoproteins. The effect of RA treatment on [3H]fucose, [3H]galactose, and [3H]glucosamine incorporation was investigated by metabolic labeling followed by analysis of labeled cellular glycoproteins using polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and fluorography. RA treatment dramatically increased the incorporation of the labeled monosaccharides into one glycoprotein of Mr 160,000 (gp160), which has been previously implicated in the growth-inhibitory effect of RA on these cells. Following RA treatment, cell-surface sialic acid residues on gp160 were also more intensely labeled by NaIO4 oxidation and subsequent NaB[3H]4 reduction than were those on gp160 of untreated cells. The activities of fucosyl- and galactosyltransferase increased about 1.5 to 1.9 times after RA treatment. These results suggest that the increased activities of the two glycosyltransferases is responsible for the increased incorporation of fucose and galactose into gp160.     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

In vivo metabolism of topically applied retinol and all-trans retinoic acid by the rabbit cornea

Corneas of normal and vitamin A-deficient rabbits were treated topically with [11, 12-3H] retinol or [11, 12-3H] all-trans retinoic acid. Methanol extracts of these corneas were analyzed by high pressure liquid chromatography. Radiolabeled compounds were extracted from the corneas which co-migrated chromatographically with known retinoid standards. In agreement with studies on other tissues and organs, retinol was metabolized to retinoic acid and more polar compounds by corneas of normal and vitamin A-deficient rabbits. All-trans retinoic acid was isomerized to 13-cis retinoic acid in normal rabbit corneas; however, this trans-cis isomerization did not occur in vitamin A-deficient, xerophthalmic corneas.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices

Slices were prepared from rat forebrains and the incorporation of [³H]mannose and [³⁵S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [³H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man₈GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc₃Man₉GlcNAc₂ accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [³H]mannose or [³⁵S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [³H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [³⁵S] methioninelabeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes.To whom to address reprint requests.     

Enhancement of glycosylation of cellular glycoconjugates in the squamous carcinoma cell line MDA886Ln by β-all-trans retinoic acid

Retinoids have been shown to inhibit the growth and modulate the glycosylation of head and neck squamous cell carcinoma (HNSCC) cells including the MDA886Ln cells. To examine the effects of -all-trans retinoic acid (RA) on glycoconjugates in HNSCC MDA886Ln cells, the cells were grown in the absence or presence of 1 µM RA and then labeled with tritiated monosaccharides, extracted and analysed by polyacrylamide gel electrophoresis and fluorography. RA increased markedly the incorporation of [³H]-glucosamine, [³H]-galactose, and [³H]-mannose into numerous cellular glycoconjugates, however, the incorporation of [³H]-fucose and [³H]-leucine was almost unaffected by RA. RA increased the incorporation of glucosamine and galactose but not mannose into high molecular weight (HMW) glycoconjugates of about 220 and 500–600 kDa. To analyse the steady state level of glycoconjugates by lectin blotting, extracts of unlabeled cells were separated by gel electrophoresis and the gels were probed with¹²⁵I-labeled wheat germ agglutinin (WGA) andMaackia amurensis (MA) agglutinin. Both lectins were found to bind to numerous glycoconjugates including the HMW glycoconjugates, whereas¹²⁵I-peanut agglutinin bound only to the HMW glycoconjugates. RA treatment increased the binding of all three lectins to the HMW glycoconjugates. These findings demonstrate that RA enhanced the incorporation of specific monosaccharides into a variety of glycoconjugates and in particular into HMW mucin-like glycoconjugates. This effect of RA may be the result of induction of a more normal differentiation state of the HNSCC cells.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices.

1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000--150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.     

Effect of glucose deprivation on the incorporation of 3H precursors into pituitary glycoproteins and on basal LH, FSH and prolactin release

Glucose can serve as a precursor for all of the components of the oligosaccharides of glycoproteins. The incorporation of various 3H precursors into glycoproteins was examined by incubating rat anterior pituitaries in vitro for 4h in the presence and absence of glucose. Substituting succinate for glucose enhanced the incorporation of D-(2-3H)mannose six-fold, the incorporation of D-(1,6-3H)-glucosamine two-fold and the incorporation of D-(6-3H) galactose was enhanced slightly. The incorporation of other 3H precursors was unaltered or reduced by glucose deprivation. Rat anterior pituitaries incorporated minimal quantities of N-acetyl-D-(6-3H)mannosamine, the immediate precursor of N-acetyl-D-neuraminic acid, into glycoproteins. Glucose deprivation also stimulated the 4h basal release of LH and FSH by 58% and 95%, respectively, as well as stimulated net intracellular degradation of prolactin. Thus, glucose deprivation enhances the incorporation of some, but not all, 3H precursors into glycoproteins as well as stimulates the basal release of LH and FSH.     

Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells

Intact murine L1210 leukemic cells incorporated significant quantities of [³H]-N-acetylneuraminic acid directly from CMP-N-acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N-acetyl-neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP-N-acetyl-neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N-acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP-N-acetylneuraminic acid during incubations with L 1210 cells was rather insignificant. Electron microscope autoradiography of cells incubated with CMP-N-acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP-N-acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells. Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP-[³H]-galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP-[³H]-galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L-1210 cells incubated in UDP-[³H]-galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto-syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-101), B-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [³H]concanavalin A, and a substantial decrease in the binding of [³H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [³H]leucine or [³H]proline into protein. There was, however, a significant inhibition in the incorporation of [³H]fucose, [³H]glucosamine, and [³H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

Synergistic effect of vitamin D derivatives and retinoids on C2C12 skeletal muscle cells

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.     

Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-³H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-³H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-³H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Testicular glycoprotein biosynthesis stimulated byin vivo administration of follicle stimulating hormone

Summary In vivo administration of follicle-stimulating hormone causes the increase of the in vitro incorporation of D-[1-³H] glucose, D-[¹⁴C-(U)] glucosamine or D-[2-³H] mannose into glycoproteins of normal or Sertoli cell enriched testes of immature rats. This effect is blocked by preincubation of the testes with tunicamycin.Abbreviations FSH follicle stimulating hormone - KRB Krebs Ringer bicarbonate buffer - TCA trichloroacetic acid - SCE testes Sertoli cell enriched testes