Inhibitory effects of tunicamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired. while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

Inhibitory effects of tunacamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired, while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.     

Developmental Changes in β-Citryl-L-G1utamate Concentration and Its Synthetic and Hydrolytic Activities in Neuronal Cells Cultured from Chick Embryo Optic Lobes

Developmental changes in the concentration of beta-citryl-L-glutamate(beta-CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by beta-CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of beta-CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into beta-CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of beta-CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.     

Addition of mannose to both the amino- and carboxy-terminal propeptides of type II procollagen occurs without formation of a triple helix

Matrix-free cells obtained from chick embryo cartilage were incubated in the presence of α,α′-dipyridyl and radioactive mannose in order to examine the incorporation of mannose into the propeptide extensions of Type II procollagen. Cell proteins were digested with bacterial collagenase and the digests were examined by polyacrylamide gel electrophoresis. Radioactive mannose was found in fragments from both the N- and C-propeptides, and therefore the results provided the first indication that both these propeptides of Type II procollagen contain mannose. The results also supported previous indications that addition of carbohydrate to the propeptides of procollagen does not require folding of the collagen domain into a triple helix.     

Proliferative response of the mesencephalic matrix areas in the reparation of the optic tectum of Triturus cristatus carnifex

The localization and proliferative response of optic tectum matrix cells has been studied in adult newt following an experimental lesion on an optic lobe. The results show that 15 days after the lesion the cells in division, autoradiographically labelled, are located in the periventricular layer. Thirty days after the lesion the labelled cells are also found in the innermost grey layers; at 90 days the injured optic tectum regains the cytoarchitecture characteristic of this centre, with labelled cells, whether in the external or in the internal pyriform layers. In all the stages the labelled cells are also found in the periventricular layers of the controlateral optic tectum, in the dorsal pallium and in the striatum. The quantitative data exhibit the existence of a direct relationship between the number of proliferating cells in the injured optic lobe and the extent of the lesion. These data show the possibility of active cellular proliferation for the reconstruction of the lesioned nervous area and for restoration of the characteristic histological structure.     

Utilization in vivo of glucose carbon in the optic lobes and the cerebellum of the chick during postnatal growth.

1. The incorporation of glucose carbon in vivo into amino acids was studied in the chick optic lobes and cerebellum during postnatal growth after subcutaneous injection of [U-14C]glucose. 2. The rapid incorporation of glucose carbon into free amino acids appears between the 1st and the 2nd day of postnatal growth in the optic lobes and between the 1st and the 4th day after hatching in the cerebellum. 3. The period during which the properties of mature brain metabolism are obtained is characterized in both structures during the first 48 hr of postnatal growth by changes in the specific radioactivity of some amino acids such as aspartate and alpha-alanine, and also by transient increases of glucose and glutamine concentrations. 4. The gamma-aminobutyrate content in the optic lobes is very high; the cerebellum on the contrary is characterized by its low gamma-aminobutyrate concentration linked to a very fast metabolism of this amino acid.     

Cyclic AMP mediates beta-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.     

The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.     

Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.     

SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells.

Migration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose-dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N- and C-terminal Ca(2+)-binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell-associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high-affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]-thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum-free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.     

Catabolic Regulation of the Expression of the Major Myelin Glycoprotein by Schwann Cells in Culture

Previous studies have suggested that neonatal Schwann cell cultures deprived of axonal contact do not express components of the myelin membrane, including the major myelin glycoprotein, P0. In contrast, Schwann cells from permanently transected, adult nerve exhibit continued biosynthesis of P0 after culture, suggesting that the ability to express the myelin glycoprotein may depend on the degree of cellular differentiation. To examine further the ability of Schwann cell cultures to express P0 as a function of age, we have performed precursor incorporation studies on endoneurial explants from 4- to 12-day-old rat sciatic nerves after 5 days in culture. The data reveal that explants from 12-day-old animals synthesize detectable levels of this integral myelin protein when assayed by [3H]mannose incorporation, even though there is no apparent myelin assembly in the cultures. Pulse-chase analysis of cultures from 12-day-old rats demonstrates that [3H]mannose-labeled P0 is substantially degraded within 3 h. This catabolism largely can be prevented by the addition of swainsonine, ammonium chloride, or L-methionine methyl ester to the pulse-chase media. The former agent alters oligosaccharide processing whereas the latter two compounds inhibit lysosomal function. The P0 synthesized by the 12-day explant cultures following the addition of swainsonine is readily fucosylated, implying that the protein has progressed at least as far as the medial Golgi before its exit and subsequent catabolism. If cultures from 4-, 6-, and 8-day-old animals are analyzed for P0 biosynthesis by [3H]mannose incorporation in the presence of swainsonine, detectable levels of the glycoprotein are seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.     

Changes in the rate of protein synthesis in parenchymatous liver cells during stimulation of the mononuclear phagocyte system

In vivo stimulation of mononuclear phagocyte system (MPS) by zymosan, dextrane sulfate, and prodigiosan caused almost a two-fold increase in hepatic protein synthesis. The rate of 14C-leucine incorporation increased both into total and soluble proteins. To define the cellular locus of these changes, preparations of hepatic parenchymal and nonparenchymal cells were obtained from the control and LPS-stimulated rats. The results indicate that the treatment of rats with prodigiosan stimulate protein synthesis in hepatocytes. No effect on protein synthesis of non-parenchymal cells was observed. Stimulation of MPS also caused a significant increase in 14C-leucine incorporation into serum lipoproteins. The results suggest that MPS may be involved in regulation of protein synthesis in hepatic parenchymal cells.     

Pigment-dispersing factor sets the night state of the medulla bilateral neurons in the optic lobe of the cricket,Gryllus bimaculatus

Pigment-dispersing factor (PDF) is an octadeca-neuropeptide widely distributed in the insect brain and suggested to be involved in the insect circadian systems. We have examined its effects on the neuronal activity of the brain efferents in the optic stalk including medulla bilateral neurons (MBNs) in the cricket, Gryllus bimaculatus. The MBNs are visually responding interneurons connecting the bilateral medulla, which show a clear day/night change in their light responsiveness that is greater during the night. Microinjection of PDF into the optic lobe induced a significant increase in the spontaneous activity of the brain efferents and the photo-responsiveness of the MBNs during the day, while little change was induced during the night. The enhancing effects began to occur about 20 min after the injection and another 10 min was necessary to reach the maximal level. The effects of PDF were dose-dependent. When 22 nl of anti-Gryllus-PDF (1:200) IgG was injected into the medulla, the photo-responsiveness of the MBNs was suppressed in both the day and the night with greater magnitude during the night. No significant suppression was induced by injection of the same amount of IgG from normal rabbit serum. These results suggest that in the cricket optic lobe, PDF is released during the night and enhances MBNs' photo-responsiveness to set their night state.     

Several Cell Responses to Insulin of Cultured Cells Derived from Micromeres, Isolated from Sea Urchin Embryos at the 16 Cell Stage

In micromere-derived cells of sea urchin embryos, treatment with insulin started for up to 24 h during culture at 20°C resulted in augmentation of ³²P incorporation into protein (protein phosphorylation) followed by activation of ³²P incorporation into RNA (RNA synthesis) and then induced pseudopodial cable growth, accompanied by considerable decreases in the rates of protein phosphorylation and RNA synthesis. This augmentation of RNA synthesis and cable growth induced by insulin were blocked by H-7, which inhibited protein phosphorylation, and were also inhibited by actinomycin D without any inhibition of protein phosphorylation. Similar results were obtained on treatment with horse serum, found to contain insulin-like compounds. In cells treated with horse serum treated cells, high rates of protein phosphorylation and RNA synthesis were maintained even after the initiation of cable growth and about 5 h later, spicule rods were produced. Insulin treatment did not induce spicule rod formation. In cells treated with horse serum, actinomycin D treatment started at the time of initiation of cable growth, cables were formed but formation of spicule rods was blocked. These results suggest that horse serum contains some other substance besides insulin-like ones, which induces expression of genes that are indispensable for spicule rod formation.