Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

The effects of cortisol and amino acids on the metabolic activity of rat skin stored in dimethyl sulfoxide buffer at −196 °C

Addition of amino acids to the DMSO buffer reduces the intracellular amino acid depletion of rat skin tissue frozen and stored at ?196 °C.Although prolonged exposure to DMSO progressively inhibits the [2-¹⁴C]glycine and l-[U-¹⁴C]leucine incorporation into the proteins, cortisol and amino acid additions to the buffer medium protect the protein-synthesizing activity. These factors also stimulate the incorporation of [6-³H]-thymidine into DNA. The stimulatory characteristics of cortisol and of amino acids separately are enhanced when both components are added together to the preserving buffer. These effects are noticeable in tissue only exposed to the DMSO buffer without freezing as well as in skin frozen and stored at ?196 °C and subsequently thawed at 40 °C.A stimulatory effect of cortisol and of a free amino acid, supplement to the medium on the α-amino-[1-¹⁴C]isobutyric acid uptake by the cells is only observed in skin exposed for a short period of time to the DMSO buffer, but it is not detectable after longer exposure and after freezing.     

Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO

Patterns of nuclear protein synthesis and phosphorylation have been investigated in Friend erythroleukemia cells. The rate of incorporation of [³H]leucine and [³²P]phosphate remains relatively constant during the first 48 h of dimethylsulfoxide (DMSO) stimulation, when more than 90% of the cells commit to erythroid differentiation, but falls to 20% by 120 h. Histone H2A phosphorylation is greatly increased during DMSO treatment, but no significant changes were found in the non-histone phosphoprotein patterns as determined by gel electrophoresis. There is also a small, but reproducible, change in the relative amounts of the two sub-fractions of histone H2A. There are no striking changes in the electrophoretic patterns of [¹⁴C]leucine-labelled nuclear proteins during the first 48 h, but the amount and the synthesis of two proteins of 46 000 and 280 000 D are increased somewhat during this period. Another protein, of molecular weight 65 000, appears to be induced in low amounts.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Lack of effect of GABA on [3H]leucine incorporation into a rat oviduct ribosomal system

A ribosomal system for [³H]leucine incorporation was isolated from the rat oviduct in order to study the possible effect of GABA on [³H]leucine incorporation during the estrous cycle. The system showed an absolute requirement for Mg²⁺ and about 50% dependence on an energy source. Optimal [³H]leucine incorporation occurred under 3–6 mM Mg²⁺ and 100 mM K⁺ and was higher indiestrous-1 than in estrous or proestrous. GABA (10 mM) had no effect on [³H]leucine incorporation in any of the three estrous phase studied.     

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.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

Distribution of the inducer of differentiation bis-acetyl-diaminopentane in murine erythroleukemia cells

Exposure of murine leukemia cells in culture to bis-acetyl-diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [³H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [³H]BADP was capable of binding to the plasma membrane-enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.     

Differential Labeling of Components in Human Erythrocyte Membranes Associated with the Transport of Glucose

The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human crythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed the basis for a double isotopic differential labeling technique using [¹⁴C]- and [³H]FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins.

Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band.

Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane.

These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.     

Molar proportions and relative rates of synthesis of histones in rat testis

The molar proportions and relative rates of synthesis of histones in normal and hypophysectomized rat testis seminiferous epithelial cells were determined. After hypophysectomy the molar proportions of histones H1, H2B and (H2A + protein A24) in seminiferous epithelial cells of rat testis increased while their corresponding variants TH1-x, TH2B-x and X₂ decreased, but the molar proportions of major-class histones (i.e., sum of subfractions) remained relatively constant and similar to the proportions in somatic cells. The apparent molar proportions of the labeled histones, determined immediately after 2-h periods of [³H]leucine incorporation, were much higher relative to H4 than the proportions of total histones determined by dye binding. The values, however, approached the molar proportions of total histones when rats were killed 11 days after the [³H]leucine injection. Two-dimensional gel electrophoresis confirmed that the high initial molar proportions relative to H4 by [³H]leucine incorporation were not due to the possible contamination by highly-labeled non-histone proteins. The specific activity of histone H4 relative to the specific activity of DNA, determined immediately after 3-h periods of [³H]leucine and [¹⁴C]thymidine incorporations was similar to the value when rats were killed 13 days after the injections. It is proposed that histones of seminiferous epithelial cells are synthesized disproportionally relative to H4 and in excess of the quantities required for polynucleosome assembly. The excess histones are subsequently displaced or degraded slowly.     

Isolation of anucleate cells using a fluorescence activated cell sorter (FACS II)

Human fibroblasts or mouse teratocarcinoma cells were enucleated by density gradient centrifugation in the presence of cytochalasin B (CB). The resulting mixed population of nucleated and anucleate cells was further purified by flow sorting, using the dye Hoechst 33342 as a fluorescent label for the nucleated cells. The purity of the anucleate cells obtained with this technique was at least 99%, as was shown by histological staining of the sorted fractions. Sorted enucleated fibroblasts were shown to have an intact cell membrane as indicated by their ability to convert fluorescein diacetate into fluorescein and to accumulate this product. They were found to attach and spread when cultured and showed protein synthesis immediately after enucleation, evidenced by the incorporation of [³H]leucine. Sorted enucleated teratocarcinoma cells also had an intact cell membrane, but they did not attach when cultured.     

Chronic Ethanol Increases the Cannabinoid Receptor Agonist Anandamide and Its Precursor N-Arachidonoylphosphatidylethanolamine in SK - N - SH Cells

Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [³H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [³H]AnNH and [³H]N-ArPE (p < 0.05) (1.43-fold for [³H]AnNH and 1.65-fold for [³H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [³H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [³H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [³H]AnNH. The formation of [³H]AnNH and [³H]N-ArPE by EtOH was enhanced by the Ca²⁺ ionophore A23187 or by the depolarizing agent veratridine and the K⁺ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [³H]AnNH and [³H]N-ArPE was inhibited by exogenous AnNH, whereas only [³H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and G_i/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Drug-resistant mutants of chinese hamster ovary cells possess an altered cell surface carbohydrate component

Surface label experiments using the galactose oxidase-[³ H] -borohydride technique reveal that cells from drug-resistant Chinese hamster ovary clones possess a surface carbohydrate component of apparent molecular weight 165,000 which is absent from wild-type cells. The component may also be demonstrated by [¹⁴C] glucosamine incorporation but not by [³ H] leucine incorporation or by the lactoperoxidase surface labeling reaction.     

Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.     

Effect of corynetoxin isolated from parasitized annual ryegrass on albumin and transferrin synthesis and secretion by cultured fetal rat hepatocytes

A group of glycolipid toxins, corynetoxin (CT), isolated from parasitized annual ryegrass, was shown to suppress the synthesis of both albumin and transferrin by cultured fetal rat hepatocytes. Based on [³H]leucine incorporation, inhibition of transferrin synthesis was greater than that of both albumin and total protein synthesis. As a result, the secretion of albumin and transferrin was decreased. The incorporation of [³H]N-AcGlc into cellular glycoproteins was only marginally affected by CT, although a dramatic reduction was observed with respect to the secreted proteins. Transferrin secreted into the culture medium was substantially non-glycosylated, judging by the absence of [³H]N-AcGlc. These studies suggested that the toxin preferentially affects the synthesis, and hence the secretion of glycoproteins, although it did not block the secretion of the proteins albumin and transferrin, as these did not accumulate intercellularly. Since transferrin labelled with [³H]leucine but not [³H]N-AcGlc is detected in the culture medium of hepatocytes exposed to CT, it was concluded that glycosylation of the protein is not required for secretion. This study shows that the effects of CT on protein synthesis and secretion in cultured hepatocytes are similar to those reported for tunicamycin (TM).