Prevalence of Equine Hepacivirus Infections in France and Evidence for Two Viral Subtypes Circulating Worldwide.

Like hepatitis C virus (HCV) in humans, the newly identified equine hepacivirus (NPHV) displays a predominating liver tropism that may evolve into chronic infections. The genomes of the two viruses share several organizational and functional features and are phylogenetically closest amongst the Hepacivirus genus. A limited amount of data is available regarding the spread of hepacivirus infections in horses. In this study, we asked whether in a more representative sample the prevalence and distribution of NPHV infections in France would resemble that reported so far in other countries. A total of 1033 horses sera from stud farms throughout France were analysed by qRT-PCR to determine the prevalence of ongoing NPHV infections and viral loads; in positive samples, partial sequences of NPHV's genome (5'UTR, NS3 and NS5B genes) were determined. Serum concentrations of biliary acids, glutamate dehydrogenase (GLDH) and L-gamma-glutamyl transferase (γ-GT) were measured for most horses. We detected NPHV infections in 6.2% of the horses, a prevalence that reached 8.3% in thoroughbreds and was significantly higher than in other breeds. The presence of circulating virus was neither significantly associated with biological disturbances nor with clinical hepatic impairment. Our phylogenetic analysis was based on both neighbour-joining and maximum-likelihood approaches. Its result shows that, like almost everywhere else in the world so far, two major groups of NPHV strains infect French domestic horses. Based on genetic distances, we propose a classification into two separate NPHV subtypes. Viral loads in the serum of horses infected by the main subtype were, in average, four times higher than in those infected by the second subtype. We hypothesize that amino acid substitutions in the palm domain of NS5B between NPHV subtypes could underlie viral phenotypes that explain this result.

Overexpression and overactivation of Akt in thyroid carcinoma.

Enhanced activation of Akt occurs in Cowden's disease, an inherited syndrome of follicular thyroid, breast, colon, and skin tumors, via inactivation of its regulatory protein, PTEN. Whereas PTEN inactivation is uncommon in sporadic thyroid cancer, activation of growth factor pathways that signal through Akt is frequently identified. We hypothesized that Akt overactivation could be a common finding in sporadic thyroid cancer and might be important in thyroid cancer biology. We examined thyroid cancer cells lines and benign and malignant thyroid tissue for total Akt activation and isoform-specific Akt expression. In thyroid cancer cells, Akt 1, 2, and 3 proteins were expressed, total Akt was activated by insulin phosphatidylinositol 3'-kinase, and inhibition of phosphatidylinositol 3'-kinase reduced cell viability. In human thyroid tissue, increased levels of phosphorylated total Akt were identified in follicular but not papillary cancers compared with normal tissue. Levels of Akt 1 and 2 proteins and Akt 2 RNA were elevated only in the follicular cancers. In paired samples, Akt 1, 2, 3, and phospho-Akt levels were higher in five of six cancers, including three of three follicular cancers. These data suggest that Akt activation may play a role in the pathogenesis or progression of sporadic thyroid cancer.

The expression of thyrotropin receptor in the brain.

The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.

Graves' disease: a host defense mechanism gone awry.

In this report we summarize evidence to support a model for the development of Graves' disease. The model suggests that Graves' disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-kappaB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8- cells which normally suppresses a population of CD4+ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.

The differential regulation of group II(A) and group V low molecular weight phospholipases A(2) in cultured rat astrocytes.

In astrocytes, cytokines stimulate the release of secretory phospholipase A(2) (sPLA(2)) activity and group II(A) sPLA(2) expression. This paper reports that two sPLA(2) isoforms, group II(A) and group V, are in fact expressed by astrocytes. Our studies showed that tumor necrosis factor alpha (TNFalpha) enhanced the mRNA of both isoforms, but the time courses of enhancement differed; group V was induced much faster than group II(A). Moreover, TNFalpha stimulated both the NF-kappaB and mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase, c-Jun NH(2)-terminal kinase, and p38 MAP kinase) signaling pathways in astrocytes. Interestingly, PI 3-kinase activity also was enhanced by TNFalpha, and NF-kappaB pathway was involved in mediating its effect. Specific inhibitors were used to show that both extracellular signal-regulated kinase and p38 MAP kinase may contribute to the effect of TNFalpha and that blocking phosphatidylinositol 3-kinase activity fully reversed the effect of TNFalpha. Furthermore, in astrocytes, TNFalpha-induced release of sPLA(2) activity was partially reversed by thyroid hormone and almost abolished by growth factors. This phenomenon was accompanied by a less marked increase in both group II(A) and group V sPLA(2) mRNA. In the presence of growth factors, the increase in group V mRNA was inhibited early and transiently, in contrast to what was observed with group II(A), which was more persistently inhibited. Although a transcriptional effect of thyroid hormone or growth factors in astrocytes cannot be definitively excluded, both types of factor interfered with sPLA(2) expression in a manner suggesting the existence of regulation of post-transcriptional events.

Mediation by arachidonic acid metabolites of the H2O2-induced stimulation of mitogen-activated protein kinases (extracellular-signal-regulated kinase and c-Jun NH2-terminal kinase).

Reactive oxygen species modulate major cellular functions by mechanisms which are still poorly understood. Recently, H2O2 has been reported to stimulate the activity of the mitogen-activated protein kinases (MAPKs) ERK and JNK, and the expression of the proto-oncogenes c-fos and c-jun. As their expression is enhanced by H2O2 in astrocytes, we studied whether these MAPKs were stimulated by H2O2 in primary cultured astrocytes. The result was positive, a maximum of stimulation being reached with 200 microM H2O2 (0.3 pmol H2O2/cell) for both ERK and JNK. ERK was previously reported to stimulate cytosolic phospholipase A2 phosphorylation and activity. H2O2 stimulated the release of arachidonic acid in astrocytes, as already reported in other cell types. We found also that cPLA2 phosphorylation was increased by H2O2. Moreover, the stimulation by H2O2 of ERK and JNK was decreased by phospholipase A2 activity inhibitors. When astrocytes were incubated first with eicosatetraynoic acid, a structural analogue competing in arachidonic acid metabolism, the stimulation of JNK by H2O was also inhibited, suggesting the involvement of arachidonic acid metabolites. Cyclooxygenase or cytochrome P450 monooxygenase inhibitors failed in decreasing the MAPK stimulation by H2O2, whereas lipoxygenase inhibitors completely abolished that of JNK. Mitogenicity has been reported to be stimulated by H2O2 in other cell types. Although ERK was strongly and durably stimulated by 200 microM H2O2 in astrocytes, at the same extent as by mitogenic growth factors, basal thymidine incorporation rate was decreased by more than 80% after 12-15 h. Moreover, the stimulation of thymidine incorporation induced by basic fibroblast growth factor was transiently abolished by H2O2. Furthermore, H2O2 likely induced the expression of CL100/PAC1/MKP-1, a dual specificity phosphatase which has been implicated in ERK and JNK inactivation in the nucleus. Finally, the prior treatment of astrocytes with MK886, a 5-lipoxygenase-activating protein inhibitor, prevented JNK from stimulation, but did not prevent thymidine incorporation from inhibition, both induced by H2O2. These results strongly suggest an involvement of arachidonic acid and/or its metabolites in the stimulation of both ERK and JNK following the oxidative stress evoked by H2O2, which induced a cell cycle arrest probably independent of the stimulation of JNK.

Cell surface protein disulfide-isomerase is involved in the shedding of human thyrotropin receptor ectodomain.

In human thyroid glands the TSH receptor undergoes a cleavage reaction which yields to an extracellular alpha subunit and a membrane spanning beta subunit linked together by disulfide bridges. A similar reaction is observed in transfected L cells although some uncleaved monomers persist in these cells. We have recently shown that the alpha subunit of the TSH receptor undergoes partial shedding in human thyroid cells and heterologous cells permanently transfected with an expression vector encoding the receptor. This shedding is a two-step process. The first step consists in the cleavage of the proreceptor at the cell surface probably by a matrix metalloprotease and the second step in the reduction of the disulfide bridge(s) (Couet, J., Sar, S., Jolivet, A., Vu Hai, M. T., Milgrom, E., & Misrahi, M. 1996, J. Biol. Chem. 271, 4545-4552). We have used the transfected L cells to study the second step involved in sTSHR shedding. The membrane impermeant sulfhydryl reagent DTNB (5,5'-dithiobis(2-nitrobenzoic acid) allowed us to confirm that the reduction of the TSH receptor disulfide bonds occurred at the cell surface. The antibiotic bacitracin even at low concentrations also elicited a marked inhibition of TSH receptor shedding. This led us to implicate the enzyme protein disulfide isomerase (PDI, EC 5.3.4.1) in this process. We thus tested the inhibitory activity of specific monoclonal antibodies raised against PDI. All antibodies elicited a marked inhibition of sTSHR shedding. This confirmed that cell surface PDI is involved in the shedding of the TSH receptor ectodomain. The shed alpha subunit may be at the origin of circulating TSH receptor ectodomain detected in human blood.

Stimulation of mitogen-activated protein kinase by thyrotropin in primary cultured human thyroid follicles.

In the thyroid, thyrotropin (TSH) stimulates both growth and function, and stimulates the production of cAMP which reproduces most of the effects of TSH. Here, we report evidence that TSH stimulates the mitogen-activated protein (MAP) kinase cascade through a cAMP-independent pathway, in human thyroid. TSH stimulated MAP kinase activity (4-9-fold the basal level) measured in the cytosolic fractions of primary cultured thyroid follicles. Maximal activity was reached after 20 min and remained sustained for 1-3 h, TSH being as potent as EGF; EC50 was 1.5 nM TSH. Only a single isoform of MAP kinase (p42) was detected in the follicles. p42 was phosphorylated on tyrosine residues and showed a reduced electrophoretic mobility in follicles stimulated by TSH. All these effects on MAP kinase were decreased by preincubation of the follicles with human anti-TSH receptor antibodies. The stimulation of MAP kinase by TSH was neither blocked by pertussis toxin nor reproduced by forskolin, cholera toxin, or 8-bromo-cAMP. In conclusion, in human thyroid cells, in contrast with previous observations on dog thyroid cells, TSH stimulates strongly MAP kinase through a pertussis toxin-insensitive and cAMP-independent pathway.

Stimulation of mitogen-activated protein kinase by thyrotropin in astrocytes.

We have recently reported the expression of the thyrotropin (TSH) receptor and the stimulation by TSH of type-II iodothyronine 5'-deiodinase in astrocytes. In these cells, TSH stimulated arachidonate release, but neither cAMP production, nor phosphatidylinositolbisphosphate hydrolysis, as described in the human thyroid gland. Here we report, in contrast to a recent observation made in dog thyroid cells, that TSH stimulates mitogen-activated protein kinase (MAP kinase) in astrocytes. Indeed, TSH increases the tyrosine phosphorylation of the two isoforms of MAP kinase expressed in these cells, in correlation with both a slower electrophoretic migration of the tyrosine phosphorylated species and an enhanced enzymic activity measured on a specific substrate peptide. This stimulation of MAP kinase by TSH was specifically inhibited by incubation of astrocytes in the presence of human blocking anti-(TSH receptor) IgG, and by immunoprecipitation of TSH with monoclonal anti-TSH IgG. In astrocytes, TSH was neither mitogenic by itself, nor modified significantly the basic-fibroblast-growth-factor-induced mitogenesis. The stimulation of MAP kinase by TSH was not affected by treatment with pertussis toxin, suggesting guanine-nucleotide-binding-regulatory protein i/o was not implicated in this TSH effect. Our model will allow the study of the stimulation of MAP kinase by TSH without interference either from cAMP or from phosphoinositide signalling pathways.

Processing of the precursors of the human thyroid-stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus-infected insect cells).

The complementary DNA for human thyroid-stimulating hormone (TSH) receptor encodes a single protein with a deduced molecular mass of 84.5 kDa. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (alpha subunit of approximately 53 kDa and beta subunit of approximately 38 kDa) held together by disulfide bridges [Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. & Miligrom, E. (1992) Proc. Natl Acad. Sci. USA 89, 3765-3769]. A similar processing occurs in an L cell line permanently expressing the human TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95-kDa high-mannose precursor which is present only in trace amounts in the thyroid. Pulse-chase experiments show the successive appearance in the L cells of two precursors: initially the approximately 95-kDa high-mannose glycoprotein followed by a approximately 120-kDa species containing mature oligosaccharides. This latter precursor is then processed into the alpha and beta subunits. In primary cultures of human thyrocytes precursors of similar size are detected. Spodoptera frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthesize a monomeric protein of about 90 kDa soluble only in denaturing conditions. Comparison with the product of in vitro transcription-translation experiments (approximately 80 kDa), suggests that it may be incompletely or improperly glycosylated. The TSH receptor expressed in these cells is unable to bind the hormone. Immunoelectron microscopy studies show that in human thyrocytes most of the receptor is present on the cell surface; in L cells the receptor is detected on the cell surface, as well as in the endoplasmic reticulum and in the Golgi apparatus (this intracellular pool of receptor molecules probably corresponding to the high-mannose precursor); in insect cells nearly all the receptor molecules are trapped in the endoplasmic reticulum. These differences in receptor distribution are concordant with the differences observed for receptor processing.

Evidence for cAMP-independent thyrotropin effects on astroglial cells.

Thyroid hormones are essential for normal brain development and function. Brain astroglial cells express type II iodothyronine 5'-deiodinase which converts thyroxine into 3,5,3'-triiodothyronine. This type II deiodinase is regulated through various signalling pathways, allowing probably for the local adaptation of the level of 3,5,3'-triiodothyronine. Our results demonstrated that thyrotropin was able to induce type II deiodinase activity in astrocytes. A thyrotropin receptor was demonstrated. It was not coupled, as in thyroid, to adenylyl cyclase and phospholipase C, but it stimulated cytosolic phospholipase A2. The stimulation by thyrotropin of both thyroxine synthesis in thyroid and its local activation in astrocytes, could protect the brain from variations in the level of 3,5,3'-triiodothyronine.

Effect of bromolevamisole and other imidazo [2,1-b] thiazole derivatives on adenylate cyclase activity.

We studied the effect of bromolevamisole (BL) and other imidazo [2,1-b] thiazole derivatives--bromodexamisole (BD) and levamisole (LV)--on adenylate cyclase (AC) activity. BL and BD both inhibited forskolin-activated human thyroid AC, while LV had no effect. This inhibition was non-stereospecific and the IC50 values, as measured with 1 mM ATP and 40 microM forskolin, were 0.95 and 0.80 mM for BL and BD, respectively. In contrast, human thyroid alkaline phosphatase (ALP) inhibition was stereospecific, with IC50 values of 0.0012 mM for BL and 0.9 mM for BD. LV was a 10-fold weaker inhibitor of ALP than BL. These results show that ALP inhibition is not correlated with forskolin-activated AC inhibition. Furthermore, in the presence of a competitive inhibitor of GTP (0.1 mM guanosine 5'-O-(2-thiodiphosphate), BL retained its antagonizing effect on forskolin-activated AC which suggests a direct action on the catalytic subunit. The inhibition was of the mixed type, indicating a complex interaction between BL and AC. Glucagon-activated AC activity in rat liver membranes was also inhibited by BL, although to a slightly lesser degree than thyroid stimulating hormone (TSH)-activated AC from human thyroid for a given BL concentration. In cultured human thyroid cells, BL (0.25 mM) induced a potent decrease in cAMP accumulation after 2 hr of stimulation by TSH. Taken together, these results show that BL inhibits AC and that this inhibition is not organ-specific.

Alteration of the functional activity of Gs protein in thyrotropin-desensitized pig thyroid cells.

Changes in the sensitivity of adenylyl cyclase observed in pig thyroid cells cultured 2 days in the presence of thyroid-stimulating hormone (TSH) or forskolin were assessed by examining the properties of Gs protein. Chronic treatment of thyroid cells with various concentrations of TSH (0.01-1 mU/ml) or forskolin (0.1-10 microM) increased the response of adenylyl cyclase to a further stimulation by forskolin or NaF + AlCl3 ([AlF4]-). In contrast, the enzyme activation promoted by guanosine 5'-(beta,gamma-imido) triphosphate (Gpp(NH)p) was markedly affected. There was a significant increase in adenylyl cyclase activation by Gpp(NH)p in membranes from cells treated with low concentrations of TSH (less than or equal to 0.1 mU/ml) or forskolin (less than or equal to 1 microM) but a significant decrease in membranes from cells cultured with a higher concentration of TSH (1 mU/ml) or forskolin (10 microM). This decrease in Gpp(NH)p-stimulated adenylyl cyclase activity was mimicked by 8-bromo-cAMP but not by 1,9-dideoxyforskolin, a forskolin analogue which has lost its ability to activate adenylyl cyclase. There was a good correlation with the ability of Gs protein to be ADP-ribosylated by cholera toxin: labeling of Gs protein decreased following chronic treatment of thyroid cells with TSH (1 mU/ml) or forskolin (10 microM). In contrast, under the same experimental culture conditions a slight but significant increase in the quantity of Gs subunits was observed by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP regulation of Gs protein. Thyrotropin and forskolin increase the quantity of stimulatory guanine nucleotide-binding proteins in cultured thyroid follicles.

This study was carried out to clarify the way in which thyrotropin (TSH) and forskolin regulate the adenylylcyclase complex in thyroid follicle cells. We examined the effects of chronic treatment of pig thyroid follicles with TSH or forskolin on the state of G proteins by (a) assaying adenylylcyclase activity, (b) analyzing the ADP-ribosylation of stimulatory G protein (Gs) by cholera toxin, and (c) quantifying the Gs subunits by Western blotting with antipeptide antibodies. Chronic exposure (18 h) of thyroid follicles to a low concentration of TSH (0.01-0.1 milliunit/ml) enhanced the subsequent response of adenylylcyclase to TSH. Higher concentration of TSH (1 milliunit/ml) induced a homologous desensitization of this response. In cells pretreated with forskolin, the TSH-stimulated adenylylcyclase activity was higher than in control cells. The forskolin-or guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p)-stimulated adenylylcyclase activity was always significantly increased after chronic treatment of cells with TSH or forskolin. Treatment of cultured thyroid follicle membranes with [32P]NAD and cholera toxin resulted in labeling of the Gs alpha (45-52-kDa) component. Culturing follicles with TSH (0.001-1 milliunit/ml) or forskolin (0.01-10 microM) greatly affected the cholera toxin-mediated ADP-ribosylation of the Gs alpha subunit. Gs alpha labeling increased progressively to level off at 1 milliunit/ml TSH or 1 microM forskolin (150-200%). Gs alpha immunoreactivity was increased in parallel (200-300%). The immunoreactivity of G beta subunits in cells cultured with TSH or forskolin was also increased compared with control cells. Cycloheximide abolished the effects of TSH and forskolin on the follicles, suggesting that new protein synthesis is required. These results indicate that Gs protein subunits are up-regulated by TSH and forskolin and suggest that their synthesis in thyroid cells is mediated, at least in part, by a cyclic AMP-dependent mechanism.

Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells.

The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane.

Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells.

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.

Comparison between 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin as inhibitors of oligosaccharide processing in intestinal epithelial cells.

The alpha-glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) inhibits the synthesis of N-linked complex oligosaccharides in rat intestinal epithelial cells to the same extent as reported previously for 1-deoxynojirimycin (DJN) [Saunier, Kilker, Tkacz, Quaroni & Herscovics (1982) J. Biol. Chem. 257, 14155-14161]. Analysis of each of the endo-beta-N-acetylglucosaminidase H (endo H)-sensitive oligosaccharides separated by h.p.l.c. with yeast glucosidase I, which specifically removes the terminal glucose residue from oligosaccharides containing three glucose residues, and with jack-bean (Canavalia ensiformis) alpha-mannosidase, indicates that both inhibitors cause the accumulation of a mixture of glucosylated oligosaccharides containing one to three glucose residues and seven to nine, and even possibly six, mannose residues. About 70% of the endo H-sensitive oligosaccharides formed in the presence of MDJN contain three glucose residues, compared with only about 20% of the corresponding oligosaccharides of the DJN treated cells. It is concluded that both compounds inhibit the formation of N-linked complex oligosaccharides by interfering with the processing glucosidases. These compounds are valuable in the study of the role of oligosaccharides in glycoproteins.