Inhibition Of Glycoprotein Synthesis Research Articles

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Hypoxia Modulates A431 Cellular Pathways Association to Tumor Radioresistance and Enhanced Migration Revealed by Comprehensive Proteomic and Functional Studies

Tumor hypoxia induces cancer cell angiogenesis, invasiveness, treatment resistance, and contributes to poor clinical outcome. However, the molecular mechanism by which tumor hypoxia exerts a coordinated effect on different molecular pathways to enhance tumor growth and survival and lead to poor clinical outcome is not fully understood. In this study, we attempt to elucidate the global protein expression and functional changes in A431 epithelial carcinoma cells induced by hypoxia and reoxygenation using iTRAQ quantitative proteomics and biochemical functional assays. Quantitative proteomics results showed that 4316 proteins were quantified with FDR<1%, in which over 1200 proteins were modulated >1.2 fold, and DNA repair, glycolysis, integrin, glycoprotein turnover, and STAT1 pathways were perturbed by hypoxia and reoxygenation-induced oxidative stress. For the first time, hypoxia was shown to up-regulate the nonhomologous end-joining pathway, which plays a central role in DNA repair of irradiated cells, thereby potentially contributing to the radioresistance of hypoxic A431 cells. The up-regulation of Ku70/Ku80 dimer, a key molecular complex in the nonhomologous end-joining pathway, was confirmed by Western blot and liquid chromatography/tandem mass spectrometry-MRM methods. Functional studies confirmed that up-regulation of glycolysis, integrin, glycoprotein synthesis, and down-regulation of STAT1 pathways during hypoxia enhanced metastastic activity of A431 cells. Migration of A431 cells was dramatically repressed by glycolysis inhibitor (2-Deoxy-d-glucose), glycoprotein synthesis inhibitor (1-Deoxynojirimycin Hydrochloride), and STAT1α overexpression that enhanced the integrin-mediated cell adhesion. These results revealed that hypoxia induced several biological processes involved in tumor migration and radioresistance and provided potential new targets for tumor therapy.

Inhibition of glycoprotein synthesis in the endoplasmic reticulum as a novel anticancer mechanism of (−)-epigallocatechin-3-gallate

(-)-Epigallocatechin-3-gallate (EGCG) has been found to trigger the unfolded protein response (UPR) likely due to the inhibition of glucosidase II, a key enzyme of glycoprotein processing and quality control in the endoplasmic reticulum (ER). These findings strongly suggest that EGCG interferes with glycoprotein maturation and sorting in the ER. This hypothesis was tested in SK-Mel28 human melanoma cells by assessing the effect of EGCG and deoxynojirimycin (DNJ) on the synthesis of two endogenous glycoproteins. Both tyrosinase and vascular endothelial growth factor (VEGF) protein levels were remarkably reduced despite unaltered mRNA expression in EGCG- or DNJ-treated cells compared to control. The hindrance of tyrosinase and VEGF protein synthesis could be prevented by proteasome inhibitor, lactacystine. Collectively, our results support that glucosidase II inhibitor EGCG interferes with protein processing and quality control in the ER, which diverts tyrosinase, VEGF, and likely other glycoproteins towards proteasomal degradation. This mechanism provides a novel therapeutic approach in dermatology and might play an important role in the antitumor effect or hepatotoxicity of EGCG.

Phase II study of clinical activity of pegaspargase in women with relapsed or refractory epithelial ovarian, fallopian tube, and/or primary peritoneal cancer.

TPS258 Background: Although the bacterial enzyme L-asparaginase (L-ASP) has been used in combination with traditional cytotoxic therapy for decades, its use has been limited mainly to its FDA-approved treatment of acute lymphoblastic leukemia. By catalyzing the hydrolysis of asparagine to aspartate, L-ASP depletes serum levels of asparagine resulting in direct cytotoxicity of sensitive cells. The noncytotoxic effects of L-ASP, such as the inhibition of glycoprotein synthesis, have been known for decades. Our ongoing preclinical work has demonstrated that L-ASP reduces the ability of endothelial cells to form capillary-like tubes, reduces the invasive potential of endothelial cells and cancer cells, and prevents heterotypic interaction between ovarian cancer and endothelial cells. These results suggest that L-ASP may provide therapeutic benefit by inhibiting angiogenesis, invasion and metastasis. Our finding that L-ASP is capable of modifying the tumor microenvironment led us to hypothesize that pegaspargase may be effective as a single agent in providing disease response or stabilization. Methods: Objectives: 1) To evaluate the antitumor activity of pegaspargase, 2,000 IU/ m2 every two weeks IV in patients with recurrent or refractory ovarian cancer, fallopian tube cancer, and/or primary peritoneal cancer. 2) To explore pharmacodynamic endpoints such as changes in circulating angiogenic cytokines, signal transduction pathway alterations, and tumor vascularity. Eligibility: Adults with histologically documented recurrent ovarian, fallopian tube, or peritoneal cancer for whom standard curative therapies do not exist or are no longer effective. Design: This is an open-label phase II, and will be conducted as a two- stage trial, in order to evaluate whether a sufficiently high proportion of patients are able to attain a 6 month progression free interval using pegaspargase 2,000 IU/m2 IV q 2 wk. Patients will undergo functional imaging and biopsy or fluid aspiration prior to and at 6 weeks of therapy for translational endpoints. The first stage accrual is 13 patients with an estimated 30 total patients needed to achieve the objectives of this trial. No significant financial relationships to disclose.

The extracellular matrix of porcine mature oocytes: Origin, composition and presumptive roles

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

Long-lasting substance-P-mediated modulation of NMDA-induced rhythmic activity in the lamprey locomotor network involves separate RNA- and protein-synthesis-dependent stages.

Bath application of the tachykinin neuropeptide substance P (1 microm) for 10 min causes long-lasting (> 24 h) modulation of the frequency and regularity of NMDA-evoked locomotor bursts in the lamprey. The change in burst frequency has an induction phase (< 2 h), which depends on the potentiation of NMDA responses and an increase in intracellular calcium levels, and a maintenance phase (> 2 h), that is blocked by translational protein synthesis inhibitors. Here, the maintenance phase has been examined further. Unlike translation inhibitors, the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside (DRB) failed to reverse the change in burst frequency 2-3 h after substance P application, suggesting that the protein synthesized at this time does not require de novo RNA synthesis. Transcription inhibitors, however, reversed the change in burst frequency 15-24 h after substance P application, as did brefeldin A, which disrupts the Golgi complex and thus interferes with the post-translational transport of proteins. The change in burst regularity was unaffected by transcription or translation inhibitors, but was partially reversed by protein kinase A inhibitors applied 2.5-8 h after substance P. The glycoprotein synthesis inhibitor 2-deoxygalactose did not affect the changes in burst frequency or burst regularity. These results suggest that there are two phases to the maintenance of the change in burst frequency: an intermediate protein-, but not RNA-, synthesis-dependent phase, and a final RNA-synthesis-dependent phase. The change in burst regularity is protein-synthesis-independent, but may depend on activation of protein kinase A for at least 8 h after substance P application.

Influences of Metabolic Inhibitors and Hydrolytic Enzymes on the Adhesion of Appressoria ofPyricularia oryzaeto Wax-coated Cover-glasses

For discovering the components that contributed to the bonding strength of the glue substances produced by appressoria of Pyricularia oryzae on wax-coated cover-glasses, the influences of metabolic inhibitors and hydrolytic enzymes were investigated. The bonding strength of appressoria was assessed by the ratio of the remaining appressoria after sonication to the appressoria formed before sonication. Remaining appressoria decreased with increasing concentrations of cerulenin, an inhibitor of lipid synthesis, but isoprothiolane and compactin showed no influence on bonding strength. Tunicamycin, an inhibitor of glycoprotein synthesis, weakened the bonding strength of appressoria, but castanospermin had no effect. Of the hydrolytic enzymes tested, protease particularly weakened the bonding strength of appressoria. On the surfaces of substrata, the appressoria's bonding strength was higher on the hydrophobic surfaces than on the hydrophilic. These results suggest that lipid components and glycoprotein were closely associated with appressoria bonding strength to the surface of wax-coated cover-glass.

Two critical periods of protein and glycoprotein synthesis in memory consolidation for visual categorization learning in chicks.

A protein synthesis inhibitor, anisomycin (ANI), and an inhibitor of glycoprotein synthesis, 2-deoxygalactose (2-D-gal), were used to investigate memory consolidation following visual categorization training in 2-day-old chicks. ANI (0.6 micromole/chick) and 2-D-gal (40 micromoles/chick) were injected intracerebrally at different time intervals from 1 hr before to 23 hr after the training. Retention was tested 24 hr post-training. Both ANI and 2-D-gal injections revealed two periods of memory sensitivity to pharmacological intervention. ANI impaired retention when injected from 5 min before to 30 min after the training or from 4 hr to 5 hr post-training, thus demonstrating that consolidation of long-term memory in this task requires two periods of protein synthesis. 2-D-Gal first produced an amnesia when it was injected in the interval from 5 min before to 5 min after the training. Injections made between 5 min and 5 hr post-training were without effect on the retention. The second period of memory impairment by 2-D-gal started at 5 hr post-training and lasted until 21 hr after the training. Administration of 2-D-gal made 23 hr after the training did not influence retention in the test at either 24 hr or 26 hr. These results are consistent with the hypothesis that two waves of protein and glycoprotein synthesis are necessary for the formation of long-term memory. The prolonged duration of performance impairment by 2-D-gal in the present task might reflect an extended memory consolidation period for a categorization form of learning.

Bowman's Gland in Nasal Tissues: As A Possible

We have investigated the Bowman's gland of the nasal cavity after in vivo study in the Sprague-Dawley rats to asses its biomarker potential in vitamin A nutritional study. Fourty-eight male weanling SpragueDawley rats at 3-4 weeks of age were allotted to four groups of 12 each and were fed diets either vitamin A-deficient or supplemented with 30.000 IU vitamin A/kg for 150 days. Rats in their respective groups were also given fume of vehicle for 150 days. Fumigation was done 2x/day for 1 hour each. Rats given vitamin A-deficient diet and fume of vehicle had the most extensive inhibition of glycoprotein synthesis in cells of Bowman's gland in the olfactory region of the nasal cavities as determined by severe loss of Alcian blue-periodic acid-Schiff staining material. Histochemical lesions were not seen in nasal cavity of vitamin A-supplemented rats

Tunicamycin potentiates drug cytotoxicity and vincristine retention in multidrug resistant cell lines.

Tunicamycin (TM), an inhibitor of glycoprotein processing, was investigated for its potential to reverse the multiple drug resistance (MDR) phenotype. When TM was added in vitro to drug-resistant NIH-3T3-MDR and KB-8-5-11 cells, they developed an increased sensitivity to doxorubicin, epirubicin, vincristine and colchicine. Similarly, the sensitivity of NIH-3T3-MDR cells to cisplatin was also enhanced by TM. In the presence of TM, drug-sensitive NIH-3T3-parental cells exhibited greater susceptibility to the toxic effects of doxorubicin, epirubicin, vincristine (marginally significant), and colchicine, but not to cisplatin. Tunicamycin-treated drug-sensitive KB-3-1 cells showed an increased response to vincristine, but not to the other anticancer drugs. Pretreatment with TM inhibited glycoprotein synthesis in all the cell lines. Neither prior exposure to, nor co-incubation with TM, influenced the uptake of vincristine (VCR) in the various cell lines. However, NIH-3T3-MDR cells accumulated less VCR than their drug-sensitive controls and also exhibited reduced efflux of the drug when treated with TM. There were no significant differences in the levels of intracellular VCR uptake between drug-sensitive KB-3-1 and KB-8-5-11 cells. Tunicamycin increased intracellular VCR retention in KB-8-5-11 and NIH-3T3-MDR cells, but not in NIH-3T3-parental cells. However, drug-sensitive KB-3-1 cells expressed reduced VCR retention in response to TM exposure, indicating that correlations between VCR toxicity and its retention in the presence of TM should be made with caution. The results suggest that the enhancement of intracellular VCR retention in MDR cells lines caused by TM is likely to be the result of inhibition of VCR efflux. Inhibition of glycoprotein synthesis during TM exposure may account for the changes in VCR efflux and retention observed in the MDR cell lines. The enhancement of cisplatin cytotoxicity in NIH-3T3-MDR cells after exposure to TM is an interesting observation, since it is generally believed that agents which modify the MDR phenotype do not show a sensitising effect to cisplatin. These findings may have applications in the reversal of drug resistance.

A role for a chicken homolog of the neural cell adhesion molecule L1 in consolidation of memory for a passive avoidance task in the chick.

Intracranial injection of antibodies directed against the neural cell adhesion molecule L1 resulted in amnesia for passive avoidance training in day-old chicks tested 24 hr subsequently. L1 antibodies were amnesic when administered at one of two time windows: 30 min pretraining and 5.5-8 hr post-training. No amnesia was apparent if injections were made at times before, between, or after these time windows (-2, +1, +3, +4, or +12 hr relative to training). A fragment of the L1 molecule derived from the external fibronectin domains FN1-5 produced amnesia only when injected at the 5.5-hr timepoint, whereas a fragment of the immunoglubin-like domains Ig I-VI produced amnesia only when injected 30 min prior to training. We have shown previously that long-term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training, and the second some 6 hr thereafter. The glycoprotein synthesis inhibitor 2-deoxygalactose results in amnesia if injected at either time, whereas the neural cell adhesion molecule (N-CAM) is specifically involved only in the second wave. The coincidence of the time course of memory disruption resulting from injection of L1 antibodies with that occurring with 2-deoxygalactose supports the hypothesis that establishment of an enduring memory for the experience of passive avoidance training requires two waves of glycoprotein synthesis, each wave being biochemically and functionally discrete. The differential effects of the two L1 fragments suggests that separate mechanisms of synaptic stabilization are involved at the two time points.

Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro.

The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A polypeptides in eukaryotic cells and examined their localization using indirect immunofluorescence and a direct in vitro membrane-binding assay. Results presented here demonstrate that both 3AB and 3A are capable of localizing in the endoplasmic reticulum and the Golgi apparatus in transfected HeLa cells in the absence of any other poliovirus protein. We have also shown that the carboxy-terminal 18 amino acids of 3A that constitute an amphipathic domain are important in membrane binding of 3A and 3AB. Additionally, we demonstrate that a significant fraction of both 3A and 3AB can be glycosylated in a membrane-dependent fashion during in vitro translation in reticulocyte lysate. We demonstrate that 6-diazo-5-oxo-L-norleucine, an inhibitor of glycoprotein synthesis, significantly inhibits poliovirus RNA synthesis in vivo. The implications of glycosylation of 3AB (and 3A) in viral replication are discussed.

2-Deoxygalactose interferes with an intermediate processing stage of memory

The effect of 2-deoxygalactose (2-D-gal), an inhibitor of glycoprotein synthesis, on memory formation was investigated with the day-old chick trained on a single-trial passive discrimination task. 2-D-gal (10 mumol/chick) was shown to inhibit memory formation at a time before the emergence of an antibiotic-sensitive long-term memory stage. The amnestic effect of 2-D-gal was successfully prevented by galactose, and more significantly by noradrenaline. In contrast, anisomycin-induced amnesia was resistant to challenge by either galactose or noradrenaline. The results are consistent with the view that some glycoprotein involvement in memory formation occurs prior to the formation of protein synthesis-dependent long-term memory, and this role of glycoproteins may be associated with the triggering of long-term memory formation by noradrenaline.

Signals derived from glucose metabolism are required for glucose regulation of pancreatic islet GLUT2 mRNA and protein.

Pancreatic islet GLUT2 mRNA is known to be regulated in vitro and in vivo by glucose. We have investigated several potential mechanisms mediating the response of islet GLUT2 to glucose. GLUT2 mRNA and protein were measured from isolated rat islets cultured for up to 24 h under selected conditions. Glucose at 11 mM stimulated GLUT2 mRNA 10-fold compared with 2 mM glucose, with no additional increase at 16.7 mM glucose, whereas maximal 4-fold induction of the protein was attained with 16 mM glucose. Time course studies showed a 2.5-fold induction of GLUT2 mRNA apparent after only 8 h of culture at 16.7 mM glucose. Glycolysis inhibitor mannoheptulose suppressed the stimulatory effect of 16.7 mM glucose on GLUT2 mRNA and protein. Metabolizable sugars mannose and glyceraldehyde enhanced transporter mRNA levels, in contrast with the lack of stimulation by nonmetabolizable 2-deoxy-D-glucose. Stimulation by different sugars and glycolysis inhibition led to analogous changes of proinsulin mRNA, suggesting that common signaling mechanisms are shared in glucose regulation of proinsulin and GLUT2 gene expression. Preexposure to mannoheptulose, however, failed to suppress glucose-stimulated insulin release. Tunicamycin, a glycoprotein synthesis inhibitor, did not block the effect of 16 mM glucose on GLUT2 mRNA levels. RNA and protein synthesis inhibitors actinomycin and cycloheximide abolished the enhancing effects of high glucose on GLUT2 mRNA. These findings indicate that glucose metabolism, but not glycoprotein synthesis or substrate interaction with the transporter protein, is instrumental in the stimulatory effects of glucose on beta-cell GLUT2 mRNA accumulation. In addition, ongoing RNA and protein synthesis are required for this effect.

Mucin exocytosis: a major target for Helicobacter pylori.

To determine whether Helicobacter pylori impairs the secretory function of mucous cells. The mucus secreting human cell line CL. 16E, maintained as confluent monolayers on nitrocellulose filters, was infected with H pylori strain CIP 101260. After three hours of incubation with H pylori the monolayers were washed and reincubated with fresh culture medium for various time periods (24, 48, or 72 hours) before evaluating both the morphology and function of mucous cells. For morphological studies, epithelial monolayers were fixed in situ and processed for both standard histochemistry on paraffin wax sections, and electron microscopy. To measure mucins secreted from cultured cells, the cells were metabolically labelled with 3H-glucosamine. Undegraded mucins were quantitated as the radioactive glycoproteins blocked at the stacker gel interface after sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the secretory glycoproteins. Control cultures of CL. 16E cells grew on filters as homogeneous monolayers of polarised mucous cells secreting a visco-elastic gel of mucins at the apical surface. In infected monolayers H pylori was in close contact with the apical surface of mucous cells. Cell counts and histological evaluation of the monolayers did not reveal any significant deleterious effect of H pylori on the mucous cells. H pylori induced only a modest inhibition of baseline mucus secretion from CL. 16E cells, this inhibition being significant only at 24 hours. In contrast, the mucus secretory response to two agents that raise intracellular cAMP and calcium--forskolin and ionophore A23187--was strongly inhibited. The inhibitory effect of H pylori on the exocytotic response was not paralleled by an inhibition of glycoprotein synthesis. Considering the fact that the exocytotic response to a variety of secretagogues constitutes the primary line of defence of the gastric mucosa in an emergency, it is suggested that H pylori exerts its deleterious effects by weakening this important physiological defence.

Functional and morphological changes induced by tunicamycin in dividing and confluent endothelial cells

Cultured bovine aortic endothelial cells treated with tunicamycin, an inhibitor of glycoprotein synthesis, developed a concentration-dependent inhibition of N-acetylglucosamine-1-phosphate transferase activity, and this inhibition was correlated with a substantial decrease in [ 3H]mannose incorporation by the cells. Endothelial cells were very sensitive to tunicamycin, and changes in their morphology occurred as a result of the inhibition of glycoprotein synthesis. The cells became elongated, the surface irregular, roughened, and granular, and there was an increase in the interstitial space between the cells. Electron dense material was accumulated within and dilated the rough endoplasmic reticulum, and the distribution of the glycoproteins laminin and fibronectin throughout the endothelial cell monolayer was modified. These morphological changes coincided with functional impairment with the permeability of endothelial cell monolayers to both 125I-albumin and [ 8H]inulin being increased by treatment with tunicamycin (10 −6 M) for 24 h. These results indicate that the synthesis of glycoproteins is crucial for cell-cell adhesion and the functional properties of the endothelial lining of blood vessels.

The effect of inhibition of glycoprotein synthesis in cultured bovine aortic endothelial cells

The effect of inhibition of glycoprotein synthesis in cultured bovine aortic endothelial cells

A herpes simplex virus type 1 mutant resistant to benzhydrazone, an inhibitor of glycoprotein synthesis in herpesvirus-infected cells. Preliminary mapping of benzhydrazone-resistance and of a novel syncytial mutation.

Benzhydrazone (BH) is an inhibitor of glycoprotein biosynthesis. It acts selectively in Herpes simplex virus (HSV)-infected cells and does not significantly affect glycoprotein synthesis in uninfected cells and in cells infected with other viruses. Previously, we reported on a syncytial (syn) mutant, designated HSV-1(13)S11, resistant to BH, and showed that BH-resistance is encoded in the mutant virus DNA and therefore can be transferred into the genome of wild type HSVs. The present paper reports on a preliminary mapping in HSV-1(13)S11 genome of the loci which confer resistance to BH and of three distinct syn mutations present simultaneously in this mutant. Two of them were mapped in previously described syn loci localized in BamHI fragment L (map units 0.707-0.745) (locus syn 1) and BamHI fragment Q (map units 0.296-0.317) (locus syn 5). A third mutation not described before and mapping in BamHI fragment SP (c.a. map units 0.81-0.85) conferred the syn phenotype to both HEp-2 and Vero cells. This novel mutation has been designated herein locus syn 6. Transfer of BH-resistance could be achieved in cotransfection experiments involving two HSV-1(13)S11 fragments, BamHIL and BamHISP.