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Background & Aims Potential celiac disease is characterized by positive results from serologic tests for tissue transglutaminase antibodies (anti-TG2) but normal duodenal architecture (Marsh stages 0–1). There is controversy over the best way to manage these patients. We investigated risk factors associated with the development of villous atrophy in children with potential celiac disease. Methods We performed a prospective study of 280 children (ages 2–18 years) in Italy with suspected celiac disease, followed for up to 12 years (range, 18–150 months; median 60 months). The subjects had 2 consecutive positive results from tests for anti-TG2, tested positive for the endomysial antibody (anti-EMA), had total serum levels of immunoglobulin A in the normal range, normal duodenal architecture (Marsh stages 0–1) in 5 biopsies, and HLA DQ2- or DQ8-positive haplotypes. The children underwent serologic tests and clinical analyses every 6 months and a small bowel biopsy was taken every 2 years. A total of 210 patients of the original cohort were assessed at the 9-year follow-up evaluation. We performed multivariate analyses of clinical, genetic, and histologic data to identify factors associated with progression to villous atrophy. Results During the follow-up period, 42 (15%) of 280 children developed villous atrophy, whereas 89 (32%) children no longer tested positive for anti-TG2 or anti-EMA. The cumulative incidence of progression to villous atrophy was 43% at 12 years. In multivariate analysis, the baseline factors most strongly associated with development of villous atrophy were numbers of γδ intraepithelial lymphocyte cells followed by age and homozygosity for the HLA DQB1*02. In discriminant analysis, these baseline factors identified 80% of the children who developed baseline atrophy. Conclusions In a long-term study of 280 children with suspected celiac disease (based on anti-TG2 and anti-EMA) on gluten-containing diets, the cumulative incidence of progression to villous atrophy was 43% over a 12-year period. We identified factors that can be used to identify children at highest risk for villous atrophy. This approach might be used to determine whether children with suspected celiac disease should immediately start a gluten-free diet or be monitored on their regular diet.

Background Small bowel biopsy is the gold standard for Celiac Disease (CD) diagnosis, nevertheless serum assays are the first step in ascertaining a diagnosis of CD. New ESPGHAN Criteria 2012 (European Society of Pediatric Gastroenterology Hepatology and Nutrition) suggest using exclusively anti-tissue Transglutaminase IgA antibodies (anti-tTGA) as initial approach to symptomatic subjects. The aim of our study was to evaluate the diagnostic accuracy of anti-tTGA as initial screening assay for CD in a large cohort of pediatric patients. Methods We selected 730 subjects aged between 6 months and 4 years (“Group A”) and 348 subjects younger than 2 years (which are part of the 730 subjects) (“Group B”). We performed anti-Deamidated Gliadin Peptides IgA and IgG antibodies (a-DGP IgA/IgG) and anti-tTGA assays by ELISA test. We evaluated the agreement between anti-tTGA and a-DGP IgA/IgG assays and compared the diagnostic accuracy of a-DGP IgA/IgG with that of anti-tTGA in both groups of patients. Results There was a substantial agreement between anti-tTGA and a-DGP IgA in “Group A” and an almost perfect agreement in “Group B”; the strength of agreement between anti-tTGA and a-DGP IgG was moderate in “Group A” and substantial in “Group B”. anti-tTGA were more sensitive and specific than a-DGP IgA/IgG in both groups. Conclusions anti-tTGA could be used as initial screening assay for CD in all subjects from 6 months of age according to ESPGHAN Criteria 2012.

Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.

Celiac disease (CD) is a gluten-sensitive enthesopathy occurring in genetically predisposed individuals that is caused by a permanent intolerance to gluten the major environmental factor associated with the risk of developing celiac-related complications is persistent exposure to dietary gluten the aim of this study was to determine the prevalence of lower limb enthesopathy in CD patients at first diagnosis compared with CD patients on a gluten-free diet (GFD). Fifty-five untreated CD patients (group A) and 55 CD patients on a GFD from at least 1 year (group B), matched for age and sex, attending gastroenterology outpatient clinic of the University Federico II of Naples, were enrolled in this study. All patients underwent clinical and ultrasonography examination. Among group A, 27 (49.8 %) patients presented at least one entheseal alteration as compared with 15 patients (27.2 %) of group B (prevalence rate ratio 1.83, I.C. 95 %∈=∈0.48-7.01; p∈

Transmission ratio distortion (TRD) is the departure from the expected Mendelian ratio in offspring, a poorly investigated biological phenomenon in livestock species. Given the current availability of specific parametric methods for the analysis of segregation data, this study focused on the screening of TRD in 602 402 single nucleotide polymorphisms covering all autosomal chromosomes in seven Spanish beef cattle breeds. On average, 0.13% (n = 786) and 0.01% (n = 29) of genetic markers evidenced sire- or dam-specific TRD respectively. There were no single nucleotide polymorphisms accounting for both sire- and dam-specific TRD at the same time, and only one marker (rs43147474) accounted for (sire-specific) TRD in all seven breeds. It must be noted that rs43147474 is located in the fourth intronic region of the GTP-binding protein 10 gene, and this locus has been previously linked to the maintenance of mitochondria and nucleolar architectures. Alternatively, other candidate genes surround this hot-spot for sire-specific TRD in the cattle genome, and they are related to embryonic and postnatal lethality as well as prostate cancer, among others. This research characterized the distribution of TRD in the bovine genome, highlighting heterogeneous results when comparing across breeds. © 2016 Stichting International Foundation for Animal Genetics

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the putamen of patients suffering from PD showed a significant 35% reduction of this transcript, compared to healthy subjects. Interestingly, in line with observations obtained in humans, we found 27% decrease in Rhes mRNA levels in the putamen of MPTP-treated primates. Based on the established inhibitory influence of Rhes on dopamine-related responses, we hypothesize that its striatal downregulation in PD patients and animal models of PD might represent an adaptive event of the dopaminergic system to functionally counteract the reduced nigrostriatal innervation.

The spatiotemporal control of mitotic exit is crucial for faithful chromosome segregation during mitosis. In budding yeast, the mitotic exit network (MEN) drives cells out of mitosis, whereas the spindle position checkpoint (SPOC) blocks MEN activity when the anaphase spindle is mispositioned. How the SPOC operates at a molecular level remains unclear. Here, we report novel insights into how mitotic signalling pathways orchestrate chromosome segregation in time and space. We establish that the key function of the central SPOC kinase, Kin4, is to counterbalance MEN activation by the cdc fourteen early anaphase release (FEAR) network in the mother cell compartment. Remarkably, Kin4 becomes dispensable for SPOC function in the absence of FEAR. Cells lacking both FEAR and Kin4 show that FEAR contributes to mitotic exit through regulation of the SPOC component Bfa1 and the MEN kinase Cdc15. Furthermore, we uncover controls that specifically promote mitotic exit in the daughter cell compartment., The mitotic exit network (MEN) triggers mitotic exit and can be blocked by the spindle position checkpoint (SPOC). Here the authors show that SPOC kinase Kin4 counterbalances MEN activation by the Cdc fourteen early anaphase release (FEAR) network in the mother cell and that in the absence of FEAR mitotic exit requires daughter cell-confined factors.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

Coeliac disease pathogenesis is characterized by an immune response triggered, in genetically predisposed subjects, by ingested gluten and its withdrawal from the diet is the only available therapy. However, enzymatic modification of gluten through the insertion of lysine to avoid antigen presentation could represent a new therapeutical approach for patients. Sixty-six duodenal biopsies from 17 coeliac patients were cultured for 48 h with gluten or enzymatically-modified gluten (treated with human recombinant transglutaminase type 2 or bacterial transglutaminase, with or without lysine). Interferonγ, anti endomisium and anti transglutaminase IgA antibodies, lactate dehydrogenase and transglutaminase activity were measured in the culture medium. Transglutaminase type 2 expression was evaluated on biopsies by immunohistochemistry. Gluten and transglutaminase-treated gluten increased by 13-15 fold interferon γ release, as well as antibodies, transglutaminase activity, and the immunohistochemical expression of transglutaminase type 2. Addition of lysine to the enzymatic modification of gluten normalized interferon γ, antibodies, transglutaminase activity and immunohistochemical expression of transglutaminase type 2. Lactate dehydrogenase did not differ among the studied groups. Enzymatic modification of gluten by transglutaminase plus lysine prevents the immunologic effects on cultured duodenal biopsies from coeliac patients and could be tested as an alternative therapy in coeliac disease.

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

Anti-tissue transglutaminase (tTG) antibodies are specifically produced in the small-intestinal mucosa of celiac disease (CD) patients. It is now recognized that these antibodies, acting on cell-surface tTG, may play an active role in CD pathogenesis triggering an intracellular response via the activation of different signal transduction pathways. In this study, we report that anti-tTG antibodies, both commercial and from a CD patient, induce a rapid Ca(2+) mobilization from intracellular stores in Caco-2 cells. We characterized the mechanism of Ca(2+) release using thapsigargin and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which are able to deplete specifically endoplasmic reticulum and mitochondria of Ca(2+), respectively. Our data highlight that both pathways of calcium release were involved, thus indicating that the spectrum of cellular responses downstream can be very wide. In addition, we demonstrate that the increased Ca(2+) level in the cells evoked by anti-tTG antibodies was sufficient to activate tTG, which is normally present as a latent protein due to the presence of low Ca(2+) and to the inhibitory effect of GTP/GDP. Herein, we discuss the importance of intracellular tTG activation as central in the context of CD pathogenesis.

Celiac disease is an autoimmune illness characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals. The immune response is based on both cellular and humoral components, although the former seem to be more important in the pathogenesis. The autoantibody response is directed at the enzyme tissue transglutaminase, tTG or TG2, which possibly play a role in the onset of the disease. In this study we sought to develop an animal model in which to analyze the immunological regulation and significance of anti-TG2 antibodies, by expressing specific human single-chain antibody fragments in mice using adeno-associated virus vectors. Upon vector injection in the skeletal muscles, high and persistent systemic levels of anti-TG2 antibodies were obtained. Mice injected with vectors encoding antibodies also recognizing rodent TG2, also developed a strong anti-idiotypic response. This finding raises the question of whether an anti-idiotypic response to anti-TG2 antibodies is a factor associated with celiac disease.

Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such may define functional organelle identity. Most rabs are ubiquitously expressed. However, some rabs are preferentially expressed in epithelial cells where they appear intimately associated with the epithelial-specific transcytotic pathway and/or tight junctions. This review discusses the role of rabs in epithelial membrane transport.

SummaryAdipocytes are cells able to produce and secrete several active substances (adipokines) with direct effects on vascular cells. Apelin, one of the most recently identified adipokines has been studied in cardiovascular system physiology in regard to vessel vasodilation and myocardial contraction, but it has not yet completely characterised for its pathophysiological role in cardiovascular disease and especially in acute coronary syndromes (ACS). Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of apelin 12 and apelin 13 on TF in human umbilical endothelial cells (HUVECs) and monocytes. Cells were stimulated with increasing concentrations of apelin 12 or apelin 13 and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and pro-coagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that apelin 13 but not apelin 12 induces transcription of mRNA for TF. In addition, we show that this adipokine promotes surface expression of TF that is functionally active. Apelin 13 effects on TF appear modulated by the activation of the G-protein-transcription factor nuclear factor (NF)-ΚB axis since G-protein inhibitors suppressed NF-ΚB mediated TF expression. Data of the present study, although in vitro, indicate that apelin-13, induces a procoagulant phenotype in HUVECs and monocytes by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease.

Tissue transglutaminase 2 (TG2) is ubiquitously expressed in normal tissues and plays an important role in the pathophysiology of wound healing. An increase in periodontal tissues has been previously reported in cyclosporine-induced gingival overgrowth. Purpose: The aim of this study was to explore associations between TG2 expression and the vascularization and maturation processes of peri-implant soft tissues over time. Materials and Methods: Edentulous patients proposed for mandibular implant-retained overdentures were included in the study. Biopsies of the peri-implant mucosa were performed at the first surgical stage and at 4, 8, and 12 months after prosthetic load. A follow-up program was directed to record plaque indexes, bleeding on probing data, and pocket probing depth around implants. An evaluation of the vessels’ density was carried out by digital virtual microscopy and using an immunohistochemistry approach (antibodies anti-CD31, anti-TG2). A robust multivariable regression model was implemented. Results: According to model results, blood vessel count and probing (as a marker of gingival overgrowth in absence of plaque) significantly decrease over time and are associated with TG2, particularly for values above the median. Conclusion: The association of an increased TG2 expression in the extracellular matrix might have a significant impact in the development of gingival overgrowth around a loaded implant.

SummaryTo successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits.

Using an RT-PCR approach a rac-related cDNA clone, designated Ms-rac1, was isolated from Medicago sativa (alfalfa). Ms-rac1 encodes a putative protein of 197 amino acids, which is closely related to known Rac-related GTP-binding proteins from Pisum sativum and Arabidopsis thaliana. RT-PCR analysis demonstrated that Ms-rac1 is ubiquitously expressed in various tissues in alfalfa. Expression of Ms-rac1 in suspension cultures occurred independently of treatment with elicitor, indicating that it is constitutively expressed. Heterologous expression of an antisense Ms-rac1 cDNA construct in transgenic tobacco plants was associated with poor growth and retarded flowering. Following infiltration with yeast elicitor, transgenic tobacco plants transformed with either the empty vector or Ms-rac1 in sense orientation developed brown necrotic lesions and subsequently cell death was observed within the infiltrated tissues. In contrast, the majority of the antisense transformants neither formed necrotic lesions nor showed any other visible defence reactions, demonstrating that Rac-related GTPases play an important role in the establishment of plant defence reactions.

Inductive interactions between different cell layers have an extremely important role in early embryogenesis. One of the most intensively studied and best characterised of these is the induction of neural tissue from ectodermal cells by the dorsal mesoderm. The competence of ectodermal cells to respond to neural induction varies according to dorsal-ventral position; with dorsal ectoderm (much of which forms the neural plate) having a far higher competence. Here we show that overexpression of the nucleotide exchange factor lfc increases ectodermal competence for neural induction as well as the amount of neural tissue in the whole embryo. Lfc is expressed pan ectodermally soon after gastrulation and may respond to an early determinant of dorsal ectoderm. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Inductive interactions between different cell layers have an extremely important role in early embryogenesis. One of the most intensively studied and best characterised of these is the induction of neural tissue from ectodermal cells by the dorsal mesoderm. The competence of ectodermal cells to respond to neural induction varies according to dorsal-ventral position; with dorsal ectoderm (much of which forms the neural plate) having a far higher competence. Here we show that overexpression of the nucleotide exchange factor lfc increases ectodermal competence for neural induction as well as the amount of neural tissue in the whole embryo. Lfc is expressed pan ectodermally soon after gastrulation and may respond to an early determinant of dorsal ectoderm. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) vβ3, β1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.

We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin–myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

Activating point mutations in the Ras oncogene occur in a large number of human tumors, especially of epithelial origin. In thyroid follicular cells, ectopic expression of oncogenic H-Ras results in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice. In fibroblasts concomitant activation of the MAP kinase cascade and the small GTPase Rac-1 leads to full malignant transformation. We have tested the effects of these key downstream mediators of Ras in thyroid epithelial cells, by stably expressing either a constitutively active form of MEK-1 (MEK(deltaN3/S218E/S222D)), a constitutively active form of Rac-1 (Val12-Rac), or both. While the activation of one molecule or the other results in a weak phenotype, concomitant activation of both MEK-1 and Rac-1 in thyroid cells leads to growth factor-independent proliferation, morphological transformation and anchorage-independent growth. However, in contrast to Ras-transformed thyroid cells, the ones expressing the constitutively active forms of MEK-1 and Rac-1 maintain their differentiate phenotype and fail to form tumors when injected into nude mice. Thus, in thyroid epithelial cells, concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects and is not sufficient to cause full malignant transformation. Significantly, Ras-mediated increased proliferation and loss of differentiation can be dissociated in these cells.

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 +/- 3.1 microg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the Vmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+ (154 +/- 13 microM). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.