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D Corda

Publications and source records attributed to D Corda.

At least 19 recordsLinked to original sources
PubMed2005

Two novel mutations of the AIRE protein affecting its homodimerization properties.

We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.

Adult↗
PubMed2002

Molecular aspects of membrane fission in the secretory pathway.

Membrane fission is essential in various intracellular dissociative transport steps. The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated. Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface. Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Maintenance of PtdIns45P2 pools under limiting inositol conditions, as assessed by liquid chromatography-tandem mass spectrometry and PtdIns45P2 mass evaluation in Ras-transformed cells.

Inositol-containing molecules are involved in important cellular functions, including signalling, membrane transport and secretion. Our interest is in lysophosphatidylinositol and the glycerophosphoinositols, which modulate cell proliferation and G-protein-dependent activities such as adenylyl cyclase and phospholipase A(2). To investigate the role of glycerophosphoinositol (GroPIns) in the modulation of Ras-dependent pathways and its correlation to Ras transformation, we employed a novel liquid chromatography-tandem mass spectrometry technique to directly measure GroPIns in cell extracts. The cellular levels of GroPIns in selected parental and Ras-transformed cells, and in some carcinoma cells, ranged from 44 to 925 microM, with no consistent correlation to Ras transformation across all cell lines. Moreover, the derived cellular inositol concentrations revealed a wide range ( approximately 150 microM to approximately 100 mM) under standard [(3)H]-inositol-loading, suggesting a complex relationship between the inositol pool and the phosphoinositides and their derivatives. We have investigated these pools under specific loading conditions, designing a further HPLC analysis for GroPIns, combined with mass determinations of cellular phosphatidylinositol 4,5-bisphosphate. The data demonstrate that limiting inositol conditions identify a preferred pathway of inositol incorporation and retention into the polyphosphoinositides pool. Thus, under conditions of increased metabolic activity, such as receptor stimulation or cellular transformation, the polyphosphoinositide levels will be maintained at the expense of phosphatidylinositol and the turnover of its aqueous derivatives.

Cell Transformation, Neoplastic↗
PubMed2001

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes.

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.

CD28 Antigens↗
PubMed2001

Incidence of drug-induced aggravation in benign epilepsy with centrotemporal spikes.

PURPOSE: Benign epilepsy with centrotemporal spikes (BECTS) is characterized by an excellent prognosis. Drug therapy is necessary in only a minority of patients. Carbamazepine (CBZ) and phenobarbital (PB) have been reported to cause electroclinical aggravation in some cases. The incidence of drug-induced aggravation in BECTS has never been established. METHODS: We retrospectively studied 98 consecutive cases of BECTS, examined at the Centre Saint Paul between 1984 and 1999; 82 patients had received one or more treatments, often successively and in association. RESULTS: We found only one case of electroclinical aggravation with CBZ among 40 patients exposed to CBZ (35 in monotherapy, five in polytherapy). An additional case showed a marked EEG aggravation on CBZ + PB among 14 patients taking PB (nine with monotherapy and five with polytherapy), and PB was apparently responsible. No patient treated with valproate or benzodiazepines showed aggravation. CONCLUSIONS: Aggravation of BECTS caused by antiepileptic drugs happens only rarely. There is a minor risk of aggravation with CBZ and also probably with PB. Drug-induced aggravation may occur only during certain periods coinciding with spontaneous worsening of BECTS.

Acute Disease↗
PubMed2000

Endogenous ADP-ribosylation of the G protein beta subunit prevents the inhibition of type 1 adenylyl cyclase.

Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein beta subunit-specific polyclonal antibody, indicating that this protein is the G protein beta subunit. The ADP-ribosylation of the beta subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited by meta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated beta subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be an in vivo modulator of the interaction of betagamma with specific effectors. Indeed, we found that the ADP-ribosylated betagamma subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the beta subunit occurs in intact Chinese hamster ovary cells, where the NAD(+) pool was labeled with [(3)H]adenine. These results show that the ADP-ribosylation of the betagamma subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction.

Adenosine Diphosphate Ribose↗
PubMed1999

Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A.

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein alpha (PI-TPalpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPalpha. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPalpha. Overexpression of PI-TPalpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13-14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G(1) phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar. Upon equilibrium labeling of the cells with myo-[(3)H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4, 5-bisphosphate were the same as those in control cells. The addition of PI-TPalpha to a total lysate of myo-[(3)H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca(2+) further increased this formation. Based on these observations, we propose that PI-TPalpha is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.

3T3 Cells↗
PubMed1999

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid.

Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.

Acyl Coenzyme A↗
PubMed1999

PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes.

We reported that an inhibitor of sphingolipid biosynthesis, D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25-38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component beta-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.

Adenosine Diphosphate↗
PubMed1999

Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure.

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex.

ADP-Ribosylation Factors↗
PubMed1999

Signaling pathways involved in thyroid hyperfunction and growth in Graves' disease.

The elucidation of the multiple signaling cascades coupled to the TSH receptor has offered new approaches in the understanding of the pathogenesis of Graves' disease. Here we review findings showing that immunoglobulins from Graves' patients are heterogeneous, bind to different epitopes and, similarly to TSH, activate different signaling pathways, including adenylyl cyclase, phospholipase C and phospholipase A2. Evidence that the multiplicity of signals correlates with the different manifestations of the disease is also summarized. We believe that the dissection of the molecular mechanisms involved in the pathogenesis of Graves' disease offers the basis for developing novel therapeutical approaches to this disease.

Animals↗
PubMed1999

ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex.

The small GTPase ADP-ribosylation factor (ARF) regulates the structure and function of the Golgi complex through mechanisms that are understood only in part, and which include an ability to control the assembly of coat complexes and phospholipase D (PLD). Here we describe a new property of ARF, the ability to recruit phosphatidylinositol-4-OH kinase-beta and a still unidentified phosphatidylinositol-4-phosphate-5-OH kinase to the Golgi complex, resulting in a potent stimulation of synthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate; this ability is independent of its activities on coat proteins and PLD. Phosphatidylinositol-4-OH kinase-beta is required for the structural integrity of the Golgi complex: transfection of a dominant-negative mutant of the kinase markedly alters the organization of the organelle.

1-Phosphatidylinositol 4-Kinase↗
PubMed1999

Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate.

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras-induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein-stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3-kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.

3T3 Cells↗
PubMed1999

Graves' immunoglobulins activate phospholipase A2 by recognizing specific epitopes on thyrotropin receptor.

Thyroid-stimulating IgG from Graves' patients bind to the TSH receptor and activate both adenylyl cyclase (AC) and phospholipase A2 (PLA2) in FRTL5 thyroid cells. Both activities have been associated with increased thyroid cell growth and function; evidence exists that subpopulations of Graves' IgG can stimulate either AC or PLA2 cascades and that the activation of both is associated with the largest goiters in patients. Studies using chimeras of the human TSHR receptor (hTSHR) and the LH-CG receptor show that most patients with Graves' disease have cAMP-stimulating IgG that require epitopes on the N-terminal portion of the TSHR extracellular domain; epitopes associated with PLA2 activation are not clear. To address this question we used stably transfected Chinese hamster ovary (CHO) cells containing the wild-type hTSHR and the hTSHR chimera with residues 8-165 (Mc1+2) substituted by equivalent residues of the LH-CG receptor. PLA2 activity, measured as arachidonic acid (AA) release, was determined in 32 patients with Graves' disease. We show that 72% of Graves' patients have IgG able to stimulate PLA2 in CHO cells transfected with the TSHR and that AA release induced by Graves' IgG was significantly reduced (P = 0.022) in the CHO-Mc1+2-transfected cells (193 +/- 88% vs. 131 +/- 67%, respectively). Unlike IgG, the effect of TSH was not modified in the CHO-Mc1+2-transfected cells. When we compared the AC- and PLA2-stimulating activities of these 32 IgG in wild-type TSHR transfectants, we found that 63% of Graves' patients have antibodies able to stimulate both PLA2 and AC, whereas some patients' IgG were active only in AC or PLA2 assays. Of the patients with IgG having activity in both assays in wild-type TSHR transfectants, 50% of the IgG lost their stimulatory activities in both AA release and cAMP assays in Mc1+2 cells. Of the remainder, some IgG maintained their activity in one (AA release) or the other (cAMP) assay when measured in Mc1+2 chimeras. Thus, our data show that the N-terminal portion of extracellular domain of the TSHR is required for PLA2 as well as AC activation by IgG from patients with Graves' disease. These data also demonstrate that patients with Graves' disease have heterogeneous autoantibodies that selectively activate AC and PLA2 pathways and suggest that patients with autoantibodies active in both assays have more severe disease, with higher thyroid hormone levels and larger goiters.

Adult↗
PubMed1999

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport.

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50. A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA. Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.

Adenosine Diphosphate Ribose↗
PubMed1998

Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis.

Magnesium homeostasis in HL-60 promyelocytic leukemia cells was compared to that in neutrophyl-like HL-60 cells obtained by 1.3% DMSO treatment. Magnesium homeostasis was studied by the characterization of magnesium efflux, the identification of intracellular magnesium pools, and the regulation of intracellular ionized Mg2+. In both undifferentiated and neutrophyl-like HL-60 cells, magnesium efflux occurred via the Na-Mg antiporter which was inhibited by imipramine and stimulated by db cAMP and forskolin. Receptor-mediated signals such as ATP, IFN-alpha, or PGE1, which can trigger cAMP-dependent magnesium efflux, were ineffective in undifferentiated HL-60 cells but induced 60-70% increase of magnesium efflux in neutrophyl-like HL-60 cells. Selective membrane permeabilization by the cation ionophore A23187 induced a large magnesium release when cells were treated with rotenone. In both cell populations, the addition of glucose to rotenone-treated cells restored magnesium release to the control level. Permeabilization by 0.005% digitonin provoked the release of 90% cell total magnesium in both cell types. Intracellular [Mg2+]i was 0.15 and 0.26 mM in undifferentiated and neutrophyl-like HL-60 cells, respectively. Stimuli that triggered magnesium efflux, such as db cAMP in undifferentiated and IFN-alpha in neutrophyl-like HL-60 cells, induced a slow but consistent increase of [Mg2+]i which was independent from Ca2+ movements. Overall, these data indicate that magnesium homeostasis is regulated by receptor-mediated magnesium efflux which was modified during differentiation of HL-60 cells. Stimulation of magnesium efflux is paralleled by an increase of [Mg2+]i which reflects a release of magnesium from the bound cation pool.

Adenosine Triphosphate↗
PubMed1998

Human Trop-2 is a tumor-associated calcium signal transducer.

Trop-2/EGP-1/GA733-1 is a recently identified cell surface glycoprotein highly expressed by human carcinomas. The cytoplasmic tail of Trop-2 possesses potential serine and tyrosine phosphorylation sites and a phosphatidyl-inositol binding consensus sequence. Thus, we investigated whether Trop-2 might be a functional signaling molecule. Using the fluorescent probe Fura-2, we assayed the cytoplasmic calcium levels in human cancer cells stimulated with anti-Trop-2 or control antibodies. Three anti-Trop-2 MAbs, Rs7-7G11, MOv16 and 162-46.2 specifically induced a transient intracellular calcium level increment in up to 40% of the experiments performed. Polyclonal antisera recognizing recombinant Trop-2 molecules possessed a much lower stimulation efficiency. The average latency of antibody-induced Ca2+ rise for OvCa-432 cells was 64+/-26 sec. Internal Ca2+ concentrations reached peaks of 190+/-24 nM vs. basal levels of 61+/-4 nM and returned to baseline within 193+/-37 sec. Similar values were obtained in MCF-7 cells. For comparison, stimulation of P2-purinergic receptors on MCF-7 and OvCa-432 cells induced a Ca2+ rise in most cases, leading to average internal Ca2+ concentrations of 297+/-41 and 391+/-39 nM, respectively. Our findings show that Trop-2 transduces an intracellular calcium signal, are consistent with the hypothesis that it acts as a cell surface receptor and support a search for a physiological ligand.

Adenosine Triphosphate↗