Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two... more
Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells. In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Com...
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Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells... more
Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by ...
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Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these... more
Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes ...
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Gangliosides are membrane-bound glycosphingolipids with oligosaccharide chains which contain one or several residues of sialic acid, a negatively charged sugar (Figure 1). In animal tissues the most abundant gangliosides are... more
Gangliosides are membrane-bound glycosphingolipids with oligosaccharide chains which contain one or several residues of sialic acid, a negatively charged sugar (Figure 1). In animal tissues the most abundant gangliosides are monosialoganglioside GM1, disialogangliosides, GD1a and GD1b, and trisialoganglioside, GT. Most of them are localized on the outer surface of the plasma membrane and are ubiquitous but minor components of the cell surface, with the major exception of the mammalian central nervous system where they comprise up to 5–10% of the total lipid, and where lipid-bound sialic acid often exceeds that bound to glycoproteins1,2. Gangliosides have been shown to interact with several bacterial toxins3, peptide hormones and other external ligands4,5 probably through association with membrane proteins6. Most likely they are involved in functional activities of the cell surface, such as regulation of growth and structural plasticity. All these functions are likely to be mediated by ganglioside head group dynamics with a strong tendency for cooperative associations with each other and with glycoproteins6,8,9,10. This behavior appears to be strongly enhanced by divalent cations and affected by crosslinking agents6,8, as observed with both native and articial membrances.
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In Paramecium, internal Ca2+ concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is... more
In Paramecium, internal Ca2+ concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca2+ influx, and the addition of drugs that block the Ca2+ current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABAB receptors in P. primaurelia swimming control. The presence of GABAB immunoanalogue in Paramecium ...
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Bradykinin (BK) mediates acute allergic asthma and airway remodeling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study B2R and NF-kB expression was... more
Bradykinin (BK) mediates acute allergic asthma and airway remodeling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls examining the role of NF-kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects (HNBFb and HABFb). B2R and NF-kB (total and nuclear) expression was analyzed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 hours after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5(+) and αSMA(+) mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/HNBFb incubated with interleukin (IL)-4/IL-13 with/without BK, and after NF-kB inhibitor, by Western blotting. Bronchial mucosa B2R and nuclear NF-kB expression was h...
Research Interests: Immunology, Asthma, Immunohistochemistry, Medicine, Gene expression, and 15 moreHumans, Female, Male, NF-kappa B, Young Adult, Allergens, Risk factors, Adult, Public health systems and services research, Risk Factors, Cross Sectional Studies, Interleukin, Bradykinin, fibroblasts, and Respiratory function tests
Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that... more
Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 ...
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Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H]D-aspartate ([H]D-Asp) in a LY341495-sensitive manner. Spinal cord... more
Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H]D-aspartate ([H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HTheteroreceptors. Actually, the 15 mM KCl-evoked [H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HTagonist (±)DOI, an effect reversed by the 5-HTantagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HTreceptors colocalize and cross-talk in these terminals and if 5-HTligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HTreceptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HTreceptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord ...
Research Interests: Psychology, Nonparametric Statistics, Neuropharmacology, Confocal Microscopy, Signal Transduction, and 15 moreCerebral Cortex, Female, Animals, Metabotropic Glutamate Receptors, Spinal Cord, Male, Immunoprecipitation, Rats, Exocytosis, SYNAPTOSOMES, Neurosciences, Glutamic Acid, Biotinylation, Nerve Endings, and Pharmacology and pharmaceutical sciences
We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their... more
We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. We studied the effect of LY566332, an mGlu2 receptor positive allosteric modulator (PAM), and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3 H]-D-aspartate]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are pr...
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment.... more
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1(G93A) mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1(G93A) mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1(G93A) mice with reduced expression of mGluR5 (SOD1(G93A)Grm5(-/+)) by crossing the SOD1(G93A) mutant mouse with the mGluR5 heterozigous Grm5(-/+) mouse. SOD1(G93A)Grm5(-/+) mice showed prolonged s...
Research Interests: Psychology, Neuropharmacology, Amyotrophic Lateral Sclerosis, Humans, Female, and 15 moreAnimals, Spinal Cord, Microglia, Male, Cell Death, Astrocytes, Superoxide Dismutase, Sex Factors, Disease Progression, Cell Survival, Neurosciences, Glutamic Acid, Motor Skills, Motor Neurons, and Pharmacology and pharmaceutical sciences
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There is increasing evidence for the neuronal coexistence of classical transmitters. Implications in favor of cotransmission have often been represented by the identification, in the same neuron, of the putative cotransmitters, their... more
There is increasing evidence for the neuronal coexistence of classical transmitters. Implications in favor of cotransmission have often been represented by the identification, in the same neuron, of the putative cotransmitters, their synthetic enzymes and/or their vesicular transporters. In contrast, coexpression of neurotransmitter transporters on the plasma membrane of the same nerve terminal, although a potentially important indication for cotransmission, has received poor attention. We here used preparations of isolated nerve endings to functionally identify transporters coexpressed on the plasma membrane of the same terminal, in order to verify if such transporter coexpression indeed exists in neuronal systems in which cotransmission has already been established or reasonably suspected through other technical approaches. We could observe that functional transporters for glycine and glutamate are coexpressed on nerve terminals in the cerebellum; transporters for dopamine and GABA coexist on striatal terminals; transporters for glycine and GABA, previously found to coexist as cotransmission markers on nerve terminals of spinal cord and cerebellum, are not coexpressed in neocortex and hippocampus, where cotransmission has not been proposed to occur; transporters for GABA, glycine and glutamate are colocalized on nerve terminals of the spinal cord. Confocal microscopy experiments were performed to substantiate functional data, highlighting the presence of the co-existing transporters under study on MAP-2 positive synaptosomes. It is concluded that investigating the colocalization of functional neurotransmitter transporters on the plasma membrane of nerve terminals can provide useful information on the possibility of cotransmission.
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Research Interests: Calcium, Mitochondria, Endoplasmic Reticulum Stress, Signal Transduction, Humans, and 14 moreMutation, Reactive Oxygen Species, Phosphorylation, Glycolysis, Proteins, Primary Cell Culture, Ribosomes, Exocrine Pancreatic Insufficiency, Protein Biosynthesis, Bone Marrow Cells, Gene Expression Regulation, Adenosine Triphosphate, lipomatosis, and Leucine
Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in central nervous system (CNS). They represent suitable targets for therapeutic approaches to central diseases that are typified by... more
Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in central nervous system (CNS). They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows to further characterize these autoreceptors. This study aims at investigating the pharmacological profile of mGlu2/3 receptors in selected CNS regions and at evaluating their functions in mice suffering from experimental autoimmune encephalomyelitis (EAE). The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by analyzing the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3) H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. Experiments were also carried out to analyze mGlu2/3 autoreceptor-mediated releasing functions in EAE animals and whether in vivo LY379268 administration can restore impaired glutamate release in these mice. Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possess LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 exist in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors lost completely their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 (1-0.01 mg kg(-1) ) administration restored glutamate exocytosis capability in spinal cord but not in cortical terminals. We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord, respectively. The mGlu3-preferring autoreceptors could represent a target for new pharmacological approach for demyelinating diseases.
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Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the... more
Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the dihydropyridine nimodipine reversibly inhibited the maximum current by 25% and the dose dependence showed IC50 close to 50 nM. omega-Conotoxin GVIA (cgtx, 5 microM) and omega-agatoxin IVA (agatx, 200 nM) irreversibly inhibited the current by 17% and by 47%, respectively. The effect of nimodipine was additive with that of the toxins. The GABAB agonist (+/-)baclofen, or (-)baclofen (100 microM), reduced the calcium current by 30 +/- 5%, with a IC50 4 microM. The effect was mediated by a pertussis toxin-sensitive G-protein. In cells treated with cgtx during the experiment or preincubated with the toxin for 30 min, the effect of baclofen was significantly reduced. However, the action of baclofen was not confined to cgtx-sensitive channels: application of nim...
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Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane... more
Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) ...
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Research Interests: Signal Transduction, Cell line, Brain, G protein-coupled receptors, Female, and 13 moreAnimals, Microglia, NF-kappa B, Nicotinic Acetylcholine Receptors, Clinical Sciences, SYNAPSES, Experimental Autoimmune Encephalomyelitis, Sirtuin, Neurosciences, Glutamic Acid, Neuroprotective Agents, Excitatory Postsynaptic Potentials, and Tissue culture techniques
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Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the... more
Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the dihydropyridine nimodipine reversibly inhibited the maximum current by 25% and the dose dependence showed IC50 close to 50 nM. omega-Conotoxin GVIA (cgtx, 5 microM) and omega-agatoxin IVA (agatx, 200 nM) irreversibly inhibited the current by 17% and by 47%, respectively. The effect of nimodipine was additive with that of the toxins. The GABAB agonist (+/-)baclofen, or (-)baclofen (100 microM), reduced the calcium current by 30 +/- 5%, with a IC50 4 microM. The effect was mediated by a pertussis toxin-sensitive G-protein. In cells treated with cgtx during the experiment or preincubated with the toxin for 30 min, the effect of baclofen was significantly reduced. However, the action of baclofen was not confined to cgtx-sensitive channels: application of nim...
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Coexpression of transmembrane NAD+-exporting activity (via connexin 43 hemichannels) and of ectocellular ADP-ribosyl cyclase activity (via CD38 and BST-1) on stromal cells in the bone marrow microenvironment potentially enables the... more
Coexpression of transmembrane NAD+-exporting activity (via connexin 43 hemichannels) and of ectocellular ADP-ribosyl cyclase activity (via CD38 and BST-1) on stromal cells in the bone marrow microenvironment potentially enables the extracellular production of cyclic ...
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CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43)... more
CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombina...
Research Interests: Kinetics, Calcium, Biological Chemistry, Cell Division, Biological Sciences, and 14 moreMice, Animals, Biological, T cells, CHEMICAL SCIENCES, Multienzyme complexes, Transfection, Connexin, NAD, Recombinant Proteins, Cell Membrane, binding sites, coculture techniques, and Medical and Health Sciences
CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from... more
CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38(+) cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that t...
Research Interests: Physiology, Calcium, Signal Transduction, Humans, Mice, and 15 moreAnimals, Medical Physiology, Calcium Homeostasis, Active site, HeLa cells, Cyclic nucleotides, NAD, High Efficiency, Second Messengers, Ligands, Catalytic Activity, Nucleotides, Biochemistry and cell biology, Intracellular Calcium, and Cell Membrane
CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP-ribose (cADPR) from NAD+ and the degradation of cADPR to ADP-ribose by means... more
CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP-ribose (cADPR) from NAD+ and the degradation of cADPR to ADP-ribose by means of its ADP-ribosyl cyclase and cADPR-hydrolase activities, respectively. The cyclase also converts NGD+ to cyclic GDP-ribose (cGDPR), which is refractory to cADPR-hydrolase. cADPR, but not cGDPR, is a potent calcium mobilizer from intracellular stores. It has been demonstrated to be a new second messenger involved in the regulation of calcium homeostasis in many cell types, from plants to mammals. The number of physiological processes shown to be regulated by cADPR is steadily increasing. A topological paradox exists because ectocellularly generated cADPR acts intracellularly. Here we demonstrate that the catalytic functioning of CD38 is accompanied by a cADPR (cGDPR) -transporting activity across natural and artificial membranes. In resealed membra...
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CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The... more
CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also ...
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1. We investigated the effect of nimodipine on the calcium current in dissociated cerebellar granule cells from 8-day-old rats. We measured the whole cell current and the depolarization-induced internal calcium elevation in Fura2-loaded... more
1. We investigated the effect of nimodipine on the calcium current in dissociated cerebellar granule cells from 8-day-old rats. We measured the whole cell current and the depolarization-induced internal calcium elevation in Fura2-loaded cells exposed to high-potassium solutions. 2. Nimodipine maximally depressed the peak calcium current from holding potential (Vh) = -80 mV by 25% and the inactivation resistant residual current from Vh = -50 mV by 44%. The nimodipine-sensitive current had the same amplitude under both conditions and the half-maximal inhibition concentration (IC50) was close to 50 nM. 3. In contrast to other components of the calcium current, the nimodipine-sensitive current did not inactivate significantly during 1-s depolarization and it was weakly sensitive to the holding potential and to depolarizing conditioning prepulses. The effect of nimodipine was higher on the current elicited by small depolarizations and the current at -30 mV was depressed by < or = 70%....
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Ganglion cells in larval tiger salamander retina were differentiated into different types according to their light response and morphology. Sustained hyperpolarizing responses were recorded from ganglion cells branching in the sclerad... more
Ganglion cells in larval tiger salamander retina were differentiated into different types according to their light response and morphology. Sustained hyperpolarizing responses were recorded from ganglion cells branching in the sclerad half of the inner plexiform layer (IPL). Sustained depolarizing responses were elicited in cells with dendrites confined in the vitread portion of the IPL. Transient responses were associated with two types of morphological units sharing level of branching, in the middle of the IPL, but differing for soma location (inner plexiform or inner nuclear layer).
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Retinae of teleosts, the marine sparid Boops boops and the fresh water cyprinid Carassius auratus, and amphibians, the anuran Rana pipiens and the urodele Ambystoma tigrinum, were stained in vitro with DAPI (4'6-diamidino... more
Retinae of teleosts, the marine sparid Boops boops and the fresh water cyprinid Carassius auratus, and amphibians, the anuran Rana pipiens and the urodele Ambystoma tigrinum, were stained in vitro with DAPI (4'6-diamidino 2-phenylindole). In all the preparations tested DAPI consistently stained nuclei in the ganglion cell layer, and three levels of nuclei could be observed from IPL (inner plexiform layer) to INL (inner nuclear layer) in the bogue, goldfish and frog retinae. In the goldfish retina a dense mosaic of stained horizontal cell nuclei was also observed. Both single and double cones were stained in fish; no photoreceptor staining could be found in amphibian retinae. Goldfish and frog retinae were incubated with 5'7'-DHT (dihydroxytryptamine) to compare the distribution of DAPI-stained cells with that of putative serotoninergic neurones. Fluorescent cells were found in the ganglion cell layer, and at two levels distally. In fixed retinae only a regular array of c...
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Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow... more
Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentra...
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We performed two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence emission after irradiation at lambda = 413 nm... more
We performed two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence emission after irradiation at lambda = 413 nm (1). This work aims to evaluate the use of two- photon interactions for activation of the molecules. Therefore experiments were performed using fixed and living cells which