42 results on '"Ciaffoni, F"'
Search Results
2. Polymorphism of galactose-1-phosphate uridyltransferase in the Albanian and Croatian settlements of Molise (Italy)
- Author
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Vaccaro, Anna Maria, Mandara, I., Ciaffoni, F., and Biondi, G.
- Published
- 1984
3. Polymorphism of Erythrocyte Galactose-1-Phosphate Uridyl-Transferase in Italy: Segregation Analysis in 693 Families
- Author
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Vaccaro, A.M., Mandara, I., Muscillo, M., Ciaffoni, F., De Pellegrin, S., Benincasa, A., Novelletto, A., and Terrenato, L.
- Published
- 1984
4. The thrombopoietin/MPL axis is activated in the Gata1low mouse model of myelofibrosis and is associated with a defective RPS14 signature
- Author
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Zingariello, M, primary, Sancillo, L, additional, Martelli, F, additional, Ciaffoni, F, additional, Marra, M, additional, Varricchio, L, additional, Rana, R A, additional, Zhao, C, additional, Crispino, J D, additional, and Migliaccio, A R, additional
- Published
- 2017
- Full Text
- View/download PDF
5. P15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment
- Author
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Funnell, A., Prontera, P., Ottaviani, V., Piccione, M., Giambona, A., Maggio, A., Ciaffoni, F., Stehling-Sun, S., Marra, M., Masiello, F., Varricchio, L., Stamatoyannopoulos, J., Migliaccio, A., and Papayannopoulou, T.
- Subjects
Settore MED/38 - Pediatria Generale E Specialistica ,Settore MED/03 - Genetica Medica ,Adolescent ,Carrier Proteins ,Child ,Female ,Fetal Hemoglobin ,Humans ,Male ,Nervous System Diseases ,Nuclear Proteins ,Up-Regulation ,Chromosome Deletion ,Chromosomes, Human, Pair 2 ,Hematology ,Biochemistry ,Cell Biology ,Immunology - Published
- 2015
6. Structural analysis of saposin C and B. Complete localization of disulfide bridges
- Author
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Vaccaro, A. M., Salvioli, R., Barca, A., Tatti, M., Ciaffoni, F., Maras, B., ROSAANNA SICILIANO, Zappacosta, F., Amoresano, A., Pucci, P., Vaccaro, Am, Salvioli, R, Barca, A, Tatti, M, Ciaffoni, F, Maras, B, Siciliano, R, Zappacosta, F, Amoresano, Angela, and Pucci, Pietro
- Subjects
Sphingolipid Activator Proteins ,Protein Conformation ,Lipid Bilayers ,Molecular Sequence Data ,Phosphatidylserines ,Mass Spectrometry ,Saposins ,Enzyme Activation ,Animals ,Glucosylceramidase ,Cattle ,Amino Acid Sequence ,Disulfides ,Glycoproteins - Abstract
Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.
- Published
- 1995
7. The N370S mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C
- Author
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Salvioli, R, Tatti, M, Scarpa, Susanna, Moavero, Sm, Ciaffoni, F, Felicetti, F, Kaneski, Cr, Brady, Ro, and Vaccaro, Am
- Published
- 2005
8. Evaluation of the spontaneous reversibility of carbon tetrachloride-induced liver cirrhosis in rabbits
- Author
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Bravo, E, primary, D'Amore, E, additional, Ciaffoni, F, additional, and Mammola, C L, additional
- Published
- 2012
- Full Text
- View/download PDF
9. Saposin D solubilizes anionic phospholipid-containing membranes.
- Author
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Ciaffoni, F, Salvioli, R, Tatti, M, Arancia, G, Crateri, P, and Vaccaro, A M
- Abstract
Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.
- Published
- 2001
- Full Text
- View/download PDF
10. Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids
- Author
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Salvioli, R., Tatti, M., Ciaffoni, F., and Vaccaro, A. M.
- Published
- 2000
- Full Text
- View/download PDF
11. Effect of saposins A and C on the enzymatic hydrolysis of liposomal glucosylceramide.
- Author
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Vaccaro, A M, Tatti, M, Ciaffoni, F, Salvioli, R, Barca, A, and Scerch, C
- Abstract
The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV). The separate addition of either Sap A or Sap C to the LUV system partially stimulated the sphingolipid degradation while saposins B and D had no effect. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV. The characteristics of glucosylceramidase activation by Sap C were different from those of Sap A. Sap C increased the rate of hydrolysis of both the artificial water soluble substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, and the lipid substrate, glucosylceramide, while Sap A only stimulated degradation of the sphingolipid. Also the binding properties of Saps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidase with the membrane. In contrast, Sap A had poor affinity for the membrane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase binding. In conclusion, our results show that both Sap A and Sap C are required for maximal hydrolysis of glucosylceramide inserted in PS-containing LUV, that their effects are synergistic, and that their mode of action is different. Sap C is responsible for the membrane binding of glucosylceramidase, while Sap A stimulation is possibly related to its effect on the conformation of the enzyme. It can be envisaged that Sap A in conjunction with Sap C might have a physiological role in glucosylceramide degradation.
- Published
- 1997
12. Structural analysis of saposin C and B. Complete localization of disulfide bridges.
- Author
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Vaccaro, A M, Salvioli, R, Barca, A, Tatti, M, Ciaffoni, F, Maras, B, Siciliano, R, Zappacosta, F, Amoresano, A, and Pucci, P
- Abstract
Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.
- Published
- 1995
13. Polymorphism of Erythrocyte Galactose-1-Phosphate Uridyl-Transf erase in Italy: Segregation Analysis in 693 Families
- Author
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Vaccaro, A.M., primary, Mandara, I., additional, Muscillo, M., additional, Ciaffoni, F., additional, De Pellegrin, S., additional, Benincasa, A., additional, Novelletto, A., additional, and Terrenato, L., additional
- Published
- 1984
- Full Text
- View/download PDF
14. 2p15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment
- Author
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Manuela Marra, Paolo Prontera, Fiorella Ciaffoni, Valentina Ottaviani, Francesca Masiello, Anna Rita Migliaccio, John A. Stamatoyannopoulos, Thalia Papayannopoulou, Sandra Stehling-Sun, Aurelio Maggio, Antonino Giambona, Alister P. W. Funnell, Lilian Varricchio, Maria Piccione, Funnell, A., Prontera, P., Ottaviani, V., Piccione, M., Giambona, A., Maggio, A., Ciaffoni, F., Stehling-Sun, S., Marra, M., Masiello, F., Varricchio, L., Stamatoyannopoulos, J., Migliaccio, A., Papayannopoulou, T., Funnell, Ap, Prontera, P, Ottaviani, V, Piccione, M, Giambona, A, Maggio, A, Ciaffoni, F, Stehling-Sun, S, Marra, M, Masiello, F, Varricchio, L, Stamatoyannopoulos, Ja, FRANCO MIGLIACCIO, ANNA RITA, and Papayannopoulou, T10.
- Subjects
Male ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Adolescent ,Immunology ,Biology ,Biochemistry ,Settore MED/38 - Pediatria Generale E Specialistica ,Red Cells, Iron, and Erythropoiesis ,Internal medicine ,hemic and lymphatic diseases ,Fetal hemoglobin ,medicine ,Gene silencing ,Humans ,Child ,Nervous System Disease ,Fetal Hemoglobin ,Nuclear Protein ,Hematology ,Nuclear Proteins ,Cell Biology ,medicine.disease ,Phenotype ,Sickle cell anemia ,Up-Regulation ,Transplantation ,Repressor Proteins ,Settore MED/03 - Genetica Medica ,Chromosomes, Human, Pair 2 ,2p15-p16.1 microdeletions BCL11A HbF neurologic ,Female ,Stem cell ,Chromosome Deletion ,Nervous System Diseases ,Carrier Protein ,Haploinsufficiency ,Carrier Proteins ,Human - Abstract
Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as β-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental g-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype.
- Published
- 2015
15. Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis
- Author
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Elena Cassella, Margherita Massa, Anna Rita Migliaccio, Fiorella Ciaffoni, Lilian Varricchio, Giovanni Barosi, Ciaffoni, F, Cassella, E, Varricchio, L, Massa, M, Barosi, G, and FRANCO MIGLIACCIO, ANNA RITA
- Subjects
Adult ,Male ,Pathology ,medicine.medical_specialty ,Autoimmunity ,Spleen ,Biology ,Article ,Transforming Growth Factor beta1 ,Mice ,Megakaryocyte ,non-canonical TGF-β1 signaling myelofibrosis ,Bone Marrow ,Fibrosis ,medicine ,Animals ,Humans ,Myelofibrosis ,Molecular Biology ,Inflammation ,Ineffective Hematopoiesis ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Haematopoiesis ,medicine.anatomical_structure ,Primary Myelofibrosis ,Molecular Medicine ,Female ,Bone marrow ,Megakaryocytes ,Signal Transduction - Abstract
Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-β1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-β1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-β1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-β1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-β1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF.
- Published
- 2015
16. Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis
- Author
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Laura Sancillo, Rosa Alba Rana, Francesca Masiello, Fabrizio Martelli, Giovanni Barosi, Maria Zingariello, Judith D. Goldberg, Xiaochun Li, Fiorella Ciaffoni, Margherita Massa, Anna Rita Migliaccio, Barbara Ghinassi, Emanuela D'Amore, Zingariello, M, Martelli, F, Ciaffoni, F, Masiello, F, Ghinassi, B, D'Amore, E, Massa, M, Barosi, G, Sancillo, L, Li, X, Goldberg, Jd, Rana, Ra, and Franco Migliaccio, Anna Rita
- Subjects
Adult ,Male ,Vascular Endothelial Growth Factor A ,Hematopoiesis and Stem Cells ,Blotting, Western ,Immunology ,Spleen ,Biology ,Real-Time Polymerase Chain Reaction ,Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF ,Biochemistry ,Transforming Growth Factor beta1 ,Mice ,Bone Marrow ,Fibrosis ,Biomarkers, Tumor ,medicine ,Animals ,Humans ,GATA1 Transcription Factor ,RNA, Messenger ,RNA, Small Interfering ,Myelofibrosis ,PI3K/AKT/mTOR pathway ,Oligonucleotide Array Sequence Analysis ,Ineffective Hematopoiesis ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,TOR Serine-Threonine Kinases ,Cell Biology ,Hematology ,Middle Aged ,Flow Cytometry ,medicine.disease ,Chemokine CXCL12 ,Extramedullary hematopoiesis ,Disease Models, Animal ,Haematopoiesis ,medicine.anatomical_structure ,Primary Myelofibrosis ,Case-Control Studies ,Cancer research ,Cytokines ,Bone marrow ,Signal Transduction - Abstract
Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.
- Published
- 2013
17. Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera
- Author
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Valentina Tirelli, Agostino Tafuri, Anna Rita Migliaccio, Giuliana Alimena, Giulia Federici, Ann Zeuner, Todd Hricik, Lilian Varricchio, Gabriella Girelli, Fiorella Ciaffoni, Francesca Masiello, Emanuel F. Petricoin, Ross L. Levine, Stefania Vaglio, Hricik, T, Federici, G, Zeuner, A, Alimena, G, Tafuri, A, Tirelli, V, Varricchio, L, Masiello, F, Ciaffoni, F, Vaglio, S, Petricoin, Ef, Girelli, G, Levine, Rl, and FRANCO MIGLIACCIO, ANNA RITA
- Subjects
Adult ,Male ,Proteomics ,Myeloid ,Erythroblasts ,erythroid-differentiation ,erythropoietin receptor ,rich akt substrate ,protein-kinase-c ,factor gata-1 ,40 kda pras40 ,gene-expression ,hematopoietic stem ,phosphorylation ,progenitor cells ,Biology ,Article ,Dexamethasone ,Receptors, G-Protein-Coupled ,Polycythemia vera ,erythroblasts expanded vitro polycythemia vera ,Proto-Oncogene Proteins ,hemic and lymphatic diseases ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,Humans ,Erythropoiesis ,Erythropoietin ,Polycythemia Vera ,Transcription factor ,Cells, Cultured ,T-Cell Acute Lymphocytic Leukemia Protein 1 ,Aged ,Regulation of gene expression ,Stem Cell Factor ,rab4 GTP-Binding Proteins ,Kinase ,Gene Expression Profiling ,GATA1 ,Hematology ,Middle Aged ,Phosphoproteins ,medicine.disease ,medicine.anatomical_structure ,Gene Expression Regulation ,Case-Control Studies ,Cancer research ,Female ,Interleukin-3 ,Signal transduction ,Signal Transduction - Abstract
Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.
- Published
- 2013
18. Caveolin-1 Endows Order in Cholesterol-Rich Detergent Resistant Membranes.
- Author
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Raggi C, Diociaiuti M, Caracciolo G, Fratini F, Fantozzi L, Piccaro G, Fecchi K, Pizzi E, Marano G, Ciaffoni F, Bravo E, Fiani ML, and Sargiacomo M
- Subjects
- Animals, Caveolae metabolism, Humans, X-Ray Diffraction, Caveolin 1 metabolism, Cholesterol metabolism, Proteomics methods
- Abstract
Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered ( L
o ) phase typical of the raft structure, namely "cholesterol-rich Lo phase," with a phase fully missing in Cav-1 KO that we named "caveolin-induced Lo phase." Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a "caveolin-induced Lo phase," as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources., Competing Interests: The authors declare no conflicts of interest.- Published
- 2019
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- View/download PDF
19. Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their ex vivo Expansion Highly Dependent on Plasma Lipoproteins.
- Author
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Zingariello M, Bardelli C, Sancillo L, Ciaffoni F, Genova ML, Girelli G, and Migliaccio AR
- Abstract
Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly in vivo . The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs. To test this hypothesis, we first compared the expression profiling of erythroblasts generated with or without dexamethasone. This analysis revealed differences in expression of 55 genes, 6 of which encoding proteins involved in lipid metabolism. These were represented by genes encoding the mitochondrial proteins 3-Hydroxymethyl-3-Methylglutaryl-CoA lyase, upregulated, and 3-Oxoacid CoA-Transferase1 and glycerol-3-phosphate acyltransferase1, both downregulated, and the proteins ATP-binding cassette transporter1 and Hydroxysteroid-17-Beta-Dehydrogenase7, upregulated, and cAMP-dependent protein kinase catalytic subunit beta, downregulated. This profiling predicts that dexamethasone, possibly by interfering with mitochondrial functions, impairs the intrinsic lipid metabolism making the synthesis of the plasma membrane of erythroid cells depend on lipid-uptake from external sources. Optical and electron microscopy analyses confirmed that the mitochondria of erythroblasts generated with dexamethasone are abnormal and that their plasma membranes present pebbles associated with membrane ruptures releasing exosomes and micro-vesicles. These results indicate that the lipid supplements of media currently available are not adequate for cRBCs. To identify better lipid supplements, we determined the number of erythroblasts generated in synthetic media supplemented with either currently used liposomes or with lipoproteins purified from human plasma [the total lipoprotein fraction (TL) or its high (HDL), low (LDL) and very low (VLDL) density lipoprotein components]. Both LDL and VLDL generated numbers of erythroid cells 3-2-fold greater than that observed in controls. These greater numbers were associated with 2-3-fold greater amplification of erythroid cells due both to increased proliferation and to resistance to stress-induced death. In conclusion, dexamethasone impairs lipid metabolism making ex vivo expansion of erythroid cells highly dependent on lipid absorbed from external sources and the use of LDL and VLDL as lipid supplements improves the generation of cRBCs.
- Published
- 2019
- Full Text
- View/download PDF
20. Disruption of IFN-I Signaling Promotes HER2/Neu Tumor Progression and Breast Cancer Stem Cells.
- Author
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Castiello L, Sestili P, Schiavoni G, Dattilo R, Monque DM, Ciaffoni F, Iezzi M, Lamolinara A, Sistigu A, Moschella F, Pacca AM, Macchia D, Ferrantini M, Zeuner A, Biffoni M, Proietti E, Belardelli F, and Aricò E
- Subjects
- Animals, Breast Neoplasms genetics, Cell Line, Tumor, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic metabolism, Female, Gene Expression Profiling, Humans, Immunophenotyping, Mice, Knockout, Mice, Transgenic, Neovascularization, Pathologic genetics, Neovascularization, Pathologic metabolism, Receptor, ErbB-2 genetics, Tumor Stem Cell Assay, Breast Neoplasms metabolism, Breast Neoplasms pathology, Interferon Type I metabolism, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Receptor, ErbB-2 metabolism, Signal Transduction
- Abstract
Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu
+/- mice (neuT mice), IFNAR1-/- neu+/- mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658-70. ©2018 AACR ., (©2018 American Association for Cancer Research.)- Published
- 2018
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21. The thrombopoietin/MPL axis is activated in the Gata1 low mouse model of myelofibrosis and is associated with a defective RPS14 signature.
- Author
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Zingariello M, Sancillo L, Martelli F, Ciaffoni F, Marra M, Varricchio L, Rana RA, Zhao C, Crispino JD, and Migliaccio AR
- Subjects
- Animals, Disease Models, Animal, Female, GATA1 Transcription Factor genetics, Humans, Male, Mice, Primary Myelofibrosis pathology, GATA1 Transcription Factor metabolism, Primary Myelofibrosis genetics, Ribosomal Proteins genetics, Thrombopoietin metabolism
- Abstract
Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1
low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.- Published
- 2017
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22. 2p15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment.
- Author
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Funnell AP, Prontera P, Ottaviani V, Piccione M, Giambona A, Maggio A, Ciaffoni F, Stehling-Sun S, Marra M, Masiello F, Varricchio L, Stamatoyannopoulos JA, Migliaccio AR, and Papayannopoulou T
- Subjects
- Adolescent, Child, Female, Humans, Male, Nervous System Diseases blood, Repressor Proteins, Up-Regulation, Carrier Proteins genetics, Chromosome Deletion, Chromosomes, Human, Pair 2, Fetal Hemoglobin metabolism, Nervous System Diseases genetics, Nuclear Proteins genetics
- Abstract
Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as β-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental γ-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype., (© 2015 by The American Society of Hematology.)
- Published
- 2015
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23. Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis.
- Author
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Ciaffoni F, Cassella E, Varricchio L, Massa M, Barosi G, and Migliaccio AR
- Subjects
- Adult, Animals, Bone Marrow immunology, Female, Humans, Inflammation immunology, Inflammation pathology, Male, Megakaryocytes immunology, Megakaryocytes pathology, Mice, Primary Myelofibrosis pathology, Spleen immunology, Spleen pathology, Autoimmunity, Bone Marrow pathology, Primary Myelofibrosis immunology, Signal Transduction, Transforming Growth Factor beta1 immunology
- Abstract
Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-β1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-β1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-β1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-β1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-β1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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24. Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.
- Author
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Hricik T, Federici G, Zeuner A, Alimena G, Tafuri A, Tirelli V, Varricchio L, Masiello F, Ciaffoni F, Vaglio S, Petricoin EF, Girelli G, Levine RL, and Migliaccio AR
- Subjects
- Adult, Aged, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Case-Control Studies, Cells, Cultured, Dexamethasone pharmacology, Erythroblasts drug effects, Erythroblasts pathology, Erythropoietin pharmacology, Female, Gene Expression Profiling, Gene Expression Regulation, Humans, Interleukin-3 pharmacology, Male, Middle Aged, Phosphoproteins metabolism, Polycythemia Vera metabolism, Polycythemia Vera pathology, Proteomics, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Signal Transduction, Stem Cell Factor pharmacology, T-Cell Acute Lymphocytic Leukemia Protein 1, rab4 GTP-Binding Proteins genetics, rab4 GTP-Binding Proteins metabolism, Erythroblasts metabolism, Erythropoiesis genetics, Phosphoproteins genetics, Polycythemia Vera genetics
- Abstract
Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera., (Copyright © 2013 Wiley Periodicals, Inc.)
- Published
- 2013
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25. Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.
- Author
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Zingariello M, Martelli F, Ciaffoni F, Masiello F, Ghinassi B, D'Amore E, Massa M, Barosi G, Sancillo L, Li X, Goldberg JD, Rana RA, and Migliaccio AR
- Subjects
- Adult, Animals, Biomarkers, Tumor genetics, Biomarkers, Tumor metabolism, Blotting, Western, Bone Marrow metabolism, Bone Marrow pathology, Case-Control Studies, Chemokine CXCL12 genetics, Chemokine CXCL12 metabolism, Cytokines metabolism, Flow Cytometry, Gene Expression Profiling, Humans, Male, Mice, Middle Aged, Oligonucleotide Array Sequence Analysis, Primary Myelofibrosis etiology, Primary Myelofibrosis metabolism, RNA, Messenger genetics, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Spleen metabolism, Spleen pathology, TOR Serine-Threonine Kinases genetics, TOR Serine-Threonine Kinases metabolism, Transforming Growth Factor beta1 antagonists & inhibitors, Transforming Growth Factor beta1 genetics, Vascular Endothelial Growth Factor A genetics, Vascular Endothelial Growth Factor A metabolism, Disease Models, Animal, GATA1 Transcription Factor physiology, Primary Myelofibrosis pathology, Signal Transduction, Transforming Growth Factor beta1 metabolism
- Abstract
Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.
- Published
- 2013
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26. Hepatic VLDL assembly is disturbed in a rat model of nonalcoholic fatty liver disease: is there a role for dietary coenzyme Q?
- Author
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Cano A, Ciaffoni F, Safwat GM, Aspichueta P, Ochoa B, Bravo E, and Botham KM
- Subjects
- Animals, Antioxidants metabolism, Apolipoproteins B metabolism, Lipid Metabolism physiology, Lipoproteins, VLDL blood, Lipoproteins, VLDL isolation & purification, Male, Microsomes, Liver enzymology, Microsomes, Liver metabolism, RNA, Messenger biosynthesis, RNA, Messenger genetics, Rats, Rats, Wistar, Reverse Transcriptase Polymerase Chain Reaction, Ubiquinone analogs & derivatives, Diet, Fatty Liver metabolism, Lipoproteins, VLDL metabolism, Liver metabolism, Ubiquinone metabolism
- Abstract
The overproduction of very-low-density lipoprotein (VLDL) is a characteristic feature of nonalcoholic fatty liver disease (NAFLD). The aim of this study was to use a high-fat diet-induced model of NAFLD in rats to investigate 1) the influence of the disease on hepatic VLDL processing in the endoplasmic reticulum and 2) the potential modulatory effects of dietary coenzyme Q (CoQ). Rats were fed a standard low-fat diet (control) or a diet containing 35% fat (57% metabolizable energy). After 10 wk, high-fat diet-fed animals were divided into three groups: the first group was given CoQ9 (30 mg*kg body wt(-1)*day(-1) in 0.3 ml olive oil), the second group was given olive oil (0.3 ml/day) only, and the third group received no supplements. Feeding (3 high-fat diets and the control diet) was then continued for 8 wk. In all high-fat diet-fed groups, the content of triacylglycerol (TG) and cholesterol in plasma VLDL, the liver, and liver microsomes was increased, hepatic levels of apolipoprotein B48 were raised, and the activities of microsomal TG transfer protein and acyl CoA:cholesterol acyltransferase were reduced. These findings provide new evidence indicating that VLDL assembly and the inherent TG transfer to the endoplasmic reticulum are altered in NAFLD and suggest a possible explanation for both the overproduction of VLDL associated with the condition and the disease etiology itself. Dietary CoQ caused significant increases in apolipoprotein B mRNA and microsomal TG levels and altered the phospholipid content of microsomal membranes. These changes, however, may not be beneficial as they may lead to the secretion of larger, more atherogenic VLDL.
- Published
- 2009
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27. Saposin B binds and transfers phospholipids.
- Author
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Ciaffoni F, Tatti M, Boe A, Salvioli R, Fluharty A, Sonnino S, and Vaccaro AM
- Subjects
- Chromatography, Gel, Humans, Hydrogen-Ion Concentration, Liposomes chemistry, Solubility, Sphingolipids metabolism, Phospholipids metabolism, Saposins metabolism
- Abstract
Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids. The capacity of Sap B to bind and transfer phospholipids occurs under conditions mimicking the interior of the late endosomal/lysosomal compartment and thus might have physiological relevance.
- Published
- 2006
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28. The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C.
- Author
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Salvioli R, Tatti M, Scarpa S, Moavero SM, Ciaffoni F, Felicetti F, Kaneski CR, Brady RO, and Vaccaro AM
- Subjects
- Amino Acid Substitution, Anions, Cell Line, Fibroblasts enzymology, Gaucher Disease enzymology, Gaucher Disease genetics, Glucosylceramidase metabolism, Humans, Membranes chemistry, Mutation, Glucosylceramidase chemistry, Glucosylceramidase genetics, Phospholipids chemistry, Saposins chemistry
- Abstract
The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (approximately 20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.
- Published
- 2005
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29. Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C.
- Author
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Salvioli R, Scarpa S, Ciaffoni F, Tatti M, Ramoni C, Vanier MT, and Vaccaro AM
- Subjects
- Blotting, Western, Cell Line, Endosomes metabolism, Fibroblasts metabolism, Glycoproteins chemistry, Humans, Lipid Metabolism, Lysophospholipids chemistry, Lysosomes metabolism, Microscopy, Fluorescence, Monoglycerides, Mutation, Phospholipids metabolism, Precipitin Tests, Saposins, Time Factors, Cholesterol metabolism, Glucosylceramidase biosynthesis, Glucosylceramidase chemistry, Niemann-Pick Diseases metabolism
- Abstract
Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability.
- Published
- 2004
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30. Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.
- Author
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Ciaffoni F, Tatti M, Salvioli R, and Vaccaro AM
- Subjects
- Anions, Cell Membrane metabolism, Humans, Saposins, Glycoproteins metabolism, Phospholipids metabolism, Sphingolipids metabolism
- Abstract
Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles. The Sap D-particle complexes formed in an acidic milieu can be dissociated by an increase in pH, suggesting a dynamic association of Sap D with membranes. The presence of anionic phospholipids is required also for the Sap D-induced perturbation and solubilization of membranes containing a neutral sphingolipid such as ceramide or a ganglioside such as G(M1). At appropriate Sap D concentrations Cer and G(M1) are solubilized as constituents of small phospholipid particles. Our findings imply that most functions of Sap D are dependent on its interaction with anionic phospholipids, which mediate the Sap D effect on other components of the membrane such as sphingolipids. On consideration of the properties of Sap D we propose that Sap D might have a role in the definition of the structure and function of membranes, such as the intra-endolysosomal membranes, that are rich in anionic phospholipids.
- Published
- 2003
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31. Structural and membrane-binding properties of saposin D.
- Author
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Tatti M, Salvioli R, Ciaffoni F, Pucci P, Andolfo A, Amoresano A, and Vaccaro AM
- Subjects
- Amino Acid Sequence, Cell Membrane metabolism, Ceramides pharmacology, Dose-Response Relationship, Drug, Gaucher Disease metabolism, Glycoproteins pharmacology, Glycosylation, Humans, Hydrogen-Ion Concentration, Mass Spectrometry, Molecular Sequence Data, Phospholipids metabolism, Protein Structure, Tertiary, Saposins, Sphingomyelins pharmacology, Structure-Activity Relationship, Glycoproteins chemistry
- Abstract
Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.
- Published
- 1999
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32. Saposins and their interaction with lipids.
- Author
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Vaccaro AM, Salvioli R, Tatti M, and Ciaffoni F
- Subjects
- Amino Acid Sequence, Glycoproteins chemistry, Glycosphingolipids metabolism, Humans, Hydrogen-Ion Concentration, Lipid Bilayers metabolism, Membranes, Artificial, Molecular Sequence Data, Phospholipids metabolism, Protein Binding, Glycoproteins metabolism, Lipid Metabolism
- Abstract
The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), 'Entamoeba histolytica' pore-forming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called 'saposin fold'. Up to now a precise fold, comprising five alpha-helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the alpha-helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.
- Published
- 1999
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33. pH-dependent conformational properties of saposins and their interactions with phospholipid membranes.
- Author
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Vaccaro AM, Ciaffoni F, Tatti M, Salvioli R, Barca A, Tognozzi D, and Scerch C
- Subjects
- Detergents, Electrophoresis, Polyacrylamide Gel, Gaucher Disease metabolism, Glycoproteins isolation & purification, Humans, Hydrogen-Ion Concentration, Kinetics, Lysosomes metabolism, Lysosomes ultrastructure, Octoxynol, Polyethylene Glycols, Saposins, Sphingolipid Activator Proteins, Spleen metabolism, Glucosylceramidase metabolism, Glycoproteins chemistry, Glycoproteins metabolism, Liposomes, Phosphatidylcholines, Protein Conformation
- Abstract
Saposins A, B, C, and D are small lysosomal glycoproteins released by proteolysis from a single precursor polypeptide, prosaposin. We have presently investigated the conformational states of saposins and their interaction with membranes at acidic pH values similar to those present in lysosomes. With the use of phase partitioning in Triton X-114, experimental evidence was provided that, upon acidification, saposins (Sap) A, C, and D acquire hydrophobic properties, while the hydrophilicity of Sap B is apparently unchanged. The pH-dependent exposure of hydrophobic domains of Sap C and D paralleled their pH-dependent binding to large unilamellar vesicles composed of phosphatidylcholine, phosphatidylserine, and cholesterol. In contrast, the binding of Sap A to the vesicles was very restricted, in spite of its increased hydrophobicity at low pH. A low affinity for the vesicles was also shown by Sap B, a finding consistent with its apparent hydrophilicity both at neutral and acidic pH. At the acidic pH values needed for binding, Sap C and D powerfully destabilized the phospholipid membranes, while Sap A and B minimally affected the bilayer integrity. In the absence of the acidic phospholipid phosphatidylserine, the induced destabilization markedly decreased. Of the four saposins, only Sap C was able to promote the binding of glucosylceramidase to phosphatidylserine-containing membranes. This result is consistent with the notion that Sap C is specifically required by glucosylceramidase to exert its activity. Our finding that an acidic environment induces an increased hydrophobicity in Sap A, C, and D, making the last two saposins able to interact and perturb phospholipid membranes, suggests that this mechanism might be relevant to the mode of action of saposins in lysosomes.
- Published
- 1995
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34. Saposin C induces pH-dependent destabilization and fusion of phosphatidylserine-containing vesicles.
- Author
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Vaccaro AM, Tatti M, Ciaffoni F, Salvioli R, Serafino A, and Barca A
- Subjects
- Animals, Cattle, Chromatography, Gel, Enzyme Activation, Glycoproteins chemistry, Lipid Bilayers chemistry, Liposomes, Membrane Fluidity, Membrane Fusion, Phosphatidylserines chemistry, Saposins, beta-Glucosidase chemistry, Glycoproteins metabolism, Lipid Bilayers metabolism, Phosphatidylserines metabolism, beta-Glucosidase metabolism
- Abstract
We have previously shown that saposin C (Sap C), a glucosylceramidase activator protein, interacts with phosphatidylserine (PS) large unilamellar vesicles (LUV), promoting the glucosylceramidase binding to the bilayer [(1993) FEBS Lett. 336, 159-162]. In the present paper the consequences of the Sap C interaction on the lipid organization of the vesicles are reported. It was found that Sap C perturbs the PS bilayer as shown by the release of an encapsulated fluorescent dye. Three different procedures, resonance energy transfer, gel filtration and electron microscopy, indicated that the activator protein is also able to make PS liposomes fuse. The effects of Sap C on PS vesicles were observed at low but not at neutral pH. The lipid composition of the bilayer also affected the Sap C-induced destabilization; in fact, the presence of PS in mixed LUV was essential for significant leakage to occur. These results demonstrate for the first time that Sap C is a protein capable of destabilizing and fusing acidic phospholipid-containing membranes in a pH-dependent fashion.
- Published
- 1994
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35. Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes.
- Author
-
Vaccaro AM, Tatti M, Ciaffoni F, Salvioli R, Maras B, and Barca A
- Subjects
- Enzyme Activation, Humans, Liposomes, Saposins, Glucosylceramidase metabolism, Glycoproteins physiology, Phosphatidylserines metabolism
- Abstract
The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.
- Published
- 1993
- Full Text
- View/download PDF
36. Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature.
- Author
-
Vaccaro AM, Tatti M, Ciaffoni F, Salvioli R, Barca A, and Roncaioli P
- Subjects
- Enzyme Activation, Humans, Lipid Bilayers chemistry, Liposomes chemistry, Oleic Acid, Oleic Acids pharmacology, Particle Size, Glucosylceramidase metabolism, Lipid Bilayers metabolism, Liposomes metabolism, Phosphatidylserines metabolism
- Abstract
The influence of phosphatidylserine (PS) liposome size on their capacity to activate and bind purified glucosylceramidase was investigated. Gel filtration and flotation experiments showed that large unilamellar vesicles (LUV) of either pure PS or PS in admixture with phosphatidylcholine (PC) are unable to tightly bind purified glucosylceramidase, and thus, to fully stimulate its activity. By contrast, small unilamellar vesicles (SUV) of PS adsorb glucosylceramidase can either be favoured or inhibited by factors affecting the bilayer curvature of PS liposomes. An increase of PS vesicle size induced by a fusogenic agent such as poly(ethylene glycol) (PEG), decreased enzyme binding and activity. On the contrary, the reduction of PS LUV size by sonication increased their stimulating ability. Enzyme association with PS SUV is reversible. In fact, glucosylceramidase bound to PS SUV was released from the lipid surface when the SUV were transformed into larger vesicles by PEG; dissociation from the vesicles resulted in a dramatic decrease of enzyme activity. Although PS LUV are unable to reconstitute glucosylceramidase, their association with oleic acid (OA) promotes the interaction with glucosylceramidase. This phenomenon is best explained in terms of OA-induced surface defects of PS LUV, with consequent exposure of the more hydrophobic part of the membrane and hence the improved binding of hydrophobic region/s of glucosylceramidase. Our data indicate that the physical organization of the PS-containing liposomes is of critical importance of glucosylceramidase reconstitution. The observation that physical changes of the lipid surface can markedly affect the enzyme activity offers a new approach to the study of glucosylceramidase regulation.
- Published
- 1993
- Full Text
- View/download PDF
37. Reconstitution of glucosylceramidase on binding to acidic phospholipid-containing vesicles.
- Author
-
Vaccaro AM, Tatti M, Ciaffoni F, Salvioli R, and Roncaioli P
- Subjects
- Centrifugation, Density Gradient, Chromatography, Hydrogen-Ion Concentration, Hymecromone metabolism, Glucosides metabolism, Glucosylceramidase metabolism, Hymecromone analogs & derivatives, Liposomes metabolism, Phospholipids metabolism
- Abstract
Studies were conducted to investigate the mechanism by which acidic phospholipid-containing vesicles stimulate purified placental glucosylceramidase activity towards the water-soluble substrate 4-methylumbelliferyl-beta-D-glucopyranoside (MUGlc). Vesicles composed of pure phosphatidic acid (PA) or pure phosphatidylserine (PS) stimulated the activity of the enzyme about 20-fold. The inclusion of cholesterol and phosphatidylcholine (PC), beside PA, into the vesicles slightly improved their stimulatory effect. Further addition of oleic acid (OA) markedly increased the stimulation (50-fold). By ultracentrifugation and gel permeation procedures it was shown that, under optimal conditions for stimulation of the MUGlc hydrolysis by acidic phospholipid-containing vesicles, purified glucosylceramidase spontaneously binds to their surface. Interestingly, the molar fraction of the acidic phospholipid into the mixed vesicles, rather than its concentration in the assay, is the crucial parameter for activation and binding of the enzyme. The importance of glucosylceramidase association with appropriate vesicles for enzyme activation was indicated by observing that the presence of 0.2 M citrate-phosphate buffer (pH 5.5), that prevented the binding to PA-containing surfaces, also inhibited the enzyme activity. Our results indicate that the reconstitution of glucosylceramidase activity occurs through the spontaneous tight association of the enzymatic protein with preformed acidic phospholipid-containing vesicles.
- Published
- 1992
- Full Text
- View/download PDF
38. Effect of experimental conditions on the appearance of distinct forms of placental glucosylceramidase: use of gel filtration analysis as a means of ascertaining their occurrence.
- Author
-
Vaccaro AM, Salvioli R, Gallozzi E, Ciaffoni F, and Tatti M
- Subjects
- Chromatography, Gel, Chromatography, High Pressure Liquid, Female, Glucosylceramidase metabolism, Guanidine, Guanidines pharmacology, Humans, Hydrogen-Ion Concentration, Lysosomes enzymology, Microsomes enzymology, Placenta ultrastructure, Pregnancy, Glucosylceramidase analysis, Placenta enzymology
- Abstract
We have found that, under some experimental conditions, the placental glucosylceramidase shows an anomalous behaviour on gel filtration chromatography. At pH 5.6, the optimal pH of the enzymatic assay, the purified enzyme remains bound to either Superose 6 or TSK-40-XL HPLC columns, while the interaction of the crude glucosylceramidase contained in the water extract of the lysosome-mitochondrial fraction of placenta with the two HPLC gel matrices is much weaker. The quite different behaviour of the crude compared to the purified enzyme may be explained by the formation in the crude preparation of associated form(s) of glucosylceramidase with suitable endogenous compound(s), which compete with the gel matrices for the binding to the enzyme. The most likely one component of the enzyme complex is the placental activating factor, previously reported by us (Vaccaro et al. (1985) Biochim. Biophys. Acta 836, 157-166), as indicated by the negligible stimulation of the crude enzyme activity on addition of the factor, either before or after passage through the HPLC columns. On the assumption that the behaviour of crude glucosylceramidase on gel filtration becomes similar to that of the purified enzyme when its interaction with endogenous substance(s) is impaired, we have identified some conditions which prevent the formation of the enzyme associated form(s): (a) the addition of guanidine chloride (0.2 M), a cahotropic agent, to the crude preparation; and (b) the increase of pH up to 8. In conclusion, taking advantage of the anomalous behaviour of glucosylceramidase on gel filtration chromatography, evidence has been obtained that placental glucosylceramidase may occur under several forms which had not been previously reported; a difference in experimental conditions can promote the formation of one or another form, by possibly affecting the composition and/or the stoichiometry and/or the stability of the enzyme complex.
- Published
- 1990
- Full Text
- View/download PDF
39. Correlation between the activity of glucosylceramidase and its binding to glucosylceramide-containing liposomes. Role of acidic phospholipids and fatty acids.
- Author
-
Vaccaro AM, Tatti M, Salvioli R, Ciaffoni F, and Gallozzi E
- Subjects
- Binding Sites, Chemical Phenomena, Chemistry, Physical, Humans, Hydrogen-Ion Concentration, Oleic Acid, Protein Binding, Ultracentrifugation, Cerebrosides metabolism, Glucosidases metabolism, Glucosylceramidase metabolism, Glucosylceramides metabolism, Liposomes metabolism, Oleic Acids pharmacology, Phosphatidic Acids pharmacology
- Abstract
Optimal enzymatic hydrolysis of glucosylceramide inserted into liposomes has been obtained when both acidic phospholipids and the appropriate fatty acids were added to glucosylceramide-containing liposomes. In fact, the stimulation of glucosylceramidase by acidic phospholipids was synergistically enhanced by fatty acids, whose effect was dependent upon chain length and increased on unsaturation. By following the partition of glucosylceramidase between the aqueous phase and the liposome-associated state with a flotation procedure, it has been found that phosphatidic acid (PA) and oleic acid (OA), as representatives of acidic phospholipids and activating fatty acids, respectively, were both required not only for optimal glucosylceramidase activity, but also for a tight binding of the enzyme to the liposomes. The binding was significantly less effective in the absence of either PA or OA. In the absence of both PA and OA no physical interaction between the enzyme and the liposomes was observed. Under all conditions, the glucosylceramidase activity directly correlated with the enzyme binding to the substrate-containing liposomes. Additionally, we have obtained evidence that the site(s) of the enzyme involved in the binding to the liposomes is distinct from the catalytic site; in fact, the enzyme could still associate with liposomes containing PA and OA but devoid of glucosylceramide, while it was incapable of binding to glucosylceramide-containing liposomes in the absence of PA and OA. In conclusion, the presence in liposomes of acidic phospholipids together with the appropriate fatty acids plays a key role in promoting the binding of glucosylceramidase. Consequently, when glucosylceramide is also included in the liposomes, its hydrolysis is markedly enhanced by these acidic lipids.
- Published
- 1990
- Full Text
- View/download PDF
40. [Efficacy of dietetic treatment in a case of galactosemia diagnosed late].
- Author
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Sebastio G, Albini F, di Martino L, Magurno T, Baffa E, and Ciaffoni F
- Subjects
- Female, Galactosemias genetics, Heterozygote, Humans, Infant, UTP-Hexose-1-Phosphate Uridylyltransferase deficiency, Galactosemias diet therapy
- Abstract
The authors describe a six months old girl affected by galactosemia, due to Galacto-1-phosphate Uridyl Transferase deficiency. The patient presented with hepatosplenomegaly and failure to thrive, without neurological impairment or cataracts. In this case removal of galactose from diet, although lately performed, resulted in normal growth and development. The authors emphasize the importance of ruling out galactosemia, even if clinical picture is unusual.
- Published
- 1982
41. Factors affecting the binding of glucosylceramidase to its natural substrate dispersion.
- Author
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Vaccaro AM, Tatti M, Ciaffoni F, and Salvioli R
- Subjects
- Binding Sites, Centrifugation, Density Gradient, Gaucher Disease enzymology, Glucosylceramidase isolation & purification, Glycoside Hydrolases metabolism, Humans, Kinetics, Lysosomes enzymology, Mitochondria enzymology, Protein Binding, Spleen enzymology, Glucosidases metabolism, Glucosylceramidase metabolism
- Abstract
This paper reports the results of ultracentrifugation experiments devised for investigating the interactions occurring in the conditions of the enzymatic assay between glucosylceramidase and the components of the substrate dispersion. This dispersion contains, besides glucosylceramide, taurocholate and oleic acid. It has been found that glucosylceramide aggregates with oleic acid, while taurocholate is unable to associate with the sphingolipid, but improves the stability of the dispersion. When a crude glucosylceramidase placental preparation is incubated with the assay mixture the enzyme is almost totally bound to the glucosylceramide-oleic acid particles. The binding between glucosylceramidase and the substrate-containing particles is dramatically depressed by changes of experimental conditions which negatively influence also the enzyme activity such as: (1) a decrease in the molarity of the citrate/phosphate buffer; (2) an increase of the buffer pH, and (3) an increase of the taurocholate concentration. An excess of oleic acid neither inhibits the binding nor the activity. These results strongly suggest that glucosylceramidase activity is directly correlated with the binding of the enzyme to the lipid interface of the substrate-containing particles. We conclude that the enzymatic mechanism of glucosylceramide hydrolysis involves at least two steps: first the physical localization of the enzyme at the lipid-water interface, second the hydrolysis of the substrate glucosidic bond.
- Published
- 1989
- Full Text
- View/download PDF
42. Presence of activator proteins for the enzymatic degradation of glucosylceramide in several human tissues.
- Author
-
Vaccaro AM, Ciaffoni F, Mandara I, and Suzuki K
- Subjects
- Cells, Cultured, Enzyme Activation, Female, Fibroblasts enzymology, Gaucher Disease enzymology, Glucosylceramides metabolism, Humans, Liver enzymology, Placenta enzymology, Pregnancy, Spleen enzymology, Glucosidases metabolism, Glucosylceramidase metabolism
- Abstract
Glucosylceramidase (EC 3.2.1.45) protein activators, similar to the 'placental factor' previously identified by us in human placenta, have also been found in human liver, normal and Gaucher fibroblasts and Gaucher spleen. They stimulate enzymatic hydrolysis of the natural substrate, glucosylceramide, but not that of the artificial substrate, 4-MU-beta-D-glucopyranoside. They are present in the tissues over the minimum amount necessary for full activation of the enzyme and must be eliminated from crude enzyme preparations in order to observer their effect on glucosylceramidase activity. The factors are not tissue-specific in that the factors from any one of the sources can activate glucosylceramidase from either placenta or liver. The presence of taurocholate or phosphatidylserine in the assay is essential for the factor efficiency. A normal level of the activator proteins was found in fibroblasts from subjects affected with Gaucher disease type I, type II and type III.
- Published
- 1988
- Full Text
- View/download PDF
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