29 results on '"Bianco, I. D."'
Search Results
2. Ability of the polysaccharide chitosan to inhibit proliferation of CD4+ lymphocytes from mucosal inductive sites, in vitro and in vivo
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Porporatto, C., Canali, M. M., Bianco, I. D., and Correa, S. G.
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- 2009
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3. The biocompatible polysaccharide chitosan enhances the oral tolerance to type II collagen
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Porporatto, C., Canali, M. M., Bianco, I. D., and Correa, S. G.
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- 2009
4. The biocompatible polysaccharide chitosan enhances the oral tolerance to type II collagen
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Porporatto, C, primary, Canali, M M, additional, Bianco, I D, additional, and Correa, S G, additional
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- 2008
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5. Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.
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Balsinde, J, primary, Bianco, I D, additional, Ackermann, E J, additional, Conde-Frieboes, K, additional, and Dennis, E A, additional
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- 1995
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6. Arachidonic acid mobilization in P388D1 macrophages is controlled by two distinct Ca(2+)-dependent phospholipase A2 enzymes.
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Balsinde, J, primary, Barbour, S E, additional, Bianco, I D, additional, and Dennis, E A, additional
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- 1994
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7. Treatment of rheumatoid arthritis by oral administration of bovine tracheal type II collagen.
- Author
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Ausar, S. F., Beltramo, D. M., Castagna, L. F., Quintana, S., Silvera, E., Kalayan, G., Revigliono, M., Landa, C. A., and Bianco, I. D.
- Subjects
AUTOIMMUNE diseases ,RHEUMATOID arthritis ,IMMUNOGLOBULINS ,BLOOD plasma ,CYTOKINES ,TUMOR necrosis factors ,GROWTH factors - Abstract
We evaluated the efficacy and safety of orally administered bovine tracheal type II collagen (CGII) in the treatment of rheumatoid arthritis (RA). Twenty RA patients received 0.5 mg/day of CGII for 12 weeks. Eighteen of them had improvements in the clinical parameters studied (swollen and tender joint counts, 15-m walking time, duration of morning stiffness, and physician's global assessment of disease activity). Anti-CGII antibodies were positive in 57% and rheumatoid factor (RF) in 71% of the patients with a short history of RA (≤2 years), whereas only 23% of those with long histories (>2 years) presented autoantibodies to CGII and 38% had positive RF. After the treatment, four patients showed reduced RF levels and all those with detectable serum tumor necrosis factor α (TNF-α) experienced its return to normal or levels below those at study entry. Although a placebo effect cannot be discounted, the oral administration of bovine tracheal CGII induced clinical benefits in 90% of the patients, without the side effects usually associated with treatment. This is the first study showing that feeding CGII can induce reductions in RF and TNF-α. The data justify further controlled studies to assess the long-term efficacy of this treatment approach. [ABSTRACT FROM AUTHOR]
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- 2001
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8. Inhibition of Human Platelet Aggregation by Gangliosides
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Guglielmone, H. A., Daniele, J. J., Bianco, I. D., Fernandez, E. J., and Fidelio, G. D.
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- 2000
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9. Effect of glycerol on the molecular properties of cerebrosides, sulphatides and gangliosides in monolayers
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Bianco, I D, Fidelio, G D, and Maggio, B
- Abstract
The presence of glycerol, free from surface-active impurities, modifies the molecular area, surface potential/molecule and thermodynamic parameters of compression of monolayers of galactosylceramide, sulphatide and gangliosides GM1, GD1a and GT1b. This may be due to changes of the composition and structural properties of the glycosphingolipid solvation shell with an influence on the intermolecular organization.
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- 1988
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10. Modulation of the inflammatory response by corticotropin-releasing factor
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Correa, S. G., Riera, C. M., Spiess, J., and Bianco, I. D.
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- 1997
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11. Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers
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Bianco, I D, Fidelio, G D, and Maggio, B
- Abstract
The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.
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- 1989
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12. Gangliosides Raise the Intracellular Ca^2^+ Level in Different Cell Types
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Isasi, Sadi Cossy, Bianco, I. D., and Fidelio, G. D.
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- 1995
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13. Surface characterization and interfacial activity of chitinase chi18-5 against chitosan in langmuir monolayers.
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Villanueva ME, Salinas SR, Vico RV, and Bianco ID
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- Microscopy, Atomic Force, Surface Properties, Chitosan
- Abstract
One of the challenges for producing active chitinase formulations relies on the gap between the laboratory tests and the biological scenarios where the enzyme will perform its function. In this work, we have employed different Langmuir monolayer arrays to evaluate the interfacial behavior of a recently purified recombinant chitinase, Chi18-5. We have demonstrated that two conformations exist for the chitinase at pH values close to its pI, showing very distinct structural properties at the air/aqueous interface. Enzyme activity was assessed by implementing different kinetic approaches and using a chitosan-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed film as organized substrate model membrane. Combining these strategies, we demonstrated that better catalytic efficiencies can be obtained for Chi18-5 at pH 5. Moreover, the chitinase activity at the air/aqueous interface can be tuned by introducing in situ pH modifications over the surrounding milieu. We also studied the changes in the topography at the mesoscale level using Brewster Angle Microscopy (BAM). We found that Chi18-5 segregated onto the chitosan domains of the membrane, showing differences in homogeneity depending on the pH imposed. Alternatively, pure Chi18-5 was tested for immobilization onto a hydrophilic activated solid support using the Langmuir-Blodgett technique. Atomic Force Microscopy (AFM) analyses showed successfully stabilization and preservation of molecular features attributed to the pH at which the enzyme deposition was performed., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ismael D. Bianco reports financial support was provided by Universidad Nacional de la Rioja. Silvina R. Salinas reports financial support was provided by CONICET., (Copyright © 2023 Elsevier B.V. All rights reserved.)
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- 2023
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14. Reversible exposure of hydrophobic residues on albumin as a novel strategy for formulation of nanodelivery vehicles for taxanes.
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Garro AG, Beltramo DM, Alasino RV, Leonhard V, Heredia V, and Bianco ID
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- Cell Line, Tumor, Chromatography, Ion Exchange, Drug Stability, Humans, Hydrogen-Ion Concentration, Hydrophobic and Hydrophilic Interactions, Nanoparticles chemistry, Nanoparticles ultrastructure, Osmolar Concentration, Particle Size, Pharmaceutical Vehicles administration & dosage, Pharmaceutical Vehicles chemistry, Polyethylene Glycols chemistry, Protein Unfolding, Serum Albumin administration & dosage, Solubility, Temperature, Paclitaxel administration & dosage, Paclitaxel chemistry, Serum Albumin chemistry
- Abstract
Background: We report herein a novel strategy for the preparation of protein-based nanodelivery vehicles for hydrophobic active pharmaceutical ingredients., Methods: The procedure consisted of three steps, ie, exposure of hydrophobic residues of a protein to a pH-induced partial unfolding: interaction between hydrophobic residues on the protein and the hydrophobic active pharmaceutical ingredient, and a final step where the structure of the protein was reversed to a native-like state by returning to neutral pH. As proof of concept, the interaction of paclitaxel with partially unfolded states of human serum albumin was evaluated as a potential method for the preparation of water-soluble complexes of the taxane with albumin., Results: We found that paclitaxel readily binds to pH-induced partially unfolded albumin, leading to the formation of optically clear water-soluble complexes. The complexes thus formed were more stable in solution when the albumin native state was at least partially restored by neutralization of the solution to a pH around 7. It was also observed that the hydrodynamic radius of human serum albumin was only slightly increased after the cycle of pH changes, remaining in a monomeric state with a size according to paclitaxel binding. Furthermore, paclitaxel binding did not affect the overall exposure of charged groups of human serum albumin, as evaluated by its interaction with an ionic exchange resin., Conclusion: The in vitro biological activity of the complexes formed was qualitatively equivalent to that of a Cremophor(®)-based formulation.
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- 2011
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15. Room temperature vulcanizing silicone sheaths on a reusable support for progesterone delivery in estrous synchronization treatments in cattle.
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Heredia V, Bianco ID, Tríbulo H, Cuesta G, Chesta P, Bó GA, Tríbulo R, Mega VI, and Beltramo DM
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- Administration, Intravaginal, Animals, Delayed-Action Preparations, Drug Delivery Systems methods, Female, Male, Pregnancy, Progesterone blood, Random Allocation, Cattle physiology, Dimethylpolysiloxanes, Drug Delivery Systems veterinary, Estrus Synchronization methods, Progesterone administration & dosage
- Abstract
High temperature vulcanizing silicone elastomers have been widely used in controlled delivery systems of steroid hormones with the aim of controlling estrous cycle in livestock. This paper reports experiments conducted to evaluate the possibility of using room temperature vulcanizing (RTV) silicone elastomers for the intravaginal administration of progesterone to cattle. In vitro studies showed that RTV silicones and high-temperature vulcanizing silicone release progesterone at a similar rate. Y-shaped inserts made of different polymers were designed as supports of RTV silicone sheaths to test the in vivo release of progesterone. Field evaluation showed that RTV silicone sheaths containing 0.75 g of progesterone were at least as effective at estrous synchronization as commercially available intravaginal inserts.
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- 2008
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16. Improvement of HDL- and LDL-cholesterol levels in diabetic subjects by feeding bread containing chitosan.
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Ausar SF, Morcillo M, León AE, Ribotta PD, Masih R, Vilaro Mainero M, Amigone JL, Rubin G, Lescano C, Castagna LF, Beltramo DM, Diaz G, and Bianco ID
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- Blood Glucose metabolism, Body Weight drug effects, Chitin adverse effects, Chitosan, Diabetes Mellitus, Type 2 blood, Diabetes Mellitus, Type 2 complications, Food Additives pharmacology, Food Additives therapeutic use, Glycated Hemoglobin analysis, Humans, Hyperlipidemias blood, Hyperlipidemias complications, Treatment Outcome, Triglycerides blood, Bread, Chitin administration & dosage, Chitin analogs & derivatives, Cholesterol, HDL blood, Cholesterol, LDL blood, Diabetes Mellitus, Type 2 diet therapy, Hyperlipidemias diet therapy
- Abstract
In this work we evaluated the efficacy and safety of a bread formulation containing chitosan in dyslipidemic type 2 diabetic subjects. For this purpose a total of 18 patients were allowed to incorporate to their habitual diets 120 g/day of bread containing 2% (wt/wt) chitosan (chitosan group, n= 9) or standard bread (control group, n= 9). Before the study and after 12 weeks on the modified diet, the following parameters were evaluated: body weight, plasma cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triglyceride, and hemoglobin A(1c) (HbA(1c)). Compared with the control group, the patients receiving chitosan-containing bread decreased their mean levels of LDL-cholesterol and significantly increased their mean levels of HDL-cholesterol at the end of the study. There were no significant differences in the body weight, serum triglyceride, and HbA(1c). These results suggest that chitosan incorporated into bread formulations could improve the lipoprotein balance similar to typical biliary salts trappers, increasing the HDL- and lowering the LDL-cholesterol, without changing the triglyceride levels. These results warrant further studies over a longer period of time to evaluate if a persistent improvement in levels of lipoproteins can be attained with this strategy.
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- 2003
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17. Hydrolysis of a chitosan-induced milk aggregate by pepsin, trypsin and pancreatic lipase.
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Ausar SF, Landa CA, Bianco ID, Castagna LF, and Beltramo DM
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- Animals, Cattle, Chitin analogs & derivatives, Chitosan, Dietary Fats analysis, Digestion, Food Additives, Hydrolysis, In Vitro Techniques, Lipase, Pepsin A, Swine, Triglycerides chemistry, Trypsin, Milk chemistry
- Abstract
The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggregated. The release of soluble peptides from the aggregate was independent of the presence of chitosan. A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent. The results indicate that a milk aggregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.
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- 2001
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18. Characterization of casein micelle precipitation by chitosans.
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Ausar SF, Bianco ID, Badini RG, Castagna LF, Modesti NM, Landa CA, and Beltramo DM
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- Animals, Biopolymers, Chelating Agents administration & dosage, Chemical Phenomena, Chemistry, Physical, Chitin analogs & derivatives, Chitosan, Colloids, Hydrogen-Ion Concentration, Molecular Weight, Static Electricity, Temperature, Water, Caseins analysis, Chitin administration & dosage, Micelles, Milk chemistry
- Abstract
We have found that the addition of chitosan, a cationic polymer, on whole or skim milk produces destabilization and coagulation of casein micelles that takes place without changes in the milk pH or the stability of most whey proteins. The amount of lipids recovered in the chitosan-casein aggregates was similar or higher than that obtained with rennet or acid precipitation. Approximately 70% of milk Ca2+ (approximately 750 mg/L) was found in the chitosan-induced aggregates, which is 10 and 50% higher than the amounts observed with acid or rennet coagulations, respectively. Purified alpha, beta-, and kappa-caseins were extensively precipitated by different molecular weight chitosans at pH 6.8. The phosphate groups of caseins seem not to be relevant in this interaction because dephosphorylated alpha- and beta-caseins were equally precipitated with chitosans. Analysis by optical microscopy of the chitosan-casein complex reveals that the size of the aggregates increase as the molecular weight of chitosans increase. Hydrophobic and electrostatic interactions particpate in the association and coagulation of casein micelles with chitosans of different molecular weights. The phenomenon is observed over a broad range of temperature (4 to 70 degrees C) with a reduction in the concentration of chitosan needed to precipitate the caseins that parallels a reduction in the viscosity of the chitosan solutions. Taken together, the results indicate that the electrostatic interactions may contribute energetically to the association between the two biopolymers, but the hydrophobicity of the complex would be the key determinant in the overall energetics of the reaction.
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- 2001
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19. Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages.
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Bianco ID, Balsinde J, Beltramo DM, Castagna LF, Landa CA, and Dennis EA
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- Animals, Cell Line, Chitin pharmacology, Chitosan, Enzyme Activation, Enzyme Inhibitors pharmacology, Indoles pharmacology, Macrophages enzymology, Macrophages metabolism, Mice, Phospholipases A antagonists & inhibitors, Phospholipases A2, Arachidonic Acid metabolism, Chitin analogs & derivatives, Macrophages drug effects, Phospholipases A metabolism
- Abstract
We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.
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- 2000
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20. Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.
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Daniele JJ, Fidelio GD, and Bianco ID
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- Animals, Chick Embryo, Culture Media, Humans, In Vitro Techniques, Neutrophils metabolism, Rats, Arachidonic Acid metabolism, Calcium metabolism, Phospholipids metabolism
- Abstract
Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.
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- 1999
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21. A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarará chica) venom with novel kinetic and chromatographic properties.
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Daniele JJ, Bianco ID, Delgado C, Carrillo DB, and Fidelio GD
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- Amino Acid Sequence, Amino Acids analysis, Animals, Antineoplastic Agents isolation & purification, Antineoplastic Agents pharmacology, Chromatography, Gel, Kinetics, Mice, Molecular Sequence Data, Phospholipases A2, Sequence Homology, Amino Acid, Tumor Cells, Cultured, Bothrops, Crotalid Venoms enzymology, Isoenzymes isolation & purification, Phospholipases A isolation & purification
- Abstract
A new phospholipase A2 isoform, called P-3, isolated from Bothrops neuwiedii (Yarará chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity. In contrast to previously reported B. neuwiedii phospholipase A2 isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide. The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers. P-3 had an optimum activity towards dilauroylphosphatidylcholine monolayers of 27 mN/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m. P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single dose of 5 micrograms/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 micrograms/ml for P-3 and of 15 micrograms/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 micrograms/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 micrograms/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2.
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- 1997
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22. Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles.
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Daniele JJ, Maggio B, Bianco ID, Goñi FM, Alonso A, and Fidelio GD
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- Lipid Bilayers, Micelles, Substrate Specificity, Type C Phospholipases metabolism, Bacillus cereus enzymology, Enzyme Inhibitors pharmacology, Gangliosides pharmacology, Type C Phospholipases antagonists & inhibitors
- Abstract
The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.
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- 1996
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23. Identification of two specific lysines responsible for the inhibition of phospholipase A2 by manoalide.
- Author
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Bianco ID, Kelley MJ, Crowl RM, and Dennis EA
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- Base Sequence, Enzyme Activation, Molecular Sequence Data, Mutagenesis, Site-Directed, Phospholipases A genetics, Phospholipases A2, Substrate Specificity, Lysine chemistry, Phospholipases A antagonists & inhibitors, Terpenes pharmacology
- Abstract
Manoalide, a natural product of sponge, irreversibly inhibits phospholipase A2 (PLA2) by reacting with lysine residues. Cobra venom PLA2 mutants were constructed in which four of the six lysine residues were independently replaced by arginine or methionine, which cannot react with manoalide. The mutants were overexpressed in Escherichia coli, renatured, and purified. The enzyme mutants lacking Lys-6 (K6R and K6M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the native cobra venom PLA2 was inhibited 80% under the same conditions. This means that the manoalide modification of either Lys-6 or Lys-79 accounted for only half of the manoalide inhibition. The double mutant (K6R79R) was not inhibited by manoalide at all. Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of the inhibitory effects produced by manoalide. These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA2. The inhibition of PLA2 and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface. Furthermore, as with native PLA2, the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are essential for this activation.
- Published
- 1995
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24. Kinetic and pharmacologic characterization of phospholipases A2 from Bothrops neuwiedii venom.
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Daniele JJ, Bianco ID, and Fidelio GD
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- Amino Acid Sequence, Animals, Bothrops genetics, Crotalid Venoms genetics, Edema etiology, Humans, In Vitro Techniques, Isoelectric Point, Isoenzymes genetics, Isoenzymes metabolism, Isoenzymes pharmacology, Kinetics, Mice, Molecular Sequence Data, Molecular Weight, Neutrophils drug effects, Neutrophils metabolism, Phospholipases A genetics, Phospholipases A pharmacology, Phospholipases A2, Reactive Oxygen Species metabolism, Sequence Homology, Amino Acid, Bothrops metabolism, Crotalid Venoms metabolism, Phospholipases A metabolism
- Abstract
Two phospholipases A2 (PLA2) (EC 3.1.1.4) were purified from Bothrops neuwiedii venom (isoenzymes P-1 and P-2). The molecular weights of P-1 and P-2 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 15,000 and 16,200 and the isoelectric points were 4.8 and 4.6, respectively. The N-terminal 14-amino-acid sequences determined were Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly and Ser-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly for P-1 and P-2, respectively. Since both show sequence almost identical to that of a PLA2 from Crotalus atrox it was tentatively classified as being of group II. The enzymatic activity of P-1 and P-2 toward lipid monolayers was studied. The hydrolysis of dilauroylphosphatidylcholine (dlPC) shows a broad optimum between 7 and 18 mN m-1 and a cut-off pressure of 22 mN m-1. The activity toward dlPC displays a maximum at pH 8 and is dependent on the presence of Ca2+ with an apparent Kd of 0.1 mM, for both enzymes. P-1 and P-2 are heat-stable enzymes, unable to hydrolyze dilauroylphosphatidic acid monolayers. The enzymes are not lethal to mice at doses up to 5 micrograms/g body weight by intraperitoneal injection and they do not show myotoxic (up to 40 micrograms) or hemolytic activity (up to 8.5 micrograms/ml). Both lack anti-coagulant activity, determined by absence of changes in the recalcification time of platelet poor plasma (up to 100 micrograms/ml), and are not able to induce platelet aggregation (up to 50 micrograms/ml). However, both isoenzymes exhibit an important edema-inducing activity that is not altered at short times by the irreversible chemical inactivation of the hydrolytic activity with phenacyl bromide. P-1 and P-2 are able to release arachidonic acid from membrane phospholipids of neutrophils, property that is lost by the inactivation of the enzyme. This suggests that the edema-inducing activity of the active but not the inactive forms may be partly due to arachidonic acid-derived mediators. The edema-inducing activity of the active or inactive forms of the enzymes is inhibited by antagonists of histamine, suggesting that histamine plays an important role in both the active and the inactive B. neuwiedii PLA2s-induced edema. The results suggest that the inflammatory and the catalytic activity of these enzymes constitute separate properties.
- Published
- 1995
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25. Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers.
- Author
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Maggio B, Bianco ID, Montich GG, Fidelio GD, and Yu RK
- Subjects
- 1,2-Dipalmitoylphosphatidylcholine chemistry, Animals, Enzyme Activation drug effects, Kinetics, Lipid Bilayers metabolism, Pancreas enzymology, Phosphatidylcholines chemistry, Phospholipases A chemistry, Phospholipases A2, Swine, 1,2-Dipalmitoylphosphatidylcholine metabolism, Gangliosides pharmacology, Phosphatidylcholines metabolism, Phospholipases A metabolism, Sulfoglycosphingolipids pharmacology
- Abstract
The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.
- Published
- 1994
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26. Concerted modulation by myelin basic protein and sulfatide of the activity of phospholipase A2 against phospholipid monolayers.
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Bianco ID, Fidelio GD, Yu RK, and Maggio B
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- Animals, Galactosylceramides metabolism, Phospholipases A2, Swine, Myelin Basic Protein metabolism, Phosphatidylcholines metabolism, Phospholipases A metabolism, Sulfoglycosphingolipids metabolism
- Abstract
The effect of myelin basic protein (MBP) on the activity of phospholipase A2 (PLA2, EC 3.1.1.4) against monolayers of dilauroylphosphatidylcholine (dlPC) or dilauroylphosphatidic acid (dlPA) containing different proportions of sulfatide (Sulf) and galactocerebroside (GalCer) was investigated. MBP was introduced into the interface by direct spreading as an initial constitutive component of the lipid-protein film or by adsorption and penetration from the subphase into the preformed lipid monolayers. The effect of MBP on PLA2 activity depends on the type of phospholipid and on the proportion of MBP at the interface. At a low mole fraction of MBP, homogeneously mixed lipid-protein monolayers are formed, and the PLA2 activity against dlPC is only slightly modified while the degradation of dlPA is markedly inhibited. This is probably due to favorable charge-charge interactions between dlPA and MBP that interfere with the enzyme action. The PLA2 activity against either phospholipid is increased when the mole fraction of MBP exceeds the proportion at which immiscible surface domains are formed. GalCer has little effect on the modulation by MBP of the phospholipase activity. The effect of Sulf depends on its proportions in relation to MBP. The individual effects of both components balance each other, and a finely tuned modulation is regulated by the interactions of MBP with Sulf or with the phospholipid.
- Published
- 1992
- Full Text
- View/download PDF
27. Anti-inflammatory effect of gangliosides in the rat hindpaw edema test.
- Author
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Correa SG, Bianco ID, Riera CM, and Fidelio GD
- Subjects
- Animals, Carrageenan, Dextrans, Edema pathology, Exudates and Transudates cytology, Foot pathology, Phospholipases A, Phospholipases A2, Rats, Rats, Inbred Strains, Anti-Inflammatory Agents, Non-Steroidal, Edema drug therapy, Gangliosides pharmacology
- Abstract
The influence of total brain gangliosides on acute inflammation was investigated using the rat hind paw edema test. Total gangliosides (10 micrograms/paw) inhibited the edema produced by the injection of bee venom phospholipase A2 (5 micrograms/paw) when the lipids were co-injected or injected 15 min before the phospholipase A2. Sulphatide (10 micrograms/paw) did not inhibit the edema but potentiated it. Gangliosides (40 micrograms/paw) inhibited the edema induced by carrageenin 1% when they were injected 1 h before the agent. However, gangliosides (up to 200 micrograms/paw) failed to inhibit the dextran-induced edema. The edema test was also used to investigate the effect of gangliosides on the production of mediators of inflammation by peritoneal adherent macrophages. Gangliosides inhibited the production of mediators of inflammation only when they were incubated with these cells before the stimulation with phospholipase A2 or carrageenin. Gangliosides did not inhibit the production of mediators of inflammation when arachidonic acid was added to the cells. These results suggest that the anti-inflammatory effect observed with gangliosides is mediated by inhibition at or before endogenous phospholipase activity.
- Published
- 1991
- Full Text
- View/download PDF
28. Degradation of dilauroylphosphatidylcholine by phospholipase A2 in monolayers containing glycosphingolipids.
- Author
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Bianco ID, Fidelio GD, Yu RK, and Maggio B
- Subjects
- Animals, G(M1) Ganglioside pharmacology, Kinetics, Models, Theoretical, Pancreas enzymology, Phospholipases A2, Swine, Glycosphingolipids pharmacology, Liposomes, Phosphatidylcholines metabolism, Phospholipases A metabolism
- Abstract
The ability of phospholipase A2 from porcine pancreas to degrade all of the available dilauroylphosphatidylcholine in mixed monolayers with galactocerebroside, sulfatide, or ganglioside GM1 was investigated at different constant surface pressures. Under the conditions used the interfacial glycosphingolipid composition was continuously enriched as the enzyme action proceeded. The total percentage of phospholipid degradation depends on the surface pressure and on the type of glycosphingolipid. The presence of sulfatide activates the enzyme while galactocerebroside and ganglioside GM1 are inhibitory. The extent of phospholipid hydrolysis is independent of the effect of glycosphingolipids on the enzyme velocity. This is so when the latter is measured either in conditions of constant glycosphingolipid composition and zero-order kinetics [Bianco, I.D., Fidelio, G.D., & Maggio, B. (1989) Biochem. J. 258, 95-99] or under variable surface composition as in the present work. The modulation of phospholipase A2 activity by glycosphingolipids operates at two independent levels. One controls the rate of enzyme activity, and the other modulates the total extent of substrate degradation. This depends on the initial interaction of the enzyme with the interface. The glycosphingolipid effect on the activity is different depending on whether the enzyme has access to the substrate from the subphase or is already adsorbed to the lipid interface.
- Published
- 1991
- Full Text
- View/download PDF
29. Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study.
- Author
-
Bianco ID, Fidelio GD, and Maggio B
- Subjects
- Adsorption, Animals, Chemical Phenomena, Chemistry, Physical, Clostridium perfringens enzymology, In Vitro Techniques, Kinetics, Pancreas enzymology, Phosphatidic Acids metabolism, Phosphatidylcholines metabolism, Phospholipases A2, Swine, Gangliosides pharmacology, Phospholipases metabolism, Phospholipases A metabolism, Sulfoglycosphingolipids pharmacology, Type C Phospholipases metabolism
- Abstract
The effect of sulfatide and gangliosides GM1, GD1a and GT1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside GM1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides GD1a and GT1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.
- Published
- 1990
- Full Text
- View/download PDF
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