The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Bruno Lomonte - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of myotoxin ii from atropoides bothrops nummifer snake venom a new lys49 phospholipase a2 homologue
The International Journal of Biochemistry & Cell Biology, 2000Co-Authors: Yamileth Angulo, Timoteo Olamendiportugal, Lourival D Possani, Bruno LomonteAbstract:Abstract Myotoxic Phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure–activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin II is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 μg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II Phospholipases A2. Sequence comparison with other Phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.
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Immunochemical Characterization and Role in Toxic Activities of Region 115–129 of Myotoxin II, a Lys49 Phospholipase A2fromBothrops asperSnake Venom
Archives of biochemistry and biophysics, 1998Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region 115–129 of myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic Phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic Phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic Phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of myotoxin II, and possibly of related class II Lys49 phospholipase A2myotoxins, is proposed.
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immunochemical characterization and role in toxic activities of region 115 129 of myotoxin ii a lys49 phospholipase a2frombothrops aspersnake venom
Archives of Biochemistry and Biophysics, 1998Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region 115–129 of myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic Phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic Phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic Phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of myotoxin II, and possibly of related class II Lys49 phospholipase A2myotoxins, is proposed.
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Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-49 phospholipase A2
Archives of biochemistry and biophysics, 1991Co-Authors: Brian R. Francis, Bruno Lomonte, Ivan I. KaiserAbstract:Abstract A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper Phospholipases A2, except that it shows only 0.1% of the phospholipase activity against l -α-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2+-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.
Leonel Calderon - One of the best experts on this subject based on the ideXlab platform.
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Immunochemical Characterization and Role in Toxic Activities of Region 115–129 of Myotoxin II, a Lys49 Phospholipase A2fromBothrops asperSnake Venom
Archives of biochemistry and biophysics, 1998Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region 115–129 of myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic Phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic Phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic Phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of myotoxin II, and possibly of related class II Lys49 phospholipase A2myotoxins, is proposed.
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immunochemical characterization and role in toxic activities of region 115 129 of myotoxin ii a lys49 phospholipase a2frombothrops aspersnake venom
Archives of Biochemistry and Biophysics, 1998Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region 115–129 of myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic Phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic Phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic Phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of myotoxin II, and possibly of related class II Lys49 phospholipase A2myotoxins, is proposed.
W V Scheuer - One of the best experts on this subject based on the ideXlab platform.
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Suppression of cytokine synthesis, integrin expression and chronic inflammation by inhibitors of cytosolic phospholipase A2.
European journal of pharmacology, 1997Co-Authors: E Amandi-burgermeister, U Tibes, B M Kaiser, W G Friebe, W V ScheuerAbstract:To define the isoform of Phospholipases A2 active in inflammation we evaluated the effects of low-molecular-weight inhibitors of secretory and cytosolic Phospholipases A2. We found that inhibitors of cytosolic phospholipase A2 had therapeutic efficacy in an in vivo model of chronic inflammation (rat adjuvant arthritis), whereas inhibitors of secretory phospholipase A2 had no beneficial effect. In vitro, inhibitors of cytosolic phospholipase A2 diminished surface expression of Mac-1 (CD11b/CD18) beta2-integrin on calcium ionophore-stimulated human blood granulocytes and suppressed synthesis of interleukin-1beta in lipopolysaccharide-stimulated human blood monocytes and U937 cells by reducing mRNA levels. Lipid mediators promote Mac-1 exocytosis and transcription of interleukin-1beta, which further enhances cytosolic phospholipase A2 activity and expression. Thus, superinduction of cytosolic phospholipase A2 may establish a positive feedback loop, converting acute inflammation into chronic inflammation. Consequently, inhibitors of cytosolic phospholipase A2 may prevent inflammation in vivo by interfering with cellular activation and infiltration. We conclude that cytosolic phospholipase A2 but not secretory phospholipase A2 is the predominant enzyme in inflammatory signalling.
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Suppression of cytokine synthesis, integrin expression and chronic inflammation by inhibitors of cytosolic phospholipase A2
European Journal of Pharmacology, 1997Co-Authors: E Amandi-burgermeister, U Tibes, B M Kaiser, W G Friebe, W V ScheuerAbstract:Abstract To define the isoform of Phospholipases A2 active in inflammation we evaluated the effects of low-molecular-weight inhibitors of secretory and cytosolic Phospholipases A2. We found that inhibitors of cytosolic phospholipase A2 had therapeutic efficacy in an in vivo model of chronic inflammation (rat adjuvant arthritis), whereas inhibitors of secretory phospholipase A2 had no beneficial effect. In vitro, inhibitors of cytosolic phospholipase A2 diminished surface expression of Mac-1 (CD11b/CD18) β2-integrin on calcium ionophore-stimulated human blood granulocytes and suppressed synthesis of interleukin-1β in lipopolysaccharide-stimulated human blood monocytes and U937 cells by reducing mRNA levels. Lipid mediators promote Mac-1 exocytosis and transcription of interleukin-1β, which further enhances cytosolic phospholipase A2 activity and expression. Thus, superinduction of cytosolic phospholipase A2 may establish a positive feedback loop, converting acute inflammation into chronic inflammation. Consequently, inhibitors of cytosolic phospholipase A2 may prevent inflammation in vivo by interfering with cellular activation and infiltration. We conclude that cytosolic phospholipase A2 but not secretory phospholipase A2 is the predominant enzyme in inflammatory signalling.
Michael H. Gelb - One of the best experts on this subject based on the ideXlab platform.
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intracellular actions of group iia secreted phospholipase a2 and group iva cytosolic phospholipase a2 contribute to arachidonic acid release and prostaglandin production in rat gastric mucosal cells and transfected human embryonic kidney cells
Journal of Biological Chemistry, 2006Co-Authors: Zhanglin Ni, Brian P. Smart, Nicole M. Okeley, Michael H. GelbAbstract:Abstract Gastric epithelial cells liberate prostaglandin E2 in response to cytokines as part of the process of healing of gastric lesions. Treatment of the rat gastric epithelial cell line RGM1 with transforming growth factor-α and interleukin-1β leads to synergistic release of arachidonate and production of prostaglandin E2. Results with highly specific and potent phospholipase A2 inhibitors and with small interfering RNA show that cytosolic phospholipase A2-α and group IIA secreted phospholipase A2 contribute to arachidonate release from cytokine-stimulated RGM1 cells. In the late phase of arachidonate release, group IIA secreted phospholipase A2 is induced (detected at the mRNA and protein levels), and the action of cytosolic phospholipase A2-α is required for this induction. Results with RGM1 cells and group IIA secreted phospholipase A2-transfected HEK293 cells show that the group IIA phospholipase acts prior to externalization from the cells. RGM1 cells also express group XIIA secreted phospholipase A2, but this enzyme is not regulated by cytokines nor does it contribute to arachidonate release. The other eight secreted Phospholipases A2 were not detected in RGM1 cells at the mRNA level. These results clearly show that cytosolic and group IIA secreted Phospholipases A2 work together to liberate arachidonate from RGM1 cell phospholipids in response to cytokines.
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Inhibition of phospholipase A2.
The FASEB Journal, 1994Co-Authors: Michael H. Gelb, Mahendra Kumar Jain, Otto G BergAbstract:Phospholipases A2 are involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Inhibitors of these enzymes are proving useful in determining the biological roles of Phospholipases A2 in complex cellular processes and may also have therapeutic potential. Inhibition of these lipolytic enzymes is more difficult to characterize as the enzymatic reaction occurs at a lipid/water interface. This review focuses on the description of a number of classes of rationally designed phospholipase A2 inhibitors. The development of a theoretical framework for the proper analysis of inhibitors is presented. Structural studies of phospholipase A2-inhibitor complexes suggest how the lipolysis reaction is catalyzed. Finally, some recent results on the use of phospholipase A2 inhibitors in living cells and tissues are revealed.
Philippe De Medina - One of the best experts on this subject based on the ideXlab platform.
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exosomes account for vesicle mediated transcellular transport of activatable Phospholipases and prostaglandins
Journal of Lipid Research, 2010Co-Authors: Caroline Subra, David Grand, Karine Laulagnier, Alexandre Stella, Gerard Lambeau, Michael Paillasse, Philippe De MedinaAbstract:Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of Phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable Phospholipases and lipid mediators from cell to cell.