The Experts below are selected from a list of 17541 Experts worldwide ranked by ideXlab platform

Xavier Santarelli - One of the best experts on this subject based on the ideXlab platform.

  • large scale production bacterial localization assessment and immobilized metal affinity chromatography purification of a human single chain fv antibody against alphaiib Beta3 Integrin
    International Journal of Biological Macromolecules, 2006
    Co-Authors: Remy Robert, Gisele Clofentsanchez, Agnes Hocquellet, Mariejosee Jacobinvalat, Abdelmajid Noubhani, Danièle Daret, Xavier Santarelli
    Abstract:

    Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-Beta3 (αIIbβ3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

  • large scale production bacterial localization assessment and immobilized metal affinity chromatography purification of a human single chain fv antibody against alphaiib Beta3 Integrin
    International Journal of Biological Macromolecules, 2006
    Co-Authors: Remy Robert, Gisele Clofentsanchez, Agnes Hocquellet, Mariejosee Jacobinvalat, Abdelmajid Noubhani, Danièle Daret, Xavier Santarelli
    Abstract:

    Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-Beta3 (alphaIIbBeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

Donna M Peters - One of the best experts on this subject based on the ideXlab platform.

  • regulation of cross linked actin network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 Integrin pathways
    Investigative Ophthalmology & Visual Science, 2009
    Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M Peters
    Abstract:

    Purpose To determine the beta1/Beta3 Integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavBeta3 Integrin coreceptor/thrombospondin-1 receptor) and Integrins alphavBeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 Integrin ligand) in the absence or presence of the Beta3 Integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and Integrins alphavBeta3 and beta1. Beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 Integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased Beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 Integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavBeta3 Integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas Beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both Integrin pathways were Src dependent.

  • regulation of cross linked actin network clan formation in human trabecular meshwork htm cells by convergence of distinct beta1 and Beta3 Integrin pathways
    Investigative Ophthalmology & Visual Science, 2009
    Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M Peters
    Abstract:

    Purpose To determine the beta1/Beta3 Integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavBeta3 Integrin coreceptor/thrombospondin-1 receptor) and Integrins alphavBeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 Integrin ligand) in the absence or presence of the Beta3 Integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and Integrins alphavBeta3 and beta1. Beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 Integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased Beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 Integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavBeta3 Integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas Beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both Integrin pathways were Src dependent.

Michael Detmar - One of the best experts on this subject based on the ideXlab platform.

  • stimulation of endothelial cell migration by vascular permeability factor vascular endothelial growth factor through cooperative mechanisms involving the alphavBeta3 Integrin osteopontin and thrombin
    American Journal of Pathology, 1996
    Co-Authors: Donald R Senger, S R Ledbetter, Kevin P Claffey, A Papadopoulossergiou, C A Peruzzi, Michael Detmar
    Abstract:

    We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and Beta3 Integrin subunits resulting in increased levels of the alphavBeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavBeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavBeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavBeta3 Integrin, the alphavBeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.

  • stimulation of endothelial cell migration by vascular permeability factor vascular endothelial growth factor through cooperative mechanisms involving the alphavBeta3 Integrin osteopontin and thrombin
    American Journal of Pathology, 1996
    Co-Authors: Donald R Senger, Kevin P Claffey, A Papadopoulossergiou, C A Peruzzi, Michael Detmar
    Abstract:

    We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and Beta3 Integrin subunits resulting in increased levels of the alphavBeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavBeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavBeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavBeta3 Integrin, the alphavBeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.

Remy Robert - One of the best experts on this subject based on the ideXlab platform.

  • large scale production bacterial localization assessment and immobilized metal affinity chromatography purification of a human single chain fv antibody against alphaiib Beta3 Integrin
    International Journal of Biological Macromolecules, 2006
    Co-Authors: Remy Robert, Gisele Clofentsanchez, Agnes Hocquellet, Mariejosee Jacobinvalat, Abdelmajid Noubhani, Danièle Daret, Xavier Santarelli
    Abstract:

    Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-Beta3 (αIIbβ3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

  • large scale production bacterial localization assessment and immobilized metal affinity chromatography purification of a human single chain fv antibody against alphaiib Beta3 Integrin
    International Journal of Biological Macromolecules, 2006
    Co-Authors: Remy Robert, Gisele Clofentsanchez, Agnes Hocquellet, Mariejosee Jacobinvalat, Abdelmajid Noubhani, Danièle Daret, Xavier Santarelli
    Abstract:

    Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-Beta3 (alphaIIbBeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

Mark S Filla - One of the best experts on this subject based on the ideXlab platform.

  • regulation of cross linked actin network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 Integrin pathways
    Investigative Ophthalmology & Visual Science, 2009
    Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M Peters
    Abstract:

    Purpose To determine the beta1/Beta3 Integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavBeta3 Integrin coreceptor/thrombospondin-1 receptor) and Integrins alphavBeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 Integrin ligand) in the absence or presence of the Beta3 Integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and Integrins alphavBeta3 and beta1. Beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 Integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased Beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 Integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavBeta3 Integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas Beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both Integrin pathways were Src dependent.

  • regulation of cross linked actin network clan formation in human trabecular meshwork htm cells by convergence of distinct beta1 and Beta3 Integrin pathways
    Investigative Ophthalmology & Visual Science, 2009
    Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M Peters
    Abstract:

    Purpose To determine the beta1/Beta3 Integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavBeta3 Integrin coreceptor/thrombospondin-1 receptor) and Integrins alphavBeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 Integrin ligand) in the absence or presence of the Beta3 Integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and Integrins alphavBeta3 and beta1. Beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 Integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased Beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 Integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavBeta3 Integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas Beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both Integrin pathways were Src dependent.