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Martin E Hemler - One of the best experts on this subject based on the ideXlab platform.
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palmitoylation supports assembly and function of integrin tetraspanin complexes
Journal of Cell Biology, 2004Co-Authors: Xiuwei Yang, Oleg V Kovalenko, Christoph Claas, Christopher S Stipp, Wei Tang, Martin E HemlerAbstract:As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (α3, α6, and β4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of β4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient β4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core α6β4–CD151 complex formation was unaltered. There is also a functional connection between CD9 and β4 integrins, as evidenced by anti-CD9 antibody effects on β4-dependent cell spreading. Notably, β4 palmitoylation neither increased localization into “light membrane” fractions of sucrose gradients nor decreased solubility in nonionic detergents—hence it does not promote lipid raft association. Instead, palmitoylation of β4 (and of the closely associated tetraspanin CD151) promotes CD151–α6β4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.
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evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts
Journal of Biological Chemistry, 2001Co-Authors: Christoph Claas, Christopher S Stipp, Martin E HemlerAbstract:Abstract Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with α3β1 (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and α3 integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-α3β1 and CD81-α3β1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-α3β1, CD81-α3β1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 °C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-α3β1, CD81-α3β1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.
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a novel link between integrins transmembrane 4 superfamily proteins cd63 and cd81 and phosphatidylinositol 4 kinase
Journal of Biological Chemistry, 1997Co-Authors: Fedor Berditchevski, Kimberly F Tolias, Karen Wong, Christopher L Carpenter, Martin E HemlerAbstract:Abstract Enzymatic and immunochemical assays show a phosphatidylinositol 4-kinase in novel and specific complexes with proteins (CD63 and CD81) of the transmembrane 4 superfamily (TM4SF) and an integrin (α3β1). The size (55 kDa) and other properties of the phosphatidylinositol 4-kinase (PI 4-K) (stimulated by nonionic detergent, inhibited by adenosine, inhibited by monoclonal antibody 4CG5) are consistent with PI 4-K type II. Not only was PI 4-K associated with α3β1-CD63 complexes in α3-transfected K562 cells, but also it could be co-purified from CD63 in untransfected K562 cells lacking α3β1. Thus, TM4SF proteins may link PI 4-K activity to the α3β1 integrin. The α5β1 integrin, which does not associate with TM4SF proteins, was not associated with PI 4-K. Notably, α3β1-CD63-CD81-PI 4-K complexes are located in focal complexes at the cell periphery rather than in focal adhesions. The novel linkage between integrins, transmembrane 4 proteins, and phosphoinositide signaling at the cell periphery may play a key role in cell motility and provides a signaling pathway distinct from conventional integrin signaling through focal adhesion kinase.
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characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains tm4 proteins
Molecular Biology of the Cell, 1996Co-Authors: Fedor Berditchevski, Mary M Zutter, Martin E HemlerAbstract:Here we identified several new integrin/TM4 protein complexes on the cell surface. By immunoprecipitation using nonstringent conditions, and by reciprocal immunoprecipitation, we found that alpha 3 beta 1 and alpha 6 beta 1 integrins but not alpha 2 beta 1, alpha 5 beta 1, or alpha 6 beta 4 integrins associated with CD9 and CD81 in alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, alpha 6 beta 1/CD81, and alpha 6 beta 1/CD9 complexes. Also, cross-linking experiments established that alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, and alpha 3 beta 1/CD63 associations occur on the surface of intact cells and suggested that a critical interaction site is located within extracellular domains. Cross-linking in conjunction with reimmunoprecipitation indicated that larger multi-component alpha 3 beta 1/TM4/TM4 complexes (alpha 3 beta 1/CD9/CD63, alpha 3 beta 1/CD81/CD63, and alpha 3 beta 1/CD9/CD81) also could be detected on the cell surface. Immunofluorescent staining showed redistribution of alpha 3 beta 1/TM4 complexes toward the periphery of cells plated on various extracellular matrix substrates and also showed that these complexes were localized in cell footprints. Staining of human tissues yielded additional results consistent with co-localization of alpha 3 beta 1 and CD9, CD63, and CD81 proteins. In conclusion we suggest that the prevalence of integrin/TM4 complexes in diverse cellular environments is indicative of their general physiological importance.
Petr Kucera - One of the best experts on this subject based on the ideXlab platform.
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updates on the surface antigens of basophils cd16 on basophils of patients with respiratory or insect venom allergy and the rejection of cd203c and cd63 externalization decoupling by bisindolylmaleimides
Clinical & Experimental Allergy, 2019Co-Authors: Petr Heneberg, Kamila Riegerova, Adela řihova, Daniela Simcikova, Petr KuceraAbstract:BACKGROUND: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FceRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. OBJECTIVE: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. METHODS: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. RESULTS: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FceRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. CONCLUSIONS & CLINICAL RELEVANCE: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim . Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.
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updates on the surface antigens of basophils cd16 on basophils of patients with respiratory or insect venom allergy and the rejection of cd203c and cd63 externalization decoupling by bisindolylmaleimides
Clinical & Experimental Allergy, 2019Co-Authors: Petr Heneberg, Kamila Riegerova, Adela řihova, Daniela Simcikova, Petr KuceraAbstract:BACKGROUND: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FceRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. OBJECTIVE: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. METHODS: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. RESULTS: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FceRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. CONCLUSIONS & CLINICAL RELEVANCE: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim . Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.
Victor L Serebruany - One of the best experts on this subject based on the ideXlab platform.
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aspirin inhibits surface glycoprotein iib iiia p selectin cd63 and cd107a receptor expression on human platelets
Blood Coagulation & Fibrinolysis, 2003Co-Authors: Marcus E Mckenzie, Alex I Malinin, Christopher R Bell, Alex Dzhanashvili, Eric D Horowitz, Benjamin Oshrine, Dan Atar, Victor L SerebruanyAbstract:Platelet inhibition after aspirin therapy reduces the risk for the development of acute coronary syndromes. However, the mechanism by which aspirin affect platelets other than by prostaglandin blockade is unclear. We sought to determine the in vitro effects of aspirin on the surface expression of nine platelet receptors using whole blood flow cytometry. Blood from 24 healthy volunteers was incubated for 30 min with 1.8 and 7.2 mg/l phosphate-buffered saline-diluted acetylsalicylic acid in the presence or absence of apyrase. Platelet serotonin release, and the surface expression of platelet receptors with or without apyrase were determined using the following monoclonal antibodies: anit-CD41 [glycoprotein (GP)IIb/IIIa], CD42b (GPIb), CD62p (P-selectin), CD51/CD61 (vitronectin receptor), CD31 [platelet/endothelial cellular adhesion molecule-1 (PECAM-1)], CD107a [lysosomal associated membrane protein (LAMP)-1], CD107b (LAMP-2), CD63 (LIMP or LAMP-3), and CD151 (PETA-3). Samples were then immediately fixed with 2% paraformaldehyde, and run on the flow cytometer within 48 h. Aspirin does not affect serotonin release from human platelets. Dose-dependent inhibition of GPIIb/IIIa, P-selectin, CD63, and CD107a receptor expression was observed in the aspirin-treated whole-blood samples. Apyrase potentiates the effects of aspirin, and independently inhibits PECAM-1. In addition to the known effect of irreversibly inhibiting platelet cyclooxygenase-1, thereby blocking thromboxane A(2) synthesis, it appears that aspirin exhibits direct effects on selective major platelet receptors.
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heterogeneity of platelet aggregation and major surface receptor expression in patients with acute myocardial infarction
American Heart Journal, 1998Co-Authors: Victor L Serebruany, Paul A Gurbel, Andrew R Shustov, E M Ohman, Eric J TopolAbstract:Abstract Background Platelets play an important role in the natural history of acute myocardial infarction (AMI). Methods and Results Platelet aggregation and receptor expression were studied in 23 patients with AMI before reperfusion therapy and compared with 10 healthy control subjects. Platelet aggregation was induced with 5 μmol/L adenosine 5′–diphosphate, 10 μmol/L ADP, 1 μg/mL collagen, 1 mg/mL thrombin, and 1.25 mg/mL ristocetin. Receptor expression was measured by flow cytometry with monoclonal antibodies to p24 (CD9), Ib (CD42b), IIb (CD41b), IIIa (CD61), IIb/IIIa (CD41b/CD61), very late antigen-2 (CD49b), P-selectin (CD62p), platelet/endothelial cell adhesion molecule-1 (CD31); and vitronectin (CD51/CD61). The percentage of platelet aggregation was higher in patients with AMI when induced by 5 μmol/L ADP (64.1 ± 12.7 vs 52.0 ± 6.7; P = .04), by 10 μmol/L ADP (71.7 ± 13.0 vs 59.2 ± 7.2, P = .003), by thrombin (75.8 ± 10.9 vs 60.5 ± 6.9, P = .01), and by ristocetin (92.5 ± 7.8 vs 71.3 ± 7.4, P = .0001). Collagen-induced platelet aggregation did not differ between groups. Expression of P-selectin (log amplification of fluorescence intensity) (31.5 ± 5.0 vs 25.1 ± 2.6, P = .003) and platelet/endothelial cell adhesion molecule-1 (56.8 ± 17.7 vs 44.5 ± 3.7, P = .04) were significantly increased in patients with AMI. The expression of IIb (28.4 ± 2.5 vs 37.2 ± 1.7, P = .0001) and Ib (103.6 ± 29.9 vs 133.8 ± 8.0, P = .007) were reduced in patients with AMI. Conclusions Platelets are not necessarily systemically activated during the prereperfusion phase of AMI. For each agonist used and surface antigen measured, there was a cohort of patients with AMI within the normal or even below normal range of platelet status. (Am Heart J 1998;136:398-405.)
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dietary coenzyme q10 supplementation alters platelet size and inhibits human vitronectin cd51 CD61 receptor expression
Journal of Cardiovascular Pharmacology, 1997Co-Authors: Victor L Serebruany, Jose V Ordonez, William R Herzog, Morten Rohde, S A Mortensen, Karl Folkers, Paul A GurbelAbstract:Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10, but a comprehensive explanation of its cardioprotective properties is still lacking. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet size and surface antigens was examined in human volunteers. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days. Receptor expression was measured by flow cytometry with monoclonal murine anti-human antibodies CD9 (p24), CD42B (Ib), CD41b (IIb), CD61 (IIIa), CD41a (IIb/IIIa), CD49b (VLA-2), CD62p (P selectin), CD31 (PECAM-1), and CD51/CD61 (vitronectin). An increase of total serum CoQ10 level (from 0.6 +/- 0.1 to 1.8 +/- 0.3 micrograms/ml; p < 0.001) was found at protocol termination. Fluorescence intensity was higher for the large platelets when compared with the whole platelet population. Significant inhibition of vitronectin-receptor expression was observed consistently throughout ubiquinone treatment. Reduction of platelet size was observed at the end of CoQ10 supplementation. Inhibition of the platelet vitronectin receptor and a reduction of the platelet size are direct evidence of a link between dietary CoQ10 intake and platelets. These findings may not be fully explained by the known antioxidant and bioenergetic properties of CoQ10. Diminished vitronectin-receptor expression and reduced platelet size resulting from CoQ10 therapy may contribute to the observed clinical benefits in patients with cardiovascular diseases.
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could coenzyme q10 affect hemostasis by inhibiting platelet vitronectin cd51 CD61 receptor
Molecular Aspects of Medicine, 1997Co-Authors: Victor L Serebruany, Paul A Gurbel, Jose V Ordonez, William R Herzog, Morten Rohde, S A Mortensen, Karl FolkersAbstract:Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet surface antigens, and certain hemostatic parameters was examined in 15 humans and 10 swine. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days resulting in a three-fold increase of total serum CoQ10 level. We observed a decline in plasma fibronectin (-20.2%), thromboxane B2 (-20.6%), prostacyclin (-23.2%), and endothelin-1 (-17.9%) level. Significant inhibition of vitronectin receptor expression was observed consistently throughout ubiquinone treatment. Inhibition of the platelet vitronectin receptor is a direct evidence of a link between dietary CoQ10 intake, platelets, and hemostasis. These findings may contribute to the observed clinical benefits by a diminished incidence of thrombotic complications in such patients.
Petr Heneberg - One of the best experts on this subject based on the ideXlab platform.
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updates on the surface antigens of basophils cd16 on basophils of patients with respiratory or insect venom allergy and the rejection of cd203c and cd63 externalization decoupling by bisindolylmaleimides
Clinical & Experimental Allergy, 2019Co-Authors: Petr Heneberg, Kamila Riegerova, Adela řihova, Daniela Simcikova, Petr KuceraAbstract:BACKGROUND: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FceRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. OBJECTIVE: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. METHODS: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. RESULTS: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FceRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. CONCLUSIONS & CLINICAL RELEVANCE: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim . Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.
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updates on the surface antigens of basophils cd16 on basophils of patients with respiratory or insect venom allergy and the rejection of cd203c and cd63 externalization decoupling by bisindolylmaleimides
Clinical & Experimental Allergy, 2019Co-Authors: Petr Heneberg, Kamila Riegerova, Adela řihova, Daniela Simcikova, Petr KuceraAbstract:BACKGROUND: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FceRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. OBJECTIVE: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. METHODS: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. RESULTS: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FceRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. CONCLUSIONS & CLINICAL RELEVANCE: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim . Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.
Richard I. Christopherson - One of the best experts on this subject based on the ideXlab platform.
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immunophenotypic changes induced on human hl60 leukaemia cells by 1α 25 dihydroxyvitamin d3 and 12 o tetradecanoyl phorbol 13 acetate
Leukemia Research, 2005Co-Authors: Sarah L White, Nicole Barber, Larissa Belov, Philip D Hodgkin, Richard I. ChristophersonAbstract:1alpha,25-Dihydroxyvitamin D3 (1,25D3) induces HL60 cells to acquire a monocyte-like phenotype, while cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) resemble macrophages. Using a microarray of 82 CD antibodies, 24 cluster of differentiation (CD) antigens were detected on HL60 cells. 1,25D3 induced the following antigens in decreasing order of the change: CD14, CD11c, CD11b, CD54, CD86, CD38 and CD66c, with repression of CD117, CD71, CD95, CD45 and CD64. TPA induced the following antigens in decreasing order of the change: CD11c, CD9, CD11b, CD54, CD38, CD45RO and CD66c, with repression of CD4, CD117, CD95, CD71 and CD64. The results presented provide a basis for monitoring differentiation therapy of myeloid leukaemias in patients.