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P. M. Strasberg - One of the best experts on this subject based on the ideXlab platform.
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Increased Globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells
Journal of Molecular Neuroscience, 1994Co-Authors: J. Pereira, B. Boyd, J. Newbigging, C. Lingwood, P. M. StrasbergAbstract:Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in Globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived from E. coli . In Vero and HeLa cells, which are susceptible to verotoxin, the expression of Globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0°C at this boundary is indicative of the amount of Globotriaosylceramide exposed on the cell surface. Above 0°C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-Globotriaosylceramide interactions in synchronized FD and normal cells at this boundary.^125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10^−2–10^−7 µg/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD_50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.
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Increased Globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells. Verotoxin binding studies.
Journal of molecular neuroscience : MN, 1994Co-Authors: J. Pereira, B. Boyd, J. Newbigging, C. Lingwood, P. M. StrasbergAbstract:Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in Globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived fromE. coli. In Vero and HeLa cells, which are susceptible to verotoxin, the expression of Globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0°C at this boundary is indicative of the amount of Globotriaosylceramide exposed on the cell surface. Above 0°C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-Globotriaosylceramide interactions in synchronized FD and normal cells at this boundary.125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10−2–10−7 µg/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.
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Increased Globotriaosylceramide in familial dysautonomia.
Lipids, 1992Co-Authors: P. M. Strasberg, Herman Yeger, Irene WarrenAbstract:Familial Dysautonomia (FD) is an autosomal recessive disease of unknown etiology, occurring primarily in Ashkenazi Jews. Patients are neurologically impaired, with deficits primarily in autonomic and sensory functions. The biochemical and genetic defects have remained elusive, precluding carrier detection and prenatal diagnosis. High-performance liquid chromatography data indicated up to a threefold increase in the neutral glycosphingolipid Globotriaosylceramide in Dysautonomic fibroblasts and lymphoblasts. Total ganglioside values, measured by colorimetric, fluorometric or specific sodium borohydride incorporation, were decreased. Affected fibroblasts exhibited a range of pleomorphic phenotypes, such that the usual swirl-like confluent growth pattern of normal fibroblasts was distorted to varying degrees, suggesting abnormalities in the FD plasma membrane, possibly affecting cell-cell contacts. The glycosphingolipid increase could not be accounted for on the basis of markedly decreased alpha-galactosidase activity, as in Fabry's disease, where patients also display decreased autonomic function.
C.a. Lingwood - One of the best experts on this subject based on the ideXlab platform.
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Expression of the verotoxin receptor glycolipid, Globotriaosylceramide, in ovarian hyperplasias
Oncology research, 1997Co-Authors: Arab S, Russel E, B Rosen, C.a. LingwoodAbstract:The presence of cell surface receptor glycolipid, Globotriaosylceramide (Gb 3 ), is essential to confer susceptibility to the E. coli-derived verotoxin (VT). Our earlier studies showed that Gb 3 is expressed in ovarian carcinoma cell lines. The Gb 3 content of normal ovary, benign and malignant primary ovarian tumors, and their metastases have now been compared by verotoxin thin-layer chromatogram (TLC) overlay of the glycolipid tissue extracts. FITC-labeled VT1 B subunit binding to frozen tumor sections was also monitored histochemically. Low to undetectable levels of Gb 3 were found in normal ovarian tissue. Gb 3 was markedly increased in both benign and malignant tumors, suggesting that increased Gb 3 may be related to proliferation, rather than malignancy per se. Mucinous tumors showed the least Gb 3 elevation; serous tumors were variable, showing higher levels of Gb 3 in less differentiated malignant tumors. By far the highest Gb 3 content was observed for secondary ovarian metastases and tumors refractory to chemotherapy. Frozen sections of neoplastic ovarian tissue overlaid with fluorescein-conjugated VTI B subunit show extensive binding to tumor cells, particularly in poorly differentiated samples and blood vessels adjacent to, and within, the tumor mass. Tumor foci were stained but stromal tissue was consistently negative both in primary tumors and metastases. VT staining of well-differentiated primary ovarian tumor sections was weak, corresponding to their low Gb 3 content, but strong staining was observed in sections from a highly differentiated primary tumor from a patient who was unexpectedly refractory to clinical chemotherapy. These studies suggest that verotoxin/Gb 3 targeting may provide the basis for new treatments for ovarian cancer.
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Glycolipid modification of alpha 2 interferon binding. Sequence similarity between the alpha 2 interferon receptor and verotoxin (Shiga-like toxin) B-subunit.
The Biochemical journal, 1992Co-Authors: C.a. Lingwood, S K YiuAbstract:Previous studies have implicated the glycolipid receptor for the Escherichia coli-derived verotoxin, Globotriaosylceramide (Gb3; Gal alpha 1-4Gal beta 1-4Glc-ceramide), in the mechanism of alpha 2 interferon signal transduction. Comparison of the amino acid sequence of the human alpha 2 interferon receptor with that of the B (receptor-binding)-subunit of verotoxin shows three regions of similarity which may provide a structural basis for alpha 2-interferon-receptor/Gb3 interaction.
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Glycolipid modification of α2 interferon binding. Sequence similarity between the α2 interferon receptor and verotoxin (Shiga-like toxin) B-subunit
Biochemical Journal, 1992Co-Authors: C.a. Lingwood, S K YiuAbstract:Previous studies have implicated the glycolipid receptor for the Escherichia coli-derived verotoxin, Globotriaosylceramide (Gb3; Gal alpha 1-4Gal beta 1-4Glc-ceramide), in the mechanism of alpha 2 interferon signal transduction. Comparison of the amino acid sequence of the human alpha 2 interferon receptor with that of the B (receptor-binding)-subunit of verotoxin shows three regions of similarity which may provide a structural basis for alpha 2-interferon-receptor/Gb3 interaction.
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Verotoxin-resistant cell clones are deficient in the glycolipid globotriosylceramide: differential basis of phenotype.
Archives of Biochemistry and Biophysics, 1991Co-Authors: A. Pudymaitis, Glen D. Armstrong, C.a. LingwoodAbstract:Escherichia coli-derived verotoxin is an extremely toxic protein and is highly selective toward certain primate cells. Two susceptible cell lines are the Daudi cell line (human Burkitt lymphoma) and the Vero cell line (Green African monkey kidney). Both of these cell lines contain significant levels of the verotoxin binding glycolipid globotriosylceramide (Gb3) (1 nmol/10(7) cells and 3 nmol/10(6) cells, respectively). A clone was selected from the Vero cell line for resistance to Verotoxin 2, while a mutant from the Daudi cell line was selected for resistance to Verotoxin 1. Both were found to be deficient in globotriosylceramide with a corresponding increase in the precursor glycolipid lactosylceramide. Cell free assay of alpha-galactosyltransferase activity revealed that the Vero cell clone (VRP) contained significantly reduced enzyme activity, whereas in the case of the Daudi mutant (VT20), no significant decrease in activity was noted in vitro. These observations suggest a complex regulation of Gb3 biosynthesis which is considered in relation to P blood group antigen expression.
T. Oguchi - One of the best experts on this subject based on the ideXlab platform.
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Urinary excretion of the vitronectin receptor (integrin alpha V beta 3) in patients with Fabry disease.
Clinica chimica acta; international journal of clinical chemistry, 1999Co-Authors: K. Utsumi, Kenichiro Tanoue, Y. Katagiri, R. Kase, T. Ozawa, Michie Shimmoto, K Itoh, N Yamamoto, M Kotani, T. OguchiAbstract:A renal disorder is one of the important manifestations of Fabry disease, but the details of the pathogenesis have not been clarified yet. We examined the possibility that the vitronectin receptor (VNR, integrin alpha V beta 3), one of the integrins, is involved in the progression of the renal injury in Fabry disease. We measured the urinary excretion of beta 3 originating from VNR in Fabry patients by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA). Immunofluorescent microscopic analyses for VNR and Globotriaosylceramide were performed on urinary sediments from Fabry patients. Furthermore, beta 3 and vitronectin in kidney tissues were analyzed immunohistochemically. Immunoblotting analysis and ELISA showed that the urinary excretion of beta 3 originating from VNR was significantly increased in the Fabry group compared with both the pathological and healthy control groups. Immunofluorescent microscopy revealed the expression of VNR and accumulation of Globotriaosylceramide in urinary sediments from the Fabry patients. Increased expression of beta 3 was observed in glomerular epithelial cells, and in Bowman's capsular epithelial layer and tubular cells, and the amount of vitronectin was moderately increased in the kidney tissues from the Fabry patients. The urinary excretion of VNR was increased, and the expression of VNR was observed in Fabry kidney tissues. The expression of VNR may be involved in the progression of the renal injury in this disease.
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Urinary excretion of the vitronectin receptor (integrin αVβ3) in patients with Fabry disease
Clinica Chimica Acta, 1999Co-Authors: K. Utsumi, Kenichiro Tanoue, Y. Katagiri, R. Kase, T. Ozawa, Michie Shimmoto, K Itoh, N Yamamoto, M Kotani, T. OguchiAbstract:Abstract A renal disorder is one of the important manifestations of Fabry disease, but the details of the pathogenesis have not been clarified yet. We examined the possibility that the vitronectin receptor (VNR, integrin α V β 3 ), one of the integrins, is involved in the progression of the renal injury in Fabry disease. We measured the urinary excretion of β 3 originating from VNR in Fabry patients by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA). Immunofluorescent microscopic analyses for VNR and Globotriaosylceramide were performed on urinary sediments from Fabry patients. Furthermore, β 3 and vitronectin in kidney tissues were analyzed immunohistochemically. Immunoblotting analysis and ELISA showed that the urinary excretion of β 3 originating from VNR was significantly increased in the Fabry group compared with both the pathological and healthy control groups. Immunofluorescent microscopy revealed the expression of VNR and accumulation of Globotriaosylceramide in urinary sediments from the Fabry patients. Increased expression of β 3 was observed in glomerular epithelial cells, and in Bowman's capsular epithelial layer and tubular cells, and the amount of vitronectin was moderately increased in the kidney tissues from the Fabry patients. The urinary excretion of VNR was increased, and the expression of VNR was observed in Fabry kidney tissues. The expression of VNR may be involved in the progression of the renal injury in this disease.
C. Lingwood - One of the best experts on this subject based on the ideXlab platform.
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Comparison of detection methods for cell surface Globotriaosylceramide.
Journal of immunological methods, 2011Co-Authors: Minji Kim, C. Lingwood, Beth Binnington, Darinka Sakac, Kimberly R Fernandes, Sheryl P Shi, Donald R BranchAbstract:The cell surface-expressed glycosphingolipid (GSL), Globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3).
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Inhibition of multidrug resistance by adamantylgb3, a Globotriaosylceramide analog.
The Journal of biological chemistry, 2007Co-Authors: María Fabiana De Rosa, Cameron Ackerley, Bernice Wang, Shinya Ito, David M. Clarke, C. LingwoodAbstract:Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/Globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.
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Increased Globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells
Journal of Molecular Neuroscience, 1994Co-Authors: J. Pereira, B. Boyd, J. Newbigging, C. Lingwood, P. M. StrasbergAbstract:Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in Globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived from E. coli . In Vero and HeLa cells, which are susceptible to verotoxin, the expression of Globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0°C at this boundary is indicative of the amount of Globotriaosylceramide exposed on the cell surface. Above 0°C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-Globotriaosylceramide interactions in synchronized FD and normal cells at this boundary.^125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10^−2–10^−7 µg/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD_50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.
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Increased Globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells. Verotoxin binding studies.
Journal of molecular neuroscience : MN, 1994Co-Authors: J. Pereira, B. Boyd, J. Newbigging, C. Lingwood, P. M. StrasbergAbstract:Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in Globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived fromE. coli. In Vero and HeLa cells, which are susceptible to verotoxin, the expression of Globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0°C at this boundary is indicative of the amount of Globotriaosylceramide exposed on the cell surface. Above 0°C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-Globotriaosylceramide interactions in synchronized FD and normal cells at this boundary.125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10−2–10−7 µg/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.
J. Pereira - One of the best experts on this subject based on the ideXlab platform.
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Increased Globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells
Journal of Molecular Neuroscience, 1994Co-Authors: J. Pereira, B. Boyd, J. Newbigging, C. Lingwood, P. M. StrasbergAbstract:Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in Globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived from E. coli . In Vero and HeLa cells, which are susceptible to verotoxin, the expression of Globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0°C at this boundary is indicative of the amount of Globotriaosylceramide exposed on the cell surface. Above 0°C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-Globotriaosylceramide interactions in synchronized FD and normal cells at this boundary.^125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10^−2–10^−7 µg/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD_50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.
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Increased Globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells. Verotoxin binding studies.
Journal of molecular neuroscience : MN, 1994Co-Authors: J. Pereira, B. Boyd, J. Newbigging, C. Lingwood, P. M. StrasbergAbstract:Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in Globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived fromE. coli. In Vero and HeLa cells, which are susceptible to verotoxin, the expression of Globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0°C at this boundary is indicative of the amount of Globotriaosylceramide exposed on the cell surface. Above 0°C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-Globotriaosylceramide interactions in synchronized FD and normal cells at this boundary.125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10−2–10−7 µg/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.