The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Shinichi Ichikawa - One of the best experts on this subject based on the ideXlab platform.
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wp1066 a small molecule inhibitor of the jak stat3 pathway inhibits Ceramide Glucosyltransferase activity
Biochemical and Biophysical Research Communications, 2017Co-Authors: Hirotaka Tsurumaki, Yoshio Hirabayashi, Hikaru Katano, Kousuke Sato, Ryou Imai, Satomi Niino, Shinichi IchikawaAbstract:Abstract WP1066 is a well-known inhibitor of the JAK/STAT3 signaling pathway. By a screen of known small molecule inhibitors of various enzymes and protein factors, we identified WP1066 as a Ceramide Glucosyltransferase inhibitor. Ceramide Glucosyltransferase catalyzes the first glycosylation step during glycosphingolipid synthesis. We found that WP1066 inhibited the activity of Ceramide Glucosyltransferase with an IC50 of 7.2 μM, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of Ceramide Glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose.
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A small molecule inhibitor of Bcl-2, HA14-1, also inhibits Ceramide Glucosyltransferase
Biochemical and biophysical research communications, 2013Co-Authors: Satomi Niino, Yoshio Hirabayashi, Yutaka Nakamura, Michiyo Nagano-ito, Shinichi IchikawaAbstract:HA14-1 is a Bcl-2 inhibitor that is widely used for studies of apoptosis. In the course of searching for a Ceramide Glucosyltransferase inhibitor that catalyzes the first glycosylation step of glycosphingolipid synthesis, we unexpectedly found that HA-14-1 also has the ability to inhibit Ceramide Glucosyltransferase. The IC50 value of HA14-1 against Ceramide Glucosyltransferase is 4.5μM, which is lower than that reported for Bcl-2 in vitro. Kinetic analyses revealed that HA14-1 is a competitive and mixed-type inhibitor with respect to C6-NBD-Ceramide and UDP-glucose, respectively.
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up regulation of Ceramide Glucosyltransferase during the differentiation of u937 cells
Journal of Biochemistry, 2011Co-Authors: Junya Aida, Yoshio Hirabayashi, Syouta Higuchi, Yoshie Hasegawa, Michiyo Naganoito, Aoi Banba, Takamitsu Shimizu, Asako Kikuchi, Mayuko Saga, Shinichi IchikawaAbstract:The phorbol ester tetradecanoylphorbol acetate (TPA) induces promyelocytic leukaemia cells to differentiate to macrophage-like cells in vitro. During the course of this differentiation, the cells adhere to the bottom of the culture dish, a process that requires an increase in cell surface glycosphingolipids (GSLs). We examined the cellular content of glucosylCeramide (GlcCer), the simplest of the GSLs, in a TPA-treated leukaemia cell line, U937. Following TPA treatment, we observed a 3.5-fold increase in GlcCer levels that was caused by enhanced activity of Ceramide Glucosyltransferase (GlcT-1), which catalyses Ceramide glycosylation. Furthermore, in TPA-treated cell GlcT-1 amounts were increased at both the mRNA and protein levels. We also found decreased activity of lactosylCeramide synthase in TPA-treated cells, which could also contribute to the increase in cellular GlcCer content.
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glccer synthase udp glucose Ceramide Glucosyltransferase ugcg
2002Co-Authors: Yoshio Hirabayashi, Shinichi IchikawaAbstract:Glycosphingolipids (GSLs) occur in vertebrates and lower animals, as well as in plants. They have been regarded as an enigmatic class of membrane lipids. Recent progress in biomembrane research indicates the existence of GSL microdomains (or “rafts”) in cell surface membranes (Simon and Ikonen 1997) GSLs form microdomains by a clustering of cholesterol and glycosyl-phosphatidyl inositol (GPI)-anchored proteins. The microdomains are thought to involve a variety of biological events such as cell signaling and cell-cell interaction. Another characteristic feature of GSLs is their chemical diversity: over 400 GSLs with different sugar chain structures have been isolated and determined. The structural studies to date indicate that almost all GSLs are derived from the simplest GSL, glucosylCeramide. This is synthesized by a Ceramidespecific β-Glucosyltransferase (GlcT-I). Recent success in the molecular cloning of the gene encoding GlcT-I has provided new insights into the previously unrecognized roles of GSLs (Ichikawa et al. 1996). When the GlcT-I function is knocked out in mice by gene-targeting technology, the mouse dies at embryonic day 8 (Yamashita et al. 1999). This study clearly shows that GlcT-I plays an essential role during embryonic development and differentiation.
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developmental patterns of Ceramide Glucosyltransferase glct 1 expression in the mouse in situ hybridization using dig labeled rna probes
Molecules and Cells, 2001Co-Authors: Youngkug Choo, Shinichi Ichikawa, Yoshio HirabayashiAbstract:Glycosphingolipids (GSLs) play significant roles in a variety of cell membrane events, including cellular interactions, signaling, and trafficking. Ceramide Glucosyltransferase (glucosylCeramide synthase, GlcT-1, EC 2.4.1.80) catalyzes the initial step in GSL synthesis, the transfer of glucose from UDP-glucose to Ceramide. The reaction product of glucosylCeramide serves as a core structure for over 400 species of GSLs. The enzyme is a key regulatory factor controlling intracellular levels of Ceramide and GSLs. Appearance and differential distribution of GlcT-1 mRNA during mice postimplantation embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes, coupled with alkaline phosphatase detection. On E9, tissues of the mesencephalon, myelecephalon, diencephalons, and telencephalon expressed GlcT-1. On E11, it was expressed to a detectable extent in various tissues including mesencephalon, myelecephalon, diencephalon, telencephalon, nose, lung, liver, vertebra, tail, spinal cord, and tongue. The expression patterns of E13 were similar to those of E11, except that the heart and stomach became positive. On E15, a specific signal for GlcT-1 was detected in all organs of the embryo. These results provide the first evidence that GlcT-1 is differentially expressed during postimplantation embryogenesis.
Yoshio Hirabayashi - One of the best experts on this subject based on the ideXlab platform.
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wp1066 a small molecule inhibitor of the jak stat3 pathway inhibits Ceramide Glucosyltransferase activity
Biochemical and Biophysical Research Communications, 2017Co-Authors: Hirotaka Tsurumaki, Yoshio Hirabayashi, Hikaru Katano, Kousuke Sato, Ryou Imai, Satomi Niino, Shinichi IchikawaAbstract:Abstract WP1066 is a well-known inhibitor of the JAK/STAT3 signaling pathway. By a screen of known small molecule inhibitors of various enzymes and protein factors, we identified WP1066 as a Ceramide Glucosyltransferase inhibitor. Ceramide Glucosyltransferase catalyzes the first glycosylation step during glycosphingolipid synthesis. We found that WP1066 inhibited the activity of Ceramide Glucosyltransferase with an IC50 of 7.2 μM, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of Ceramide Glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose.
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A small molecule inhibitor of Bcl-2, HA14-1, also inhibits Ceramide Glucosyltransferase
Biochemical and biophysical research communications, 2013Co-Authors: Satomi Niino, Yoshio Hirabayashi, Yutaka Nakamura, Michiyo Nagano-ito, Shinichi IchikawaAbstract:HA14-1 is a Bcl-2 inhibitor that is widely used for studies of apoptosis. In the course of searching for a Ceramide Glucosyltransferase inhibitor that catalyzes the first glycosylation step of glycosphingolipid synthesis, we unexpectedly found that HA-14-1 also has the ability to inhibit Ceramide Glucosyltransferase. The IC50 value of HA14-1 against Ceramide Glucosyltransferase is 4.5μM, which is lower than that reported for Bcl-2 in vitro. Kinetic analyses revealed that HA14-1 is a competitive and mixed-type inhibitor with respect to C6-NBD-Ceramide and UDP-glucose, respectively.
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up regulation of Ceramide Glucosyltransferase during the differentiation of u937 cells
Journal of Biochemistry, 2011Co-Authors: Junya Aida, Yoshio Hirabayashi, Syouta Higuchi, Yoshie Hasegawa, Michiyo Naganoito, Aoi Banba, Takamitsu Shimizu, Asako Kikuchi, Mayuko Saga, Shinichi IchikawaAbstract:The phorbol ester tetradecanoylphorbol acetate (TPA) induces promyelocytic leukaemia cells to differentiate to macrophage-like cells in vitro. During the course of this differentiation, the cells adhere to the bottom of the culture dish, a process that requires an increase in cell surface glycosphingolipids (GSLs). We examined the cellular content of glucosylCeramide (GlcCer), the simplest of the GSLs, in a TPA-treated leukaemia cell line, U937. Following TPA treatment, we observed a 3.5-fold increase in GlcCer levels that was caused by enhanced activity of Ceramide Glucosyltransferase (GlcT-1), which catalyses Ceramide glycosylation. Furthermore, in TPA-treated cell GlcT-1 amounts were increased at both the mRNA and protein levels. We also found decreased activity of lactosylCeramide synthase in TPA-treated cells, which could also contribute to the increase in cellular GlcCer content.
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glccer synthase udp glucose Ceramide Glucosyltransferase ugcg
2002Co-Authors: Yoshio Hirabayashi, Shinichi IchikawaAbstract:Glycosphingolipids (GSLs) occur in vertebrates and lower animals, as well as in plants. They have been regarded as an enigmatic class of membrane lipids. Recent progress in biomembrane research indicates the existence of GSL microdomains (or “rafts”) in cell surface membranes (Simon and Ikonen 1997) GSLs form microdomains by a clustering of cholesterol and glycosyl-phosphatidyl inositol (GPI)-anchored proteins. The microdomains are thought to involve a variety of biological events such as cell signaling and cell-cell interaction. Another characteristic feature of GSLs is their chemical diversity: over 400 GSLs with different sugar chain structures have been isolated and determined. The structural studies to date indicate that almost all GSLs are derived from the simplest GSL, glucosylCeramide. This is synthesized by a Ceramidespecific β-Glucosyltransferase (GlcT-I). Recent success in the molecular cloning of the gene encoding GlcT-I has provided new insights into the previously unrecognized roles of GSLs (Ichikawa et al. 1996). When the GlcT-I function is knocked out in mice by gene-targeting technology, the mouse dies at embryonic day 8 (Yamashita et al. 1999). This study clearly shows that GlcT-I plays an essential role during embryonic development and differentiation.
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glycosphingolipids are required for sorting melanosomal proteins in the golgi complex
Journal of Cell Biology, 2001Co-Authors: Hein Sprong, Yoshio Hirabayashi, Peter Van Der Sluijs, Sophie Degroote, Tijs Claessens, Judith Van Drunen, Viola Oorschot, Ben H C Westerink, Judith Klumperman, Gerrit Van MeerAbstract:A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of Ceramide Glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.
Satomi Niino - One of the best experts on this subject based on the ideXlab platform.
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wp1066 a small molecule inhibitor of the jak stat3 pathway inhibits Ceramide Glucosyltransferase activity
Biochemical and Biophysical Research Communications, 2017Co-Authors: Hirotaka Tsurumaki, Yoshio Hirabayashi, Hikaru Katano, Kousuke Sato, Ryou Imai, Satomi Niino, Shinichi IchikawaAbstract:Abstract WP1066 is a well-known inhibitor of the JAK/STAT3 signaling pathway. By a screen of known small molecule inhibitors of various enzymes and protein factors, we identified WP1066 as a Ceramide Glucosyltransferase inhibitor. Ceramide Glucosyltransferase catalyzes the first glycosylation step during glycosphingolipid synthesis. We found that WP1066 inhibited the activity of Ceramide Glucosyltransferase with an IC50 of 7.2 μM, and that its action was independent of JAK/STAT3 pathway blockade. Moreover, the modes of inhibition of Ceramide Glucosyltransferase were uncompetitive with respect to both C6-NBD-cermide and UDP-glucose.
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A small molecule inhibitor of Bcl-2, HA14-1, also inhibits Ceramide Glucosyltransferase
Biochemical and biophysical research communications, 2013Co-Authors: Satomi Niino, Yoshio Hirabayashi, Yutaka Nakamura, Michiyo Nagano-ito, Shinichi IchikawaAbstract:HA14-1 is a Bcl-2 inhibitor that is widely used for studies of apoptosis. In the course of searching for a Ceramide Glucosyltransferase inhibitor that catalyzes the first glycosylation step of glycosphingolipid synthesis, we unexpectedly found that HA-14-1 also has the ability to inhibit Ceramide Glucosyltransferase. The IC50 value of HA14-1 against Ceramide Glucosyltransferase is 4.5μM, which is lower than that reported for Bcl-2 in vitro. Kinetic analyses revealed that HA14-1 is a competitive and mixed-type inhibitor with respect to C6-NBD-Ceramide and UDP-glucose, respectively.
Frances M Platt - One of the best experts on this subject based on the ideXlab platform.
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cellular effects of deoxynojirimycin analogues uptake retention and inhibition of glycosphingolipid biosynthesis
Biochemical Journal, 2004Co-Authors: Howard R Mellor, David C A Neville, David J Harvey, Frances M Platt, Raymond A Dwek, Terry D ButtersAbstract:Deoxynojirimycin (DNJ) analogues are inhibitors of Ceramide Glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40–50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following Ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.
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n butyldeoxygalactonojirimycin a more selective inhibitor of glycosphingolipid biosynthesis than n butyldeoxynojirimycin in vitro and in vivo
Biochemical Pharmacology, 2000Co-Authors: Ulrika Andersson, Terry D Butters, Raymond A Dwek, Frances M PlattAbstract:N-Butyldeoxynojirimycin (NB-DNJ) inhibits the Ceramide Glucosyltransferase which catalyses the first step in glycosphingolipid (GSL) biosynthesis. It has the potential to be used for the treatment of the GSL lysosomal storage diseases and is currently in clinical trials for the treatment of type 1 Gaucher's disease. However, NB-DNJ is also a potent inhibitor of other enzymes, including alpha-glucosidase I and II, which could potentially cause side effects in patients receiving life-long therapy. Wetherefore evaluated a potentially more selective GSL biosynthesis inhibitor, N-butyldeoxygalactonojirimycin (NB-DGJ), in vitro and in vivo. The distribution and degree of GSL depletion in the liver of mice treated with NB-DGJ or NB-DNJ were equivalent. Mice treated with NB-DGJ had normal body weights and lymphoid organ sizes, whereas NB-DNJ-treated mice showed weight loss and partial lymphoid organ shrinkage. NB-DNJ inhibited glycogen catabolism in the liver, whereas NB-DGJ did not. NB-DNJ was also a potent inhibitor of sucrase and maltase in vitro but not of lactase, while NB-DGJ inhibited lactase but not sucrase or maltase. NB-DGJ is therefore more selective than NB-DNJ, and deserves to be evaluated for human therapy.
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molecular requirements of imino sugars for the selective control of n linked glycosylation and glycosphingolipid biosynthesis
Tetrahedron-asymmetry, 2000Co-Authors: Terry D Butters, L A G M Van Den Broek, George W J Fleet, Thomas Martin Krulle, Mark R Wormald, R A Dwek, Frances M PlattAbstract:Abstract N -Butyl-deoxynojirimycin (NB-DNJ) has been approved for clinical trials as a potential therapy for Gaucher disease, a glycolipid lysosomal storage disorder. As this compound has both glycoprotein processing α-glucosidase and Ceramide Glucosyltransferase inhibitory activity, we have sought to determine the molecular basis for these two activities. NB-DNJ is known to resemble the positively charged oxocarbonium-like transition state for α-glucosidase I and the structure–function relationships we present now help to define the recognition epitope for the enzyme. Inhibition of Ceramide Glucosyltransferase by NB-DNJ was competitive for Ceramide ( K i =7.4 μM) and non-competitive for UDP-glucose, indicating inhibitory activity is by Ceramide mimicry. The presence of an N -alkyl chain was obligatory for transferase inhibition and increases in alkyl chain length provided a modest increase in inhibitory potency. By contrast, α-glucosidase inhibition was independent of the N -alkyl chain and changes in chain length. The effects of ring substitutions identified the C 3 hydroxyl group as being critical for both enzymes but C 1 and C 6 modifications led to a loss of transferase inhibition only. Attempts to rationalise these data for transferase inhibition using an energy minimised molecular model of NB-DNJ and Ceramide predicted structural homology of three stereogenic centres and the N -alkyl chain of NB-DNJ, with the trans -alkenyl and N -acyl chain of Ceramide. On the basis of these studies, modifications to imino sugar inhibitors can be suggested that allow a more selective approach for molecular inhibition of both Ceramide Glucosyltransferase and α-glucosidase I, leading to improved compounds for the potential treatment of lysosomal glycosphingolipid storage disorders and viral infections, respectively.
Genji Imokawa - One of the best experts on this subject based on the ideXlab platform.
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A reconstructed human epidermal keratinization culture model to characterize Ceramide metabolism in the stratum corneum.
Archives of dermatological research, 2012Co-Authors: Naoki Yoshida, Eri Sawada, Genji ImokawaAbstract:To examine factors that regulate Ceramide production during keratinization of the human stratum corneum (SC), we developed a reconstructed human epidermal keratinization model in which a fresh layer of SC is newly formed within 1 week. Addition of the UDP-glucose: Ceramide Glucosyltransferase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol significantly diminished SC Ceramide levels (expressed as µg/mg protein) with decreased glucosylCeramide levels. Desipramine hydrochloride, an inhibitor of sphingomyelinase, also significantly reduced SC Ceramide levels. Similarly, conduritol B epoxide, an inhibitor of β-glucocerebrosidase, significantly down-regulated SC Ceramide levels and significantly increased glucosylCeramide levels. These results indicate the reliability of this model to elucidate Ceramide synthesis regulating factors. Using this model, we assessed the effects of the inflammatory cytokine interleukin-1α (IL-1α), several bioactive sphingolipids and all-trans retinoic acid (RA) on Ceramide levels in the SC. Whereas treatment with IL-1α (at 10 nM) significantly down-regulated Ceramide levels, treatment with sphingosylphosphorylcholine (at 50 µM) or sphingosine-1-phosphate (at 10 or 20 µM) distinctly up-regulated Ceramide levels. Interestingly, RA (at low as 10 nM) significantly up-regulated Ceramide levels without affecting the formation of the SC or levels of keratinization-related proteins in the epidermis. The increased levels of Ceramide were accompanied by a significantly increased secretion of granulocyte-macrophage colony-stimulating factor as well as by a significantly down-regulated expression of acid-ceramidase at both the gene and protein levels. Taken together, our results underscore the superiority of this reconstructed human epidermal keratinization model to analyze factors that regulate Ceramide synthesis, especially in human SC.
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a rapid and simple assay method for udp glucose Ceramide Glucosyltransferase
Biochimica et Biophysica Acta, 1992Co-Authors: Noburo Matsuo, Tomoko Nomura, Genji ImokawaAbstract:We have developed a simple rapid method for measuring UDP-glucose: Ceramide glucsoyltransferase; the method utilizes Ceramide immobilized on the surface of silica gell and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylCeramide formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4–6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS). Apparent Km values of 8.7 μM for UDP-glucose and 292 μM for Ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2–5 times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.