Browsing by Author "Sillence, Daniel J."
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Item Metadata only 1H NMR-Linked Urinary Metabolic Profiling of Niemann-Pick Class C1 (NPC1) Disease: Identification of Potential New Biomarkers using Correlated Component Regression (CCR) and Genetic Algorithm (GA) Analysis Strategies(Bentham Science, 2014-11-14) Ruiz-Rodado, Victor; Luque-Baena, R. M.; te Vruchte, D. J.; Probert, Fay; Lachmann, R. H.; Hendriksz, Christian J.; Wraith, James E.; Imrie, Jackie; Elizondo, David; Sillence, Daniel J.; Clayton, P.; Platt, Frances M.; Grootveld, MartinNiemann-Pick Class 1 (NPC1) disease is a rare, debilitating neurodegenerative lysosomal storage disease; however, urinary biomarkers available for it and its prognosis are currently limited. In order to identify and establish such biomarkers, we employed high-resolution 1H NMR analysis coupled to a range of multivariate (MV) analysis approaches, i.e. PLS-DA, RFs and uniquely the cross-validated correlated component regression (CCR) strategy in order to discern differences between the urinary metabolic profiles of 13 untreated NPC1 disease and 47 heterozygous (parental) carrier control participants. Novel computational intelligence techniques (CITs) involving genetic algorithms (GAs) were also employed for this purposeItem Metadata only Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport.(American Society for Biochemistry and Molecular Biology, 2004-06-18) Sillence, Daniel J.; Lloyd-Evans, E.; Veldman, R. J.; Vruchte, D. T.; Neville, David C. A.; Platt, Frances M.; van Blitterswijk, Wim J.Item Metadata only Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling(Elsevier, 2004) Neville, David C. A.; Coquard, S. M.; Priestman, D. A.; te Vruchte, D. J.; Sillence, Daniel J.; Dwek, R. A.; Platt, Frances M.; Butters, T. D.Item Metadata only Apoptosis and signalling in acid sphingomyelinase deficient cells(2000) Sillence, Daniel J.BACKGROUND: Recent evidence suggests that the activation of a non-specific lipid scramblase during apoptosis induces the flipping of sphingomyelin from the cell surface to the cytoplasmic leaftet of the plasma membrane. Inner leaflet sphingomyelin is then cleaved to ceramide by a neutral sphingomyelinase. The production of this non-membrane forming lipid induces blebbing of the plasma membrane to aid rapid engulfment by professional phagocytes. However contrary evidence suggests that cells which are deficient in acid sphingomyelinase are defective in apoptosis signalling. This data has been interpreted as support for the activation of acid sphingomyelinase as an early signal in apoptosis. HYPOTHESIS: An alternative explanation is put forward whereby the accumulation of intracellular sphingomyelin in sphingomyelinase deficient cells leads to the formation of intracellular rafts which lead to the sequestration of important signalling molecules that are normally present on the cell surface where they perform their function. TESTING THE HYPOTHESIS: It is expected that the subcellular distribution of important signalling molecules is altered in acid sphingomyelinase deficient cells, leading to their sequestration in late endosomes/lysosomes. Other sphingolipid storage diseases such as Niemann-Pick type C which have normal acid sphingomyelinase activity would also be expected to show the same phenotype. IMPLICATIONS OF THE HYPOTHESIS: If true the hypothesis would provide a mechanism for the pathology of the sphingolipid storage diseases at the cellular level and also have implications for the role of ceramide in apoptosis.Item Metadata only Assay for the transbilayer distribution of glycolipids; selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals(2000) Sillence, Daniel J.; Raggers, R. J.; Neville, David C. A.; Harvey, D. J.; van Meer, G.In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine- egg phosphatidylethanolamine;-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A(2) confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing <1 mol% radiolabeled glucosylceramide or short-chain C(6)-glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C(6)-glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 +/- 1%), 94% of the remaining C(6)-glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0 degrees C, the reaction reached completion by 20 min.Item Metadata only Assays for the transmembrane movement of sphingolipids(Elsevier, 2000) Sillence, Daniel J.; Raggers, R. J.; van Meer, G.Item Metadata only The association of shiga-like toxin with detergent-resistant membranes is modulated by glucosylceramide and is an essential requirement in the endoplasmic reticulum for cytotoxic effect.(American Society for Cell Biology, 2006-03-01) Sillence, Daniel J.; Jarvis, R. M.; Falguires, T.; Smith, D. C.; Johannes, Ludger; Lord, J. Michael; Platt, Frances M.; Roberts, Lynne M.Item Open Access Cytosolic Glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease(Elsevier, 2019-03-12) Sillence, Daniel J.; Bhardwaj, Meenakshi; Tongue, Paige; Schmid, Ralf; Haberkant, Per; Wheeler, SimonNiemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.Item Open Access Effect of antidepressant drugs on the brain sphingolipid system(2020-05-14) Zetterstrom, Tyra; Jaddoa, Estabraq; Masania, Jinit; Masiero, Eva; Sgamma, Tiziana; Arroo, R. R. J.; Sillence, Daniel J.Background: Major depression is a common mood disorder and the central sphingolipid system has been identified as a possible drug target of this condition. Here we investigated the action of antidepressant drugs on sphingolipid levels in rat brain regions, plasma and in cultured mouse macrophages. Methods: Two antidepressant drugs were tested; the serotonin reuptake inhibitor paroxetine and the noradrenaline reuptake inhibitor desipramine, either following acute or chronic treatments. Content of sphingosine and ceramide were analysed using LC-MS or HPLC-UV respectively. This from samples of brain, plasma and cultured mouse macrophages. Antidepressant induced effects on mRNA expression for two key genes of the sphingolipid pathway, SMPD1 and ASAH1 were also measured by using quantitative Real-Time PCR. Results: Chronic but not acute administration of paroxetine or desipramine reduced sphingosine levels in the prefrontal cortex and hippocampus (only paroxetine) but not in the striatum. Ceramide levels were also measured in the hippocampus following chronic paroxetine and likewise to sphingosine this treatment reduced its levels. The corresponding collected plasma samples from chronically treated animals did not show any decrease of sphingosine compared to the corresponding controls. Both drugs failed to reduce sphingosine levels from cultured mouse macrophages. The drug-induced decrease of sphingolipids coincided with reduced mRNA expression of two enzymes of the central sphingolipid pathway, i.e. acid sphingomyelinase (SMPD1) and acid ceramidase (ASAH1). Conclusions: This study supports the involvement of brain sphingolipids in the mechanism of action by antidepressant drugs and for the first time highlights their differential effects on brain versus plasma levels.Item Metadata only Evidence against an early signalling role for ceramide in Fas-mediated apoptosis(1997) Sillence, Daniel J.; Allan, D.We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.Item Metadata only Glucosylceramide modulates endolysosomal pH in Gaucher disease(Elsevier, 2013) Sillence, Daniel J.Item Metadata only Glucosylceramide modulates membrane traffic along the endocytic pathway.(American Society for Biochemistry and Molecular Biology., 2002) Sillence, Daniel J.; Puri, V.; Marks, D. L.; Butters, T. D.; Dwek, R. A.; Pagano, R. E.; Platt, Frances M.Item Metadata only Glycosphingolipid storage leads to the enhanced degradation of the B cell receptor in Sandhoff disease mice.(Springer, 2010) te Vruchte, D. J.; Jeans, A.; Platt, Frances M.; Sillence, Daniel J.Item Metadata only Glycosphingolipids in endocytic membrane transport.(Elsevier, 2004) Sillence, Daniel J.; Platt, Frances M.Item Metadata only Hydrolysis of cell surface phosphatidylinositol leads to the delayed stimulation of inositol phospholipid synthesis in Bovine Aortic Endothelial Cells(1993) Sillence, Daniel J.; Low, M. G.In order to address the issue of how inositol phospholipid synthesis is controlled in a resting cell we looked for enhanced [3H]phosphatidylinositol (PtdIns) labelling in response to the hydrolysis of cell surface PtdIns. Bacillus thuringiensis PtdIns-PLC when added to intact bovine aortic endothelial (BAE) cells rapidly hydrolysed 9.1 +/- 1% of the total cellular PtdIns. This result suggests that BAE cells have a cell surface pool of PTdIns. Hydrolysis of cell surface PtdIns, in contrast to the agonist-stimulated hydrolysis of inner leaflet PtdIns, did not lead to a rapid (minutes) stimulation of PtdIns resynthesis. Prolonged incubation of BAE cells with PtdIns-PLC led to further hydrolysis of PtdIns (up to 20% of total cellular PtdIns). This second phase of PtdIns-PLC induced hydrolysis was inhibited by the addition of brefeldin A suggesting that it was dependent on vesicular traffic to the plasma membrane from the endoplasmic reticulum. Furthermore, the above result suggests that prolonged incubation of intact cells with PtdIns-PLC leads to the slow depeletion of intracellular PtdIns stores. This second phase of PtdIns-PLC induced hydrolysis was associated with PtdIns resynthesis since prolonged incubation with PtdIns-PLC, but not B. cereus PtdCho-PLC (which does not hydrolyse PtdIns), led to enhanced PtdIns labelling. The results indicate that extracellular PtdIns-PLC induced PtdIns resynthesis may occur due to PtdIns-PLC induced intracellular PtdIns depletion.Item Metadata only Introduction: Glycosphingolipids in cell biology and disease.(Elsevier, 2004) Sillence, Daniel J.; Platt, Frances M.Item Metadata only Lipid Rafts; Properties, Controversies and Roles in Signal Transduction(Nova Science Ltd, 2014-03-01) Sillence, Daniel J.Item Open Access Lipid–Protein Interactions in Niemann–Pick Type C Disease: Insights from Molecular Modeling(MDPI, 2019-02-07) Wheeler, Simon; Schmid, R.; Sillence, Daniel J.The accumulation of lipids in the late endosomes and lysosomes of Niemann–Pick type C disease (NPCD) cells is a consequence of the dysfunction of one protein (usually NPC1) but induces dysfunction in many proteins. We used molecular docking to propose (a) that NPC1 exports not just cholesterol, but also sphingosine, (b) that the cholesterol sensitivity of big potassium channel (BK) can be traced to a previously unappreciated site on the channel’s voltage sensor, (c) that transient receptor potential mucolipin 1 (TRPML1) inhibition by sphingomyelin is likely an indirect effect, and (d) that phosphoinositides are responsible for both the mislocalization of annexin A2 (AnxA2) and a soluble NSF (N-ethylmaleimide Sensitive Fusion) protein attachment receptor (SNARE) recycling defect. These results are set in the context of existing knowledge of NPCD to sketch an account of the endolysosomal pathology key to this disease.Item Metadata only Lithium treatment of affective disorders; effects on the cyclic AMP and inositol phospholipid signalling pathways’(Elsevier, 1992) Sillence, Daniel J.; Downes, C. P.The effects of lithium (Li+) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li+ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC50 of approx. 20 mM. By contrast, half-maximal effects of Li+ on inositol monophosphate (InsP) accumulation in [3H]inositol labelled tissue slices occurred at about 1 mM. A similar IC50 for Li+ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [3H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated cortical slices using a novel mass assay for InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li+ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li+ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are compatible with the inositol depletion hypothesis of Li+ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.Item Open Access Mechanisms of Gaucher Disease Pathogenesis(Annals of Translational Medicine, 2015-02-06) Wheeler, Simon; Sillence, Daniel J.Gaucher disease is caused by mutations in the Gba1 gene encoding an acid β-glucocerebrosidase (GBA1), the lysosomal hydrolase which breaks down glucosylceramide (GlcCer). In Gaucher type 1 disease the accumulation of this simple glycolipid is mainly restricted to tissue phagocyte lysosomes resulting ultimately in hepatomegaly, splenomegaly and osteopenia. Lower residual GBA1 levels leads to neuronal storage, in types 2 and 3 neurological symptoms are characterised by acute (death at age 2) or sub-acute onset, respectively. The links between cellular changes and clinical manifestations are largely unknown but are the key to the development and monitoring of new therapies. The newcomer to Gaucher disease is likely attracted to the apparent simplicity of an autosomal recessive disorder which promises to unravel the critical GlcCer function in normal cells (GlcCer is widespread, it’s even present in some bacteria—also, mouse and fly GlcCer knockouts die at embryo stage). However, closer acquaintance reveals not a classic Mendelian disorder—sometimes even monozygotic twins have different symptoms—and studies at the cellular level have so far failed to reveal clear GlcCer functions. Now a team led by Ellen Sidransky at the NIH has taken what appears to be a big step forward by producing two in vitro models of Gaucher cells (1).