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Cynthia Q Sun, Ulrike Hubl, Petra Hoefakker, Madhusudan K Vasudevamurthy, Keryn D Johnson
A new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC and monosialoganglioside sialyltransferase (SAT IV, EC is described. The assay utilised the commercially available fluorophore labelled sphingolipids, boron dipyrromethene difluoride (BODIPY) lactosylceramide (LacCer), and BODIPY-monosialotetrahexosylganglioside (GM1) as the acceptor substrates, for SAT I and SAT IV, respectively. HPLC coupled with fluorescence detection was used to analyse product formation...
2014: PloS One
David C Essaka, Jillian Prendergast, Richard B Keithley, Monica M Palcic, Ole Hindsgaul, Ronald L Schnaar, Norman J Dovichi
Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed...
March 20, 2012: Analytical Chemistry
Scott A Sarver, Richard B Keithley, David C Essaka, Hidenori Tanaka, Yayoi Yoshimura, Monica M Palcic, Ole Hindsgaul, Norman J Dovichi
Several glycosphingolipids were labeled with the fluorphore Bodipy-Fl and analyzed using capillary electrophoresis with laser-induced fluorescence detection. GM1-, LacCer-, and Cer-Bodipy-Fl were prepared through acylation using the N-hydroxysuccinimide ester of Bodipy-Fl. Several other glycosphingolipids including GT1a-, GD1a-, GM2-, GM3-, GD3-, and GlcCer-Bodipy-Fl were enzymatically synthesized. Micellar electrokinetic capillary chromatography with a TRIS/CHES/SDS/α-cyclodextrin buffer produced better separation than an established borate/deoxycholate/methyl-β-cyclodextrin buffer...
March 16, 2012: Journal of Chromatography. A
Ludovic D'auria, Patrick Van der Smissen, Frédéric Bruyneel, Pierre J Courtoy, Donatienne Tyteca
BACKGROUND: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. METHODOLOGY/PRINCIPAL FINDINGS: We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries...
2011: PloS One
Eugenia Trushina, Raman Deep Singh, Roy B Dyer, Sheng Cao, Vijay H Shah, Robert G Parton, Richard E Pagano, Cynthia T McMurray
We show that the mutant Huntington's disease (HD) protein (mhtt) specifically inhibits endocytosis in primary striatal neurons. Unexpectedly, mhtt does not inhibit clathrin-dependent endocytosis as was anticipated based on known interacting partners. Instead, inhibition occurs through a non-clathrin, caveolar-related pathway. Expression of mhtt inhibited internalization of BODIPY-lactosylceramide (LacCer), which is internalized by a caveolar-related mechanism. In contrast, endocytosis of Alexa Fluor 594-transferrin (Tfn) and epidermal growth factor, internalized through clathrin pathway, was unaffected by mhtt expression...
December 15, 2006: Human Molecular Genetics
Yidong Liu, Robert Bittman
The intracellular distribution of synthetic glycosphingolipids (GSLs) bearing a fluorophore can be monitored in living cells by fluorescence microscopy. We reported previously that variation in the length of the long-chain base and in the structure of the carbohydrate-containing polar head group of (2S,3R) (or D-erythro-)-beta-lactosylceramide (LacCer) did not alter the mechanism of endocytic uptake from the plasma membrane of various mammalian cell types [Singh, R.D., Puri, V., Valiyaveettil, J.T., Marks, D...
July 2006: Chemistry and Physics of Lipids
Daniel G Milis, Messiah K Moore, Barbara P Atshaves, Friedhelm Schroeder, John R Jefferson
The influence of sterol carrier protein-2 (SCP-2) on the cellular metabolism of sphingolipids was examined in control mouse L-cells and stably transfected clones expressing the protein SCP-2. Three approaches were used to examine for differences; (1) compositional analysis of endogenous sphingolipid classes, (2) metabolism of NBD-ceramide, and (3) live cell labelling via endocytic uptake of BODIPY-sphingomyelin. SCP-2 over expression significantly altered the endogenous levels of both neutral and acidic sphingolipid classes...
February 2006: Molecular and Cellular Biochemistry
Deepak K Sharma, Jennifer C Brown, Zhijie Cheng, Eileen L Holicky, David L Marks, Richard E Pagano
Glycosphingolipids are known to play roles in integrin-mediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10 degrees C causes the formation of glycosphingolipid-enriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells...
September 15, 2005: Cancer Research
Deepak K Sharma, Amit Choudhury, Raman Deep Singh, Christine L Wheatley, David L Marks, Richard E Pagano
We have previously demonstrated that glycosphingolipids are internalized from the plasma membrane of human skin fibroblasts by a clathrin-independent, caveolar-related mechanism and are subsequently transported to the Golgi apparatus by a process that is dependent on microtubules, phosphatidylinositol 3-kinase, Rab7, and Rab9. Here we characterized the early steps of intracellular transport of a fluorescent glycosphingolipid analog, BODIPY-lactosylceramide (LacCer), and compared this to fluorescent transferrin (Tfn), a well established marker for the clathrin pathway...
February 28, 2003: Journal of Biological Chemistry
R Watanabe, K Asakura, M Rodriguez, R E Pagano
We studied the formation of early endosomes in differentiating oligodendrocytes and type-2 astrocytes, which are derived from common precursor cells in rat neonates, using fluorescent analogues of lactosylceramide (LacCer) and sulfatide labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-pentanoic acid (BODIPY FL C5). These sphingolipid analogues exhibit a concentration-dependent shift in their fluorescence emission maximum from green to red wavelengths that can be used to estimate the relative concentration of an analogue in the intracellular membranes of living cells by quantitative fluorescence microscopy...
October 1999: Journal of Neurochemistry
C S Chen, M C Patterson, C L Wheatley, J F O'Brien, R E Pagano
BACKGROUND: Lipid-storage diseases are collectively important because they cause substantial morbidity and mortality, and because they may present as dementia, major psychiatric illness, developmental delay, or cerebral palsy. At present, no single assay can be used as an initial general screen for lipid-storage diseases. METHODS: We used a fluorescent analogue of lactosylceramide, called N-[5-(5,7-dimethylborondipyrromethenedifluoride)-1-pentanoyl]D- lactosylsphingosine (BODIPY-LacCer), the emission of which changes from green to red wavelengths with increasing concentrations in membranes, to examine the intracellular distribution of the lipid within living cells...
September 11, 1999: Lancet
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