Download - GLYCOSPHINGOLIPID LECTURE
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Dr. Subroto Chatterjee
GLYCOSPHINGOLIPID LECTURE
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The most common characteristic component of glycosphingolipids is the aliphatic amino alcohol discovered by Thudichum.Thudichum named it Sphingosine after the enigmatic Sphinx from Egypt having a head of Pharaoh and body of a lion.
WHAT ARE
GLYCOSPHINGOLIPIDS?
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STRUCTURE OF LACTOSYLCERAMIDE
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STRUCTURE OF GLYCOSPHINGOLIPIDS
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LYSOSOMAL STORAGE DISORDERSSeveral metabolic basis of inherited
diseases in man occur due to the lack/deficiency of enzymes which catabolize glycosphingolipids
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LYSOSOMAL STORAGE DISORDERS
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Lysosomal Storage Disorders PrevalenceAdrenoleukodystrophy (ADL) Approx. 1 in 20,000 or 13,600 people
in USANiemann Pick (Type A,B, and C) Type A & B: Approx. 1 in 250,000
Type C: Approx. 1 in 150,000
Gaucher
Approx. 1 in 200 for general populationHigh as 1 in 10 in Jewish people with East. European ancestry
Krabbe Approx. 1 in 100,000 peopleMetachromatic leukodystrophy (MLD)
Approx. 1 in 625,000 people
Tay-Sachs Approx. 1 in 27 Jewish people in USAApprox. 1 in 250 for general population
Fabry
Approx. 1 in 40,000 malesApprox. 1 in 117,000 people for general population
PREVALENCE OF GLYCOSPHINGOLIPIDS DISORDER
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FunctionMethods for Determining FunctionExtraction and PurificationQuantitationStructural DeterminationLocalization ImagingMetabolism
GLYCOSPHINGOLIPID LECTURE TOPICS
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1) Superoxide generation and CAM expression2) Inhibition of nitric oxide production in endothelial
cells3) As mediators of growth factors contributing to cell
proliferation4) As receptors for toxins and bacteria
FUNCTION OF GLYCOSPHINGOLIPIDS
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1) Generation of superoxide in arterial smooth muscle cells and expression of cell adhesion molecules.
FUNCTION OF GLYCOSPHINGOLIPIDS IN ATHEROSCLEROSIS AND
VASCULAR BIOLOGY
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LACTOSYLCERAMIDE MEDIATES TNF Α-INDUCED ICAM-1 EXPRESSION IN
ENDOTHELIAL CELLS
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LACTOSYLCERAMIDE STIMULATES SUPEROXIDE GENERATION IN HUMAN
ENDOTHELIAL CELLS
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LACTOSYLCERAMIDE STIMULATES SUPEROXIDE GENERATION IN HUMAN ENDOTHELIAL CELLS
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2) Inhibition of nitric oxide production in endothelial cells.
FUNCTION OF GLYCOSPHINGOLIPIDS IN ATHEROSCLEROSIS AND
VASCULAR BIOLOGY
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EFFECT OF LACCER ON ENDOTHELIUM DEPENDENT VASO-RELAXATION AND
PORCINE CORONARY ARTERY
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EFFECTS OF LacCer ON eNOS mRNA LEVELS IN HCAECs.
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3. As mediators of growth factors contributing to cell proliferation and angiogenesis
FUNCTION OF GLYCOSPHINGOLIPIDS IN ATHEROSCLEROSIS AND
VASCULAR BIOLOGY
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5) Serve as receptors to various toxins, e.g. cholera toxin and other bacteria
FUNCTION OF GLYCOSPHINGOLIPIDS
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METHODS FOR DETERMINING GSL
FUNCTIONCellular assays
ProliferationAdhesionAngiogenesisMigrationApoptosis
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METHODS FOR DETERMINING GSL
FUNCTION
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METHODS FOR DETERMINING GSL
FUNCTION
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1) Bligh and Dyer2) Folch Partitioning
GSL EXTRACTION
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EXTRACTION OF GLYCOSPHINGOLIPIDS FROM HEART TISSUE
Analysis of Glycosphingolipids Subroto ChatterjeeOctober 7, 2013
• Modified Bligh & Dyer Methodo extraction in
chloroform:methanol 2:1
o homogenized by hand
o lipids extracted from organic phase
https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcSoXJZC7o-xxP_UJqYeqM-GpLvFH_2Vq-18XdegMaMqdjDx9_V2
http://www.sfu.ca/bisc/bisc-429/folch.gif
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Folch Partitioning
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Alkaline methanolysis The Glycolipid fraction from the silicic acid column is
treated with mild base to remove contaminating phospholipids. This treatment does not affect glycolipids or gangliosides unless they contain an O-acyl group. The following quantities are used for 1-10mg of glycolipid fraction. Add 1ml of chloroform and 1ml of 0.6 N NaOH in methanol to the dry fraction and allow the mixture to react at room temperature for 1 hour. Then add 1.2 ml of 0.5 N HCL in methanol, 1.7ml of water, and 3.4ml of chloroform, mix well, centrifuge, and remove the lower layer containing the glycolipids. Was the lower layer three times with methanol:water (1:1) and then evaporate it to dryness in vacuo.
PURIFICATION GLYCOSPHINGOLIPIDS
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A) Mixture of compounds in lane C through F
B) Monohexosyl ceramide, gal- and glc-ceramide
C) Dihexosyl ceramide, gal(1->4) glc-ceramide and gal(1->4)gal-ceramide
D) Trihexosyl ceramide, gal(1->4)gal(1->4)glc-cermaide
E) Tetrahexosyl ceramide galNAc(1->3)gal(1->4)gal(1->4)glc-ceramide
F) On a si l ica gel H plate developed with chloroform-methanol-water (100:42:6) and visualized with alpha-napthol spray
HPTLC ANALYSIS OF NEUTRAL GSL
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HPTLC ANALYSIS OF GANGLIOSIDES
Thin-layer chromatogramA) disaloganglioside, NANA
(2->3)gal(1->3)galNAc(14)[NANA(2->3)]gal(1->4)glc-ceramide
B) Monosialoganglioside, galNAc(1->4)[NANA(2->3)gal(1->4)glc-ceramide
C) On a silica gel G (0.25mm) plate developed two times with chloroform-methanol-2.5N NH4OH (65:45:9) and visualized with alpha-naphtol spray
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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TRIMETHYLSILYLATION AND GAS-LIQUID CHROMATOGRAPHY OF METHYL
GLYCOSIDES
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QUANTITATION OF GLYCOSPHINGOLIPIDS
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QUANTITATION OF GLYCOSPHINGOLIPIDS
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QUANTITATION OF GLYCOSPHINGOLIPIDS
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QUANTITATION OF GLYCOSPHINGOLIPIDS
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QUANTITATION OF GLYCOSPHINGOLIPIDS
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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Mass spectrometry of intact TMS derivatives of glycolipids gives information about the sugar groups, the fatty acid and the sphingosine portion of glycosphingolipids. Bis (trimethylsiyl) trifluroroacetamide (100microliter) and pyridine (50 microliter) are added to 20-200microgram of the purified glycospingolipid in a small capped vial and heated at 60 degrees F for about 30 minutes. An aliquot containing 10-20 microgram of the TMS glycolipid is evaporated to dryness under nitrogen in a mass spectrometer direct probe tube. The samples are volati l ized in the mass spectrometer ion source at temperatures ranging 100 degrees to 180 degrees depending on the size of the oligosaccharide unit.
The following information can be obtained by comparison of the resulting mass spectra with those of reference samples: (1) whether the terminal residue is a hexose or hexosamine; (2) the number of and nature of N-acetylneuramine acid groups (i.e., terminal or branched); (3) whether N-acetyl and/or N-glycolylneuraminate is present; (4) information regarding the number of glycosyl residues present and the fatty acid and sphingosine composition. It is essential, because of the limitations of this technique (e.g., the inabil ity to distinguish between hexoses), that it be used in conjunction with other techniques, such as permethylation analyses, and studies with specific glycosidases.
MASS SPECTROMETRY OF INTACT TMSI DERIVATIVES OF GLYCOLIPIDS
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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Principle: Since neutral glycosphingolpids do not
possess a characteristic chromophore that permits their quantitation by UV detection, they can be derivatized with benzoylchloride to form stable per-O,N benzoylated products. These products can be quantified by UV l ight at 280nm or at a higher sensit ivity at 230nm.
GSL(200ng) plus N-acetylpsychosine (an internal standard) are dried in N2. Perbenzoylation is carried out by adding 500uL of 10% benzoylchloride in pyridine for 16hr at 37 C. The samples are N2 dried and washed thrice with 1.8mL of methanol: water(saturated with sodium carbonate). The hexane layer is washed and finally dried in N2. The derivatives are dissolved in CCL4(100uL) and a suitable aliquot injected in to the HPLC column(Zipax,E.I DuPont column 2.1 mmx500nm).
PERBENZOYLATIONMcCluer: Methods in Enzymology 138: 1987
HPLC of male (C57BL/6J) mouse kidney perbenzoylated glycosphingolipids on a Zipax column with detection at 230nm
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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3) Endoglycoceramidase use to excise the oligosaccharides for quantification by HPLC.Wing, D. R., et al. "High-performance liquid
chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide." Analytical biochemistry 298.2 (2001): 207-217.
QUANTITATION OF GLYCOSPHINGOLIPIDS
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INCREASED GLYCOSPHINGOLIPID LEVELS IN SERUM AND AORTAE
OF APOLIPOPROTEIN E GENE KNOCKOUT MICE
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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2) deacylase treatment and quantification of lyso GSL Lipid Extraction
Modified Bligh and Dyer N-deacylation
Dried sphingolipid standards and samples were deacylated using sphingolipid ceramide N-deacylase (SCDase). To each dried sample and standard, 27uL 25mM sodium acetate buffer (pH 5.5) was added
To this solution, 5uL SCDase was added Samples and standards were enzymatically digested for 19h The reaction was stopped with 200uL chloroform-methanol (1:1, v/v) The organic layer was removed and dried under N2 gas.
Derivatization Samples were derivatized with 15uL OPA solution.
HPLC Agilent 1260 Infinity using a Zorbax SB-C18 reversed-phase column Solvent System: isocratic (methanol:water acidified with 0.2%
trifluoroacetic acid at 88:12, v/v) Flow Rate: 0.75 mL/min Ex λ: 340nm Em λ: 360nm
QUANTITATION OF GLYCOSPHINGOLIPIDS USING
DEACYLASE
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Gas chromatography of sugars TMSI derivatization
MS analysis TMSI derivatization
HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)
HPTLC and densitometric scanningHPLC analysis
deacylation (Zama)MS analysis
Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
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Mass Spectrometry Without DerivatizationPreparation
Lipid extract spotted directly onto MALDI Opti-TOF plate with DHB matrix for positive/negative ion mode analysis
GSL standards spottedAnalysis
Spectral ID for GSL ESTD precursor and product ions Comparison of ESTD to sample spectra Normalization to ISTD and protein concentration
QUANTITATION OF GLYCOSPHINGOLIPIDS
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QUANTITATION OF GLYCOSPHINGOLIPIDS
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QUANTITATION OF GLYCOSPHINGOLIPIDS
Normalize to concentration of
ISTD
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QUANTITATION OF GLYCOSPHINGOLIPIDS
Normalize to concentration of
ISTD
Calculate molar concentration (nmol/mg) using standard MW (968) and protein concentration
from Bradford
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A) Sequential digestion with exoglycosidases
B) Deacylation to determine fatty acid composition
C) Tandem mass spectrometryQTOF
D) NMR
STRUCTURAL DETERMINATION
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Sequential digestion with exoglycosidases
STRUCTURAL DETERMINATION
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STRUCTURAL DETERMINATION
Deacylation to determine fatty acid composition- Dr. Chatterjee “TMSI Slide”
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STRUCTURAL DETERMINATION
NMR-Allen Bush lecture
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Galactose oxdiase oxidizes D-galactosyl and N-acetyl-D-galactosaminyl residues at nonreducing terminals of glycoproteins and glycolipids to carbon-6 aldehydes.
These aldehydes are then reduced back to galactose/N-acetylgalactosamine with tritiated borohydride. When intact cells are treated with the enzyme, only surface-exposed glycoconjugates are oxidized subsequently reduced, because the enzyme is unable to penetrate the cell plasma membrane.
Because sialic acids often are linked to penultimate galactosyl residues, more efficient labeling is achieved by the simultaneous use of neuraminidase.
LOCALIZATION OF GSL USING GALACTOSEOXIDASE
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LOCALIZATION OF GSL
A) cell surface labeling using galactoseoxidase
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B) Immunohistochemistry
LOCALIZATION OF GSL
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ELECTRON MICROSCOPY
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METABOLIC LABELING OF GSL
use of radioactive serine, glucose, galactose , palmitate , acetate.
Use of galactose oxidase to label cell surface GSL
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A : Gangliosides of mouse embryo secondary cells
B : Neutral GSLs of mouse embryo secondary cells
AUTORADIOGRAM OF GLYCOSPHINGOLIPIDS
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1,5 – Standards of the neutral GSL fraction of human kidney were applied in channels 1 and 5.
2: 1-14C-N-acetyl-D-glucosamine
3: 1-14C-D-galactose
4: 1-14C-D-glucosamine
A. THIN-LAYER CHROMATOGRAMB.RADIOAUTOGRAM
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1,5 – Human brain gangliosides
2: 1-14C-D-glucosamine3: 1-14C-D-galactose4: 1-14C-D-glucose
A. THIN-LAYER CHROMATOGRAMB. AUTORADIOGRAM
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GSL GLYCOSYLTRANSFERASE ASSAYS
The Lactosylceramide Synthase Reaction
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An essential feature of this galactosyltransferase is the requirement for manganese ions and detergent for optimal activity. This is because this is a Golgi –bound enzyme but is also present in the cell membrane.
Radiolabeled UDP-galactose serves as the galactose donor and GlcCer as the acceptor. The pH optima is 7.8 and Tris or Cacodylate buffer is preferred.
Following incubation for 2hr at 37 C, the reaction is terminated with C:M 2:1.The upper aqueous layer is discarded. The lower phase is N2 dried mixed with unlabeled LacCer and separated by HPTLC. The LacCer band id identified by exposing the HPTLC plate to iodine vapors. Gel area corresponding to LacCer is scraped and radioactivity s measured. The data is expressed as pmol LacCer synthesized /mg protein /2hr
GSL GLYCOSYLTRANSFERASE ASSAYS
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MEASUREMENT OF GLYCOSYLTRANSFERASE MASS
Measurement of mass by:
A) western immunoblot assay
B) ELISA assay
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D-PDMP ALTERS THE EXPRESSION OF VARIOUS GLYCOSYLTRANSFERASES AND COMPONENTS OF
SIGNALING PATHWAYS LEADING TO CELL PROLIFERATION AND ANGIOGENESIS IN A MOUSE
MODEL OF RENAL CANCER.
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DELIVERY OF INHIBITORS OF GSL GLYCOSYLTRANSFERASE IN
EXPERIMENTAL ANIMAL MODELS:
Use of biopolymers, drug eluting stents
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DELIVERY OF INHIBITORS OF GSL GLYCOSYLTRANSFERASE IN
EXPERIMENTAL ANIMAL MODELS:
Use of a novel-antiproliferative compound coated on a biopolymer to mitigate platelet-derived growth factor-induced proliferation in human aortic smooth muscle cells: comparison with sirolimus
Yong-Dan Tang, Ambarish Pandey, Antonina Kolmakova, Xin-Tong Wang, Subbu S. Venkatraman, Subroto Chatterjee, Freddy Y.C. Boey: Glycoconjugate Journal 11/2008; 26(6):721-32.
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DELIVERY OF INHIBITORS OF GSL GLYCOSYLTRANSFERASE IN
EXPERIMENTAL ANIMAL MODELS:
1. Stent coating with PLGA and loading of DPDMP on the polymer Spraying fixture and enclosure Pre-spray materials preparations Metal stent preparation Airbrush or spray coating Stent sterilization and packaging for animal trial
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DELIVERY OF INHIBITORS OF GSL GLYCOSYLTRANSFERASE IN
EXPERIMENTAL ANIMAL MODELS:
2. In vitro degradation and drug release Preparation of PLGA films and coated stents Drug quantification Degradation study
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REFERENCES Bligh, EG, and WJ Dyer. "Canadian Journal of Biochemistry and
Physiology." Canadian Journal of Biochemistry and Physiology . 37.8 (1959): 911-917. Print. http://www.nrcresearchpress.com/doi/abs/10.1139/o59-099
Chatterjee, S. Methods in Enzymology Chatterjee, S. and Martin, S Methods in Enzymology Wei, H and Chatterjee, S. Methods in Enzymology Mccluer , S. Methods in Enzymology Wing, D. R., et al. "High-performance l iquid chromatography analysis
of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide." Analytical biochemistry 298.2 (2001): 207-217.
Zama, K, Y Hayashi, S Ito, Y Hirabayashi, T Inoue, K Ohno, N Okino, and M Ito. "Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phthalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase." Glycobiology . 19.7 (2009): 767-775. Print. doi: 10.1093/glycob/cwp047