Sodium nitrate, sodium acetate, N-acetylgalactosamine, glucosamine, galactose, and glucuronic acid were from Sigma–Aldrich (USA). The NaOH solution was from J.T. Baker (Netherlands). The hydrochloric acid was from Carlo Erba (Italy). The medium and the enzymes used in the biological experiments for cell cultures were from Gibco, Invitrogen (USA), unless otherwise specified. The 10 food supplements (FS), containing both CS and GlcN, and the two pharmaceuticals (Ph), containing only CS, that were from different European countries and companies, were either purchased or obtained as test/gift samples. Cell culture reagents and enzymes used in the biological experiments were from Gibco, Invitrogen (USA), unless otherwise specified.
N acetylgalactosamine
Manufactured by Merck Group
Sourced in United States
N-acetylgalactosamine is a chemical compound that is commonly used in laboratory settings. It is a monosaccharide, a type of simple sugar, that contains an acetyl group attached to a galactose molecule. N-acetylgalactosamine is often utilized in biochemical and biological research applications, but its core function is to serve as a building block for more complex carbohydrates and glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
N acetylglucosamine, by Merck Group (5 mentions)
Galactose, by Merck Group (3 mentions)
Glucuronic acid, by Merck Group (3 mentions)
N acetylneuraminic acid, by Merck Group (2 mentions)
Galacturonic acid, by Merck Group (2 mentions)
Fucose, by Merck Group (2 mentions)
Ribose, by Merck Group (2 mentions)
Rhamnose, by Merck Group (2 mentions)
Arabinose, by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
L fucose, by Merck Group (2 mentions)
D mannose, by Merck Group (2 mentions)
Sodium acetate, by Merck Group (1 mentions)
Sodium nitrate, by Merck Group (1 mentions)
Glucosamine, by Merck Group (1 mentions)
Wire software, by Renishaw (1 mentions)
Hplc grade acetonitrile, by Honeywell (1 mentions)
Lactic acid, by Merck Group (1 mentions)
Succinic acid, by Merck Group (1 mentions)
Butyric acid, by Merck Group (1 mentions)
12 protocols using n acetylgalactosamine
1
Characterization of Glycosaminoglycan Supplements
2
Raman Spectral Database of Glycan Standards
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To generate the glycosylation database, Raman spectra of glycan standards were collected. Glycan standards were mannose, fucose, N-acetyl-galactosamine, N-acetyl neuraminic acid, galactose, glucose, and N-acetyl-glucosamine (Sigma-Aldrich, Merck Group, MO, USA). Standards were dissolved in ultra-pure water at three different concentrations (12, 25, and 50 mg/ml) to assure specificity of the peaks. A 20 µl liquid droplet was placed on a stainless-steel slide for each concentration of standard. Measurements were taken in duplicate for each concentration in the non-dried form with a Raman spectrophotometer inVia Qontor (Renishaw, Gloucestershire, UK). All spectra were recorded between 400 and 1800 cm− 1 wavenumber range (1 cm− 1 spectral resolution) with a 50x objective and a 785 nm (Near infrared) laser. An integration time of 10 s was used at 50% (approximately, 55 mW) laser power at the sample surface. The baseline was automatically selected and subtracted, cosmic rays were removed and a 5th order polynomial smoothing (Slavitzky-Golay) applied with the WiRE software (Renishaw, Gloucestershire, UK). Calibration using the 520 cm− 1 peak of a silicon wafer was performed before sample spectrum acquisition. All spectra obtained were then averaged for each glycan. Only the highest peaks (reaching at least 100 intensity counts) were selected for inclusion in the database.
3
Comprehensive Metabolite Analysis Protocol
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Methanol (MeOH) of HPLC grade, 2-picolylamine
(2-PA), dipyridyl disulfide (DPDS), triphenylphosphine (TPP), acetic
acid, propionic acid, butyric acid, valeric acid, caproic acid, lactic
acid, succinic acid, isobutyric acid, isovaleric acid, 2-methylbutyric
acid, 3,3-dimethylbutyric acid, 2-methylvaleric acid, 3-methylvaleric
acid, 4-methylbutyric acid, indole-3-acetic acid, indole-3-butyric
acid, indole-3-lactic acid, 2-ethylbutyric acid (2-EtB), d4-acetic acid, glucose, galactose, fructose, arabinose, fucose, rhamnose,
glucuronic acid, galacturonic acid, Nacetylglucosamine, N-acetylgalactosamine, mannose, allose, ribose, 3-methyl-1-phenyl-2-
pyrazoline-5-one (PMP), trifluoroacetic acid (TFA), and ammonium acetate
were purchased from Sigma-Aldrich (St. Louis, MO). d2-indole-3-propionic
acid was purchased from Toronto Research Chemicals (Toronto, Canada).
Algal starch (U–13C, 98%+), 13C6 glucose, and unlabeled algal starch were purchased from Cambridge
Isotope Laboratories (Tewksbury, MA). Isopropanol of LC/MS grade was
purchased from Fisher Scientific (Waltham, MA). Acetonitrile (HPLC-grade)
was purchased from Honeywell (Muskegon, MI).
(2-PA), dipyridyl disulfide (DPDS), triphenylphosphine (TPP), acetic
acid, propionic acid, butyric acid, valeric acid, caproic acid, lactic
acid, succinic acid, isobutyric acid, isovaleric acid, 2-methylbutyric
acid, 3,3-dimethylbutyric acid, 2-methylvaleric acid, 3-methylvaleric
acid, 4-methylbutyric acid, indole-3-acetic acid, indole-3-butyric
acid, indole-3-lactic acid, 2-ethylbutyric acid (2-EtB), d4-acetic acid, glucose, galactose, fructose, arabinose, fucose, rhamnose,
glucuronic acid, galacturonic acid, Nacetylglucosamine, N-acetylgalactosamine, mannose, allose, ribose, 3-methyl-1-phenyl-2-
pyrazoline-5-one (PMP), trifluoroacetic acid (TFA), and ammonium acetate
were purchased from Sigma-Aldrich (St. Louis, MO). d2-indole-3-propionic
acid was purchased from Toronto Research Chemicals (Toronto, Canada).
Algal starch (U–13C, 98%+), 13C6 glucose, and unlabeled algal starch were purchased from Cambridge
Isotope Laboratories (Tewksbury, MA). Isopropanol of LC/MS grade was
purchased from Fisher Scientific (Waltham, MA). Acetonitrile (HPLC-grade)
was purchased from Honeywell (Muskegon, MI).
4
Measuring Alpha-Galactosidase A Activity
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In female patients, α-Gal A enzyme activity was measured using whole blood. Leukocytes were isolated [7 (link)], and α-Gal A enzyme activity was measured with a fluorometer (1420 Multi-label Counter; PerkinElmer). The substrate and inhibitor used were 4-methylumbelliferyl-α-D-galactopyranoside (Sigma-Aldrich) and N-acetylgalactosamine (Sigma-Aldrich), respectively. Enzymatic activity was evaluated using the calibration curve of 4-methylumbelliferone (Sigma-Aldrich) and expressed as nmol/hr/mg protein. In male patients, α-Gal A enzyme activity was measured from dried venous blood spots on a Whatman 903 filter paper (GE Healthcare Life Sciences). The separation and detection of α-Gal A was performed using a high-performance liquid chromatography system (HPLC system; Agilent 1200 series, Agilent) and a flow injection analysis-tandem mass spectrometry system (FIA-MS/MS, API 4000, SCIEX) operated in a multiple reaction monitoring mode using NeoLSDTM MSMS KIT (PerkinElmer). The enzyme activity was calculated and expressed as µmol/hr/L.
5
Cultivation of Gut Bacterial Species
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Akkermansia muciniphila MucT (ATTC BAA-835) was grown in a basal medium as described previously [7 (link)]. The medium was supplemented with either hog gastric mucin (0.5%, Type III; Sigma-Aldrich, St. Louis, MO, USA), a mix of sugars (D-glucose, L-fucose, N-acetylglucosamine, N-acetylgalactosamine; 2.5 mM each, Sigma-Aldrich) or glucose (10 mM, Sigma-Aldrich). The medium without mucin was supplemented with tryptone (8 g/l, Oxoid Ltd, Basingstoke, Hampshire, England) and L-threonine (2 mM, Sigma-Aldrich). Incubations were performed in serum bottles sealed with butyl-rubber stoppers at 37°C under anaerobic conditions provided by a gas phase of 182 kPa (1.5 atm) N2/CO2 (80/20 ratio). Growth was measured by spectrophotometer as optical density at 600 nm (OD600).
Faecalibacterium prausnitzii A2-165 was grown anaerobically at 37°C in YCFA medium supplemented with 33 mM glucose [23 (link)]. Lactobacillus plantarum WCFS1 was grown aerobically and Bifidobacterium breve DSM-20213 anaerobically at 37°C in Difco™ Lactobacilli MRS broth (Becton Dickinson, Sparks, USA).
Faecalibacterium prausnitzii A2-165 was grown anaerobically at 37°C in YCFA medium supplemented with 33 mM glucose [23 (link)]. Lactobacillus plantarum WCFS1 was grown aerobically and Bifidobacterium breve DSM-20213 anaerobically at 37°C in Difco™ Lactobacilli MRS broth (Becton Dickinson, Sparks, USA).
6
Motility of Vibrio and Microcystis in the Presence of Carbohydrates
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Motility in the presence of different compounds was quantified by measuring the diameter of one colony after 24 hours of growth at 37°C on a semi-solid (0.3%) agar plate. MSR media at 28°C was used for V. fischeri. M9 minimal medium + 0.1% glycerol was used to measure baseline motility. 0.1% glycerol plates were supplemented either with 0.01% porcine mucin, 0.01% N-acetylglucosamine (GlcNAc), N-acetylneuraminic acid (Neu5Ac), L-fucose, D-mannose, D-galactose, or N-acetylgalactosamine (GalNAc) to measure motility in the presence of these compounds (Sigma). Motility was also evaluated on soft agar plates containing M9 minimal medium and 0.01% hexaacetyl-chitohexaose (Megazyme). Microcystis aeruginosa was grown on MBL medium at 22°C in the presence of full light with light shaking to avoid flocculation. M. aeruginosa was added 0.01% w/v. The mucus-containing gills of Crassostrea spp. were homogenized and autoclaved. Crassostrea spp. gill homogenate was added to 0.01% w/v. Results were plotted using Prism software and the statistical significance was obtained by using student’s t-test.
7
Enzymatic and Genetic Analysis of α-Galactosidase A
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Enzymatic activity of α-galactosidase A (α-GalA) was measured following the fluorometric method described by Chamoles [11 (link)]. Briefly, the assay was performed on triplicates in buffer citrate-phosphate 0.15 M pH 4.2, using 4-methylumbeliferil-galactopiranoside substrate (2 mM, Glycosinth, UK) in the presence of N-acetylgalactosamine (70 mM, Sigma-Aldrich). α-GalA activity was expressed as micromoles of substrate per hour and liter of blood (μmol/Lh).
For genetic study, the seven exons of GLA were amplified by PCR with specific primers and were sequenced by the Sanger method, using the Chromas 2.4. software to detect point mutations and small deletions or insertions in exons and near intronic regions (±25 bp).
For genetic study, the seven exons of GLA were amplified by PCR with specific primers and were sequenced by the Sanger method, using the Chromas 2.4. software to detect point mutations and small deletions or insertions in exons and near intronic regions (±25 bp).
8
Dried Blood Spot Assay for Fabry Disease
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Dried blood spots (DBS) were prepared from venous blood samples using Guthrie's paper. Each bloodspot contained 75 µL of sample and was subsequently dried while protected from light at room temperature for 4 hours. The prepared DBS were placed in long-term storage at -4°C for future use. Protein was extracted from 3-mm punches through each DBS and incubated at 37°C with 4-methylumbelliferyl-α-D-galactopyranoside (4-MUGaL; Sigma-Aldrich, MO, USA) as a substrate. N-acetyl-galactosamine (Sigma-Aldrich) was added to inhibit α-galactosidase B, which can suppress α-galactosidase A activity. The fluorescence of the 4-methylumbelliferone product of α-GAL A was quantified using a fluorescence plate reader (BioTek, VT), with excitation at 360 nm and emission at 460 nm. Each sample was processed in duplicate. Quality control (QC) in the assay consisted of using QC-DBS pools (Center for Disease Control and Prevention, GA). A positive control sample was donated by a male patient affected by classical Fabry disease with the p.E66Q mutation. Relying on several references and the Mayo Clinic Fabry Disease Testing Algorithm, α-GAL A activity less than 1.2 nmol/(mL·hour) in males and 2.8 nmol/(mL·hour) in females were considered positive (Mayo Medical Laboratories).
9
Carbohydrate Compound Sourcing and Preparation
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D-Mannosamine hydrochloride was obtained from Sigma (M4670) or Spectrum Chemical MFG Corp (M3220). 1-Amino-1-deoxy-D-Fructose hydrochloride (D-isoglucosamine) (803278), D-(+)-Galactosamine (1287722), D-(+)-Glucosamine (1294207), N-acetyl-Mannosamine (A8176), N-acetyl-galactosamine (A2795), N-Acetyl-Glucosamine (A8625), Meglumine (M9179), Muramic acid (M2503), N-Acetylneuraminic acid (A2388), D-(+)-Glucose (D9434), D-(+)-Mannose (1375182), Meglumine (M9179), Tunicamycin from Streptomyces sp. (T7765) and SP600125 (S5567) were obtained from Sigma. Hypure cell culture grade water used to dissolve compounds (endotoxin < 0.005 EU/ml) was obtained from Hyclone. Axitinib was obtained from Santa Cruz (SC-217679). Tauroursodeoxycholic acid (TUDCA) was from Calbiochem (1180-95-6) and 4-phenylbutyric acid (4-PBA) (P21005), Castanospermine (Cas, C3784), Kifunensine (K1140), and DMSO (D2650) were from Sigma. DMSO (D2650) was used as a solvent for Cas.
10
Isolation and Characterization of Mandarin Pectin
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Orah mandarins were bought from Guangxi Hyperion International Agricultural Logistics Co., Ltd. (Nanning, China). HPLC-grade mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose, N-acetyl glucosamine and N-acetyl galactosamine were provided by Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). NK-92MI cells (natural killer cells in patients with human malignant non-Hodgkin’s lymphoma), Calu-1 cells (human lung cancer cells), MEMα medium, and McCoy’s 5A medium were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). TRIzol Reagent was obtained from Thermo Fisher Scientific (Waltham, MA, USA). HiScript II QRT SuperMix for qPCR (+gDNA wiper) and ChamQ Universal SYBR qPCR Master Mix were acquired from Vazyme Biotech Co., Ltd. (Nanjing, China). All compounds were analytical grade unless otherwise specified.
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