The production of GOSs was measured using 5% (wt/vol) lactose as a substrate in the assay buffer (20 mM sodium phosphate buffer, pH 7.0, containing 150 mM NaCl) at 25 °C. The reactions were initiated by mixing 125 μL of substrate solution containing 20% (wt/vol) lactose and 375 μL of protein solution containing 0.4 μM BgaD-D and/or an appropriate concentration of monobody (133–266 μM). Samples were withdrawn periodically and boiled for 10 min to terminate the reaction. The amounts of monosaccharides, disaccharides and GOSs were determined using a CK04S column (Mitsubishi Chemical) on an HPLC (LC-30AD, Shimadzu) equipped with an evaporative light scattering detector (ELSD-LTII, Shimadzu). The assay samples were eluted from the column using H2O at a flow rate of 0.4 mL/min at 80 °C. For separation and determination of lactose and other disaccharides (DP2), an Asahipak NH2P-40 3E column (Shodex) was used with a gradient of H2O (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min at 30 °C. Sugar concentrations were determined from peak areas. Glucose, galactose, lactose and 4′-galactosyllactose purchased from Wako Chemicals, and tetrasaccharides and larger oligosaccharides prepared as described above, were used as reference compounds for producing standard curves for these assays. Experiments were performed in triplicate.
Galactose
Manufactured by Fujifilm
Sourced in Japan
Galactose is a monosaccharide, a type of simple sugar. It is a key component in the production of important biomolecules and is utilized in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium hydroxide (naoh), by Fujifilm (3 mentions)
Proline, by Fujifilm (2 mentions)
Mannose, by Fujifilm (2 mentions)
Chloroform, by Kanto Chemical (2 mentions)
Fructose, by Fujifilm (2 mentions)
Hydrochloric acid (hcl), by Fujifilm (2 mentions)
Tetrahydrofuran, by Fujifilm (2 mentions)
Pinacol, by Fujifilm (2 mentions)
1 4 dioxane, by Kanto Chemical (2 mentions)
Acetonitrile, by Kanto Chemical (2 mentions)
Lactose, by Fujifilm (1 mentions)
Lc 30ad, by Shimadzu (1 mentions)
Elsd ltii, by Shimadzu (1 mentions)
Glycine, by Thermo Fisher Scientific (1 mentions)
Nicotinamide, by Fujifilm (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
L alanine, by Thermo Fisher Scientific (1 mentions)
Oncostatin m, by Fujifilm (1 mentions)
L glutamine, by Fujifilm (1 mentions)
Sucrose, by Fujifilm (1 mentions)
9 protocols using galactose
1
Quantitative Analysis of GOS Production
2
Hepatocyte Proliferation Enhancing Protocol
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Non-essential amino acids (glycine, 7.5 mg/L; L-alanine, 8.9 mg/L; L-asparagine, 13.2 mg/L; L-aspartic acid, 13.3 mg/L; L-glutamic acid, 14.7 mg/L; L-proline, 11.5 mg/L; and L-serine, 10.5 mg/L) and sodium pyruvate (1 mM) were purchased from Life Technologies. The apoptosis inhibitor, M5054 [100 μg/mL; 2,2′-methylenebis(1,3-cyclohexanedione)], was purchased from Merck (Billerica, MA, USA). 2-(N-(5-chloro-2-methylphenyl)methylsulfonamido)-N-(2,6-difluorophenyl)acetamide), (10 nM; hepatocyte functional proliferation enhancer, FPH1) was purchased from XcessBio (San Diego, CA, USA) [23 (link)]. Galactose (900 mg/mL), ornithine (1 mM), oncostatin M (20 ng/mL), nicotinamide (1.2 mg/mL), proline (30 ng/mL), and L-glutamine (0.3 mg/mL) were purchased from Wako Pure Chemicals (Osaka, Japan).
3
Synthesis of Cholesterol-Based Lipid Conjugates
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Cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) was synthesized as described previously (13 (link)). N-(2-(2-Hydroxyethylamino)ethyl)cholesteryl- 3-carboxamide (OH-Chol) and cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) were synthesized as described previously (14 (link)). 1,2-Dioleoyl- 3- trimethylammonium-propane methyl sulfate salt (DOTAP) was obtained from Avanti Polar Lipids Inc. Dimethyldioctadecylammonium bromide (DDAB, product name: DC-1-18) and 11- ((1,3- bis(dodecanoyloxy)- 2- ((dodecanoyloxy)methyl)propan- 2- yl) amino)- N,N,N- trimethyl-11- oxoundecan-1- aminium bromide (product name: TC-1-12) were obtained from Sogo Pharmaceutical Co., Ltd.. 1,2-Dioleoyl- sn- glycero- 3-phosphoethanolamine (DOPE, COATSOME ME-8181) was obtained from NOF Co., Ltd.. Glucose, fructose, galactose, mannose, sucrose, trehalose dihydrate, maltose monohydrate, lactose monohydrate, lactulose, cellobiose, and raffinose pentahydrate were obtained from Wako Pure Chemical Industries, Ltd.. Melezitose was purchased form Sigma-Aldrich Co. LLC. All other chemicals were of the highest grade available.
4
Structural Relaxation of Hemicellulose for Delignification
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Cellulose, monosaccharides
(glucose, xylose, arabinose, galactose, mannose), lower organic acids,
NaOH, and Na2CO3 were purchased from Wako Pure
Chemical Industries. A type of xylan, derived from beech wood, was
purchased from SERVA Electrophoresis. Ultrapurified water with an
electrical resistance of over 18.2 MΩ was prepared with a Milli-Q
water purification system (Merck, MPGP02001). Japanese cedar in the
form of particles sized 0.85–2 mm was used as the starting
material. It was subjected to a steam pretreatment at 220 °C
under atmospheric pressure. This pretreatment was performed to cause
structural relaxation of hemiCellulose and thereby promote the delignification.33 (link) The procedure of the pretreatment is described
in detail in theSupporting Information . The compositions of the original and pretreated cedars are listed
inTable 3 .
(glucose, xylose, arabinose, galactose, mannose), lower organic acids,
NaOH, and Na2CO3 were purchased from Wako Pure
Chemical Industries. A type of xylan, derived from beech wood, was
purchased from SERVA Electrophoresis. Ultrapurified water with an
electrical resistance of over 18.2 MΩ was prepared with a Milli-Q
water purification system (Merck, MPGP02001). Japanese cedar in the
form of particles sized 0.85–2 mm was used as the starting
material. It was subjected to a steam pretreatment at 220 °C
under atmospheric pressure. This pretreatment was performed to cause
structural relaxation of hemiCellulose and thereby promote the delignification.33 (link) The procedure of the pretreatment is described
in detail in the
in
5
Glycolytic and TCA Cycle Metabolites
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The compounds used in this study were galactose (Wako, Osaka, Japan), 3-deoxyglucosone (3-DG; Toronto Research Chemicals Inc., North York, Canada), citrate (Wako), fumarate, malate (Nacalai Tesque Inc., Kyoto, Japan), acetyl CoA, isocitrate, 2-oxoglutarate, succinyl CoA, succinate, oxaloacetate, phosphoenolpyruvic acid monopotassium salt, benfotiamine, fructose 1,6-bisphosphate (F1,6BP), 3-phosphoglycelic acid (3PG), mannitol (Sigma-Aldrich Co. LCC, St Louis, MO, USA), and rucaparib (Selleck Chemicals, Houston, TX, USA). The AR inhibitor, ranirestat, was provided by Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan) as a collaborative research agreement.
6
Boronic Acid Derivatization Synthesis
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4-Carboxy-3-fluorophenylboronic acid, pinacol, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), tetrahydrofuran, hydrochloric acid, sodium hydroxide, β-cyclodextrin, γ-cyclodextrin, fructose, glucose, galactose, phosphoric acid, sodium chloride, and dimethyl sulfoxide were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 1,4-Dioxane, chloroform, acetonitrile, and dimethyl sulfoxide-d6 were purchased from Kanto Chemical, Co., Inc. (Tokyo, Japan). 2-Aminoanthracene was purchased from Sigma-Aldrich Japan, Co., LLC (Tokyo, Japan). All organic solvents and reagents were commercially available with guaranteed grades and used without further purification. Water was doubly distilled and deionized using a Milli-Q water system (WG222, Yamato Scientific Co., Ltd., Tokyo, Japan, and Autopure WR-600G, Merck Millipore, MA, USA) before use.
7
Defined Xeno-Free Hepatocyte Culture Protocol
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HSM was prepared from amino acid powders following the formulation of Leibovits-15 medium (Life Technologies) [18 (link)]. This HSM lacked arginine, tyrosine, glucose, and sodium pyruvate, but was supplemented with galactose (900 mg/L), ornithine (1 mM), glycerol (5 mM), and proline (260 mM) (all from Wako Pure Chemicals). proline (30 mg/L) was added for DNA synthesis to occur [21 (link)]. Aspartic acid was not included since it is one of the products of ornithine and an arginine substrate. KSR (Life Technologies) was added at a final concentration of 10%, and used instead of fetal calf serum to establish defined xeno-free conditions.
HDI was prepared by mixing oncostatin M (20 ng/mL), FPH1, M50054 (100 μg/mL), non-essential amino acids (glycine, 7.5 mg/L; L-alanine, 8.9 mg/L; L-asparagine, 13.2 mg/L; L-aspartic acid, 13.3 mg/L; L-glutamic acid, 14.7 mg/L; L-proline, 11.5 mg/L; and L-serine, 10.5 mg/L), sodium pyruvate (1 mM), nicotinamide (1.2 mg/mL), proline (30 ng/mL), and glutamine (0.3 mg/mL). proline and nicotinamide are necessary for primary hepatocytes to proliferate [21 (link), 22 (link)].
HDI was prepared by mixing oncostatin M (20 ng/mL), FPH1, M50054 (100 μg/mL), non-essential amino acids (glycine, 7.5 mg/L; L-alanine, 8.9 mg/L; L-asparagine, 13.2 mg/L; L-aspartic acid, 13.3 mg/L; L-glutamic acid, 14.7 mg/L; L-proline, 11.5 mg/L; and L-serine, 10.5 mg/L), sodium pyruvate (1 mM), nicotinamide (1.2 mg/mL), proline (30 ng/mL), and glutamine (0.3 mg/mL). proline and nicotinamide are necessary for primary hepatocytes to proliferate [21 (link), 22 (link)].
8
Yeast Culture and Genetic Transformation
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Yeast strains were cultured in YPDA-rich medium (1% yeast extract [BD Biosciences, San Jose, CA], 2% bacto-peptone [BD], 2% glucose [Nacalai Tesque, Kyoto, Japan], and 0.01% adenine [FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan]). Strains carrying plasmids were selected in synthetic medium (SD) containing the required nutritional supplements55 . When appropriate, 0.5% casamino acids [BD] were added to SD medium without uracil [FUJIFILM Wako] (SDA-Ura). For induction of the GAL1 promoter, 3% galactose [FUJIFILM Wako] and 0.2% sucrose [Nacalai] were used as carbon sources instead of glucose (YPGA and SGA-Ura). Blue (peak at 470 nm) and red (peak at 660 nm) light-emitting diode panels [SL-150X150 series; CCS, Tokyo, Japan] were used as light sources. In all analyses, BL and RL were used at an intensity of 10 μmol m−2 s−1 unless otherwise noted. When grown on the agar medium, the yeast cells were irradiated with light vertically from a height of about 10 cm above the plate. When cultured in a liquid medium, the entire test tube was irradiated with light from a distance of about 10 cm from the side of the tube. Escherichia coli strains were cultured in LB medium [Nacalai] containing appropriate antibiotics as needed. The lithium acetate method was used to introduce plasmids into yeast cells56 (link),57 .
9
Synthesis and Characterization of Fluorescent Probes
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4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), pinacol, sodium hydroxide, hydrochloric acid, β-CyD, γ-CyD, tetrahydrofuran (THF), methanol, lithium aluminum hydride, diethyl ether, dichloromethane, sodium sulfate, magnesium sulfate, sodium azide, dimethylformamide (DMF), ammonia solution, sodium hydrogen carbonate, benzene, fructose, glucose, and galactose were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). 4-Carboxyphenylboronic acid, triethylamine, methanesulfonyl chloride, triphenylphosphine, and oxalyl chloride were purchased from Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan). 1,4-Dioxane, chloroform, acetonitrile, sulfuric acid, and acetone were purchased from Kanto Chemical, Co., Inc. (Tokyo, Japan). methanol-d4, chloroform-d, DMSO-d6, 1-pyreneacetic acid, and 1-pyrenebutyric acid were purchased from Sigma-Aldrich Japan, Co., LLC (Tokyo, Japan). 1-Pyrenebutylamine was purchased from Toronto Chemical Research Inc. (Toronto, USA). All other organic solvents and reagents were commercially available with guaranteed grades and used without further purification. Water was doubly distilled and deionized by a Milli-Q water system (WG222, Yamato Scientific Co., Ltd., Tokyo, Japan and Milli-Q Advantage, Merck Millipore, MA, USA) before use.
(Osaka, Japan). 4-Carboxyphenylboronic acid, triethylamine, methanesulfonyl chloride, triphenylphosphine, and oxalyl chloride were purchased from Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan). 1,4-Dioxane, chloroform, acetonitrile, sulfuric acid, and acetone were purchased from Kanto Chemical, Co., Inc. (Tokyo, Japan). methanol-d4, chloroform-d, DMSO-d6, 1-pyreneacetic acid, and 1-pyrenebutyric acid were purchased from Sigma-Aldrich Japan, Co., LLC (Tokyo, Japan). 1-Pyrenebutylamine was purchased from Toronto Chemical Research Inc. (Toronto, USA). All other organic solvents and reagents were commercially available with guaranteed grades and used without further purification. Water was doubly distilled and deionized by a Milli-Q water system (WG222, Yamato Scientific Co., Ltd., Tokyo, Japan and Milli-Q Advantage, Merck Millipore, MA, USA) before use.
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