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    Name:
    Gas Chromatography
    Description:
    This book presents an introduction to gas chromatography including examples of applications Shows readers malfunctions that can occur enabling them to recognize and fix faults
    Catalog Number:
    z271756
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    Millipore gas chromatograph
    Gas Chromatography
    This book presents an introduction to gas chromatography including examples of applications Shows readers malfunctions that can occur enabling them to recognize and fix faults
    https://www.bioz.com/result/gas chromatograph/product/Millipore
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    gas chromatograph - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Gas Chromatography:

    Article Title: Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli
    Article Snippet: .. All chemicals for gas chromatography (GC) standards except for ethanol and 1-pentanol were purchased from Sigma-Aldrich (St. Louis, MO). .. 1-Pentanol was purchased from Acros Organics (Belgium).

    Article Title: Crystallisation properties of amorphous cyclodextrin powders and their complexation with fish oil
    Article Snippet: .. Absolute ethanol, gas chromatography grade hexane and fish oil (derived from the Menhaden fish.) were purchased from Sigma–Aldrich (Sigma–Aldrich Co. LLC, Australia). ..

    Article Title: Combined Use of S. pombe and L. thermotolerans in Winemaking. Beneficial Effects Determined through the Study of Wines’ Analytical Characteristics
    Article Snippet: .. We used gas chromatography quality compounds as the sets of the volatile standards for this purpose (Fluka, Sigma-Aldrich Corp., Buchs SG, Standort, Switzerland). ..

    Article Title: Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide
    Article Snippet: .. Determination of inhibitory activity of the lysate of m3MST-overexpressing cells by means of gas chromatography HEK 293 cells in ice-cold isolation buffer containing 30 mM HEPES buffer (pH 7.4), 100 μM DTT and 1% protease inhibitor cocktail (Sigma, Japan) were sonicated for 10 sec twice in an Astrason 4000 sonicator (MISONIX, USA) to obtain the lysate. ..

    Chromatography:

    Article Title: The genome and phenome of the green alga Chloroidium sp. UTEX 3007 reveal adaptive traits for desert acclimatization
    Article Snippet: .. Gas chromatography-flame ionization detection (GC-FID) For the extraction, 50 mg algal material (dry weight) was used by adding 1 ml of 1 N HCl/methanol solution (Sigma-Aldrich, Darmstadt, Germany). .. An internal standard (100 µl of FA15:0, pentadecanoic acid) was added to each sample before incubation at 80°C in a water bath for 30 min. After cooling to room temperature, 1 ml of 0.9% NaCl and 1 ml of 100% hexane were added to each vial.

    Isolation:

    Article Title: Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling
    Article Snippet: .. Gas chromatography-mass spectrometry (GC-MS) metabolomics 1×108 yeast cells yeast cells were isolated by rapid vacuum filtration onto a nylon filter membrane (0.45 μm pore size, Millipore) using a Glass Microanalysis Filter Holder (Millipore), briefly washed with phosphate buffered saline (1 mL), and immediately submerged into ACN/MeOH/H2 O (2:2:1, v/v/v, 1.5 mL, pre-cooled to −20 °C) in a plastic tube. .. The time from sampling yeast from the culture to submersion in cold extraction solvent was less than 30 s. Tubes with the extraction solvent, nylon filter, and yeast were stored at −80 °C prior to analysis.

    Article Title: Rapid assessment of the authenticity of limequat fruit using the electronic nose and gas chromatography coupled with mass spectrometry
    Article Snippet: .. Gas chromatography–mass spectrometry analysis Solid phase microextraction was utilized for the isolation and enrichment of analytes using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)-coated fibre with thickness of 50/30 µm and length of 2 cm (Sigma-Aldrich). .. Extraction was done at 40 °C for 35 min. Next, the SPME fibre with extracted analytes was automatically transferred into the injector port of the gas chromatograph for thermal desorption for 6 min.

    Article Title: Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide
    Article Snippet: .. Determination of inhibitory activity of the lysate of m3MST-overexpressing cells by means of gas chromatography HEK 293 cells in ice-cold isolation buffer containing 30 mM HEPES buffer (pH 7.4), 100 μM DTT and 1% protease inhibitor cocktail (Sigma, Japan) were sonicated for 10 sec twice in an Astrason 4000 sonicator (MISONIX, USA) to obtain the lysate. ..

    Solid-phase Microextraction:

    Article Title: Rapid assessment of the authenticity of limequat fruit using the electronic nose and gas chromatography coupled with mass spectrometry
    Article Snippet: .. Gas chromatography–mass spectrometry analysis Solid phase microextraction was utilized for the isolation and enrichment of analytes using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)-coated fibre with thickness of 50/30 µm and length of 2 cm (Sigma-Aldrich). .. Extraction was done at 40 °C for 35 min. Next, the SPME fibre with extracted analytes was automatically transferred into the injector port of the gas chromatograph for thermal desorption for 6 min.

    Size-exclusion Chromatography:

    Article Title: Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide
    Article Snippet: .. Determination of inhibitory activity of the lysate of m3MST-overexpressing cells by means of gas chromatography HEK 293 cells in ice-cold isolation buffer containing 30 mM HEPES buffer (pH 7.4), 100 μM DTT and 1% protease inhibitor cocktail (Sigma, Japan) were sonicated for 10 sec twice in an Astrason 4000 sonicator (MISONIX, USA) to obtain the lysate. ..

    Suction Filtration:

    Article Title: Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling
    Article Snippet: .. Gas chromatography-mass spectrometry (GC-MS) metabolomics 1×108 yeast cells yeast cells were isolated by rapid vacuum filtration onto a nylon filter membrane (0.45 μm pore size, Millipore) using a Glass Microanalysis Filter Holder (Millipore), briefly washed with phosphate buffered saline (1 mL), and immediately submerged into ACN/MeOH/H2 O (2:2:1, v/v/v, 1.5 mL, pre-cooled to −20 °C) in a plastic tube. .. The time from sampling yeast from the culture to submersion in cold extraction solvent was less than 30 s. Tubes with the extraction solvent, nylon filter, and yeast were stored at −80 °C prior to analysis.

    Gas Chromatography-Mass Spectrometry:

    Article Title: Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling
    Article Snippet: .. Gas chromatography-mass spectrometry (GC-MS) metabolomics 1×108 yeast cells yeast cells were isolated by rapid vacuum filtration onto a nylon filter membrane (0.45 μm pore size, Millipore) using a Glass Microanalysis Filter Holder (Millipore), briefly washed with phosphate buffered saline (1 mL), and immediately submerged into ACN/MeOH/H2 O (2:2:1, v/v/v, 1.5 mL, pre-cooled to −20 °C) in a plastic tube. .. The time from sampling yeast from the culture to submersion in cold extraction solvent was less than 30 s. Tubes with the extraction solvent, nylon filter, and yeast were stored at −80 °C prior to analysis.

    Article Title: Rapid assessment of the authenticity of limequat fruit using the electronic nose and gas chromatography coupled with mass spectrometry
    Article Snippet: .. Gas chromatography–mass spectrometry analysis Solid phase microextraction was utilized for the isolation and enrichment of analytes using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)-coated fibre with thickness of 50/30 µm and length of 2 cm (Sigma-Aldrich). .. Extraction was done at 40 °C for 35 min. Next, the SPME fibre with extracted analytes was automatically transferred into the injector port of the gas chromatograph for thermal desorption for 6 min.

    Article Title: Caenorhabditis elegans Battling Starvation Stress: Low Levels of Ethanol Prolong Lifespan in L1 Larvae
    Article Snippet: .. Fatty acid analysis by gas chromatography-mass spectrometry (GC-MS) L1 larvae prepared as described above for the survival experiments were incubated for various times at 20°C at 160 rpm in M9 medium alone, M9 medium with 1 mM ethanol, or M9 medium with 1 mM fully-deuterated ethanol (Sigma, catalog no. 186414). ..

    Incubation:

    Article Title: Caenorhabditis elegans Battling Starvation Stress: Low Levels of Ethanol Prolong Lifespan in L1 Larvae
    Article Snippet: .. Fatty acid analysis by gas chromatography-mass spectrometry (GC-MS) L1 larvae prepared as described above for the survival experiments were incubated for various times at 20°C at 160 rpm in M9 medium alone, M9 medium with 1 mM ethanol, or M9 medium with 1 mM fully-deuterated ethanol (Sigma, catalog no. 186414). ..

    Activity Assay:

    Article Title: Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide
    Article Snippet: .. Determination of inhibitory activity of the lysate of m3MST-overexpressing cells by means of gas chromatography HEK 293 cells in ice-cold isolation buffer containing 30 mM HEPES buffer (pH 7.4), 100 μM DTT and 1% protease inhibitor cocktail (Sigma, Japan) were sonicated for 10 sec twice in an Astrason 4000 sonicator (MISONIX, USA) to obtain the lysate. ..

    Protease Inhibitor:

    Article Title: Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide
    Article Snippet: .. Determination of inhibitory activity of the lysate of m3MST-overexpressing cells by means of gas chromatography HEK 293 cells in ice-cold isolation buffer containing 30 mM HEPES buffer (pH 7.4), 100 μM DTT and 1% protease inhibitor cocktail (Sigma, Japan) were sonicated for 10 sec twice in an Astrason 4000 sonicator (MISONIX, USA) to obtain the lysate. ..

    Sonication:

    Article Title: Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide
    Article Snippet: .. Determination of inhibitory activity of the lysate of m3MST-overexpressing cells by means of gas chromatography HEK 293 cells in ice-cold isolation buffer containing 30 mM HEPES buffer (pH 7.4), 100 μM DTT and 1% protease inhibitor cocktail (Sigma, Japan) were sonicated for 10 sec twice in an Astrason 4000 sonicator (MISONIX, USA) to obtain the lysate. ..

    Fluorescence In Situ Hybridization:

    Article Title: Crystallisation properties of amorphous cyclodextrin powders and their complexation with fish oil
    Article Snippet: .. Absolute ethanol, gas chromatography grade hexane and fish oil (derived from the Menhaden fish.) were purchased from Sigma–Aldrich (Sigma–Aldrich Co. LLC, Australia). ..

    Derivative Assay:

    Article Title: Crystallisation properties of amorphous cyclodextrin powders and their complexation with fish oil
    Article Snippet: .. Absolute ethanol, gas chromatography grade hexane and fish oil (derived from the Menhaden fish.) were purchased from Sigma–Aldrich (Sigma–Aldrich Co. LLC, Australia). ..

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  • 84
    Millipore hepatic pgc 1α
    <t>PGC-1α</t> isoforms differentially regulate inflammatory and metabolic signaling pathways downstream of TNFα. Gene expression microarrays of mRNA isolated from primary mouse hepatocytes over-expressing either PGC-1α1, PGC-1α4, or vector control by adenoviral infection. A) Number of genes changed greater than 2-fold 48 hr following transduction in the absence or presence of 2 ng/mL TNFα (2 hr) (n = 3 biological replicates, adj. p-value
    Hepatic Pgc 1α, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore ucp1 promoter
    Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of <t>Ucp1</t> , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P
    Ucp1 Promoter, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore myotubes
    Regulation of: A , Cox4 ; B , Cpt-1β ; and C , Vegf mRNA by PGC-1α with and without STARS knockdown in C2C12 <t>myotubes</t>
    Myotubes, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore arabidopsis cell suspension culture
    Acyltransferase time courses for enzymes extracted from <t>Arabidopsis</t> and spinach. (a, b) Cell‐wall enzymes were salt‐extracted from live cell cultures of (a) Arabidopsis and (b) spinach at various ages after subculturing, then dialysed, freeze dried and redissolved at 1% (w/v) in 10 m m PIPES (Na + , pH 7.0). The enzyme preparations (10 μl) were incubated with 50 μ m [ 14 C]oxalyl‐threonate (OxT) with (+) or without (−) 5% (w/v) glucose for 4 h. Products were separated by electrophoresis at pH 6.5 and autoradiographed (OxG, oxalyl‐glucose). Non‐radiolabelled markers (Glc, glucose; LA, lactobionic acid) were stained in silver nitrate. LA is a readily silver‐stainable compound possessing 12 C atoms and 1 − charge, and is thus a useful nearby‐migrating marker for substances such as oxalyl‐disaccharides (14 C atoms, 1 − ). Radioactive markers: OxA, oxalate; OxT, oxalyl‐threonate. Pencilled: OG, orange G. [The streaking of the [ 14 C]oxalate spots in Figures 5 and 6 (compare with Figure 4 ) was caused by omission of EDTA from the electrophoresis buffer. This streaking does not interfere with interpretation of the results.] [Colour figure can be viewed at http://www.wileyonlinelibrary.com ].
    Arabidopsis Cell Suspension Culture, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PGC-1α isoforms differentially regulate inflammatory and metabolic signaling pathways downstream of TNFα. Gene expression microarrays of mRNA isolated from primary mouse hepatocytes over-expressing either PGC-1α1, PGC-1α4, or vector control by adenoviral infection. A) Number of genes changed greater than 2-fold 48 hr following transduction in the absence or presence of 2 ng/mL TNFα (2 hr) (n = 3 biological replicates, adj. p-value

    Journal: bioRxiv

    Article Title: PGC-1α isoforms coordinate to balance hepatic metabolism and apoptosis in inflammatory environments

    doi: 10.1101/703678

    Figure Lengend Snippet: PGC-1α isoforms differentially regulate inflammatory and metabolic signaling pathways downstream of TNFα. Gene expression microarrays of mRNA isolated from primary mouse hepatocytes over-expressing either PGC-1α1, PGC-1α4, or vector control by adenoviral infection. A) Number of genes changed greater than 2-fold 48 hr following transduction in the absence or presence of 2 ng/mL TNFα (2 hr) (n = 3 biological replicates, adj. p-value

    Article Snippet: Hepatic PGC-1α was immunoprecipitated from liver using anti-PGC-1α (Millipore, ST1202) in 1% Triton/TBS.

    Techniques: Pyrolysis Gas Chromatography, Expressing, Isolation, Plasmid Preparation, Infection, Transduction

    PGC-1α isoforms differentially regulate metabolic genes downstream of TNFα. A-D) mRNA expression of primary mouse hepatocytes over-expressing PGC-1α1, PGC-1α4 or vector alone following 2-hr treatment with 2 ng/mL TNFα or vehicle (n=3). *p

    Journal: bioRxiv

    Article Title: PGC-1α isoforms coordinate to balance hepatic metabolism and apoptosis in inflammatory environments

    doi: 10.1101/703678

    Figure Lengend Snippet: PGC-1α isoforms differentially regulate metabolic genes downstream of TNFα. A-D) mRNA expression of primary mouse hepatocytes over-expressing PGC-1α1, PGC-1α4 or vector alone following 2-hr treatment with 2 ng/mL TNFα or vehicle (n=3). *p

    Article Snippet: Hepatic PGC-1α was immunoprecipitated from liver using anti-PGC-1α (Millipore, ST1202) in 1% Triton/TBS.

    Techniques: Pyrolysis Gas Chromatography, Expressing, Plasmid Preparation

    Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of Ucp1 , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of Ucp1 , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: In Vitro, Infection, shRNA, Staining, Concentration Assay, Expressing, Pyrolysis Gas Chromatography, Marker, Activation Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Construct, Reporter Assay, Isolation, Mouse Assay

    The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: Expressing, In Vitro, Isolation, Mouse Assay, Pyrolysis Gas Chromatography

    The association of IRX3 and human obesity. (A) Relative mRNA levels of IRX3 in oWAT or sWAT from normal weight ( n = 9) and obese subjects ( n = 21) measured by qPCR. Gene expression was normalized to βACTIN . (B) Comparison of the frequency of the IRX3 rare variants in normal weight subjects and obese patients. (C) Sequence conservation analysis of the IRX3 orthologs related to variants identified in obese subjects and controls. Mutant sites are shown with a red box. Dark gray represents residues that are completely conserved, mild gray partially conserved, and light gray shows similar residues. (D) Protein expression levels of WT and mutant IRX3 in HEK293T cells. GFP antibody was used to indicate IRX3, and actin antibody served as a loading control. (E) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector, WT or mutant IRX3 for 48 h ( n = 3). Normalized luciferase activities are shown as fold change and compared to WT IRX3 ( n = 4). oWAT, omental white adipose tissue. sWAT, subcutaneous white adipose tissue. Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: The association of IRX3 and human obesity. (A) Relative mRNA levels of IRX3 in oWAT or sWAT from normal weight ( n = 9) and obese subjects ( n = 21) measured by qPCR. Gene expression was normalized to βACTIN . (B) Comparison of the frequency of the IRX3 rare variants in normal weight subjects and obese patients. (C) Sequence conservation analysis of the IRX3 orthologs related to variants identified in obese subjects and controls. Mutant sites are shown with a red box. Dark gray represents residues that are completely conserved, mild gray partially conserved, and light gray shows similar residues. (D) Protein expression levels of WT and mutant IRX3 in HEK293T cells. GFP antibody was used to indicate IRX3, and actin antibody served as a loading control. (E) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector, WT or mutant IRX3 for 48 h ( n = 3). Normalized luciferase activities are shown as fold change and compared to WT IRX3 ( n = 4). oWAT, omental white adipose tissue. sWAT, subcutaneous white adipose tissue. Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    IRX3/Irx3 expression is induced in browning adipose tissues. (A) Relative mRNA levels of Ucp1 and Irx3 in IWAT from mice under cold stress (4 °C) for one week ( n = 6) and at 25 °C ( n = 8). (B–C) Relative mRNA levels of Ucp1 and Irx3 in IWAT (B) and BAT (C) from mice subjected to PBS or CL-316,243 for 10 days. For A–C, gene expression was normalized to 36b4 . (D–E) Protein levels of Ucp1 and Irx3 (left) and the quantification value of Irx3 relative to Hsp90 (right) in IWAT (D) and BAT (E) in the mice subjected to PBS or CL-316,243 treatment. (F) Representative images of immunohistochemical staining in IWAT from mice subjected to PBS or CL-316,243 treatment (scale bar, 50 μm for the upper and middle panels, and 100 μm for the bottom panel). (G) PET-CT, gross image, HE staining (scale bar, 20 μm), and electron microscopy (scale bar, 5 μm) of the browning WAT from pheochromocytoma patients. (H) Representative images of immunohistochemical staining in oWAT from pheochromocytoma patients (scale bar, 100 μm for the upper panel, and 50 μm for the bottom panel). Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: IRX3/Irx3 expression is induced in browning adipose tissues. (A) Relative mRNA levels of Ucp1 and Irx3 in IWAT from mice under cold stress (4 °C) for one week ( n = 6) and at 25 °C ( n = 8). (B–C) Relative mRNA levels of Ucp1 and Irx3 in IWAT (B) and BAT (C) from mice subjected to PBS or CL-316,243 for 10 days. For A–C, gene expression was normalized to 36b4 . (D–E) Protein levels of Ucp1 and Irx3 (left) and the quantification value of Irx3 relative to Hsp90 (right) in IWAT (D) and BAT (E) in the mice subjected to PBS or CL-316,243 treatment. (F) Representative images of immunohistochemical staining in IWAT from mice subjected to PBS or CL-316,243 treatment (scale bar, 50 μm for the upper and middle panels, and 100 μm for the bottom panel). (G) PET-CT, gross image, HE staining (scale bar, 20 μm), and electron microscopy (scale bar, 5 μm) of the browning WAT from pheochromocytoma patients. (H) Representative images of immunohistochemical staining in oWAT from pheochromocytoma patients (scale bar, 100 μm for the upper panel, and 50 μm for the bottom panel). Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining, Positron Emission Tomography, Electron Microscopy

    Regulation of: A , Cox4 ; B , Cpt-1β ; and C , Vegf mRNA by PGC-1α with and without STARS knockdown in C2C12 myotubes

    Journal: The Journal of Physiology

    Article Title: Striated muscle activator of Rho signalling (STARS) is a PGC-1?/oestrogen-related receptor-? target gene and is upregulated in human skeletal muscle after endurance exercise

    doi: 10.1113/jphysiol.2011.205468

    Figure Lengend Snippet: Regulation of: A , Cox4 ; B , Cpt-1β ; and C , Vegf mRNA by PGC-1α with and without STARS knockdown in C2C12 myotubes

    Article Snippet: For myotubes, total protein was extracted using 1× RIPA buffer (Millipore, North Ryde, NSW, Australia) with 1 μl ml−1 protease inhibitor cocktail (Sigma, Castle Hill, NSW, Australia) and 10 μl ml−1 Halt Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific, Rockford, IL, USA).

    Techniques: Cycling Probe Technology, Pyrolysis Gas Chromatography

    PGC-1α and ERRα bind an ERRE upstream of Stars in C2C12 myotubes

    Journal: The Journal of Physiology

    Article Title: Striated muscle activator of Rho signalling (STARS) is a PGC-1?/oestrogen-related receptor-? target gene and is upregulated in human skeletal muscle after endurance exercise

    doi: 10.1113/jphysiol.2011.205468

    Figure Lengend Snippet: PGC-1α and ERRα bind an ERRE upstream of Stars in C2C12 myotubes

    Article Snippet: For myotubes, total protein was extracted using 1× RIPA buffer (Millipore, North Ryde, NSW, Australia) with 1 μl ml−1 protease inhibitor cocktail (Sigma, Castle Hill, NSW, Australia) and 10 μl ml−1 Halt Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific, Rockford, IL, USA).

    Techniques: Pyrolysis Gas Chromatography

    Stars is regulated via the PGC-1α/ERRα transcriptional program in C2C12 myotubes

    Journal: The Journal of Physiology

    Article Title: Striated muscle activator of Rho signalling (STARS) is a PGC-1?/oestrogen-related receptor-? target gene and is upregulated in human skeletal muscle after endurance exercise

    doi: 10.1113/jphysiol.2011.205468

    Figure Lengend Snippet: Stars is regulated via the PGC-1α/ERRα transcriptional program in C2C12 myotubes

    Article Snippet: For myotubes, total protein was extracted using 1× RIPA buffer (Millipore, North Ryde, NSW, Australia) with 1 μl ml−1 protease inhibitor cocktail (Sigma, Castle Hill, NSW, Australia) and 10 μl ml−1 Halt Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific, Rockford, IL, USA).

    Techniques: Pyrolysis Gas Chromatography

    Acyltransferase time courses for enzymes extracted from Arabidopsis and spinach. (a, b) Cell‐wall enzymes were salt‐extracted from live cell cultures of (a) Arabidopsis and (b) spinach at various ages after subculturing, then dialysed, freeze dried and redissolved at 1% (w/v) in 10 m m PIPES (Na + , pH 7.0). The enzyme preparations (10 μl) were incubated with 50 μ m [ 14 C]oxalyl‐threonate (OxT) with (+) or without (−) 5% (w/v) glucose for 4 h. Products were separated by electrophoresis at pH 6.5 and autoradiographed (OxG, oxalyl‐glucose). Non‐radiolabelled markers (Glc, glucose; LA, lactobionic acid) were stained in silver nitrate. LA is a readily silver‐stainable compound possessing 12 C atoms and 1 − charge, and is thus a useful nearby‐migrating marker for substances such as oxalyl‐disaccharides (14 C atoms, 1 − ). Radioactive markers: OxA, oxalate; OxT, oxalyl‐threonate. Pencilled: OG, orange G. [The streaking of the [ 14 C]oxalate spots in Figures 5 and 6 (compare with Figure 4 ) was caused by omission of EDTA from the electrophoresis buffer. This streaking does not interfere with interpretation of the results.] [Colour figure can be viewed at http://www.wileyonlinelibrary.com ].

    Journal: The Plant Journal

    Article Title: Oxalyltransferase, a plant cell‐wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates

    doi: 10.1111/tpj.13984

    Figure Lengend Snippet: Acyltransferase time courses for enzymes extracted from Arabidopsis and spinach. (a, b) Cell‐wall enzymes were salt‐extracted from live cell cultures of (a) Arabidopsis and (b) spinach at various ages after subculturing, then dialysed, freeze dried and redissolved at 1% (w/v) in 10 m m PIPES (Na + , pH 7.0). The enzyme preparations (10 μl) were incubated with 50 μ m [ 14 C]oxalyl‐threonate (OxT) with (+) or without (−) 5% (w/v) glucose for 4 h. Products were separated by electrophoresis at pH 6.5 and autoradiographed (OxG, oxalyl‐glucose). Non‐radiolabelled markers (Glc, glucose; LA, lactobionic acid) were stained in silver nitrate. LA is a readily silver‐stainable compound possessing 12 C atoms and 1 − charge, and is thus a useful nearby‐migrating marker for substances such as oxalyl‐disaccharides (14 C atoms, 1 − ). Radioactive markers: OxA, oxalate; OxT, oxalyl‐threonate. Pencilled: OG, orange G. [The streaking of the [ 14 C]oxalate spots in Figures 5 and 6 (compare with Figure 4 ) was caused by omission of EDTA from the electrophoresis buffer. This streaking does not interfere with interpretation of the results.] [Colour figure can be viewed at http://www.wileyonlinelibrary.com ].

    Article Snippet: Fate of OxT, cOxT and OxG in living cell‐suspension cultures Spinach or Arabidopsis cell‐suspension culture (7 days old, unless otherwise stated) was filtered on four layers of Miracloth (Calbiochem), then triplicate mini‐cultures [each 250 mg (fresh weight) of cells resuspended in 500 μl of 7‐day culture medium in flat‐bottomed glass vials] were shaken at ~120 rpm in constant light for at least 1 h before the addition of [14 C]OxT or [14 C]OxA or [14 C]OxG (~200 Bq, in 1–5 μl) at ‘time 0’, to give a concentration of ~0.67 μm .

    Techniques: Subculturing Assay, Incubation, Electrophoresis, Gas Chromatography, Staining, Marker

    Formation of oxalyl esters with polysaccharide–cellulose complexes as acceptor substrates catalysed by Arabidopsis acyltransferase. Polysaccharide–cellulose complexes (polysaccharide‐impregnated paper; 1.5 × 2.0 cm) were incubated with Arabidopsis enzyme extract plus [ 14 C]oxalyl‐threonate at pH 7 (total liquid volume 35 μl) for 0–24 h. Controls lacked enzyme. (a–e) The polysaccharides tested were: (a) xylan, (b) xyloglucan, (c) homogalacturonan, (d) methyl‐esterified pectin, or (e) untreated paper (i.e., cellulose only). After the specified incubation times the papers were washed in 70% ethanol three or four times, and then the radioactivity present in the washes and in the final washed paper was assayed. Each sample containing enzyme was in triplicate, and the enzyme‐free controls were single samples.

    Journal: The Plant Journal

    Article Title: Oxalyltransferase, a plant cell‐wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates

    doi: 10.1111/tpj.13984

    Figure Lengend Snippet: Formation of oxalyl esters with polysaccharide–cellulose complexes as acceptor substrates catalysed by Arabidopsis acyltransferase. Polysaccharide–cellulose complexes (polysaccharide‐impregnated paper; 1.5 × 2.0 cm) were incubated with Arabidopsis enzyme extract plus [ 14 C]oxalyl‐threonate at pH 7 (total liquid volume 35 μl) for 0–24 h. Controls lacked enzyme. (a–e) The polysaccharides tested were: (a) xylan, (b) xyloglucan, (c) homogalacturonan, (d) methyl‐esterified pectin, or (e) untreated paper (i.e., cellulose only). After the specified incubation times the papers were washed in 70% ethanol three or four times, and then the radioactivity present in the washes and in the final washed paper was assayed. Each sample containing enzyme was in triplicate, and the enzyme‐free controls were single samples.

    Article Snippet: Fate of OxT, cOxT and OxG in living cell‐suspension cultures Spinach or Arabidopsis cell‐suspension culture (7 days old, unless otherwise stated) was filtered on four layers of Miracloth (Calbiochem), then triplicate mini‐cultures [each 250 mg (fresh weight) of cells resuspended in 500 μl of 7‐day culture medium in flat‐bottomed glass vials] were shaken at ~120 rpm in constant light for at least 1 h before the addition of [14 C]OxT or [14 C]OxA or [14 C]OxG (~200 Bq, in 1–5 μl) at ‘time 0’, to give a concentration of ~0.67 μm .

    Techniques: Incubation, Radioactivity

    Fate of [ 14 C]oxalyl‐threonate in Arabidopsis and spinach cell culture. Samples of culture medium (50 μl) collected from spinach or Arabidopsis cell mini‐cultures (250 mg cells plus 500 μl medium) or pH 4.5 buffer (cell free) that had been incubated with 0.67 μ m [ 14 C]OxT for 0–6 h were fractionated by electrophoresis at pH 6.5. Vertical lines indicate the positions of authentic markers oxalyl‐threonate (OxT) and oxalic acid (OxA).

    Journal: The Plant Journal

    Article Title: Oxalyltransferase, a plant cell‐wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates

    doi: 10.1111/tpj.13984

    Figure Lengend Snippet: Fate of [ 14 C]oxalyl‐threonate in Arabidopsis and spinach cell culture. Samples of culture medium (50 μl) collected from spinach or Arabidopsis cell mini‐cultures (250 mg cells plus 500 μl medium) or pH 4.5 buffer (cell free) that had been incubated with 0.67 μ m [ 14 C]OxT for 0–6 h were fractionated by electrophoresis at pH 6.5. Vertical lines indicate the positions of authentic markers oxalyl‐threonate (OxT) and oxalic acid (OxA).

    Article Snippet: Fate of OxT, cOxT and OxG in living cell‐suspension cultures Spinach or Arabidopsis cell‐suspension culture (7 days old, unless otherwise stated) was filtered on four layers of Miracloth (Calbiochem), then triplicate mini‐cultures [each 250 mg (fresh weight) of cells resuspended in 500 μl of 7‐day culture medium in flat‐bottomed glass vials] were shaken at ~120 rpm in constant light for at least 1 h before the addition of [14 C]OxT or [14 C]OxA or [14 C]OxG (~200 Bq, in 1–5 μl) at ‘time 0’, to give a concentration of ~0.67 μm .

    Techniques: Cell Culture, Incubation, Electrophoresis

    Oxalyl‐sugar formation in vitro with various donor and acceptor substrates (a) Three potential donor substrates with glucose as acceptor. Arabidopsis cell‐wall enzyme (1% w/v) was incubated with 50 μ m [ 14 C]OxT, [ 14 C]cOxT or [ 14 C]OxA, plus 5% (w/v) glucose (final volume 10 μl). Controls lacked glucose and/or enzyme. Products formed after 4 h were electrophoresed, then autoradiographed. Lanes labelled ‘sub’ (substrate) represent time‐0 samples. (b) Five potential acceptor substrates with oxalyl‐threonate as donor. Incubations as in (a) with 50 μ m [ 14 C]OxT plus 5% (w/v) of the potential acceptor substrate. After 4 h, products were analysed as in (a). Controls lacked acceptor substrate and/or enzyme. Non‐radioactive markers were stained in AgNO 3 . White arrows show the predicted electrophoretic mobilities of oxalyl‐XGOs (from top to bottom: Ox‐XXXG; Ox‐XXLG; Ox‐XLLG) relative to those of Ox‐Hex 2 and glucose, as estimated from Q:M r 2/3 values (see Figure 4 ). OxT, oxalyl‐threonate; cOxT, cyclic oxalyl‐threonate; Ox‐Glc, oxalyl‐glucose; Ox‐Hex, oxalyl‐hexose; Ox‐Hex 2 , oxalyl ester of a hexose disaccharide; Ox‐GlcN, O ‐oxalyl‐glucosamine; ThrO, threonate; LA, lactobionate; Glc, glucose; XGOs, xyloglucan oligosaccharides. [Colour figure can be viewed at http://www.wileyonlinelibrary.com ].

    Journal: The Plant Journal

    Article Title: Oxalyltransferase, a plant cell‐wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates

    doi: 10.1111/tpj.13984

    Figure Lengend Snippet: Oxalyl‐sugar formation in vitro with various donor and acceptor substrates (a) Three potential donor substrates with glucose as acceptor. Arabidopsis cell‐wall enzyme (1% w/v) was incubated with 50 μ m [ 14 C]OxT, [ 14 C]cOxT or [ 14 C]OxA, plus 5% (w/v) glucose (final volume 10 μl). Controls lacked glucose and/or enzyme. Products formed after 4 h were electrophoresed, then autoradiographed. Lanes labelled ‘sub’ (substrate) represent time‐0 samples. (b) Five potential acceptor substrates with oxalyl‐threonate as donor. Incubations as in (a) with 50 μ m [ 14 C]OxT plus 5% (w/v) of the potential acceptor substrate. After 4 h, products were analysed as in (a). Controls lacked acceptor substrate and/or enzyme. Non‐radioactive markers were stained in AgNO 3 . White arrows show the predicted electrophoretic mobilities of oxalyl‐XGOs (from top to bottom: Ox‐XXXG; Ox‐XXLG; Ox‐XLLG) relative to those of Ox‐Hex 2 and glucose, as estimated from Q:M r 2/3 values (see Figure 4 ). OxT, oxalyl‐threonate; cOxT, cyclic oxalyl‐threonate; Ox‐Glc, oxalyl‐glucose; Ox‐Hex, oxalyl‐hexose; Ox‐Hex 2 , oxalyl ester of a hexose disaccharide; Ox‐GlcN, O ‐oxalyl‐glucosamine; ThrO, threonate; LA, lactobionate; Glc, glucose; XGOs, xyloglucan oligosaccharides. [Colour figure can be viewed at http://www.wileyonlinelibrary.com ].

    Article Snippet: Fate of OxT, cOxT and OxG in living cell‐suspension cultures Spinach or Arabidopsis cell‐suspension culture (7 days old, unless otherwise stated) was filtered on four layers of Miracloth (Calbiochem), then triplicate mini‐cultures [each 250 mg (fresh weight) of cells resuspended in 500 μl of 7‐day culture medium in flat‐bottomed glass vials] were shaken at ~120 rpm in constant light for at least 1 h before the addition of [14 C]OxT or [14 C]OxA or [14 C]OxG (~200 Bq, in 1–5 μl) at ‘time 0’, to give a concentration of ~0.67 μm .

    Techniques: In Vitro, Incubation, Staining, Gas Chromatography