Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Key resources.

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Recombinant, Sequencing

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Expression and function of ABCC11. ( a ) Immunoblot detection of ABCC11 protein in the plasma membrane vesicles using an anti-ABCC11 antibody. Mock means plasma membrane vesicles that were prepared from control cells transfected with an empty pcDNA3.1/hyg(−) vector. Arrowhead: matured ABCC11 as an N -linked glycosylated protein. Na + /K + -ATPase (a plasma membrane protein) was used for a loading control. ( b ) [1,2,6,7- 3 H(N)]-dehydroepiandrosterone sulfate (DHEA-S) transport activities. Plasma membrane vesicles were incubated with or without ATP for 5 min. In this assay, all incubation mixtures contained 1% dimethyl sulfoxide (DMSO). Data are expressed as the mean ± SD; n = 3. Statistical analyses for significant differences were performed using Bartlett’s test, followed by a parametric Tukey–Kramer multiple-comparison test. Different letters indicate significant differences between groups ( p < 0.05).

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Expressing, Western Blot, Clinical Proteomics, Membrane, Control, Transfection, Plasmid Preparation, Incubation, Comparison

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Screening of inhibitory effects of various plant extracts on the transport activity of ABCC11. Inhibitory effect of each plant extract on the ABCC11-mediated [1,2,6,7- 3 H(N)]-DHEA-S transport activity was investigated by the vesicle transport assay. Plasma membrane vesicles (0.375 mg/mL in the reaction mixture) were incubated with the extract (100 ppm) in the presence of 50 μM [1,2,6,7- 3 H(N)]-DHEA-S for 5 min; 1% water was used for the vehicle control. Data are expressed as % of vehicle control, and they represent averages of two independent experiments.

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Activity Assay, Plant Extract, Vesicular Transport Assay, Clinical Proteomics, Membrane, Incubation, Control

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Separation scheme used to fractionate ABCC11 inhibitors in the soybean extract. In each separation step, the fraction with the highest ABCC11-inhibitory activity is colored in red. DP: dominant peak (details are described in Materials and Methods).

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Activity Assay

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: ABCC11-inhibitory activities for each fraction of soybean extract from the first separation step with preparative medium-pressure liquid chromatography (MPLC). ( a ) A preparative MPLC chromatogram for separating the water extract of soybeans. The chromatogram was recorded at 265 nm. Red line indicates linear gradients of solvent B (0.2% formic acid in acetonitrile). ( b ) ABCC11-inhibitory activity profile of each fraction (100 ppm) obtained from the first separation process. The effects on ABCC11-mediated [1,2,6,7- 3 H(N)]-DHEA-S transport activity were investigated by the vesicle transport assay; 1% methanol was used for the vehicle control. Data are expressed as % of vehicle and the mean ± SD; n = 3. **, p < 0.01 vs. control (Dunnett’s test).

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Liquid Chromatography, Solvent, Activity Assay, Vesicular Transport Assay, Control

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Isolation of an ABCC11-inhibitory ingredient by means of recycling preparative HPLC. ( a ) Recycling preparative HPLC chromatograms for the separation of fractions Fr.#11-5-1 and Fr.#11-5-2. The upper chromatogram was recorded with a refractive index detector, and the lower one was recorded with a diode array and multiple-wavelength detector at 254 nm. After separation under the recycling mode (0–120 min), the mode was changed; Fr.#11-5-1 (123–126 min) and Fr.#11-5-2 (160–176 min) were collected, and all the wastes were collected and further processed as Fr.#11-5-3. R, recycled peaks for Fr.#11-5-1; r, recycled peaks for Fr.#11-5-2. ( b ) ABCC11-inhibitory activities of each subfraction (20 ppm) in terms of ABCC11-mediated [1,2,6,7- 3 H(N)]-DHEA-S transport activity measured by the vesicle transport assay; 1% DMSO was used for the vehicle control. Data are expressed as % of vehicle and the mean ± SD; n = 3. **, p < 0.01 vs. control (Dunnett’s test). ( c ) Purity verification of the isolated ingredient in Fr.#11-5-2 by spectrometric analyses. Left: UV chromatograms recorded at 265 nm. Right: LC-quadrupole time-of-flight-MS (LC-Q-TOF-MS) base peak chromatograms, excluding peaks derived from the plasticizing materials and injected solvent. †, a specific peak in Fr.#11-5-2 with a retention time of 5.83 min. ( d ) Full scan mass spectrum obtained in the positive ion mode of this peak (indicated by † in c ) at 5.83 min. The inset is the magnified view for ions at m / z 271.0616 and 293.0428, which corresponded to the [M + H] + and [M + Na] + of the target constituent, respectively.

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Isolation, Refractive Index, Activity Assay, Vesicular Transport Assay, Control, Derivative Assay, Injection, Solvent

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Chemical characterization of an ABCC11 inhibitory activity-guided fraction from soybean extract. Fraction (Fr.) #11-5-2 (upper panels) and authentic genistein (lower panels) were analyzed by a high-performance liquid chromatography instrument coupled with a diode array and multiple wavelength detector (DAD) and Q-TOF-MS system. ( a ) Chemical structure of genistein. ( b ) Extracted ion chromatograms (EICs) with a single peak at m / z 271.0621 in the positive ESI spectrum. ( c ) MS spectrums with a retention time of 5.83 min for the parent ion. ( d ) DAD spectrums. ( e ) Information on the fragment ions derived from MS/MS analyses.

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Activity Assay, High Performance Liquid Chromatography, Derivative Assay, Tandem Mass Spectroscopy

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: Effects of soybean flavonoids and their metabolites on the transport activity of ABCC11. ( a ) Chemical structures. Gein-7G-4’S, genistein 7-β-D-glucuronide 4’-sulfate; dein-7G-4’S, daidzein 7-β-D-glucuronide 4’-sulfate. ( b ) Inhibitory effects of each flavonoid (100 μM) on ABCC11-mediated [1,2,6,7- 3 H(N)]-DHEA-S transport. ( c ) Concentration-dependent inhibition of ABCC11-mediated DHEA-S transport by genistein. Data are expressed as % of vehicle and the mean ± SD; n = 3–6. **, p < 0.01 vs. control (Dunnett’s test).

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Activity Assay, Concentration Assay, Inhibition, Control

Journal: Nutrients

Article Title: Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids

doi: 10.3390/nu12082452

Figure Lengend Snippet: ABCC11-inhibitory activities of dietary flavonoids.

Article Snippet: Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond ® ECL TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na + /K + -ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively.

Techniques: Control