nadph  (Millipore)


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    Name:
    NADPH
    Description:
    Amorphous powder bottled under nitrogen
    Catalog Number:
    nadph-ro
    Price:
    None
    Applications:
    NADPH-RO has been used for the determination of thioredoxin reductase activity.
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    Structured Review

    Millipore nadph
    NADPH
    Amorphous powder bottled under nitrogen
    https://www.bioz.com/result/nadph/product/Millipore
    Average 99 stars, based on 128 article reviews
    Price from $9.99 to $1999.99
    nadph - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "GlnR Activation Induces Peroxide Resistance in Mycobacterial Biofilms"

    Article Title: GlnR Activation Induces Peroxide Resistance in Mycobacterial Biofilms

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01428

    Role of gpr in maintenance of redox homeostasis of M. smegmatis. (A,B) Levels of oxidized (NADP) form of nicotinamide adenine dinucleotide phosphate in biofilms of wild-type, Δ gpr and the complemented strains cultured in normal Sauton’s medium. (C) Ratio of NADPH to NADP calculated from (A,B) . Data represent mean of three biologically independent experiments ∗ , ∗∗ , and ∗∗∗ indicate p ( t -test)
    Figure Legend Snippet: Role of gpr in maintenance of redox homeostasis of M. smegmatis. (A,B) Levels of oxidized (NADP) form of nicotinamide adenine dinucleotide phosphate in biofilms of wild-type, Δ gpr and the complemented strains cultured in normal Sauton’s medium. (C) Ratio of NADPH to NADP calculated from (A,B) . Data represent mean of three biologically independent experiments ∗ , ∗∗ , and ∗∗∗ indicate p ( t -test)

    Techniques Used: Cell Culture

    2) Product Images from "Inverted stereocontrol of iridoid synthase in snapdragon"

    Article Title: Inverted stereocontrol of iridoid synthase in snapdragon

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.800979

    Identification of AmISY. a , phylogenetic tree of iridoid synthase homologues in A. majus ( Am ), Olea europaea ( Oe ), C. roseus ( Cr ), Nepeta cataria ( Nc ), and N. mussinii ( Nm ). The neighbor joining tree was built from a MuscleWS alignment using the BLOSUM62 similarity matrix in Jalview 2.10.1 ( 24 ). Numbers next to the nodes indicate evolutionary distances. Proteins with proven iridoid synthase activity are highlighted in bold font . One of the A. majus homologues (AmISY or Am18679) groups closely with the iridoid synthases from O. europaea and C. roseus. b , SDS-PAGE of nickel affinity- and gel filtration chromatography–purified AmISY. c , the 8-oxogeranial–dependent NADPH consumption of AmISY showed catalytic parameters close to those of CrISY at a fixed NADPH concentration of 50 μ m (AmISY: k cat = 0.72 ± 0.02 s −1 , K m = 1.1 ± 0.1 μ m ; CrISY: k cat = 1.6 ± 0.1 s −1 , K m = 4.5 ± 0.2 μ m ). Values are given as the mean ± S.D. of two independent measurements with different batches of protein. d , qRT-PCR shows tissue-dependent expression of ISY homologues in A. majus . Abundance of the Am29566 transcript was too low for quantification in all tissues. Expression values are given as the mean ± S.D. (four reactions). Each gene was separately normalized to the tissue with the highest expression level. Two replicates each were analyzed for two independent samples of cDNA.
    Figure Legend Snippet: Identification of AmISY. a , phylogenetic tree of iridoid synthase homologues in A. majus ( Am ), Olea europaea ( Oe ), C. roseus ( Cr ), Nepeta cataria ( Nc ), and N. mussinii ( Nm ). The neighbor joining tree was built from a MuscleWS alignment using the BLOSUM62 similarity matrix in Jalview 2.10.1 ( 24 ). Numbers next to the nodes indicate evolutionary distances. Proteins with proven iridoid synthase activity are highlighted in bold font . One of the A. majus homologues (AmISY or Am18679) groups closely with the iridoid synthases from O. europaea and C. roseus. b , SDS-PAGE of nickel affinity- and gel filtration chromatography–purified AmISY. c , the 8-oxogeranial–dependent NADPH consumption of AmISY showed catalytic parameters close to those of CrISY at a fixed NADPH concentration of 50 μ m (AmISY: k cat = 0.72 ± 0.02 s −1 , K m = 1.1 ± 0.1 μ m ; CrISY: k cat = 1.6 ± 0.1 s −1 , K m = 4.5 ± 0.2 μ m ). Values are given as the mean ± S.D. of two independent measurements with different batches of protein. d , qRT-PCR shows tissue-dependent expression of ISY homologues in A. majus . Abundance of the Am29566 transcript was too low for quantification in all tissues. Expression values are given as the mean ± S.D. (four reactions). Each gene was separately normalized to the tissue with the highest expression level. Two replicates each were analyzed for two independent samples of cDNA.

    Techniques Used: Activity Assay, SDS Page, Filtration, Chromatography, Purification, Concentration Assay, Quantitative RT-PCR, Expressing

    3) Product Images from "A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis"

    Article Title: A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis

    Journal: RNA

    doi: 10.1261/rna.7370104

    GPx-1 and TR levels are influenced by selenium and tRNA [Ser]Sec levels. CHO cells overexpressing tRNA [Ser]Sec (ST4) or a dominant-negative A37G tRNA [Ser]Sec mutant whose expression results in reduced levels of mcm 5 Um (i 6 A − ) were grown in standard medium or medium supplemented with 30 nM selenium for 3 d and the indicated selenoprotein levels were determined. Values are the average of three independent lysates ± standard deviation. ( A ) Glutathione peroxidase activity was measured by a coupled spectrophotometric assay and activity is expressed as nanomoles NADPH per minute per milligram of protein. ( B ) Thioredoxin reductase activity was measured from partially purified protein extracts by the reduction of sulfhydryl groups in DNTB. Activity is expressed as micromoles of NTB reduced per micromole of protein.
    Figure Legend Snippet: GPx-1 and TR levels are influenced by selenium and tRNA [Ser]Sec levels. CHO cells overexpressing tRNA [Ser]Sec (ST4) or a dominant-negative A37G tRNA [Ser]Sec mutant whose expression results in reduced levels of mcm 5 Um (i 6 A − ) were grown in standard medium or medium supplemented with 30 nM selenium for 3 d and the indicated selenoprotein levels were determined. Values are the average of three independent lysates ± standard deviation. ( A ) Glutathione peroxidase activity was measured by a coupled spectrophotometric assay and activity is expressed as nanomoles NADPH per minute per milligram of protein. ( B ) Thioredoxin reductase activity was measured from partially purified protein extracts by the reduction of sulfhydryl groups in DNTB. Activity is expressed as micromoles of NTB reduced per micromole of protein.

    Techniques Used: Size-exclusion Chromatography, Dominant Negative Mutation, Mutagenesis, Expressing, Standard Deviation, Activity Assay, Spectrophotometric Assay, Purification

    4) Product Images from "Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis *"

    Article Title: Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.584185

    FabI·NADH·AFN ternary complex structure. A , Ct FabI·NADH·AFN-152 ternary complex (Protein Data Bank accession code 4Q9N ). B , Sa FabI·NADPH·AFN-1252 ternary complex (Protein Data Bank accession code 4FS3 ).
    Figure Legend Snippet: FabI·NADH·AFN ternary complex structure. A , Ct FabI·NADH·AFN-152 ternary complex (Protein Data Bank accession code 4Q9N ). B , Sa FabI·NADPH·AFN-1252 ternary complex (Protein Data Bank accession code 4FS3 ).

    Techniques Used:

    5) Product Images from "Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction"

    Article Title: Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction

    Journal: Scientific Reports

    doi: 10.1038/srep27995

    PMs induce NADPH oxidase activation, generation of reactive oxygen species (ROS) and p47 phox translocation in HaCaT cells. ( A ) Cells were exposed to PMs (50 μg/cm 2 ) for the indicated times (0, 5′, 10′, 30′, 1 h and 2 h). ( B ) Cells were pre-incubated with AhRI (10 and 30 μM), APO (100 μM) or NAC (100 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. Effects of PMs and antioxidants on ROS generation (open bars) and NADPH oxidase activity (shaded bars) were measured using DCF-DA fluorescence and lucigenin chemiluminescence as described in “Materials and methods” (n = 3 in each group; *P
    Figure Legend Snippet: PMs induce NADPH oxidase activation, generation of reactive oxygen species (ROS) and p47 phox translocation in HaCaT cells. ( A ) Cells were exposed to PMs (50 μg/cm 2 ) for the indicated times (0, 5′, 10′, 30′, 1 h and 2 h). ( B ) Cells were pre-incubated with AhRI (10 and 30 μM), APO (100 μM) or NAC (100 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. Effects of PMs and antioxidants on ROS generation (open bars) and NADPH oxidase activity (shaded bars) were measured using DCF-DA fluorescence and lucigenin chemiluminescence as described in “Materials and methods” (n = 3 in each group; *P

    Techniques Used: Activation Assay, Translocation Assay, Incubation, Activity Assay, Fluorescence

    6) Product Images from "Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes"

    Article Title: Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18050952

    Inhibitory effects of dimethyllirioresinol on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with dimethyllirioresinol and reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) for 30 min at 37 °C and ●: No pre-incubation. Data represent the average ± standard deviation (SD, n = 3).
    Figure Legend Snippet: Inhibitory effects of dimethyllirioresinol on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with dimethyllirioresinol and reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) for 30 min at 37 °C and ●: No pre-incubation. Data represent the average ± standard deviation (SD, n = 3).

    Techniques Used: Incubation, Standard Deviation

    Inhibitory effects of magnolin on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with magnolin and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).
    Figure Legend Snippet: Inhibitory effects of magnolin on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with magnolin and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).

    Techniques Used: Incubation

    Inhibitory effects of epimagnolin A on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with epimagnolin A and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).
    Figure Legend Snippet: Inhibitory effects of epimagnolin A on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with epimagnolin A and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).

    Techniques Used: Incubation

    Inhibitory effects of eudesmin on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with eudesmin and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).
    Figure Legend Snippet: Inhibitory effects of eudesmin on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with eudesmin and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).

    Techniques Used: Incubation

    Inhibitory effects of fargesin on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1’-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with fargesin and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).
    Figure Legend Snippet: Inhibitory effects of fargesin on CYP1A2-mediated phenacetin O -deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, CYP2C8-mediated amodiaquine N -deethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [ S ]-mephenytoin 4′-hydroxylation, CYP2D6-mediated bufuralol 1’-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation in pooled human liver microsomes. ○: Pre-incubation of liver microsomes with fargesin and NADPH for 30 min at 37 °C, ●: No pre-incubation. Data represent the average ± SD ( n = 3).

    Techniques Used: Incubation

    7) Product Images from "Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene"

    Article Title: Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2018.01.009

    Lipid content, oxidative stress and apoptosis in the heart. The hearts of 6 month-old mice (n = 5/genotype) were assayed in triplicate for (A) triacylglycerol level; (B) fatty acid synthase (FASN) activity by [ 14 C]-malonyl-CoA incorporation; (C i ) NO content and (C ii ) NADP/NADPH as measure of oxidative stress. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice. (D) Heart lysates were analyzed by Western blot using caspase 3 antibody that detected the cleaved fragment of ∼20 kDa apparent molecular weight. Gels represent 2 separate experiments performed on different mice/genotype/treatment.
    Figure Legend Snippet: Lipid content, oxidative stress and apoptosis in the heart. The hearts of 6 month-old mice (n = 5/genotype) were assayed in triplicate for (A) triacylglycerol level; (B) fatty acid synthase (FASN) activity by [ 14 C]-malonyl-CoA incorporation; (C i ) NO content and (C ii ) NADP/NADPH as measure of oxidative stress. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice. (D) Heart lysates were analyzed by Western blot using caspase 3 antibody that detected the cleaved fragment of ∼20 kDa apparent molecular weight. Gels represent 2 separate experiments performed on different mice/genotype/treatment.

    Techniques Used: Mouse Assay, Activity Assay, Western Blot, Molecular Weight

    8) Product Images from "Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments"

    Article Title: Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagen.2013.11.018

    A) Diagram of the RexYFP structure. RexYFP consists of cpYFP (yellow) integrated between residues 79 and 80 of T-Rex (blue) via short polypeptide linkers SAG and GT (red). The diagram shows mutations in the structure of RexYFP (numbers in parentheses indicate the position for EYFP). B) Fluorescence spectra of RexYFP. Excitation spectrum has a maximum at 490 nm. Emission spectrum has a maximum at 516 nm. C) Excitation spectrum of RexYFP (250 nM) in Tris–HCl (pH 7.5) with 150 mM NaCl and 10 mM MgCl 2 upon addition of NADH (50, 250, 1000 nM) to the probe. Emission was measured at 530 nm. D) Dependence of RexYFP signal on concentrations of various nucleotides (NAD + , NADH, NADPH, ATP) in range of concentration from 10 nM to 50 μM in the probe (Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 ). The RexYFP signal is expressed as 1/F490. Plotted line for each type of nucleotide is the result of five independent experiments.
    Figure Legend Snippet: A) Diagram of the RexYFP structure. RexYFP consists of cpYFP (yellow) integrated between residues 79 and 80 of T-Rex (blue) via short polypeptide linkers SAG and GT (red). The diagram shows mutations in the structure of RexYFP (numbers in parentheses indicate the position for EYFP). B) Fluorescence spectra of RexYFP. Excitation spectrum has a maximum at 490 nm. Emission spectrum has a maximum at 516 nm. C) Excitation spectrum of RexYFP (250 nM) in Tris–HCl (pH 7.5) with 150 mM NaCl and 10 mM MgCl 2 upon addition of NADH (50, 250, 1000 nM) to the probe. Emission was measured at 530 nm. D) Dependence of RexYFP signal on concentrations of various nucleotides (NAD + , NADH, NADPH, ATP) in range of concentration from 10 nM to 50 μM in the probe (Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 ). The RexYFP signal is expressed as 1/F490. Plotted line for each type of nucleotide is the result of five independent experiments.

    Techniques Used: Fluorescence, Concentration Assay

    9) Product Images from "Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase"

    Article Title: Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.274571

    Reduction of the Fe/S clusters of complex I and the E183H F variant by nucleotides. Complex I was reduced with a 1,000-fold molar excess of both NADH ( trace a ) and NADPH ( trace b ). The E183H F variant was reduced with a 1,000-fold molar excess NADPH ( trace c ). The spectra in A were recorded at 40 K and 2 milliwatts of microwave power, and the spectra in B were recorded at 13 K and 5 milliwatts of microwave power. Other EPR conditions were as follows: microwave frequency, 9.44 GHz; modulation amplitude, 0.6 mT; time constant: 0.124 s; scan rate: 17.9 mT/min.
    Figure Legend Snippet: Reduction of the Fe/S clusters of complex I and the E183H F variant by nucleotides. Complex I was reduced with a 1,000-fold molar excess of both NADH ( trace a ) and NADPH ( trace b ). The E183H F variant was reduced with a 1,000-fold molar excess NADPH ( trace c ). The spectra in A were recorded at 40 K and 2 milliwatts of microwave power, and the spectra in B were recorded at 13 K and 5 milliwatts of microwave power. Other EPR conditions were as follows: microwave frequency, 9.44 GHz; modulation amplitude, 0.6 mT; time constant: 0.124 s; scan rate: 17.9 mT/min.

    Techniques Used: Variant Assay, Electron Paramagnetic Resonance

    10) Product Images from "Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene"

    Article Title: Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2018.01.009

    Lipid content, oxidative stress and apoptosis in the heart. The hearts of 6 month-old mice (n = 5/genotype) were assayed in triplicate for (A) triacylglycerol level; (B) fatty acid synthase (FASN) activity by [ 14 C]-malonyl-CoA incorporation; (C i ) NO content and (C ii ) NADP/NADPH as measure of oxidative stress. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice. (D) Heart lysates were analyzed by Western blot using caspase 3 antibody that detected the cleaved fragment of ∼20 kDa apparent molecular weight. Gels represent 2 separate experiments performed on different mice/genotype/treatment.
    Figure Legend Snippet: Lipid content, oxidative stress and apoptosis in the heart. The hearts of 6 month-old mice (n = 5/genotype) were assayed in triplicate for (A) triacylglycerol level; (B) fatty acid synthase (FASN) activity by [ 14 C]-malonyl-CoA incorporation; (C i ) NO content and (C ii ) NADP/NADPH as measure of oxidative stress. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice. (D) Heart lysates were analyzed by Western blot using caspase 3 antibody that detected the cleaved fragment of ∼20 kDa apparent molecular weight. Gels represent 2 separate experiments performed on different mice/genotype/treatment.

    Techniques Used: Mouse Assay, Activity Assay, Western Blot, Molecular Weight

    11) Product Images from "The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria *"

    Article Title: The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.730473

    Assessment of the NADPH-supported t -BOOH metabolism rate via the autofluorescence of NAD(P) in suspensions of isolated mitochondria. At the beginning (∼20 s) of the representative traces shown in A–C , mitochondria were added (1 mg/ml)
    Figure Legend Snippet: Assessment of the NADPH-supported t -BOOH metabolism rate via the autofluorescence of NAD(P) in suspensions of isolated mitochondria. At the beginning (∼20 s) of the representative traces shown in A–C , mitochondria were added (1 mg/ml)

    Techniques Used: Isolation

    t -BOOH metabolism supported by NADPH is dependent on the combination of substrates fed to Nnt −/− mitochondria: Nnt +/− mice mitochondria perform as Nnt +/+ . The autofluorescence of reduced NAD(P) was continuously monitored in the
    Figure Legend Snippet: t -BOOH metabolism supported by NADPH is dependent on the combination of substrates fed to Nnt −/− mitochondria: Nnt +/− mice mitochondria perform as Nnt +/+ . The autofluorescence of reduced NAD(P) was continuously monitored in the

    Techniques Used: Mouse Assay

    Independently of NNT, malate or glutamate alone can sustain NADPH-supported t -BOOH metabolism during oxidative phosphorylation: possible contributions of NADP-ME and GDH. The autofluorescence of reduced NAD(P) was continuously monitored in the representative
    Figure Legend Snippet: Independently of NNT, malate or glutamate alone can sustain NADPH-supported t -BOOH metabolism during oxidative phosphorylation: possible contributions of NADP-ME and GDH. The autofluorescence of reduced NAD(P) was continuously monitored in the representative

    Techniques Used:

    Graphical summary of the rate of t -BOOH metabolism and its relative dependence on NNT as the NADPH source under different experimental conditions. This figure summarizes the effects of the three mitochondrial respiratory states (non-phosphorylating, respiration
    Figure Legend Snippet: Graphical summary of the rate of t -BOOH metabolism and its relative dependence on NNT as the NADPH source under different experimental conditions. This figure summarizes the effects of the three mitochondrial respiratory states (non-phosphorylating, respiration

    Techniques Used:

    Mitochondrial oxidative phosphorylation induced by ADP decreases the contribution of NNT to NADPH-supported t -BOOH metabolism. The autofluorescence of reduced NAD(P) was continuously monitored in the representative experiments shown in A and B . Isolated
    Figure Legend Snippet: Mitochondrial oxidative phosphorylation induced by ADP decreases the contribution of NNT to NADPH-supported t -BOOH metabolism. The autofluorescence of reduced NAD(P) was continuously monitored in the representative experiments shown in A and B . Isolated

    Techniques Used: Isolation

    12) Product Images from "Inhibition of Bupropion Metabolism by Selegiline: Mechanism-Based Inactivation of Human CYP2B6 and Characterization of Glutathione and Peptide Adducts"

    Article Title: Inhibition of Bupropion Metabolism by Selegiline: Mechanism-Based Inactivation of Human CYP2B6 and Characterization of Glutathione and Peptide Adducts

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.112.046979

    LC-MS/MS analysis of the GSH adduct obtained when selegiline was incubated with CYP2B6 in the presence of NADPH. CYP2B6 was reconstituted with reductase and incubated with selegiline and GSH. The separation and analysis of the adduct were as described
    Figure Legend Snippet: LC-MS/MS analysis of the GSH adduct obtained when selegiline was incubated with CYP2B6 in the presence of NADPH. CYP2B6 was reconstituted with reductase and incubated with selegiline and GSH. The separation and analysis of the adduct were as described

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Incubation

    Time- and concentration-dependent inactivation of CYP2B6 by selegiline. A, purified recombinant CYP2B6 reconstituted with reductase was incubated with various concentrations of selegiline and NADPH (1.2 mM) at 30°C. Aliquots of the mixture were
    Figure Legend Snippet: Time- and concentration-dependent inactivation of CYP2B6 by selegiline. A, purified recombinant CYP2B6 reconstituted with reductase was incubated with various concentrations of selegiline and NADPH (1.2 mM) at 30°C. Aliquots of the mixture were

    Techniques Used: Concentration Assay, Purification, Recombinant, Incubation

    LC-MS/MS spectrum of the CYP2B6 tryptic peptide modified by selegiline. P4502B6 was reconstituted with reductase and selegiline in the presence or absence of NADPH and subsequently analyzed by LC-MS/MS and Sequest software as described under Materials
    Figure Legend Snippet: LC-MS/MS spectrum of the CYP2B6 tryptic peptide modified by selegiline. P4502B6 was reconstituted with reductase and selegiline in the presence or absence of NADPH and subsequently analyzed by LC-MS/MS and Sequest software as described under Materials

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Software

    13) Product Images from "Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD"

    Article Title: Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12139-3

    Metabolic Analysis of STO-609 in Human Liver Microsomes. ( A) Representative MS/MS spectra of commercial STO-609 (STO-609 C ) metabolites M1 (purple), M2 (blue) and M3 (green) compared to that of STO-609 C (black). (B) Chromatograms of STO-609 C (red) and its metabolites (M1 (purple), M2 (blue) and M3 (green)) following incubation with human liver microsomes (HLM). Triplicate incubations were conducted in 1X PBS (pH 7.4) containing STO-609 C (30 µM), HLM (1.0 mg/ml) with or without NADPH (1.0 mM). The metabolites were quantified by UHPLC-QTOFMS. The accompanying inset shows the representative amount of each STO-609 metabolite calculated as a percent of total STO-609 C . (C) Comparison of STO-609 C metabolism in human (HLM) versus mouse liver microsomes (MLM). The metabolites were quantified by UHPLC-QTOFMS. Data are graphed as the representative amount of each STO-609 metabolite calculated as a percent of total STO-609 C . (D) cDNA-expressed P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) were used to determine the role of individual CYP450 enzymes in the formation of STO-609 metabolites. All samples were analyzed by UHPLC-QTOFMS. Each STO-609 metabolite is represented as a percent of total STO-609 C : M1 (purple), M2 (blue) and M3 (green). Conditions where no metabolites of STO-609 were identified are denoted as 100% of unmetabolized STO-609 C (red). (E) Validation of CYP1A2 in the formation of M1, M2 and M3 metabolites of STO-609 C . Incubations were conducted in 1X PBS (pH 7.4) containing STO-609 C (30 µM), α-naphthoflavone (CYP1A2 inhibitor, 6.0 µM), HLM (1.0 mg/ml) and NADPH (1.0 mM). Metabolites were identified by UHPLC-QTOFMS. The peak area of the M1, M2 and M3 metabolites generated from the incubation of STO-609 C with HLM in the absence of α-naphthoflavone was set as 100%. All data are expressed as mean ± s.e.m. (n = 3). **P
    Figure Legend Snippet: Metabolic Analysis of STO-609 in Human Liver Microsomes. ( A) Representative MS/MS spectra of commercial STO-609 (STO-609 C ) metabolites M1 (purple), M2 (blue) and M3 (green) compared to that of STO-609 C (black). (B) Chromatograms of STO-609 C (red) and its metabolites (M1 (purple), M2 (blue) and M3 (green)) following incubation with human liver microsomes (HLM). Triplicate incubations were conducted in 1X PBS (pH 7.4) containing STO-609 C (30 µM), HLM (1.0 mg/ml) with or without NADPH (1.0 mM). The metabolites were quantified by UHPLC-QTOFMS. The accompanying inset shows the representative amount of each STO-609 metabolite calculated as a percent of total STO-609 C . (C) Comparison of STO-609 C metabolism in human (HLM) versus mouse liver microsomes (MLM). The metabolites were quantified by UHPLC-QTOFMS. Data are graphed as the representative amount of each STO-609 metabolite calculated as a percent of total STO-609 C . (D) cDNA-expressed P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) were used to determine the role of individual CYP450 enzymes in the formation of STO-609 metabolites. All samples were analyzed by UHPLC-QTOFMS. Each STO-609 metabolite is represented as a percent of total STO-609 C : M1 (purple), M2 (blue) and M3 (green). Conditions where no metabolites of STO-609 were identified are denoted as 100% of unmetabolized STO-609 C (red). (E) Validation of CYP1A2 in the formation of M1, M2 and M3 metabolites of STO-609 C . Incubations were conducted in 1X PBS (pH 7.4) containing STO-609 C (30 µM), α-naphthoflavone (CYP1A2 inhibitor, 6.0 µM), HLM (1.0 mg/ml) and NADPH (1.0 mM). Metabolites were identified by UHPLC-QTOFMS. The peak area of the M1, M2 and M3 metabolites generated from the incubation of STO-609 C with HLM in the absence of α-naphthoflavone was set as 100%. All data are expressed as mean ± s.e.m. (n = 3). **P

    Techniques Used: Mass Spectrometry, Incubation, Generated

    14) Product Images from "Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling"

    Article Title: Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101363

    Signaling of p38 MAPK/AP-1 modulated by the ROS level in G6PD -kd THP-1 cells. (A) Flow cytometry for determining the ROS production in PMA-differentiated control THP-1 cells and G6PD -kd THP-1 cells. Cells were treated with LPS for 3 h and nigericin for 30 min. Cells were stained with 10 μM CM-H 2 DCFDA for 30 min. (B) Superoxide production was measured by cytochrome c reduction. The PMA-differentiated control and G6PD -kd THP-1 cells were incubated in cytochrome c containing HBSS with 0.1 μg/ml LPS. The production of superoxide was measured spectrophotometrically at 550 nm. The PMA-differentiated control and G6PD -kd THP-1 cells were treated with (C, E) DPI, (G, I) H 2 O 2 or NADPH for 60 min prior to LPS treatment for 30 min for p38 detection or 180 min for pro-IL-1β detection. The expression of p-p38, p38 and pro-IL-1β was determined by a Western blot. (D), (F) p-p38 and pro-IL-1β quantitative levels of (C) and (E). (H), (J) p-p38 and pro-IL-1β quantitative levels of (G) and (I). β-Actin was used as a loading control. The results are representative of three independent experiments (n = 3, * p
    Figure Legend Snippet: Signaling of p38 MAPK/AP-1 modulated by the ROS level in G6PD -kd THP-1 cells. (A) Flow cytometry for determining the ROS production in PMA-differentiated control THP-1 cells and G6PD -kd THP-1 cells. Cells were treated with LPS for 3 h and nigericin for 30 min. Cells were stained with 10 μM CM-H 2 DCFDA for 30 min. (B) Superoxide production was measured by cytochrome c reduction. The PMA-differentiated control and G6PD -kd THP-1 cells were incubated in cytochrome c containing HBSS with 0.1 μg/ml LPS. The production of superoxide was measured spectrophotometrically at 550 nm. The PMA-differentiated control and G6PD -kd THP-1 cells were treated with (C, E) DPI, (G, I) H 2 O 2 or NADPH for 60 min prior to LPS treatment for 30 min for p38 detection or 180 min for pro-IL-1β detection. The expression of p-p38, p38 and pro-IL-1β was determined by a Western blot. (D), (F) p-p38 and pro-IL-1β quantitative levels of (C) and (E). (H), (J) p-p38 and pro-IL-1β quantitative levels of (G) and (I). β-Actin was used as a loading control. The results are representative of three independent experiments (n = 3, * p

    Techniques Used: Flow Cytometry, Cytometry, Staining, Incubation, Expressing, Western Blot

    15) Product Images from "Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense"

    Article Title: Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense

    Journal: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology

    doi: 10.1016/j.cbpb.2019.04.005

    The effect of tyrosine acetylation on metMb peroxidase activity. A. The peroxidase activity of acetylated-metMb was measured with respect to NADPH, resveratrol and TMB. Reactions consisted of 1 μM metMb or acetyl-metMb, 200 μM H 2 O 2 and 250 μM NADPH/resveratrol or 500 μM TMB. The activity of acetyl-metMb was divided by the activity of metMb (n=6). Error bars represent standard error of the mean. An ANOVA was performed for statistical analyses. * P≤0.05 compared to control. B. The effect of tyrosine on ferrylMb autoreduction was assessed spectrophotometrically. Oxidized metMb or acetylmetMb (7 μM) were incubated for 42 minutes and auto-reduction was monitored via absorbance increase at 408 nm (n=6). Error bars represent standard error of the mean. An ANOVA was performed for statistical analyses. * P≤0.05 compared to control.
    Figure Legend Snippet: The effect of tyrosine acetylation on metMb peroxidase activity. A. The peroxidase activity of acetylated-metMb was measured with respect to NADPH, resveratrol and TMB. Reactions consisted of 1 μM metMb or acetyl-metMb, 200 μM H 2 O 2 and 250 μM NADPH/resveratrol or 500 μM TMB. The activity of acetyl-metMb was divided by the activity of metMb (n=6). Error bars represent standard error of the mean. An ANOVA was performed for statistical analyses. * P≤0.05 compared to control. B. The effect of tyrosine on ferrylMb autoreduction was assessed spectrophotometrically. Oxidized metMb or acetylmetMb (7 μM) were incubated for 42 minutes and auto-reduction was monitored via absorbance increase at 408 nm (n=6). Error bars represent standard error of the mean. An ANOVA was performed for statistical analyses. * P≤0.05 compared to control.

    Techniques Used: Activity Assay, Incubation

    pH dependence of metMb peroxidase activity using NAD(P)H as substrates. MetMb peroxidase activity was measured at various pH values in the presence of 588 μM H 2 O 2 and 250 μM NADH (A) or NADPH (B) (n=10). Error bars represent standard error of the mean. An ANOVA with repeated measures was performed for both figures A and B. * P≤0.05 compared to pH 7.4.
    Figure Legend Snippet: pH dependence of metMb peroxidase activity using NAD(P)H as substrates. MetMb peroxidase activity was measured at various pH values in the presence of 588 μM H 2 O 2 and 250 μM NADH (A) or NADPH (B) (n=10). Error bars represent standard error of the mean. An ANOVA with repeated measures was performed for both figures A and B. * P≤0.05 compared to pH 7.4.

    Techniques Used: Activity Assay

    16) Product Images from "Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase †Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase † , ‡"

    Article Title: Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase †Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase † , ‡

    Journal:

    doi: 10.1021/bi7016427

    Enzyme kinetics of actKR. (A) The double reciprocal plot (1/ v versus 1/[ trans -1-decalone]) altering the concentration of trans -1-decalone and NADPH eliminates a ping-pong mechanism (which will produce parallel lines) as a possible mechanism. (B) The double
    Figure Legend Snippet: Enzyme kinetics of actKR. (A) The double reciprocal plot (1/ v versus 1/[ trans -1-decalone]) altering the concentration of trans -1-decalone and NADPH eliminates a ping-pong mechanism (which will produce parallel lines) as a possible mechanism. (B) The double

    Techniques Used: Concentration Assay

    17) Product Images from "Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach *"

    Article Title: Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.737122

    Two biochemical reaction sequences for H 2 evolution from CpI. a , driven by NADPH. Fd ox , oxidized ferredoxin; PGL , 6-phosphoglucono- d -lactone. b , driven by DTH.
    Figure Legend Snippet: Two biochemical reaction sequences for H 2 evolution from CpI. a , driven by NADPH. Fd ox , oxidized ferredoxin; PGL , 6-phosphoglucono- d -lactone. b , driven by DTH.

    Techniques Used:

    18) Product Images from "Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli"

    Article Title: Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Inductions of the NADPH-dependent nitroreductase. The induction of NADPH:furantoin nitroreductase by paraquat was explored as described in Materials and Methods . Bars 1, 3, and 5, without exposure to paraquat; bars 2, 4, and 6, with exposure to paraquat. Bars 1 and 2, parental strain; bars 3 and 4, Δ soxR strain; bars 5 and 6, soxR constitutive strain.
    Figure Legend Snippet: Inductions of the NADPH-dependent nitroreductase. The induction of NADPH:furantoin nitroreductase by paraquat was explored as described in Materials and Methods . Bars 1, 3, and 5, without exposure to paraquat; bars 2, 4, and 6, with exposure to paraquat. Bars 1 and 2, parental strain; bars 3 and 4, Δ soxR strain; bars 5 and 6, soxR constitutive strain.

    Techniques Used:

    19) Product Images from "Antimycobacterial Activity and Mechanism of Action of NAS-91 ▿"

    Article Title: Antimycobacterial Activity and Mechanism of Action of NAS-91 ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00968-07

    Inhibition of oleic acid synthesis by NAS-91 and NAS-21 in a cell-free assay. Crude extracts of M. bovis BCG were incubated in the presence of NADPH and [1- 14 C]stearoyl-CoA. Following incubation at 37°C, radiolabeled fatty acids were saponified and methyl esterified. The radiolabeled products were then resolved by argentation-TLC in petroleum ether-diethyl ether (17:3, vol/vol). Autoradiograms were obtained by overnight exposure to Kodak Biomax MR film. (A) Synthesis of oleoyl-CoA from stearoyl-CoA in the presence of M. bovis BCG lysates (lane 3). The appropriate radiolabeled standards used were stearic acid (lane 1) and oleic acid (lane 2). (B) The reaction mixtures were preincubated in the presence of increasing concentrations (μg/ml) of either NAS-21 (left panel) or NAS-91 (right panel) at 37°C for 10 min prior to the addition of [1- 14 C]stearoyl-CoA. (C) The inhibition of Δ9-desaturase activity was evaluated by scraping and counting the bands corresponding to [ 14 C]oleic acid shown in panel B. Results are from one representative experiment out of two independent experiments. Values shown are means ± SEM from triplicates.
    Figure Legend Snippet: Inhibition of oleic acid synthesis by NAS-91 and NAS-21 in a cell-free assay. Crude extracts of M. bovis BCG were incubated in the presence of NADPH and [1- 14 C]stearoyl-CoA. Following incubation at 37°C, radiolabeled fatty acids were saponified and methyl esterified. The radiolabeled products were then resolved by argentation-TLC in petroleum ether-diethyl ether (17:3, vol/vol). Autoradiograms were obtained by overnight exposure to Kodak Biomax MR film. (A) Synthesis of oleoyl-CoA from stearoyl-CoA in the presence of M. bovis BCG lysates (lane 3). The appropriate radiolabeled standards used were stearic acid (lane 1) and oleic acid (lane 2). (B) The reaction mixtures were preincubated in the presence of increasing concentrations (μg/ml) of either NAS-21 (left panel) or NAS-91 (right panel) at 37°C for 10 min prior to the addition of [1- 14 C]stearoyl-CoA. (C) The inhibition of Δ9-desaturase activity was evaluated by scraping and counting the bands corresponding to [ 14 C]oleic acid shown in panel B. Results are from one representative experiment out of two independent experiments. Values shown are means ± SEM from triplicates.

    Techniques Used: Inhibition, Cell-Free Assay, Incubation, Thin Layer Chromatography, Activity Assay

    20) Product Images from "Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase"

    Article Title: Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M809338200

    Contribution of Trx with Grx system in RNR assay. Samples with 120 μg/ml R1 and 40 μg/ml R2 were assayed with different reductants as indicated in each column. The Grx system contained 1 μ m Grx1, 4 m m GSH plus NADPH, and GR.
    Figure Legend Snippet: Contribution of Trx with Grx system in RNR assay. Samples with 120 μg/ml R1 and 40 μg/ml R2 were assayed with different reductants as indicated in each column. The Grx system contained 1 μ m Grx1, 4 m m GSH plus NADPH, and GR.

    Techniques Used:

    21) Product Images from "Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1"

    Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

    Journal: Redox Biology

    doi: 10.1016/j.redox.2013.02.001

    Effects of acrolein on enzymatic activity of mTrxR. Semisynthetic mTrxR-GCUG and mTrxRΔ3 (200 nM) were incubated with acrolein for 30 min, and thioredoxin reductase activity was assessed using the insulin assay (A), and NADPH oxidase activity was determined in the absence (white bars) or presence (black bars) of α-lipoic acid (B). Results expressed as the mean change in absorbance x 60 s −1 ±SEM, n =5. *: p
    Figure Legend Snippet: Effects of acrolein on enzymatic activity of mTrxR. Semisynthetic mTrxR-GCUG and mTrxRΔ3 (200 nM) were incubated with acrolein for 30 min, and thioredoxin reductase activity was assessed using the insulin assay (A), and NADPH oxidase activity was determined in the absence (white bars) or presence (black bars) of α-lipoic acid (B). Results expressed as the mean change in absorbance x 60 s −1 ±SEM, n =5. *: p

    Techniques Used: Activity Assay, Incubation

    22) Product Images from "The Tumor Suppressor Mst1 Promotes Changes in the Cellular Redox State by Phosphorylation and Inactivation of Peroxiredoxin-1 Protein *"

    Article Title: The Tumor Suppressor Mst1 Promotes Changes in the Cellular Redox State by Phosphorylation and Inactivation of Peroxiredoxin-1 Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.414524

    Phosphorylation at Thr-90 and Thr-183 inactivates Prdx1. A and C , peroxidase activities of WT-Prdx1, T90A, T90D, T183A, and T183D mutants of Prdx1 were determined by measuring decrease in absorbance ( Ab ) of NADPH at 340 nm for 300 s. B and D , peroxidase
    Figure Legend Snippet: Phosphorylation at Thr-90 and Thr-183 inactivates Prdx1. A and C , peroxidase activities of WT-Prdx1, T90A, T90D, T183A, and T183D mutants of Prdx1 were determined by measuring decrease in absorbance ( Ab ) of NADPH at 340 nm for 300 s. B and D , peroxidase

    Techniques Used:

    23) Product Images from "Oxytocin attenuates NADPH-dependent superoxide activity and IL-6 secretion in macrophages and vascular cells"

    Article Title: Oxytocin attenuates NADPH-dependent superoxide activity and IL-6 secretion in macrophages and vascular cells

    Journal:

    doi: 10.1152/ajpendo.90718.2008

    Effect of oxytocin on NADPH-dependent superoxide production in THP-1 monocytes ( A ) and macrophages ( B ), endothelial cells ( C ), and smooth muscle cells ( D ). NADPH oxidase activity from cells was detected by lucigenin chemiluminescence. Data are means ±
    Figure Legend Snippet: Effect of oxytocin on NADPH-dependent superoxide production in THP-1 monocytes ( A ) and macrophages ( B ), endothelial cells ( C ), and smooth muscle cells ( D ). NADPH oxidase activity from cells was detected by lucigenin chemiluminescence. Data are means ±

    Techniques Used: Activity Assay

    24) Product Images from "CYP4F2 Is a Vitamin K1 Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant"

    Article Title: CYP4F2 Is a Vitamin K1 Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant

    Journal:

    doi: 10.1124/mol.109.054833

    MRM LC/ESI + -MS/MS assay for VK1 metabolism. The figure depicts results from incubations of VK1 with recombinant CYP4F2 in the presence and absence of NADPH cofactor. The traces represent total ion chromatograms (TICs) generated from the following two
    Figure Legend Snippet: MRM LC/ESI + -MS/MS assay for VK1 metabolism. The figure depicts results from incubations of VK1 with recombinant CYP4F2 in the presence and absence of NADPH cofactor. The traces represent total ion chromatograms (TICs) generated from the following two

    Techniques Used: Mass Spectrometry, Recombinant, Generated

    VK1 metabolism in HLM is inhibited by CYP4F2 antibody. Incubations contained 500 μg of microsomal protein preincubated on ice with antibody for 30 min before initiation with the addition of saturating concentrations of VK1 and NADPH. Results
    Figure Legend Snippet: VK1 metabolism in HLM is inhibited by CYP4F2 antibody. Incubations contained 500 μg of microsomal protein preincubated on ice with antibody for 30 min before initiation with the addition of saturating concentrations of VK1 and NADPH. Results

    Techniques Used:

    Metabolic screen of VK1 metabolism catalyzed by recombinant human P450 enzymes. Only CYP4F2 provided any VK1 oxidase activity above background. Reactions were run at 50 μM VK1 with 50 pmol of P450 and 1 mM NADPH unless otherwise indicated on
    Figure Legend Snippet: Metabolic screen of VK1 metabolism catalyzed by recombinant human P450 enzymes. Only CYP4F2 provided any VK1 oxidase activity above background. Reactions were run at 50 μM VK1 with 50 pmol of P450 and 1 mM NADPH unless otherwise indicated on

    Techniques Used: Recombinant, Activity Assay

    25) Product Images from "Dynamic characteristics of GMP reductase complexes revealed by high resolution 31P field cycling NMR relaxometry"

    Article Title: Dynamic characteristics of GMP reductase complexes revealed by high resolution 31P field cycling NMR relaxometry

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00142

    Dynamic characteristics of GMPR complexes revealed by high resolution 31 P field cycling NMR relaxometry. ( A ) Multiple binding modes for substrate and cofactors. Two distinct binding modes are observed for IMP and GMP in their catalytically active NADP + complexes. The cofactor also has two distinct binding modes in these complexes as shown by the effects of D 2 O on relaxation, albeit with similar 31 P-proton relaxer distances. These binding modes are indicated by the positions of the two ellipses. When the binding mode cannot be distinguished by the values of τ/R 0 , the ellipse is positioned in the center. A third conformation, indicated by the dashed ellipse, is observed in the dIMP-cofactor complex. The extrapolated 31 P-proton relaxer distances are show in Å. ( B ) Distinct dynamic states are observed in wild-type enzyme and D219A and with ribose and deoxyribose substrates as evidenced by values of τ.
    Figure Legend Snippet: Dynamic characteristics of GMPR complexes revealed by high resolution 31 P field cycling NMR relaxometry. ( A ) Multiple binding modes for substrate and cofactors. Two distinct binding modes are observed for IMP and GMP in their catalytically active NADP + complexes. The cofactor also has two distinct binding modes in these complexes as shown by the effects of D 2 O on relaxation, albeit with similar 31 P-proton relaxer distances. These binding modes are indicated by the positions of the two ellipses. When the binding mode cannot be distinguished by the values of τ/R 0 , the ellipse is positioned in the center. A third conformation, indicated by the dashed ellipse, is observed in the dIMP-cofactor complex. The extrapolated 31 P-proton relaxer distances are show in Å. ( B ) Distinct dynamic states are observed in wild-type enzyme and D219A and with ribose and deoxyribose substrates as evidenced by values of τ.

    Techniques Used: Nuclear Magnetic Resonance, Binding Assay

    26) Product Images from "In vitro and in vivo Inhibitory Activity of NADPH Against the AmpC BER Class C β-Lactamase"

    Article Title: In vitro and in vivo Inhibitory Activity of NADPH Against the AmpC BER Class C β-Lactamase

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00441

    In vitro and in vivo restoration of susceptibility of ceftazidime-resistant bacteria by NADPH. (A) In vitro combined effect of NADPH and ceftazidime (CAZ) on a CAZ-resistant E. coli BER strain producing AmpC BER. The growth curves were constructed using data from three independent experiments. Error bars indicate standard deviations. (B) Rescue of ceftazidime (CAZ) activity by NADPH in a mouse model. Survival rates were represented as percentage from at least two independent experiments, each with five to six mice.
    Figure Legend Snippet: In vitro and in vivo restoration of susceptibility of ceftazidime-resistant bacteria by NADPH. (A) In vitro combined effect of NADPH and ceftazidime (CAZ) on a CAZ-resistant E. coli BER strain producing AmpC BER. The growth curves were constructed using data from three independent experiments. Error bars indicate standard deviations. (B) Rescue of ceftazidime (CAZ) activity by NADPH in a mouse model. Survival rates were represented as percentage from at least two independent experiments, each with five to six mice.

    Techniques Used: In Vitro, In Vivo, Construct, Activity Assay, Mouse Assay

    27) Product Images from "Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation"

    Article Title: Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation

    Journal: Scientific Reports

    doi: 10.1038/srep00377

    Characterization of Nfo, Ami, and Iso. (A) SDS-PAGE of Nfo and Ami. M, molecular size markers; lanes 1 and 2, cell extracts of E. coli BL21(DE3) expressing His 6 -Nfo; lane 3, purified His 6 -Nfo; lanes 4 and 5, cell extracts of E. coli BL21(DE3) expressing His 6 -Ami; 6, purified His 6 -Ami. (B) Nfo activity, a , complete reaction: 695 μl buffer + 5 μl NAD + 50 μl Fdh + 50 μl Nfo + 200 μl NFM; b , positive control containing HCOOH (1.65 mM) instead of Nfo and NFM; c , negative control without NFM; d , negative control without Nfo; e , negative control without Nfo and NAD. (C) Enzymatic activity of Ami, a , negative control without Ami; b , negative control, without maleamic acid and Ami; c , negative control without maleamic acid; d , complete reaction containing 480 μl buffer (50 mM Tris-HCl + 0.001 mol l −1 EDTA), 200 μl maleamic acid (0.034 mol l −1 ), 10 μl α-ketoglutaric acid (0.5 mol l −1 ), 10 μl NADPH (0.01 mol l −1 ), 100 μl Gdh (9.5 unit ml −1 ), and 200 μl amidase (0.106 mg protein ml −1 ); e , positive control, containing ammonia (30 μM NH 4 Cl) instead of maleamic acid and Ami. (D) SDS-PAGE of Iso. M, molecular size markers; lanes 1 and 2, cell extracts of expressed Iso in E. coli BL21(DE3); lanes 3 and 4, purified His 6 -tagged Iso. (E) Transformation of maleic acid by Iso. (F) Determination of K m of Iso.
    Figure Legend Snippet: Characterization of Nfo, Ami, and Iso. (A) SDS-PAGE of Nfo and Ami. M, molecular size markers; lanes 1 and 2, cell extracts of E. coli BL21(DE3) expressing His 6 -Nfo; lane 3, purified His 6 -Nfo; lanes 4 and 5, cell extracts of E. coli BL21(DE3) expressing His 6 -Ami; 6, purified His 6 -Ami. (B) Nfo activity, a , complete reaction: 695 μl buffer + 5 μl NAD + 50 μl Fdh + 50 μl Nfo + 200 μl NFM; b , positive control containing HCOOH (1.65 mM) instead of Nfo and NFM; c , negative control without NFM; d , negative control without Nfo; e , negative control without Nfo and NAD. (C) Enzymatic activity of Ami, a , negative control without Ami; b , negative control, without maleamic acid and Ami; c , negative control without maleamic acid; d , complete reaction containing 480 μl buffer (50 mM Tris-HCl + 0.001 mol l −1 EDTA), 200 μl maleamic acid (0.034 mol l −1 ), 10 μl α-ketoglutaric acid (0.5 mol l −1 ), 10 μl NADPH (0.01 mol l −1 ), 100 μl Gdh (9.5 unit ml −1 ), and 200 μl amidase (0.106 mg protein ml −1 ); e , positive control, containing ammonia (30 μM NH 4 Cl) instead of maleamic acid and Ami. (D) SDS-PAGE of Iso. M, molecular size markers; lanes 1 and 2, cell extracts of expressed Iso in E. coli BL21(DE3); lanes 3 and 4, purified His 6 -tagged Iso. (E) Transformation of maleic acid by Iso. (F) Determination of K m of Iso.

    Techniques Used: SDS Page, Expressing, Purification, Activity Assay, Positive Control, Negative Control, Transformation Assay

    28) Product Images from "Chronic NF-κB blockade improves renal angiotensin II type 1 receptor functions and reduces blood pressure in Zucker diabetic rats"

    Article Title: Chronic NF-κB blockade improves renal angiotensin II type 1 receptor functions and reduces blood pressure in Zucker diabetic rats

    Journal: Cardiovascular Diabetology

    doi: 10.1186/s12933-015-0239-7

    Effects of PDTC on Nox-2 and iNOS expression in the renal cortex of LZ and ZDF rats. mRNA level of Nox-2 ( a ) and iNOS ( b ) in the renal cortex was measured using qRT-PCR and normalized to GAPDH expression. c Protein expression of Nox-2 and iNOS in the renal cortex of LZ and ZDF rats was measured by western blot, and data were normalized using GAPDH expression. d The activity of NADPH oxidase in renal cortical homogenates was measured by using lucigenin-enhanced chemiluminescence and expressed as percentage of relative luminescence units (RLU)/μg protein. * P
    Figure Legend Snippet: Effects of PDTC on Nox-2 and iNOS expression in the renal cortex of LZ and ZDF rats. mRNA level of Nox-2 ( a ) and iNOS ( b ) in the renal cortex was measured using qRT-PCR and normalized to GAPDH expression. c Protein expression of Nox-2 and iNOS in the renal cortex of LZ and ZDF rats was measured by western blot, and data were normalized using GAPDH expression. d The activity of NADPH oxidase in renal cortical homogenates was measured by using lucigenin-enhanced chemiluminescence and expressed as percentage of relative luminescence units (RLU)/μg protein. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

    29) Product Images from "Covalent Attachment of the Heme to Synechococcus Hemoglobin Alters its Reactivity Toward Nitric Oxide"

    Article Title: Covalent Attachment of the Heme to Synechococcus Hemoglobin Alters its Reactivity Toward Nitric Oxide

    Journal: Journal of inorganic biochemistry

    doi: 10.1016/j.jinorgbio.2017.09.018

    NOD assay performed with (A) ~20 μM GlbN or (B) ~20 μM GlbN-A (50 mM Tris, pH 7.2). The Fe III bis –histidine proteins (most intense of the dashed-line spectra) were incubated with Fd and FNR; 300 μM NADPH was then added to initiate the reactions. (A) Following reduction and dioxygen binding, Fe II –O 2 GlbN (most intense of the solid-line spectra) was treated with 15 μM of MAHMA-NONOate. Rapid oxidation to Fe III bis –histidine GlbN followed (middle dashed-line spectrum) and this state persisted for ~6 min during turnover. Return to the Fe II –O 2 state occurred after NO consumption (middle solid-line spectrum). A second NONOate addition yielded the lowest intensity Fe III bis –histidine GlbN and Fe II –O 2 GlbN spectra. The inset presents the response at 550 nm (arrow). Upper asterisks indicate NO donor addition and lower asterisks mark the turnover periods. (B) Data of the same acquired on GlbN-A. Note the different x-axis in the inset.
    Figure Legend Snippet: NOD assay performed with (A) ~20 μM GlbN or (B) ~20 μM GlbN-A (50 mM Tris, pH 7.2). The Fe III bis –histidine proteins (most intense of the dashed-line spectra) were incubated with Fd and FNR; 300 μM NADPH was then added to initiate the reactions. (A) Following reduction and dioxygen binding, Fe II –O 2 GlbN (most intense of the solid-line spectra) was treated with 15 μM of MAHMA-NONOate. Rapid oxidation to Fe III bis –histidine GlbN followed (middle dashed-line spectrum) and this state persisted for ~6 min during turnover. Return to the Fe II –O 2 state occurred after NO consumption (middle solid-line spectrum). A second NONOate addition yielded the lowest intensity Fe III bis –histidine GlbN and Fe II –O 2 GlbN spectra. The inset presents the response at 550 nm (arrow). Upper asterisks indicate NO donor addition and lower asterisks mark the turnover periods. (B) Data of the same acquired on GlbN-A. Note the different x-axis in the inset.

    Techniques Used: Incubation, Binding Assay

    Reduction of Fe III –NO GlbN-A and GlbN using heme-free diaphorase and NADPH. (A) The Fe III –NO GlbN-A species (dashed trace) was generated by treatment of Fe III bis –histidine GlbN-A with excess MAHMA-NONOate. Some residual Fe III bis –histidine species is present. Addition of NADPH and heme-free diaphorase resulted in the formation of Fe II –NO GlbN-A (dash-dot line), with minimal spectral change upon addition of DT (solid trace). (B) The Fe III –NO GlbN species (dashed trace) was slowly converted to Fe II –NO GlbN (dash-dot trace) by incubation with the heme-free diaphorase and NADPH for 70 min. Addition of DT then resulted in the gradual formation of Fe II –HNO GlbN (solid trace, ~4 h after DT addition) as evidenced by the increase of the Soret peak to 417 nm.
    Figure Legend Snippet: Reduction of Fe III –NO GlbN-A and GlbN using heme-free diaphorase and NADPH. (A) The Fe III –NO GlbN-A species (dashed trace) was generated by treatment of Fe III bis –histidine GlbN-A with excess MAHMA-NONOate. Some residual Fe III bis –histidine species is present. Addition of NADPH and heme-free diaphorase resulted in the formation of Fe II –NO GlbN-A (dash-dot line), with minimal spectral change upon addition of DT (solid trace). (B) The Fe III –NO GlbN species (dashed trace) was slowly converted to Fe II –NO GlbN (dash-dot trace) by incubation with the heme-free diaphorase and NADPH for 70 min. Addition of DT then resulted in the gradual formation of Fe II –HNO GlbN (solid trace, ~4 h after DT addition) as evidenced by the increase of the Soret peak to 417 nm.

    Techniques Used: Generated, Incubation

    30) Product Images from "Synthesis, structure-activity relationships and biological evaluation of 7-phenyl-pyrroloquinolinone 3-amide derivatives as potent antimitotic agents"

    Article Title: Synthesis, structure-activity relationships and biological evaluation of 7-phenyl-pyrroloquinolinone 3-amide derivatives as potent antimitotic agents

    Journal: European journal of medicinal chemistry

    doi: 10.1016/j.ejmech.2016.10.026

    Assessment of metabolic stability of 4a in human liver microsomes. ( A ) 4a (10 μM) was incubated in the presence of human liver microsomes (1.0 mg/mL; HLMs;-△-), HLMs plus NADPH (1 mM; -●-), or buffer only (0.1 M KH 2 PO 4 , pH 7.4; –□–), for 0, 15, 30 or 60 min at 37 °C. The data are expressed as percent of parent compound ( 4a ) remaining at each time compared with time 0 min, and represent the mean ± SD of n = 3 or 4 independent determinations. ( B ) Representative stacked HPLC-fluorescence traces of supernatants from mixtures containing 4a (10 μM) and HLMs (1.0 mg/mL), incubated for 0, 15, 30 or 60 min at 37 °C. M, 4a metabolite.
    Figure Legend Snippet: Assessment of metabolic stability of 4a in human liver microsomes. ( A ) 4a (10 μM) was incubated in the presence of human liver microsomes (1.0 mg/mL; HLMs;-△-), HLMs plus NADPH (1 mM; -●-), or buffer only (0.1 M KH 2 PO 4 , pH 7.4; –□–), for 0, 15, 30 or 60 min at 37 °C. The data are expressed as percent of parent compound ( 4a ) remaining at each time compared with time 0 min, and represent the mean ± SD of n = 3 or 4 independent determinations. ( B ) Representative stacked HPLC-fluorescence traces of supernatants from mixtures containing 4a (10 μM) and HLMs (1.0 mg/mL), incubated for 0, 15, 30 or 60 min at 37 °C. M, 4a metabolite.

    Techniques Used: Incubation, High Performance Liquid Chromatography, Fluorescence

    31) Product Images from "Gene Deficiency in Activating Fc? Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice"

    Article Title: Gene Deficiency in Activating Fc? Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066754

    Responses of bone marrow-derived macrophages to FcγR stimulation. Bone marrow-derived macrophages from ApoE −/− and γ −/− ApoE −/− mice were stimulated with soluble IC (75 µg/mL). Real-time PCR analysis of M1 ( A ) and M2 ( B ) response genes at 6 h of stimulation. Expression data are normalized to control ApoE −/− values ( = 1) and displayed as arbitrary units (a.u.). ( C ) Chemokine production in supernatants was detected by ELISA. ( D ) NADPH oxidase-dependent superoxide anion production (lucigenin assay) at 1 h of stimulation. Values are expressed as chemiluminescence units per mg of cell protein. Data represent the mean±SEM of 4–6 independent experiments.* P
    Figure Legend Snippet: Responses of bone marrow-derived macrophages to FcγR stimulation. Bone marrow-derived macrophages from ApoE −/− and γ −/− ApoE −/− mice were stimulated with soluble IC (75 µg/mL). Real-time PCR analysis of M1 ( A ) and M2 ( B ) response genes at 6 h of stimulation. Expression data are normalized to control ApoE −/− values ( = 1) and displayed as arbitrary units (a.u.). ( C ) Chemokine production in supernatants was detected by ELISA. ( D ) NADPH oxidase-dependent superoxide anion production (lucigenin assay) at 1 h of stimulation. Values are expressed as chemiluminescence units per mg of cell protein. Data represent the mean±SEM of 4–6 independent experiments.* P

    Techniques Used: Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    32) Product Images from "Reversal by Growth Hormone of Homocysteine-induced Epithelial-to-Mesenchymal Transition through Membrane Raft-Redox Signaling in Podocytes"

    Article Title: Reversal by Growth Hormone of Homocysteine-induced Epithelial-to-Mesenchymal Transition through Membrane Raft-Redox Signaling in Podocytes

    Journal: Cellular Physiology and Biochemistry

    doi: 10.1159/000330078

    Inhibition by GH of Hcys-induced NADPH oxidase activation. A. Representative ESR spectra traces for SOD-dependent O2 .- producton in podocytes. B. Summarized data show O2 .- production in podocytes under different conditions, which were normalized to control
    Figure Legend Snippet: Inhibition by GH of Hcys-induced NADPH oxidase activation. A. Representative ESR spectra traces for SOD-dependent O2 .- producton in podocytes. B. Summarized data show O2 .- production in podocytes under different conditions, which were normalized to control

    Techniques Used: Inhibition, Activation Assay, Electron Paramagnetic Resonance

    33) Product Images from "Bioactivation of the Cancer Chemopreventive Agent Tamoxifen to Quinone Methides by Cytochrome P4502B6 and Identification of the Modified Residue on the Apoprotein"

    Article Title: Bioactivation of the Cancer Chemopreventive Agent Tamoxifen to Quinone Methides by Cytochrome P4502B6 and Identification of the Modified Residue on the Apoprotein

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.112.047266

    LC-MS/MS spectrum of the CYP2B6 tryptic peptide modified by tamoxifen. CYP2B6 was reconstituted with reductase and tamoxifen in the presence or absence of NADPH and digested with trypsin. The samples were analyzed by LC-MS/MS and Sequest software as described under Materials and Methods . The figure shows the profile for the doubly charged ion with m / z 678.2 for the modified peptide 188 FHYQDQE 194 . The inset shows the calculated b and y ions for the modified peptide, and the b and y ions identified in the MS/MS spectrum are shown in blue and red, respectively.
    Figure Legend Snippet: LC-MS/MS spectrum of the CYP2B6 tryptic peptide modified by tamoxifen. CYP2B6 was reconstituted with reductase and tamoxifen in the presence or absence of NADPH and digested with trypsin. The samples were analyzed by LC-MS/MS and Sequest software as described under Materials and Methods . The figure shows the profile for the doubly charged ion with m / z 678.2 for the modified peptide 188 FHYQDQE 194 . The inset shows the calculated b and y ions for the modified peptide, and the b and y ions identified in the MS/MS spectrum are shown in blue and red, respectively.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Software

    ESI-LC-MS analysis of CYP2B6 after incubation with tamoxifen. CYP2B6 was incubated with 60 μM tamoxifen in the presence and absence of NADPH. The incubation conditions and analyses are described under Materials and Methods . The figure shows the deconvoluted mass spectrum of the modified (54,761 Da) and the unmodified CYP2B6 apoprotein (54,373 Da). The inset shows the deconvoluted spectrum of the apo2B6 from a control incubation in the presence of tamoxifen and in the absence of NADPH.
    Figure Legend Snippet: ESI-LC-MS analysis of CYP2B6 after incubation with tamoxifen. CYP2B6 was incubated with 60 μM tamoxifen in the presence and absence of NADPH. The incubation conditions and analyses are described under Materials and Methods . The figure shows the deconvoluted mass spectrum of the modified (54,761 Da) and the unmodified CYP2B6 apoprotein (54,373 Da). The inset shows the deconvoluted spectrum of the apo2B6 from a control incubation in the presence of tamoxifen and in the absence of NADPH.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation, Modification

    LC-MS analysis of the GSHEE adduct formed by incubation of tamoxifen with CYP2B6 in the presence of NADPH. CYP2B6 was reconstituted with reductase and incubated with tamoxifen and GHSEE. The separation and analysis of the adduct are as described under Materials and Methods . A, the extracted ion chromatogram for the hydroxytamoxifen quinone methide adduct with m / z 719. B, the MS 2 of the peak eluting at 18.3 min with an m / z 719. C, the primary sites of fragmentation of the tamoxifen-GSHEE adduct.
    Figure Legend Snippet: LC-MS analysis of the GSHEE adduct formed by incubation of tamoxifen with CYP2B6 in the presence of NADPH. CYP2B6 was reconstituted with reductase and incubated with tamoxifen and GHSEE. The separation and analysis of the adduct are as described under Materials and Methods . A, the extracted ion chromatogram for the hydroxytamoxifen quinone methide adduct with m / z 719. B, the MS 2 of the peak eluting at 18.3 min with an m / z 719. C, the primary sites of fragmentation of the tamoxifen-GSHEE adduct.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation, Mass Spectrometry

    Proposed sequence of reactions for the metabolism of tamoxifen by CYP2B6 in the presence of GSHEE and NADPH.
    Figure Legend Snippet: Proposed sequence of reactions for the metabolism of tamoxifen by CYP2B6 in the presence of GSHEE and NADPH.

    Techniques Used: Sequencing

    LC-MS chromatogram of the GSHEE adduct formed when 4OHtam was incubated with P4502B6 in the presence of NADPH. CYP2B6 was reconstituted with reductase and incubated with 4OHtam, GSHEE, and NADPH. Incubation conditions and analysis of the adduct are as described under Materials and Methods . A, the extracted ion chromatogram of the 4OHtam quinone methide with m / z 719. B, the mass spectrum of the peak eluting at 16.9 min with m / z 719. C, the MS 2 of the m / z 719 ion.
    Figure Legend Snippet: LC-MS chromatogram of the GSHEE adduct formed when 4OHtam was incubated with P4502B6 in the presence of NADPH. CYP2B6 was reconstituted with reductase and incubated with 4OHtam, GSHEE, and NADPH. Incubation conditions and analysis of the adduct are as described under Materials and Methods . A, the extracted ion chromatogram of the 4OHtam quinone methide with m / z 719. B, the mass spectrum of the peak eluting at 16.9 min with m / z 719. C, the MS 2 of the m / z 719 ion.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation, Mass Spectrometry

    LC-MS/MS spectrum of the GSHEE adduct formed by human liver microsomes. Human liver microsomes were incubated with tamoxifen and GSHEE in the presence or absence of NADPH. The incubation conditions and the analysis are as described under Materials and Methods . A, the extracted ion chromatogram for the OHtam quinone methide-GSHEE adduct eluting at 16.6 min. B, the MS/MS spectrum of the peak eluting at 16.6 min with m / z 719. C, the MS 2 of the m / z 719 ion.
    Figure Legend Snippet: LC-MS/MS spectrum of the GSHEE adduct formed by human liver microsomes. Human liver microsomes were incubated with tamoxifen and GSHEE in the presence or absence of NADPH. The incubation conditions and the analysis are as described under Materials and Methods . A, the extracted ion chromatogram for the OHtam quinone methide-GSHEE adduct eluting at 16.6 min. B, the MS/MS spectrum of the peak eluting at 16.6 min with m / z 719. C, the MS 2 of the m / z 719 ion.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Incubation

    34) Product Images from "Antifolate Activity of Epigallocatechin Gallate against Stenotrophomonas maltophilia"

    Article Title: Antifolate Activity of Epigallocatechin Gallate against Stenotrophomonas maltophilia

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.7.2914-2920.2005

    (A) Double-reciprocal plots of the reaction of DHFR from S. maltophilia isolate 1 (3 nM) with NADPH (100 μM) and DHF (variable substrate) in the presence of EGCG at pH 7.4. EGCG concentrations were 0 (•), 10 (○), 20 (▪),
    Figure Legend Snippet: (A) Double-reciprocal plots of the reaction of DHFR from S. maltophilia isolate 1 (3 nM) with NADPH (100 μM) and DHF (variable substrate) in the presence of EGCG at pH 7.4. EGCG concentrations were 0 (•), 10 (○), 20 (▪),

    Techniques Used:

    (A) Double-reciprocal plots of the reaction of DHFR from S. maltophilia isolate 1 (3 nM) with NADPH (100 μM) and DHF (variable substrate) in the presence of TMP at pH 7.4. TMP concentrations were 10 (•), 20 (○), 40 (▪),
    Figure Legend Snippet: (A) Double-reciprocal plots of the reaction of DHFR from S. maltophilia isolate 1 (3 nM) with NADPH (100 μM) and DHF (variable substrate) in the presence of TMP at pH 7.4. TMP concentrations were 10 (•), 20 (○), 40 (▪),

    Techniques Used:

    35) Product Images from "Thioredoxin System from Deinococcus radiodurans ▿"

    Article Title: Thioredoxin System from Deinococcus radiodurans ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01046-09

    Michaelis-Menten plot of D. radiodurans TrxR, determined using D. radiodurans Trx1 as a substrate. The reaction was monitored by consumption of NADPH via a decrease in absorbance at 340 nm. Initial velocity ( V o ) was measured as μmoles of NADPH consumed per minute. Reaction mixtures contained 100 mM HEPES buffer (pH 7.4), 2 mM EDTA, 30 μM solution of bovine insulin (Sigma), 0.1 mM NADPH (Calbiochem), 0.1 μM D. radiodurans TrxR, and D. radiodurans Trx1 (0 to 35 μM).
    Figure Legend Snippet: Michaelis-Menten plot of D. radiodurans TrxR, determined using D. radiodurans Trx1 as a substrate. The reaction was monitored by consumption of NADPH via a decrease in absorbance at 340 nm. Initial velocity ( V o ) was measured as μmoles of NADPH consumed per minute. Reaction mixtures contained 100 mM HEPES buffer (pH 7.4), 2 mM EDTA, 30 μM solution of bovine insulin (Sigma), 0.1 mM NADPH (Calbiochem), 0.1 μM D. radiodurans TrxR, and D. radiodurans Trx1 (0 to 35 μM).

    Techniques Used:

    36) Product Images from "RNF34 overexpression exacerbates neurological deficits and brain injury in a mouse model of intracerebral hemorrhage by potentiating mitochondrial dysfunction-mediated oxidative stress"

    Article Title: RNF34 overexpression exacerbates neurological deficits and brain injury in a mouse model of intracerebral hemorrhage by potentiating mitochondrial dysfunction-mediated oxidative stress

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52494-x

    RNF34 overexpression potentiates the ICH-induced mitochondrial ROS generation in mouse brain. ( A – D ) The levels of GSH ( A ), SOD ( B ), MDA ( C ), and NO ( D ) in brain tissues from WT and RNF34 transgenic mice with sham or ICH surgery was determined. n = 6/group. ( E ) ROS generation in brain sections was determined by H 2 DCF-DA staining. ( F ) Quantitative analysis of DCF fluorescence intensity. ( G ) Superoxide anion was examined by dihydroethidium (DHE) staining. ( H ) Quantitative evaluation of DHE fluorescence intensity was performed. n = 4/group. ( I ) NADPH oxidase activity was measured. n = 6/group. ( J ) Mitochondrial ROS generation was determined using MitoSOX Red staining. ( K ) Quantitative evaluation of MitoSOX fluorescence intensity. n = 5/group. ( L ) ATP activity was measured. n = 6/group. ** P
    Figure Legend Snippet: RNF34 overexpression potentiates the ICH-induced mitochondrial ROS generation in mouse brain. ( A – D ) The levels of GSH ( A ), SOD ( B ), MDA ( C ), and NO ( D ) in brain tissues from WT and RNF34 transgenic mice with sham or ICH surgery was determined. n = 6/group. ( E ) ROS generation in brain sections was determined by H 2 DCF-DA staining. ( F ) Quantitative analysis of DCF fluorescence intensity. ( G ) Superoxide anion was examined by dihydroethidium (DHE) staining. ( H ) Quantitative evaluation of DHE fluorescence intensity was performed. n = 4/group. ( I ) NADPH oxidase activity was measured. n = 6/group. ( J ) Mitochondrial ROS generation was determined using MitoSOX Red staining. ( K ) Quantitative evaluation of MitoSOX fluorescence intensity. n = 5/group. ( L ) ATP activity was measured. n = 6/group. ** P

    Techniques Used: Over Expression, Multiple Displacement Amplification, Transgenic Assay, Mouse Assay, Staining, Fluorescence, Activity Assay

    37) Product Images from "Discovery of Tumor-Specific Irreversible Inhibitors of Stearoyl CoA Desaturase"

    Article Title: Discovery of Tumor-Specific Irreversible Inhibitors of Stearoyl CoA Desaturase

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.2016

    Stearoyl CoA desaturase-1 (SCD) is the target of the oxalamides and benzothiazoles. a. Silver stain of purified p37 and p30 proteins. H2122 cells were treated with SW208108 with or without 3 μM (−)-SW203668, an active competitor. Full gel image is shown in Supplementary Figure 2a . b. Immunoprecipitation of SCD from 0.1 μM SW208108-treated H2122 cell lysate following conjugation of a fluorescent azide. Full gel is shown in Supplementary Figure 2b . c. Structure of known SCD inhibitor, Abbott-28c. d. Competition of SW208108 with Abbott-28c. Full gel image is shown in Supplementary Figure 2c . e. In vitro inhibition of SCD activity by oxalamide and benzothiazole scaffolds in microsomal preparation of H2122 cells. Each data point represents one replicate. Counts shown are after subtraction of counts from a control sample lacking NADPH. f. Rescue of oxalamide toxicity by 100 μM of sodium oleate. Each point represents the average of two biological replicates. Best fit curves of H460 + oleate and HCC44 + oleate are overlapping.
    Figure Legend Snippet: Stearoyl CoA desaturase-1 (SCD) is the target of the oxalamides and benzothiazoles. a. Silver stain of purified p37 and p30 proteins. H2122 cells were treated with SW208108 with or without 3 μM (−)-SW203668, an active competitor. Full gel image is shown in Supplementary Figure 2a . b. Immunoprecipitation of SCD from 0.1 μM SW208108-treated H2122 cell lysate following conjugation of a fluorescent azide. Full gel is shown in Supplementary Figure 2b . c. Structure of known SCD inhibitor, Abbott-28c. d. Competition of SW208108 with Abbott-28c. Full gel image is shown in Supplementary Figure 2c . e. In vitro inhibition of SCD activity by oxalamide and benzothiazole scaffolds in microsomal preparation of H2122 cells. Each data point represents one replicate. Counts shown are after subtraction of counts from a control sample lacking NADPH. f. Rescue of oxalamide toxicity by 100 μM of sodium oleate. Each point represents the average of two biological replicates. Best fit curves of H460 + oleate and HCC44 + oleate are overlapping.

    Techniques Used: Silver Staining, Purification, Immunoprecipitation, Conjugation Assay, In Vitro, Inhibition, Activity Assay

    38) Product Images from "Identification of a novel inactivating mutation in Isocitrate Dehydrogenase 1 (IDH1-R314C) in a high grade astrocytoma"

    Article Title: Identification of a novel inactivating mutation in Isocitrate Dehydrogenase 1 (IDH1-R314C) in a high grade astrocytoma

    Journal: Scientific Reports

    doi: 10.1038/srep30486

    Homodimeric IDH1 R314C -GST is defective in the isocitrate-to α-KG reaction and does not produce D-2-HG. ( A ) SDS-page gel stained with coomassie brilliant blue showing expression of purified IDH1-GST constructs. ( B ) Fluorescent monitoring of NADPH formation at 340 nm shows that only purified IDH1 WT -GST is capable to convert isocitrate to α-KG under the reaction conditions tested, whereas IDH1 R132H -GST and IDH1 R314C -GST are inactive. ( C ) α-KG production by IDH1-GST enzymes was measured with LC-MS. Note that only IDH1 WT -GST is capable of isocitrate-to-α-KG conversion. (n.d. = non detectable, area
    Figure Legend Snippet: Homodimeric IDH1 R314C -GST is defective in the isocitrate-to α-KG reaction and does not produce D-2-HG. ( A ) SDS-page gel stained with coomassie brilliant blue showing expression of purified IDH1-GST constructs. ( B ) Fluorescent monitoring of NADPH formation at 340 nm shows that only purified IDH1 WT -GST is capable to convert isocitrate to α-KG under the reaction conditions tested, whereas IDH1 R132H -GST and IDH1 R314C -GST are inactive. ( C ) α-KG production by IDH1-GST enzymes was measured with LC-MS. Note that only IDH1 WT -GST is capable of isocitrate-to-α-KG conversion. (n.d. = non detectable, area

    Techniques Used: SDS Page, Staining, Expressing, Purification, Construct, Liquid Chromatography with Mass Spectroscopy

    IDH1 R314C -BAPHIS expression in glioma cell lines leads to reduced forward activity compared to IDH WT expression. ( A ) Western blots showing expression of IDH1-BAPHIS in transiently transfected HEK293t and lentivirally transduced LN229 and U251 glioma cells. Blots were stained with anti-IDH1 PAN and anti-IDH1 R132H as indicated, or with anti-GAPDH as a loading control. Since the recombinant proteins are ~3 kDa larger than endogenous IDH1 they can be distinguished from the endogenous IDH1. Control LN229 and U251 were transduced with empty vector virus (EV). ( B ) Monitoring of NADPH formation after isocitrate/NADP + addition to extracts from transfected/transduced cell lines. NADPH formation was monitored by absorbance measurements at 340 nm. Note that IDH1 R314C -BAPHIS was far less effective in oxidative carboxylation of isocitrate than IDH1 WT -BAPHIS. ( C ) Km values of IDH1-GST for NADP + . The Km value for NADP + for IDH1 R132H -GST could not be determined. ( D ) D-2-HG production by IDH1-BAPHIS expressing cell lines was measured with LC-MS. Only IDH1 R132H -BAPHIS expressing cells were capable of D-2-HG production.
    Figure Legend Snippet: IDH1 R314C -BAPHIS expression in glioma cell lines leads to reduced forward activity compared to IDH WT expression. ( A ) Western blots showing expression of IDH1-BAPHIS in transiently transfected HEK293t and lentivirally transduced LN229 and U251 glioma cells. Blots were stained with anti-IDH1 PAN and anti-IDH1 R132H as indicated, or with anti-GAPDH as a loading control. Since the recombinant proteins are ~3 kDa larger than endogenous IDH1 they can be distinguished from the endogenous IDH1. Control LN229 and U251 were transduced with empty vector virus (EV). ( B ) Monitoring of NADPH formation after isocitrate/NADP + addition to extracts from transfected/transduced cell lines. NADPH formation was monitored by absorbance measurements at 340 nm. Note that IDH1 R314C -BAPHIS was far less effective in oxidative carboxylation of isocitrate than IDH1 WT -BAPHIS. ( C ) Km values of IDH1-GST for NADP + . The Km value for NADP + for IDH1 R132H -GST could not be determined. ( D ) D-2-HG production by IDH1-BAPHIS expressing cell lines was measured with LC-MS. Only IDH1 R132H -BAPHIS expressing cells were capable of D-2-HG production.

    Techniques Used: Expressing, Activity Assay, Western Blot, Transfection, Staining, Recombinant, Transduction, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy

    39) Product Images from "Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase"

    Article Title: Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00648-18

    Biochemical analysis of the metagenomic ENR FabL2. Initial velocities were determined in triplicates as a function of NADPH concentration (A) and crotonyl-CoA concentration (B). Data were fitted to the Michaelis-Menten nonlinear regression equation using GraphPad Prism version 7. The fitted line and K m values are shown.
    Figure Legend Snippet: Biochemical analysis of the metagenomic ENR FabL2. Initial velocities were determined in triplicates as a function of NADPH concentration (A) and crotonyl-CoA concentration (B). Data were fitted to the Michaelis-Menten nonlinear regression equation using GraphPad Prism version 7. The fitted line and K m values are shown.

    Techniques Used: Concentration Assay

    40) Product Images from "Mycobacterium tuberculosis expresses methionine sulfoxide reductases A and B that protect from killing by nitrite and hypochlorite"

    Article Title: Mycobacterium tuberculosis expresses methionine sulfoxide reductases A and B that protect from killing by nitrite and hypochlorite

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2008.06548.x

    Enzyme activity of recombinant Msrs and cell lysates. A. Kinetic analysis of recombinant MsrA and MsrB against peptidyl and free methionine sulfoxide epimers. Results are representative of 3 independent experiments. B. Activity (pmoles of NADPH oxidized/min) of 200 μg of lysate from indicated strains against N -acetyl methionine-( R,S )-sulfoxide. Cmpl. AB refers to the strain deficient in both msrA and msrB after complementation with both genes. Results are the mean ± standard error from 3 independent experiments.
    Figure Legend Snippet: Enzyme activity of recombinant Msrs and cell lysates. A. Kinetic analysis of recombinant MsrA and MsrB against peptidyl and free methionine sulfoxide epimers. Results are representative of 3 independent experiments. B. Activity (pmoles of NADPH oxidized/min) of 200 μg of lysate from indicated strains against N -acetyl methionine-( R,S )-sulfoxide. Cmpl. AB refers to the strain deficient in both msrA and msrB after complementation with both genes. Results are the mean ± standard error from 3 independent experiments.

    Techniques Used: Activity Assay, Recombinant

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    Activity Assay:

    Article Title: A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis
    Article Snippet: .. The activity of TR was measured optically through the reduction of sulfhydryl groups by a timed reaction with recombinant Escherichia coli thioredoxin (Calbiochem), insulin (Sigma), and NADPH (Sigma; ). .. Cell lysates were partially purified by affinity chromatography on a 2′5′-ADP-sepharose (Pharmacia) column.

    Staining:

    Article Title: The timing and location of GDNF expression determines enteric nervous system structure and function
    Article Snippet: .. For ChAT/NADPH-d staining, 2 cm of proximal duodenum were first stained using the NADPH-d method, and then by ChAT immunohistochemistry (1:10, AB144P, Chemicon). .. For GDNF injected mice, 3 cm small bowel and 1.5 cm colon segments were analyzed.

    Recombinant:

    Article Title: A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis
    Article Snippet: .. The activity of TR was measured optically through the reduction of sulfhydryl groups by a timed reaction with recombinant Escherichia coli thioredoxin (Calbiochem), insulin (Sigma), and NADPH (Sigma; ). .. Cell lysates were partially purified by affinity chromatography on a 2′5′-ADP-sepharose (Pharmacia) column.

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  • 99
    Millipore nadp
    ]). α-Helices 4, 8, 10, and 11 are labeled for reference and colored in yellow (monomer A, helices 4, 10, and 11) and orange (monomer B and helix 8). The <t>NADP</t> + - and Ca 2+ -binding sites are expanded in the labeled sub-panels. Calcium is shown as a pink sphere. NADP + ].
    Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 217 article reviews
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    nadp - by Bioz Stars, 2020-09
    99/100 stars
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    89
    Millipore nadph oxidase inhibitor
    ET formation depends on actin polymerization and partially on <t>NADPH</t> oxidase. Coelomocytes were isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or bacteria X . bovienii (BACT). Prior to stimulation with PMA/BACT, some cells were pretreated with inhibitors as specifically indicated below. After addition of Sytox orange, the amount of extDNA was evaluated spectrofluorometerically. Effects of a) cytochalasin D inhibitor (CYTO-D), b) cytochalasin B inhibitor (CYTO-B), and c) peptidylarginine deiminase inhibitor (PAD4) on intensity of ETs formation measured by release of extDNA. d)Measurement of respiratory burst (NBT reduction) of coelomocytes isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or BACT.Prior to stimulation with PMA/BACT, some cells were pretreated with <t>DPI,</t> an inhibitor of NADPH oxidase. e) Effects of DPIon intensity of ETs formation measured by release of extDNA. Each experiment was repeated at least 3 times and in each of them 3 to 4 individuals were used. Mean+SD, data for unstimulated cells are expressed as 100% and marked with a horizontal line, different letters (e.g. a vs. b or A vs. B) indicate statistically significant differences between the groups at p
    Nadph Oxidase Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore mitochondrial nadp
    Levels of <t>NADPH,</t> activity of GR and GPx, and GSSG/tGSH in the mitochondria from <t>Idh2</t> +/+ and Idh2 −/− mice after I/R. Idh2 +/+ ( Idh2 WT) and Idh2 −/− ( Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. (A) Kidney sections were subjected to immunohistochemical staining using an anti-NAPDH antibody. Images were obtained from the outer medulla. Upper panels are at low magnification, lower panels are at high magnification of the dash-lined rectangle in upper panel. Brown indicates NADPH-positive signal. (B) Activity of GR, (C) GSSG/tGSH, and (D) the activity of GPx in the mitochondrial fractions from kidneys were determined as described in the Concise Methods section. Results are expressed as the mean±SEM ( n =6). * P
    Mitochondrial Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ]). α-Helices 4, 8, 10, and 11 are labeled for reference and colored in yellow (monomer A, helices 4, 10, and 11) and orange (monomer B and helix 8). The NADP + - and Ca 2+ -binding sites are expanded in the labeled sub-panels. Calcium is shown as a pink sphere. NADP + ].

    Journal: The Biochemical journal

    Article Title: Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants

    doi: 10.1042/BCJ20180424

    Figure Lengend Snippet: ]). α-Helices 4, 8, 10, and 11 are labeled for reference and colored in yellow (monomer A, helices 4, 10, and 11) and orange (monomer B and helix 8). The NADP + - and Ca 2+ -binding sites are expanded in the labeled sub-panels. Calcium is shown as a pink sphere. NADP + ].

    Article Snippet: NADPH (tetrasodium salt) and NADP+ (disodium salt) were purchased from Calbiochem (San Diego, CA).

    Techniques: Labeling, Binding Assay

    NADP + RMSF in WT, R132Q, R132H, and R132L IDH1 simulations with Ca 2+ bound. The RMSF per atom is colored onto each atom for ( A ) WT IDH1 (gray), ( B ) R132H IDH1 (green), ( C ) R132Q IDH1 (orange), and ( D ) R132L IDH1 (blue). The NADP + atoms that fluctuate more than 7 Å are in red, while the atoms which fluctuation less than 4 Å are in blue. Gradations between the two are modulated with white. The orientation of NADP + in the crystal structures is shown.

    Journal: The Biochemical journal

    Article Title: Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants

    doi: 10.1042/BCJ20180424

    Figure Lengend Snippet: NADP + RMSF in WT, R132Q, R132H, and R132L IDH1 simulations with Ca 2+ bound. The RMSF per atom is colored onto each atom for ( A ) WT IDH1 (gray), ( B ) R132H IDH1 (green), ( C ) R132Q IDH1 (orange), and ( D ) R132L IDH1 (blue). The NADP + atoms that fluctuate more than 7 Å are in red, while the atoms which fluctuation less than 4 Å are in blue. Gradations between the two are modulated with white. The orientation of NADP + in the crystal structures is shown.

    Article Snippet: NADPH (tetrasodium salt) and NADP+ (disodium salt) were purchased from Calbiochem (San Diego, CA).

    Techniques:

    ET formation depends on actin polymerization and partially on NADPH oxidase. Coelomocytes were isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or bacteria X . bovienii (BACT). Prior to stimulation with PMA/BACT, some cells were pretreated with inhibitors as specifically indicated below. After addition of Sytox orange, the amount of extDNA was evaluated spectrofluorometerically. Effects of a) cytochalasin D inhibitor (CYTO-D), b) cytochalasin B inhibitor (CYTO-B), and c) peptidylarginine deiminase inhibitor (PAD4) on intensity of ETs formation measured by release of extDNA. d)Measurement of respiratory burst (NBT reduction) of coelomocytes isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or BACT.Prior to stimulation with PMA/BACT, some cells were pretreated with DPI, an inhibitor of NADPH oxidase. e) Effects of DPIon intensity of ETs formation measured by release of extDNA. Each experiment was repeated at least 3 times and in each of them 3 to 4 individuals were used. Mean+SD, data for unstimulated cells are expressed as 100% and marked with a horizontal line, different letters (e.g. a vs. b or A vs. B) indicate statistically significant differences between the groups at p

    Journal: PLoS ONE

    Article Title: Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    doi: 10.1371/journal.pone.0159031

    Figure Lengend Snippet: ET formation depends on actin polymerization and partially on NADPH oxidase. Coelomocytes were isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or bacteria X . bovienii (BACT). Prior to stimulation with PMA/BACT, some cells were pretreated with inhibitors as specifically indicated below. After addition of Sytox orange, the amount of extDNA was evaluated spectrofluorometerically. Effects of a) cytochalasin D inhibitor (CYTO-D), b) cytochalasin B inhibitor (CYTO-B), and c) peptidylarginine deiminase inhibitor (PAD4) on intensity of ETs formation measured by release of extDNA. d)Measurement of respiratory burst (NBT reduction) of coelomocytes isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or BACT.Prior to stimulation with PMA/BACT, some cells were pretreated with DPI, an inhibitor of NADPH oxidase. e) Effects of DPIon intensity of ETs formation measured by release of extDNA. Each experiment was repeated at least 3 times and in each of them 3 to 4 individuals were used. Mean+SD, data for unstimulated cells are expressed as 100% and marked with a horizontal line, different letters (e.g. a vs. b or A vs. B) indicate statistically significant differences between the groups at p

    Article Snippet: Inhibitor of NADPH oxidase To determine involvement of ROS in ET production, cells were pre-treated with NADPH oxidase inhibitor, diphenyleneiodonium (DPI, 5 and 50 μM, Calbiochem, San Diego, California) in the in vitro conditions [ ].

    Techniques: Isolation, In Vitro

    Levels of NADPH, activity of GR and GPx, and GSSG/tGSH in the mitochondria from Idh2 +/+ and Idh2 −/− mice after I/R. Idh2 +/+ ( Idh2 WT) and Idh2 −/− ( Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. (A) Kidney sections were subjected to immunohistochemical staining using an anti-NAPDH antibody. Images were obtained from the outer medulla. Upper panels are at low magnification, lower panels are at high magnification of the dash-lined rectangle in upper panel. Brown indicates NADPH-positive signal. (B) Activity of GR, (C) GSSG/tGSH, and (D) the activity of GPx in the mitochondrial fractions from kidneys were determined as described in the Concise Methods section. Results are expressed as the mean±SEM ( n =6). * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Mitochondrial NADP+-Dependent Isocitrate Dehydrogenase Deficiency Exacerbates Mitochondrial and Cell Damage after Kidney Ischemia-Reperfusion Injury

    doi: 10.1681/ASN.2016030349

    Figure Lengend Snippet: Levels of NADPH, activity of GR and GPx, and GSSG/tGSH in the mitochondria from Idh2 +/+ and Idh2 −/− mice after I/R. Idh2 +/+ ( Idh2 WT) and Idh2 −/− ( Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. (A) Kidney sections were subjected to immunohistochemical staining using an anti-NAPDH antibody. Images were obtained from the outer medulla. Upper panels are at low magnification, lower panels are at high magnification of the dash-lined rectangle in upper panel. Brown indicates NADPH-positive signal. (B) Activity of GR, (C) GSSG/tGSH, and (D) the activity of GPx in the mitochondrial fractions from kidneys were determined as described in the Concise Methods section. Results are expressed as the mean±SEM ( n =6). * P

    Article Snippet: Western blotting was performed using antibodies directed against mitochondrial NADP+ -dependent IDH2, cytosolic NADP+ -dependent IDH1, MnSOD (Calbiochem, San Diego, CA), copper-zinc superoxide dismutase (CuZnSOD; Chemicon, Temecula, CA), Opa1 (BD Biosciences, San Jose, CA), Drp1 (Cell Signaling Technology, Danvers, MA), Fis1 (Sigma-Aldrich), Bax (5B7) (EMD Millipore, Billerica, MA), Bax (6A7) (Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-2 (EMD Millipore), XIAP (AnaSpec, San Jose, CA), Bcl-xL (BD Biosciences), cleaved caspase-3 (Cell Signaling Technology), 4-HNE (Abcam, Inc., Cambridge, MA), G6PD (Cell Signaling Technology), catalase (Fitzgerald, Concord, MA), Ly6G (eBioscience, San Diego, CA), cytochrome c (BD Biosciences), COX-IV (Abcam, Inc.), β -actin (Sigma-Aldrich), and GAPDH (Novus Biologicals, Littleton, CO).

    Techniques: Activity Assay, Mouse Assay, Immunohistochemistry, Staining