nicotinamide adenine dinucleotide phosphate nadph  (Millipore)


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    Name:
    Nicotinamide
    Description:

    Catalog Number:
    n3376
    Price:
    None
    Applications:
    Used as a cofactor for certain enzymes
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    Structured Review

    Millipore nicotinamide adenine dinucleotide phosphate nadph
    Nicotinamide

    https://www.bioz.com/result/nicotinamide adenine dinucleotide phosphate nadph/product/Millipore
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    nicotinamide adenine dinucleotide phosphate nadph - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1"

    Article Title: HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1

    Journal:

    doi: 10.1161/ATVBAHA.109.197822

    Increased HO-1 expression leads to inhibition of PDGF-stimulated NADPH oxidase activity
    Figure Legend Snippet: Increased HO-1 expression leads to inhibition of PDGF-stimulated NADPH oxidase activity

    Techniques Used: Expressing, Inhibition, Activity Assay

    CO inhibits PDGF-stimulated NADPH oxidase activity
    Figure Legend Snippet: CO inhibits PDGF-stimulated NADPH oxidase activity

    Techniques Used: Activity Assay

    2) Product Images from "Aldosterone signaling regulates the over-expression of claudin-4 and -8 at the distal nephron from type 1 diabetic rats"

    Article Title: Aldosterone signaling regulates the over-expression of claudin-4 and -8 at the distal nephron from type 1 diabetic rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177362

    SPL decreases diabetes-induced oxidative stress in GL and PT. To evaluate oxidative stress, superoxide anion (O 2 ●― ) production, lipid peroxidation, protein carbonylation and reduced glutathione (GSH) content were measured in isolated GL and PT. SPL treatment prevents diabetes-induced increment in O 2 ●― production (A) by using nicotinamide adenine dinucleotide phosphate (NADPH) as substrate and, diphenyleneiodonium (DPI) as inhibitor, lipid peroxidation (B) and protein carbonylation (C) and decreased GSH content (D) in GL ( S3 Fig ). Also, SPL diminished diabetes-induced increment of O 2 ●― production (E), lipid peroxidation (F) and protein carbonylation (G) and decreased GSH content (H) in PT ( S3 Fig ). Similar results were found between CTL and SPL groups. Data are mean±SEM from 5–6 rats per group. *p
    Figure Legend Snippet: SPL decreases diabetes-induced oxidative stress in GL and PT. To evaluate oxidative stress, superoxide anion (O 2 ●― ) production, lipid peroxidation, protein carbonylation and reduced glutathione (GSH) content were measured in isolated GL and PT. SPL treatment prevents diabetes-induced increment in O 2 ●― production (A) by using nicotinamide adenine dinucleotide phosphate (NADPH) as substrate and, diphenyleneiodonium (DPI) as inhibitor, lipid peroxidation (B) and protein carbonylation (C) and decreased GSH content (D) in GL ( S3 Fig ). Also, SPL diminished diabetes-induced increment of O 2 ●― production (E), lipid peroxidation (F) and protein carbonylation (G) and decreased GSH content (H) in PT ( S3 Fig ). Similar results were found between CTL and SPL groups. Data are mean±SEM from 5–6 rats per group. *p

    Techniques Used: Isolation, CTL Assay

    3) Product Images from "Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus †"

    Article Title: Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus †

    Journal: Biochemistry

    doi: 10.1021/bi701467g

    X-band EPR spectra recorded for reaction mixtures containing 80 µM IDI-2, 200 µM FMN, and 2 mM IPP. The FMN coenzyme was either photoreduced (A, light gray) or chemically reduced by 10 mM dithionite (A, black) or 10 mM NADPH (A, dark gray) prior to the addition of 2 mM IPP. After 2 min, the reactions were flash frozen and stored in liquid nitrogen until the spectra were recorded. The concentration of FMN sem was determined to be 0.39, 0.86, and 0.72 µM for dithionite reduced, NADPH reduced, and photoreduced samples, respectively. These values correspond to 0.5, 1, and 0.9% of the total enzyme concentration. (B) In contrast to these reaction mixtures, the photoreduced E•FMN ox •IPP mixture (gray) yielded 29.6 µM neutral semiquinone, corresponding to 37% of the total enzyme concentration. The spectrum of the NADPH reduced reaction mixture (black) is shown for comparison. Experimental conditions: temperature, 100 K; modulation amplitude, 5 G; microwave power, 1 mW; and spectrometer frequency, 9.601 GHz. The spectra are an average of five 3 min scans.
    Figure Legend Snippet: X-band EPR spectra recorded for reaction mixtures containing 80 µM IDI-2, 200 µM FMN, and 2 mM IPP. The FMN coenzyme was either photoreduced (A, light gray) or chemically reduced by 10 mM dithionite (A, black) or 10 mM NADPH (A, dark gray) prior to the addition of 2 mM IPP. After 2 min, the reactions were flash frozen and stored in liquid nitrogen until the spectra were recorded. The concentration of FMN sem was determined to be 0.39, 0.86, and 0.72 µM for dithionite reduced, NADPH reduced, and photoreduced samples, respectively. These values correspond to 0.5, 1, and 0.9% of the total enzyme concentration. (B) In contrast to these reaction mixtures, the photoreduced E•FMN ox •IPP mixture (gray) yielded 29.6 µM neutral semiquinone, corresponding to 37% of the total enzyme concentration. The spectrum of the NADPH reduced reaction mixture (black) is shown for comparison. Experimental conditions: temperature, 100 K; modulation amplitude, 5 G; microwave power, 1 mW; and spectrometer frequency, 9.601 GHz. The spectra are an average of five 3 min scans.

    Techniques Used: Electron Paramagnetic Resonance, Concentration Assay

    4) Product Images from "Profiling patterns of glutathione reductase inhibition by the natural product illudin S and its acylfulvene analogues"

    Article Title: Profiling patterns of glutathione reductase inhibition by the natural product illudin S and its acylfulvene analogues

    Journal: Molecular bioSystems

    doi: 10.1039/b904720d

    Inhibition of GR by AF, HMAF and illudin S. A, concentration-dependent inhibition of GR. GR (5 nM) was incubated with test compounds in the presence of NADPH (150 μM) for 30 min at 25 °C (AF (◆), 62.5, 125, 250, 625, 750, 1000,
    Figure Legend Snippet: Inhibition of GR by AF, HMAF and illudin S. A, concentration-dependent inhibition of GR. GR (5 nM) was incubated with test compounds in the presence of NADPH (150 μM) for 30 min at 25 °C (AF (◆), 62.5, 125, 250, 625, 750, 1000,

    Techniques Used: Inhibition, Concentration Assay, Incubation

    5) Product Images from "Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug Laromustine"

    Article Title: Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug Laromustine

    Journal: Molecular and cellular biochemistry

    doi: 10.1007/s11010-012-1411-y

    Inhibition of purified rat liver TrxR by Laromustine (blue), 101MDCE (red), 90CE (purple), and Carmustine (purple). Enzyme was pre-incubated with drugs for 2 hr at pH 8.0 before introducing DTNB and NADPH substrates. Activity was determined by monitoring
    Figure Legend Snippet: Inhibition of purified rat liver TrxR by Laromustine (blue), 101MDCE (red), 90CE (purple), and Carmustine (purple). Enzyme was pre-incubated with drugs for 2 hr at pH 8.0 before introducing DTNB and NADPH substrates. Activity was determined by monitoring

    Techniques Used: Inhibition, Purification, Incubation, Activity Assay

    Time course of purified TrxR pre-incubation with 7.4 µM Laromustine at pH 8.0. At the indicated times, Laromustine-treated TrxR was added to DTNB and NADPH substrates and enzyme activity was measured using the DTNB reduction assay. Data are reported
    Figure Legend Snippet: Time course of purified TrxR pre-incubation with 7.4 µM Laromustine at pH 8.0. At the indicated times, Laromustine-treated TrxR was added to DTNB and NADPH substrates and enzyme activity was measured using the DTNB reduction assay. Data are reported

    Techniques Used: Purification, Incubation, Activity Assay

    6) Product Images from "Characterization of the metabolism of benzaldehyde dimethane sulfonate (NSC 281612, DMS612)"

    Article Title: Characterization of the metabolism of benzaldehyde dimethane sulfonate (NSC 281612, DMS612)

    Journal: Cancer chemotherapy and pharmacology

    doi: 10.1007/s00280-015-2828-2

    BEN added to lysed and non-lysed RBCs. 10% RBCs (◇); 10% lysed RBCs (■); 10% lysed RBCs + NADH (▲); and 10% lysed RBCs + NADPH (◆).
    Figure Legend Snippet: BEN added to lysed and non-lysed RBCs. 10% RBCs (◇); 10% lysed RBCs (■); 10% lysed RBCs + NADH (▲); and 10% lysed RBCs + NADPH (◆).

    Techniques Used:

    7) Product Images from "A pharmacokinetic evaluation and metabolite identification of the GHB receptor antagonist NCS‐382 in mouse informs novel therapeutic strategies for the treatment of GHB intoxication. A pharmacokinetic evaluation and metabolite identification of the GHB receptor antagonist NCS‐382 in mouse informs novel therapeutic strategies for the treatment of GHB intoxication"

    Article Title: A pharmacokinetic evaluation and metabolite identification of the GHB receptor antagonist NCS‐382 in mouse informs novel therapeutic strategies for the treatment of GHB intoxication. A pharmacokinetic evaluation and metabolite identification of the GHB receptor antagonist NCS‐382 in mouse informs novel therapeutic strategies for the treatment of GHB intoxication

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.265

    In vitro NCS ‐382 metabolism in mouse liver microsomes (Top) and human liver microsomes (bottom). NCS ‐382 dehydrogenation of NCS ‐382 (left) and glucuronide conjugation (right) in the absence and presence of nicotinamide adenine dinucleotide phosphate (1 mmol/L) and in the absence and presence of uridine 5′‐diphospho‐glucuronosyltransferase (3 mmol/L). Data are presented as the mean ± SD of triplicate incubations.
    Figure Legend Snippet: In vitro NCS ‐382 metabolism in mouse liver microsomes (Top) and human liver microsomes (bottom). NCS ‐382 dehydrogenation of NCS ‐382 (left) and glucuronide conjugation (right) in the absence and presence of nicotinamide adenine dinucleotide phosphate (1 mmol/L) and in the absence and presence of uridine 5′‐diphospho‐glucuronosyltransferase (3 mmol/L). Data are presented as the mean ± SD of triplicate incubations.

    Techniques Used: In Vitro, Conjugation Assay

    8) Product Images from "Resistin-Induced Endoplasmic Reticulum Stress Contributes to the Impairment of Insulin Signaling in Endothelium"

    Article Title: Resistin-Induced Endoplasmic Reticulum Stress Contributes to the Impairment of Insulin Signaling in Endothelium

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01226

    Effect of antioxidant on resistin-induced ROS ( A , N = 7 per condition), GRP78 ( B , N = 7 per condition), pAkt (Ser473, C ; N = 7, per condition) and peNOS (Ser1177, D ; N = 7, per condition) expressions in HUVECs. Resistin (10–100 ng/mL) dose-dependently increased NADPH oxidase-derived ROS production, which was partially reduced by TUDCA (500 μg/mL). Antioxidant with either NADPH oxidase inhibitor DPI (10 μmol/L) or ROS scavenger NAC (1 μmol/L) inhibited resistin-induced GRP78 expression and improved insulin signaling pAkt (Ser473) and peNOS (Ser 1177) impaired by resistin. Loading control imagines for βactin were reused for pAkt and peNOS. ∗ p
    Figure Legend Snippet: Effect of antioxidant on resistin-induced ROS ( A , N = 7 per condition), GRP78 ( B , N = 7 per condition), pAkt (Ser473, C ; N = 7, per condition) and peNOS (Ser1177, D ; N = 7, per condition) expressions in HUVECs. Resistin (10–100 ng/mL) dose-dependently increased NADPH oxidase-derived ROS production, which was partially reduced by TUDCA (500 μg/mL). Antioxidant with either NADPH oxidase inhibitor DPI (10 μmol/L) or ROS scavenger NAC (1 μmol/L) inhibited resistin-induced GRP78 expression and improved insulin signaling pAkt (Ser473) and peNOS (Ser 1177) impaired by resistin. Loading control imagines for βactin were reused for pAkt and peNOS. ∗ p

    Techniques Used: Derivative Assay, Expressing

    9) Product Images from "Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo"

    Article Title: Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo

    Journal: Toxins

    doi: 10.3390/toxins9080249

    ( a ) Mass spectrometric profiles obtained from MRM scanning (ion transition m/z 592→503) analysis of mouse liver microsomal incubations containing DIOB, NADPH, Lys, and Cys; ( b ) Mass spectrometric profiles obtained from MRM scanning (ion transition m/z 592→503) analysis of mouse liver microsomal incubations containing DIOB and NADPH, followed by exhaustive proteolytic digestion; ( c ) High resolution mass spectrum; ( d ) MS/MS spectrum of pyrrole 12 .
    Figure Legend Snippet: ( a ) Mass spectrometric profiles obtained from MRM scanning (ion transition m/z 592→503) analysis of mouse liver microsomal incubations containing DIOB, NADPH, Lys, and Cys; ( b ) Mass spectrometric profiles obtained from MRM scanning (ion transition m/z 592→503) analysis of mouse liver microsomal incubations containing DIOB and NADPH, followed by exhaustive proteolytic digestion; ( c ) High resolution mass spectrum; ( d ) MS/MS spectrum of pyrrole 12 .

    Techniques Used: Mass Spectrometry

    Levels of pyrrole 6 ( a ), pyrrole 10 ( b ), andpyrrole 12 ( c ) detected in microsomal incubations, containing DIOB, NADPH, and liver microsomes obtained from mice pretreated with KTC (75 mg/kg/d, 2 days), vehicle, or DEX (50 mg/kg/d, 5 days) ( n = 3). * P
    Figure Legend Snippet: Levels of pyrrole 6 ( a ), pyrrole 10 ( b ), andpyrrole 12 ( c ) detected in microsomal incubations, containing DIOB, NADPH, and liver microsomes obtained from mice pretreated with KTC (75 mg/kg/d, 2 days), vehicle, or DEX (50 mg/kg/d, 5 days) ( n = 3). * P

    Techniques Used: Mouse Assay

    ( a ) Mass spectrometric profiles obtained from a four-channel scanning system analysis of mouse liver microsomal incubations containing DIOB, NADPH, Cys, and BBA; ( b ) Mass spectrometric profiles obtained from the four-channel scanning system analysis of mouse liver microsomal incubations containing DIOB, NADPH, and BBA, followed by exhaustive proteolytic digestion; ( c ) High resolution mass spectrum; ( d ) MS/MS spectrum of pyrrole 6 .
    Figure Legend Snippet: ( a ) Mass spectrometric profiles obtained from a four-channel scanning system analysis of mouse liver microsomal incubations containing DIOB, NADPH, Cys, and BBA; ( b ) Mass spectrometric profiles obtained from the four-channel scanning system analysis of mouse liver microsomal incubations containing DIOB, NADPH, and BBA, followed by exhaustive proteolytic digestion; ( c ) High resolution mass spectrum; ( d ) MS/MS spectrum of pyrrole 6 .

    Techniques Used: Mass Spectrometry

    10) Product Images from "Resistin-Induced Endoplasmic Reticulum Stress Contributes to the Impairment of Insulin Signaling in Endothelium"

    Article Title: Resistin-Induced Endoplasmic Reticulum Stress Contributes to the Impairment of Insulin Signaling in Endothelium

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01226

    Effect of antioxidant on resistin-induced ROS ( A , N = 7 per condition), GRP78 ( B , N = 7 per condition), pAkt (Ser473, C ; N = 7, per condition) and peNOS (Ser1177, D ; N = 7, per condition) expressions in HUVECs. Resistin (10–100 ng/mL) dose-dependently increased NADPH oxidase-derived ROS production, which was partially reduced by TUDCA (500 μg/mL). Antioxidant with either NADPH oxidase inhibitor DPI (10 μmol/L) or ROS scavenger NAC (1 μmol/L) inhibited resistin-induced GRP78 expression and improved insulin signaling pAkt (Ser473) and peNOS (Ser 1177) impaired by resistin. Loading control imagines for βactin were reused for pAkt and peNOS. ∗ p
    Figure Legend Snippet: Effect of antioxidant on resistin-induced ROS ( A , N = 7 per condition), GRP78 ( B , N = 7 per condition), pAkt (Ser473, C ; N = 7, per condition) and peNOS (Ser1177, D ; N = 7, per condition) expressions in HUVECs. Resistin (10–100 ng/mL) dose-dependently increased NADPH oxidase-derived ROS production, which was partially reduced by TUDCA (500 μg/mL). Antioxidant with either NADPH oxidase inhibitor DPI (10 μmol/L) or ROS scavenger NAC (1 μmol/L) inhibited resistin-induced GRP78 expression and improved insulin signaling pAkt (Ser473) and peNOS (Ser 1177) impaired by resistin. Loading control imagines for βactin were reused for pAkt and peNOS. ∗ p

    Techniques Used: Derivative Assay, Expressing

    11) Product Images from "Model combustion-generated particulate matter containing persistent free radicals redox cycle to produce reactive oxygen species"

    Article Title: Model combustion-generated particulate matter containing persistent free radicals redox cycle to produce reactive oxygen species

    Journal: Chemical research in toxicology

    doi: 10.1021/tx400227s

    Effect of co-treatment with epithelial lining fluid constituents, including ascorbate (AA), urate, (UA), N-acetylcysteine (NAC, a model thiol) and NADPH, on MCP230-mediated ROS production. Particles were suspended at 1 mg/mL in 0.9% saline (w/v) containing 0.02% Tween (v/v), and then incubated for 1h together with 100 µM NADPH, 100 µM AA, 100 µM urate, or 100 µM NAC. A, B) ROS production was indicated by an increase in DHR fluorescence. C, D) Hydroxyl radical production was measured by scavenging with 4-HBA, and then measuring products using HPLC. *Indicates p
    Figure Legend Snippet: Effect of co-treatment with epithelial lining fluid constituents, including ascorbate (AA), urate, (UA), N-acetylcysteine (NAC, a model thiol) and NADPH, on MCP230-mediated ROS production. Particles were suspended at 1 mg/mL in 0.9% saline (w/v) containing 0.02% Tween (v/v), and then incubated for 1h together with 100 µM NADPH, 100 µM AA, 100 µM urate, or 100 µM NAC. A, B) ROS production was indicated by an increase in DHR fluorescence. C, D) Hydroxyl radical production was measured by scavenging with 4-HBA, and then measuring products using HPLC. *Indicates p

    Techniques Used: Incubation, Fluorescence, High Performance Liquid Chromatography

    Effect of co-treatment with epithelial lining fluid constituents, including ascorbate (AA), urate, (UA), N-acetylcysteine (NAC, a model thiol) and NADPH, on CuO/ SiO 2 -mediated ROS production. Particles were suspended at 1 mg/mL in 0.9% saline (w/v) containing 0.02% Tween (v/v), and then incubated for 1h together with 100 µM NADPH, 100 µM AA, 100 µM urate, or 100 µM NAC. A) ROS production was indicated by an increase in DHR fluorescence. B) Hydroxyl radical production was measured by scavenging with 4-HBA, then measuring products using HPLC. * p
    Figure Legend Snippet: Effect of co-treatment with epithelial lining fluid constituents, including ascorbate (AA), urate, (UA), N-acetylcysteine (NAC, a model thiol) and NADPH, on CuO/ SiO 2 -mediated ROS production. Particles were suspended at 1 mg/mL in 0.9% saline (w/v) containing 0.02% Tween (v/v), and then incubated for 1h together with 100 µM NADPH, 100 µM AA, 100 µM urate, or 100 µM NAC. A) ROS production was indicated by an increase in DHR fluorescence. B) Hydroxyl radical production was measured by scavenging with 4-HBA, then measuring products using HPLC. * p

    Techniques Used: Incubation, Fluorescence, High Performance Liquid Chromatography

    12) Product Images from "Simultaneous Measurement of CYP1A2 Activity, Regioselectivity, and Coupling: Implications for Environmental Sensitivity of Enzyme-Substrate Binding"

    Article Title: Simultaneous Measurement of CYP1A2 Activity, Regioselectivity, and Coupling: Implications for Environmental Sensitivity of Enzyme-Substrate Binding

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2010.10.002

    Representative data for the depletion of EOMCC (a) and NADPH (b), and the generation of 7HCC (c), in the presence (○) and absence (●) of the inhibitor PBO. The data with inhibition are subtracted from the data without inhibition to yield
    Figure Legend Snippet: Representative data for the depletion of EOMCC (a) and NADPH (b), and the generation of 7HCC (c), in the presence (○) and absence (●) of the inhibitor PBO. The data with inhibition are subtracted from the data without inhibition to yield

    Techniques Used: Inhibition

    13) Product Images from "A High-Throughput Screening Compatible Assay for Activators and Inhibitors of Methionine Sulfoxide Reductase A"

    Article Title: A High-Throughput Screening Compatible Assay for Activators and Inhibitors of Methionine Sulfoxide Reductase A

    Journal: Assay and Drug Development Technologies

    doi: 10.1089/adt.2009.0263

    Effect of SeCm and NEM on the reduction of DMSO using a fluorescence assay. The oxidation of NADPH is measured using a fluorescence assay for reactions containing MsrA, DMSO, and the thioredoxin reducing system (control, ♦) plus either SeCm (▾)
    Figure Legend Snippet: Effect of SeCm and NEM on the reduction of DMSO using a fluorescence assay. The oxidation of NADPH is measured using a fluorescence assay for reactions containing MsrA, DMSO, and the thioredoxin reducing system (control, ♦) plus either SeCm (▾)

    Techniques Used: Fluorescence

    Effect of selenocystamine (SeCm) and N -ethylmaleimide (NEM) on the reduction of DMSO by MsrA. The oxidation of NADPH is measured using an absorbance assay for reactions containing MsrA, DMSO, and the thioredoxin reducing system (control, ♦) plus
    Figure Legend Snippet: Effect of selenocystamine (SeCm) and N -ethylmaleimide (NEM) on the reduction of DMSO by MsrA. The oxidation of NADPH is measured using an absorbance assay for reactions containing MsrA, DMSO, and the thioredoxin reducing system (control, ♦) plus

    Techniques Used:

    Related Articles

    Conditioned Place Preference:

    Article Title: NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward
    Article Snippet: .. Mice were injected between ZT9–11 on days 1–5 (~12–16 hrs prior to CPP) with saline or the NAD+ precursor, NMN (β-Nicotinamide mononucleotide, 500mg/kg, i.p., dissolved in saline, Sigma Aldrich, n=9–10 per group). .. The CPP score was calculated as the amount of time spent within the conditioned chamber on the test day subtracted from the amount of time spent on the same side on the pretest day.

    Cell Culture:

    Article Title: Acetylation of Pregnane X Receptor protein determines selective function independent of ligand activation
    Article Snippet: .. Cell culture media, charcoal adsorbed fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), rifampicin (rif), pregnenolone carbonitrile (PCN), 9-cis-retinoic acid (RA), nicotinamide (NAM), resveratrol (res), trichostatin A (TSA) and Nile Red were purchased from Sigma Aldrich (St. Louis, MO). .. Effectene transfection reagent and Ni-NTA-agarose were purchased from Qiagen (Valencia, CA).

    Mouse Assay:

    Article Title: NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward
    Article Snippet: .. Mice were injected between ZT9–11 on days 1–5 (~12–16 hrs prior to CPP) with saline or the NAD+ precursor, NMN (β-Nicotinamide mononucleotide, 500mg/kg, i.p., dissolved in saline, Sigma Aldrich, n=9–10 per group). .. The CPP score was calculated as the amount of time spent within the conditioned chamber on the test day subtracted from the amount of time spent on the same side on the pretest day.

    Concentration Assay:

    Article Title: Temozolomide-Perillyl alcohol conjugate impairs Mitophagy flux by inducing lysosomal dysfunction in non-small cell lung Cancer cells and sensitizes them to irradiation
    Article Snippet: .. Temozolomide (TMZ), 3-Methyladenine (3-MA), baflomycin A1 (Baf.A1), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Nicotinamide (NAM), mevalonolactone (MVL), geranylgeraniol (GGOH), catalase (CAT) and N-acetyl-L-cysteine (NAC), Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich, Shanghai, China) were dissolved in DMSO or deionized water dependently; In all cases of cell treatment, the final DMSO concentration in the culture medium never exceeded 0.5%. ..

    Nuclear Magnetic Resonance:

    Article Title: Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis *
    Article Snippet: .. CSP were measured between a reference NMR sample containing 0.1 m m NarE protein in the previously described buffer and test samples additionally containing (I) 1 m m DTT (II) 1 m m DTT, 0.1 m m FeCl3 (Sigma-Aldrich) (III) 1 m m DTT, 0.2 m m β-nicotinamide-adenine-dinucleotide (NAD; Sigma) (IV) 1 m m DTT, 0.1 m m FeCl3 , 0.2 m m NAD (V) 1 m m DTT, 0.1 m m ZnCl2 (Merck). .. Residues showing chemical shift perturbations were assumed to be less affected by the ligand binding than residues showing a signal attenuation.

    other:

    Article Title: Novel neuroprotective therapy with NeuroHeal by autophagy induction for damaged neonatal motoneurons
    Article Snippet: NeuroHeal and 5 mM nicotinamide (NAM) (Sigma-Aldrich) were added to drinking water given to the mothers on the day prior to injury to ensure that the pups began to receive drug on the day of the injury.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Temozolomide-Perillyl alcohol conjugate impairs Mitophagy flux by inducing lysosomal dysfunction in non-small cell lung Cancer cells and sensitizes them to irradiation
    Article Snippet: .. Temozolomide (TMZ), 3-Methyladenine (3-MA), baflomycin A1 (Baf.A1), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Nicotinamide (NAM), mevalonolactone (MVL), geranylgeraniol (GGOH), catalase (CAT) and N-acetyl-L-cysteine (NAC), Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich, Shanghai, China) were dissolved in DMSO or deionized water dependently; In all cases of cell treatment, the final DMSO concentration in the culture medium never exceeded 0.5%. ..

    Injection:

    Article Title: NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward
    Article Snippet: .. Mice were injected between ZT9–11 on days 1–5 (~12–16 hrs prior to CPP) with saline or the NAD+ precursor, NMN (β-Nicotinamide mononucleotide, 500mg/kg, i.p., dissolved in saline, Sigma Aldrich, n=9–10 per group). .. The CPP score was calculated as the amount of time spent within the conditioned chamber on the test day subtracted from the amount of time spent on the same side on the pretest day.

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    Millipore aldered aldh detection kit
    CAF-derived exosomes transfer H19 to colorectal cancer cells. (A) The representative images of exosomes derived from CAFs and NFs analyzed by transmission electron microscopy. Scale bar, 100 nm. (B) The size distribution of the isolated exosomes was analyzed by nanoparticle tracking analysis. (C) Western blotting analysis for exosomal markers CD63, Hsp70 and GM130 of SW480 cells and exosomes derived from CAF cells. (D-E) CAFs were pre-treated with or without GW4869. Then, the CAFs were labeled with CM-Dil (red) and co-cultured with SW480 for 18 h. Immunofluorescence analyzed the distribution of CM-Dil in cells. Scale bars, 20 µm. (F) qRT-PCR analyzed the level of H19 in the isolated exosomes derived from CAFs and NFs of five patients. (G) The SW480 cells were incubated with indicated exosomes or PBS for 48 h, and the level of H19 was analyzed by qRT-PCR. (H) The SW480 and HCT116 were incubated with exosomes (10 µg/mL from CAFs or NFs for 24 h, and the capacity of cells to form spheres was assessed. Measurements were taken digitally, and mammospheres were quantified using six pictures per well. The mean number of mammospheres > 75 μm was counted and shown. (I-J) SW480 cells were incubated with exosomes (10 µg/mL) from CAFs or NFs from three patients for 48 h, and <t>AldeRed</t> <t>ALDH</t> detection assay was performed. (K) SW480 and HCT116 cells were incubated with exosomes (10 µg/mL) from CAFs or NFs from three patients for 24 h. Cells were then treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h, and the cell viability was measured by CCK8. All representative data are from three independent experiments. Error bars, SD. *P
    Aldered Aldh Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    85
    Millipore nad glycohydrolase activity
    S . mansoni utilizes extracellular <t>NAD</t> to support intracellular NAD homeostasis and egg production. (A) Genomic reconstruction of NAD metabolism in S . mansoni . Genes (red boxes with gene names listed below) encoding enzymes predicted to participate in the NAD-biosynthesis salvage pathways (left side of cartoon) and NAD catabolism (right side of cartoon) are shown. Metabolites (blue boxes) in the pathways and inhibitors (yellow boxes) of NAMPT and SmNACE are indicated. No genes encoding candidate orthologs of enzymes in the de novo NAD synthesis pathway from tryptophan or aspartate were identified. Enzymes include: PNP (Purine Nucleotide Phosphorylase), MTAP (Nucleoside hydrolase), NAMPT (Nicotinamide phosphoribosyltransferase), NAPRT (Nicotinic acid phosphoribosyltransferase), NMNAT (Nicotinamide mononucleotide adenylyltransferase), GAT (Glutamine amidotransferase), NADS (NAD Synthetase), NADK (NAD Kinase), SmNACE ( S . mansoni NAD-catabolizing enzyme) and Poly-ADP-ribose polymerase (PARP). Metabolites include: NAD(P) (nicotinamide adenine dinucleotide (phosphate)), NAM (nicotinamide), NA (nicotinic acid), NR (nicotinamide riboside), NMN (nicotinamide mononucleotide), NaMN (nicotinic acid mononucleotide) and NAAD (nicotinic acid adenine dinucleotide). (B) S . mansoni genes encoding putative NAD salvage pathway biosynthetic enzymes are transcribed. PCR amplification of NADS, NAPRT, GAT, NAMPT, NMNAT, MTAP, NADK1 and NADK2 from cDNA prepared from RNA isolated from adult S . mansoni . Transcripts for PNP were not detected. (C-D) Recombinant SmNACE (rSmNACE, panel C) and live male or female S . mansoni (n = 1 parasites/well; panel D) catabolize extracellular NAD. NAD <t>glycohydrolase</t> activity measured by monitoring hydrolysis of the NAM-ribose bond in etheno-NAD (ε-NAD) resulting in release of NAM and ε-ADPR, which is detected using a fluorimeter. Data reported as Relative Fluorescence Units (RFU) measured over time. (E) SmNACE is expressed by male and female S . mansoni . A SmNACE standard curve, which was generated by measuring the NAD glycohydrolase activity of increasing concentrations of rSmNACE (see panel C), was used to determine the amount of enzymatically active native SmNACE expressed by live S . mansoni parasites. Data pooled from 2 experiments with n = 4 wells/group and 1 parasite/well. (F) Intracellular NAD levels in S . mansoni females cultured for 48 h in serum free media (SFM) ± 2mM extracellular NAD. Data represents n = 5 wells/group with 2 female parasites/well. (G) Native SmNACE activity is blocked by the SmNACE inhibitor CMP1. NAD glycohydrolase activity of adult schistosomes incubated with ε-NAD ± increasing concentrations of CMP1. The IC 50 of CMP1 on live parasites is indicated. n = 2 wells/group with 4 parasites/well. (H) Intracellular NAD levels in S . mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well. (I) Egg production in S . mansoni females cultured for 48h in SFM ± 2mM extracellular NAD. Data are pooled from 3 experiments with n = 15 wells/group and 2 female parasites/well. (J) Egg production in S . mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 6 wells/group with 2 female parasites/well. (K) S . mansoni egg production is restored following removal of SmNACE inhibitor. Egg production by S . mansoni females cultured 48h with 2mM extracellular NAD + CMP1 (200μM) and then washed and recultured for an additional 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well). Data are representative of 2 (C-H, J-K) or 3 (I) independent experiments and are represented as the mean ± SD (bars) of individual samples (squares) (E-F, H-K). Statistical analyses were performed using two-tailed Student’s t test.
    Nad Glycohydrolase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 3 article reviews
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    nad glycohydrolase activity - by Bioz Stars, 2020-09
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    Image Search Results


    CAF-derived exosomes transfer H19 to colorectal cancer cells. (A) The representative images of exosomes derived from CAFs and NFs analyzed by transmission electron microscopy. Scale bar, 100 nm. (B) The size distribution of the isolated exosomes was analyzed by nanoparticle tracking analysis. (C) Western blotting analysis for exosomal markers CD63, Hsp70 and GM130 of SW480 cells and exosomes derived from CAF cells. (D-E) CAFs were pre-treated with or without GW4869. Then, the CAFs were labeled with CM-Dil (red) and co-cultured with SW480 for 18 h. Immunofluorescence analyzed the distribution of CM-Dil in cells. Scale bars, 20 µm. (F) qRT-PCR analyzed the level of H19 in the isolated exosomes derived from CAFs and NFs of five patients. (G) The SW480 cells were incubated with indicated exosomes or PBS for 48 h, and the level of H19 was analyzed by qRT-PCR. (H) The SW480 and HCT116 were incubated with exosomes (10 µg/mL from CAFs or NFs for 24 h, and the capacity of cells to form spheres was assessed. Measurements were taken digitally, and mammospheres were quantified using six pictures per well. The mean number of mammospheres > 75 μm was counted and shown. (I-J) SW480 cells were incubated with exosomes (10 µg/mL) from CAFs or NFs from three patients for 48 h, and AldeRed ALDH detection assay was performed. (K) SW480 and HCT116 cells were incubated with exosomes (10 µg/mL) from CAFs or NFs from three patients for 24 h. Cells were then treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h, and the cell viability was measured by CCK8. All representative data are from three independent experiments. Error bars, SD. *P

    Journal: Theranostics

    Article Title: Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19

    doi: 10.7150/thno.25541

    Figure Lengend Snippet: CAF-derived exosomes transfer H19 to colorectal cancer cells. (A) The representative images of exosomes derived from CAFs and NFs analyzed by transmission electron microscopy. Scale bar, 100 nm. (B) The size distribution of the isolated exosomes was analyzed by nanoparticle tracking analysis. (C) Western blotting analysis for exosomal markers CD63, Hsp70 and GM130 of SW480 cells and exosomes derived from CAF cells. (D-E) CAFs were pre-treated with or without GW4869. Then, the CAFs were labeled with CM-Dil (red) and co-cultured with SW480 for 18 h. Immunofluorescence analyzed the distribution of CM-Dil in cells. Scale bars, 20 µm. (F) qRT-PCR analyzed the level of H19 in the isolated exosomes derived from CAFs and NFs of five patients. (G) The SW480 cells were incubated with indicated exosomes or PBS for 48 h, and the level of H19 was analyzed by qRT-PCR. (H) The SW480 and HCT116 were incubated with exosomes (10 µg/mL from CAFs or NFs for 24 h, and the capacity of cells to form spheres was assessed. Measurements were taken digitally, and mammospheres were quantified using six pictures per well. The mean number of mammospheres > 75 μm was counted and shown. (I-J) SW480 cells were incubated with exosomes (10 µg/mL) from CAFs or NFs from three patients for 48 h, and AldeRed ALDH detection assay was performed. (K) SW480 and HCT116 cells were incubated with exosomes (10 µg/mL) from CAFs or NFs from three patients for 24 h. Cells were then treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h, and the cell viability was measured by CCK8. All representative data are from three independent experiments. Error bars, SD. *P

    Article Snippet: The AldeRed ALDH Detection Kit (Millipore, USA) was used following the manufacturer's instructions .

    Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Western Blot, Labeling, Cell Culture, Immunofluorescence, Quantitative RT-PCR, Incubation, ALDH Detection Assay

    H19 promotes the stemness and oxaliplatin resistance of colorectal cancer. (A) SW480 cells were transfected with three small interfering RNAs. The level of H19 in the SW480 cells after transfection was analyzed by qRT-PCR. The H19 in SW480 was significantly knocked down by si-H19-1 and si-H19-3 transfection. (B-C) SW480 and HCT116 cells were transfected with si-control, mixture of si-H19-1 and si-H19-3, pcDNA3.1 or pcDNA3.1-H19, and the knockdown of H19 significantly decreased the capacity to form spheres. Representative images are presented. Scale bars, 200 µm. (D-E) The SW480 and HCT116 cells were transfected with si-control, mixture of si-H19-1 and si-H19-3, pcDNA3.1 or pcDNA3.1-H19, and an AldeRed ALDH detection assay was performed. Representative images are presented; the ALDH1 high cell population was increased by H19 overexpression and decreased by knockdown of H19. (F) H19 was overexpressed in SW480 and HCT116 cells, which were then treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h for the CCK8 assay. H19 overexpression promoted oxaliplatin resistance. (G-H) Expression of H19 were inhibited or overexpressed in SW480 cells. Then, cells were treated with 4 μM oxaliplatin for 48 h. Flow cytometry was used to analyze cell apoptosis. H19 overexpression decreased the cell apoptosis induced by oxaliplatin. All representative data are from three independent experiments. Error bars, SD. *P

    Journal: Theranostics

    Article Title: Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19

    doi: 10.7150/thno.25541

    Figure Lengend Snippet: H19 promotes the stemness and oxaliplatin resistance of colorectal cancer. (A) SW480 cells were transfected with three small interfering RNAs. The level of H19 in the SW480 cells after transfection was analyzed by qRT-PCR. The H19 in SW480 was significantly knocked down by si-H19-1 and si-H19-3 transfection. (B-C) SW480 and HCT116 cells were transfected with si-control, mixture of si-H19-1 and si-H19-3, pcDNA3.1 or pcDNA3.1-H19, and the knockdown of H19 significantly decreased the capacity to form spheres. Representative images are presented. Scale bars, 200 µm. (D-E) The SW480 and HCT116 cells were transfected with si-control, mixture of si-H19-1 and si-H19-3, pcDNA3.1 or pcDNA3.1-H19, and an AldeRed ALDH detection assay was performed. Representative images are presented; the ALDH1 high cell population was increased by H19 overexpression and decreased by knockdown of H19. (F) H19 was overexpressed in SW480 and HCT116 cells, which were then treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h for the CCK8 assay. H19 overexpression promoted oxaliplatin resistance. (G-H) Expression of H19 were inhibited or overexpressed in SW480 cells. Then, cells were treated with 4 μM oxaliplatin for 48 h. Flow cytometry was used to analyze cell apoptosis. H19 overexpression decreased the cell apoptosis induced by oxaliplatin. All representative data are from three independent experiments. Error bars, SD. *P

    Article Snippet: The AldeRed ALDH Detection Kit (Millipore, USA) was used following the manufacturer's instructions .

    Techniques: Transfection, Quantitative RT-PCR, ALDH Detection Assay, Over Expression, CCK-8 Assay, Expressing, Flow Cytometry, Cytometry

    CAF-derived exosomes might be involved in the enhanced expression of H19, stemness and oxaliplatin resistance of colorectal cancer cells. (A) In situ analysis with Cy3-labeled RNA using an H19 FISH probe in CRC tissues. The scale bar represents 50 μm. CRC tissues were also stained with pancytokeratins (cancer nest) and α-SMA (stroma) by immunofluorescence assay. (B) qRT-PCR analyzed the level of H19 in isolated CAFs and NFs. SW480 and HCT116 cells were cultured under conditioned medium derived from NFs (NF-CM) and CAFs (CAF-CM) for 4 days. (C) The capacity of cells to form spheres was assessed. Measurements were taken digitally, and mammospheres were quantified using six pictures per well. The mean number of mammospheres > 75 μm was counted and shown. (D) CAF cells were treated with GW4869, and the conditioned medium was collected. CAF1-CM (GW4869) represents the conditioned medium of CAF cells treated with GW4869. The SW480 and HCT116 cells were cultured under NF-CM, CAF-CM and CAF1-CM (GW4869) for 4 days. The AldeRed ALDH detection assay was performed to detect the intracellular ALDH enzyme activity. The ALDH1 high population was defined as cells showing a right shift in fluorescence in the absence of DEAB. (E) The percentage of the ALDH1 high cancer cell population. (F-G) SW480 and HCT116 cells were cultured under NF-CM and CAF-CM for 4 days. Cells were treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h, and cell viability was measured by CCK8. (H) SW480 cells were cultured under NF-CM and CAF-CM for 4 days. Cells were then treated with 4 μM oxaliplatin for 48 h, and cell apoptosis was measured by flow cytometry. (I) SW480 cells were cultured under NF-CM, CAF-CM and CAF1-CM (GW4869) for 4 days. The level of H19 was analyzed by qRT-PCR. (J) SW480 cells were pretreated with 10 μM galunisertib or PBS and were then treated with NF-CM, CAF-CM and CAF1-CM (GW4869) for 4 days. Levels of H19 in cells were analyzed. All representative data are from three independent experiments. Error bars, SD. *P

    Journal: Theranostics

    Article Title: Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19

    doi: 10.7150/thno.25541

    Figure Lengend Snippet: CAF-derived exosomes might be involved in the enhanced expression of H19, stemness and oxaliplatin resistance of colorectal cancer cells. (A) In situ analysis with Cy3-labeled RNA using an H19 FISH probe in CRC tissues. The scale bar represents 50 μm. CRC tissues were also stained with pancytokeratins (cancer nest) and α-SMA (stroma) by immunofluorescence assay. (B) qRT-PCR analyzed the level of H19 in isolated CAFs and NFs. SW480 and HCT116 cells were cultured under conditioned medium derived from NFs (NF-CM) and CAFs (CAF-CM) for 4 days. (C) The capacity of cells to form spheres was assessed. Measurements were taken digitally, and mammospheres were quantified using six pictures per well. The mean number of mammospheres > 75 μm was counted and shown. (D) CAF cells were treated with GW4869, and the conditioned medium was collected. CAF1-CM (GW4869) represents the conditioned medium of CAF cells treated with GW4869. The SW480 and HCT116 cells were cultured under NF-CM, CAF-CM and CAF1-CM (GW4869) for 4 days. The AldeRed ALDH detection assay was performed to detect the intracellular ALDH enzyme activity. The ALDH1 high population was defined as cells showing a right shift in fluorescence in the absence of DEAB. (E) The percentage of the ALDH1 high cancer cell population. (F-G) SW480 and HCT116 cells were cultured under NF-CM and CAF-CM for 4 days. Cells were treated with different concentrations of oxaliplatin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM) for 24 h, and cell viability was measured by CCK8. (H) SW480 cells were cultured under NF-CM and CAF-CM for 4 days. Cells were then treated with 4 μM oxaliplatin for 48 h, and cell apoptosis was measured by flow cytometry. (I) SW480 cells were cultured under NF-CM, CAF-CM and CAF1-CM (GW4869) for 4 days. The level of H19 was analyzed by qRT-PCR. (J) SW480 cells were pretreated with 10 μM galunisertib or PBS and were then treated with NF-CM, CAF-CM and CAF1-CM (GW4869) for 4 days. Levels of H19 in cells were analyzed. All representative data are from three independent experiments. Error bars, SD. *P

    Article Snippet: The AldeRed ALDH Detection Kit (Millipore, USA) was used following the manufacturer's instructions .

    Techniques: Derivative Assay, Expressing, In Situ, Labeling, Fluorescence In Situ Hybridization, Staining, Immunofluorescence, Quantitative RT-PCR, Isolation, Cell Culture, ALDH Detection Assay, Activity Assay, Fluorescence, Flow Cytometry, Cytometry

    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

    S . mansoni utilizes extracellular NAD to support intracellular NAD homeostasis and egg production. (A) Genomic reconstruction of NAD metabolism in S . mansoni . Genes (red boxes with gene names listed below) encoding enzymes predicted to participate in the NAD-biosynthesis salvage pathways (left side of cartoon) and NAD catabolism (right side of cartoon) are shown. Metabolites (blue boxes) in the pathways and inhibitors (yellow boxes) of NAMPT and SmNACE are indicated. No genes encoding candidate orthologs of enzymes in the de novo NAD synthesis pathway from tryptophan or aspartate were identified. Enzymes include: PNP (Purine Nucleotide Phosphorylase), MTAP (Nucleoside hydrolase), NAMPT (Nicotinamide phosphoribosyltransferase), NAPRT (Nicotinic acid phosphoribosyltransferase), NMNAT (Nicotinamide mononucleotide adenylyltransferase), GAT (Glutamine amidotransferase), NADS (NAD Synthetase), NADK (NAD Kinase), SmNACE ( S . mansoni NAD-catabolizing enzyme) and Poly-ADP-ribose polymerase (PARP). Metabolites include: NAD(P) (nicotinamide adenine dinucleotide (phosphate)), NAM (nicotinamide), NA (nicotinic acid), NR (nicotinamide riboside), NMN (nicotinamide mononucleotide), NaMN (nicotinic acid mononucleotide) and NAAD (nicotinic acid adenine dinucleotide). (B) S . mansoni genes encoding putative NAD salvage pathway biosynthetic enzymes are transcribed. PCR amplification of NADS, NAPRT, GAT, NAMPT, NMNAT, MTAP, NADK1 and NADK2 from cDNA prepared from RNA isolated from adult S . mansoni . Transcripts for PNP were not detected. (C-D) Recombinant SmNACE (rSmNACE, panel C) and live male or female S . mansoni (n = 1 parasites/well; panel D) catabolize extracellular NAD. NAD glycohydrolase activity measured by monitoring hydrolysis of the NAM-ribose bond in etheno-NAD (ε-NAD) resulting in release of NAM and ε-ADPR, which is detected using a fluorimeter. Data reported as Relative Fluorescence Units (RFU) measured over time. (E) SmNACE is expressed by male and female S . mansoni . A SmNACE standard curve, which was generated by measuring the NAD glycohydrolase activity of increasing concentrations of rSmNACE (see panel C), was used to determine the amount of enzymatically active native SmNACE expressed by live S . mansoni parasites. Data pooled from 2 experiments with n = 4 wells/group and 1 parasite/well. (F) Intracellular NAD levels in S . mansoni females cultured for 48 h in serum free media (SFM) ± 2mM extracellular NAD. Data represents n = 5 wells/group with 2 female parasites/well. (G) Native SmNACE activity is blocked by the SmNACE inhibitor CMP1. NAD glycohydrolase activity of adult schistosomes incubated with ε-NAD ± increasing concentrations of CMP1. The IC 50 of CMP1 on live parasites is indicated. n = 2 wells/group with 4 parasites/well. (H) Intracellular NAD levels in S . mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well. (I) Egg production in S . mansoni females cultured for 48h in SFM ± 2mM extracellular NAD. Data are pooled from 3 experiments with n = 15 wells/group and 2 female parasites/well. (J) Egg production in S . mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 6 wells/group with 2 female parasites/well. (K) S . mansoni egg production is restored following removal of SmNACE inhibitor. Egg production by S . mansoni females cultured 48h with 2mM extracellular NAD + CMP1 (200μM) and then washed and recultured for an additional 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well). Data are representative of 2 (C-H, J-K) or 3 (I) independent experiments and are represented as the mean ± SD (bars) of individual samples (squares) (E-F, H-K). Statistical analyses were performed using two-tailed Student’s t test.

    Journal: PLoS Pathogens

    Article Title: Inhibition of the NAD salvage pathway in schistosomes impairs metabolism, reproduction, and parasite survival

    doi: 10.1371/journal.ppat.1008539

    Figure Lengend Snippet: S . mansoni utilizes extracellular NAD to support intracellular NAD homeostasis and egg production. (A) Genomic reconstruction of NAD metabolism in S . mansoni . Genes (red boxes with gene names listed below) encoding enzymes predicted to participate in the NAD-biosynthesis salvage pathways (left side of cartoon) and NAD catabolism (right side of cartoon) are shown. Metabolites (blue boxes) in the pathways and inhibitors (yellow boxes) of NAMPT and SmNACE are indicated. No genes encoding candidate orthologs of enzymes in the de novo NAD synthesis pathway from tryptophan or aspartate were identified. Enzymes include: PNP (Purine Nucleotide Phosphorylase), MTAP (Nucleoside hydrolase), NAMPT (Nicotinamide phosphoribosyltransferase), NAPRT (Nicotinic acid phosphoribosyltransferase), NMNAT (Nicotinamide mononucleotide adenylyltransferase), GAT (Glutamine amidotransferase), NADS (NAD Synthetase), NADK (NAD Kinase), SmNACE ( S . mansoni NAD-catabolizing enzyme) and Poly-ADP-ribose polymerase (PARP). Metabolites include: NAD(P) (nicotinamide adenine dinucleotide (phosphate)), NAM (nicotinamide), NA (nicotinic acid), NR (nicotinamide riboside), NMN (nicotinamide mononucleotide), NaMN (nicotinic acid mononucleotide) and NAAD (nicotinic acid adenine dinucleotide). (B) S . mansoni genes encoding putative NAD salvage pathway biosynthetic enzymes are transcribed. PCR amplification of NADS, NAPRT, GAT, NAMPT, NMNAT, MTAP, NADK1 and NADK2 from cDNA prepared from RNA isolated from adult S . mansoni . Transcripts for PNP were not detected. (C-D) Recombinant SmNACE (rSmNACE, panel C) and live male or female S . mansoni (n = 1 parasites/well; panel D) catabolize extracellular NAD. NAD glycohydrolase activity measured by monitoring hydrolysis of the NAM-ribose bond in etheno-NAD (ε-NAD) resulting in release of NAM and ε-ADPR, which is detected using a fluorimeter. Data reported as Relative Fluorescence Units (RFU) measured over time. (E) SmNACE is expressed by male and female S . mansoni . A SmNACE standard curve, which was generated by measuring the NAD glycohydrolase activity of increasing concentrations of rSmNACE (see panel C), was used to determine the amount of enzymatically active native SmNACE expressed by live S . mansoni parasites. Data pooled from 2 experiments with n = 4 wells/group and 1 parasite/well. (F) Intracellular NAD levels in S . mansoni females cultured for 48 h in serum free media (SFM) ± 2mM extracellular NAD. Data represents n = 5 wells/group with 2 female parasites/well. (G) Native SmNACE activity is blocked by the SmNACE inhibitor CMP1. NAD glycohydrolase activity of adult schistosomes incubated with ε-NAD ± increasing concentrations of CMP1. The IC 50 of CMP1 on live parasites is indicated. n = 2 wells/group with 4 parasites/well. (H) Intracellular NAD levels in S . mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well. (I) Egg production in S . mansoni females cultured for 48h in SFM ± 2mM extracellular NAD. Data are pooled from 3 experiments with n = 15 wells/group and 2 female parasites/well. (J) Egg production in S . mansoni females cultured for 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 6 wells/group with 2 female parasites/well. (K) S . mansoni egg production is restored following removal of SmNACE inhibitor. Egg production by S . mansoni females cultured 48h with 2mM extracellular NAD + CMP1 (200μM) and then washed and recultured for an additional 48h with 2mM extracellular NAD ± CMP1 (200μM) or vehicle (2% DMSO). n = 5 wells/group with 2 female parasites/well). Data are representative of 2 (C-H, J-K) or 3 (I) independent experiments and are represented as the mean ± SD (bars) of individual samples (squares) (E-F, H-K). Statistical analyses were performed using two-tailed Student’s t test.

    Article Snippet: SmNACE activity and inhibition assays The NAD glycohydrolase activity of rSmNACE [ ] and native SmNACE was measured as previously described [ ] using 1,N6 -ethenonicotinamide adenine dinucleotide (ε-NAD; MilliporeSigma).

    Techniques: Polymerase Chain Reaction, Amplification, Isolation, Recombinant, Activity Assay, Fluorescence, Generated, Cell Culture, Incubation, Two Tailed Test