Journal: Frontiers in Veterinary Science

Article Title: Pharmacokinetics and Tissue Levels of Pantoprazole in Neonatal Calves After Intravenous Administration

doi: 10.3389/fvets.2020.580735

Figure Lengend Snippet: Mean plasma pantoprazole concentration (logarithmic scale) vs. time (hr) profiles for neonatal calves ( n = 9) following intravenous (IV) single dose administration of 1.0 mg/kg of pantoprazole.

Article Snippet: Pantoprazole sulfone (Toronto Research Chemicals, Ontario, Canada) became the analyte spiked into blank tissue samples to generate calibration spikes and QC samples.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Frontiers in Veterinary Science

Article Title: Pharmacokinetics and Tissue Levels of Pantoprazole in Neonatal Calves After Intravenous Administration

doi: 10.3389/fvets.2020.580735

Figure Lengend Snippet: Pantoprazole pharmacokinetic parameters following a single intravenous (1 mg/kg) administration to neonatal Holstein calves.

Article Snippet: Pantoprazole sulfone (Toronto Research Chemicals, Ontario, Canada) became the analyte spiked into blank tissue samples to generate calibration spikes and QC samples.

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Pharmacokinetics and Tissue Levels of Pantoprazole in Neonatal Calves After Intravenous Administration

doi: 10.3389/fvets.2020.580735

Figure Lengend Snippet: Tissue concentrations of pantoprazole sulfone (μg/g) in collected tissues 1, 3, and 5 days after intravenous administration of pantoprazole (1 mg/kg) from study calves.

Article Snippet: Pantoprazole sulfone (Toronto Research Chemicals, Ontario, Canada) became the analyte spiked into blank tissue samples to generate calibration spikes and QC samples.

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Pharmacokinetics and Tissue Levels of Pantoprazole in Neonatal Calves After Intravenous Administration

doi: 10.3389/fvets.2020.580735

Figure Lengend Snippet: Comparisons of pharmacokinetic parameters of pantoprazole in domestic animal species, after single dose intravenous administration.

Article Snippet: Pantoprazole sulfone (Toronto Research Chemicals, Ontario, Canada) became the analyte spiked into blank tissue samples to generate calibration spikes and QC samples.

Techniques:

Journal: Molecular Pharmaceutics

Article Title: Role of the Phospholipase A 2 Receptor in Liposome Drug Delivery in Prostate Cancer Cells

doi: 10.1021/mp500174p

Figure Lengend Snippet: Effect of PLA2R1 knockdown on chemotherapeutic-induced toxicity in PC-3 cells. Cells were treated with docetaxel (A), cisplatin (B), or Dox (C) for 72 h. MTT assays were used to determine the effect of knocking down PLA2R1 on chemotherapeutic-induced cytotoxicity. The effect of PLA2R1 on doxorubicin-induced toxicity was further assessed at 72 h using phase contrast microscopy at 10× magnification (D). Data in panels A–C are represented as the mean ± SD of at least three separate experiments ( n = 3/study). Data in panel D are indicative of at least three separate experiments.

Article Snippet: Doxorubicin (Dox) was purchased from Toronto Research Chemicals (North York, ON, Canada).

Techniques: Knockdown, Microscopy

Journal: Molecular Pharmaceutics

Article Title: Role of the Phospholipase A 2 Receptor in Liposome Drug Delivery in Prostate Cancer Cells

doi: 10.1021/mp500174p

Figure Lengend Snippet: Effect of PLA2R1 knockdown on the toxicity of doxorubicin encapsulated in SSL and SPRL in PC-3 cells. PLA2R1 knockdown cells and those expressing scrambled shRNA were treated with 2.5 μM concentrations of Dox encapsulated in SSL or SPRL. MTT assays (A) and phase contrast microscopy at 40× magnification (B) were used to determine the effect of PLA2R1 knockdown on cytotoxicity. Data in panel A are represented as the mean ± SEM of at least three separate experiments ( n = 3/study). *Indicates a significant difference ( p < 0.05) as compared to cells transfected with scrambled shRNA. Data in panel E are indicative of at least three separate experiments.

Article Snippet: Doxorubicin (Dox) was purchased from Toronto Research Chemicals (North York, ON, Canada).

Techniques: Knockdown, Expressing, shRNA, Microscopy, Transfection

Journal: Molecular Pharmaceutics

Article Title: Role of the Phospholipase A 2 Receptor in Liposome Drug Delivery in Prostate Cancer Cells

doi: 10.1021/mp500174p

Figure Lengend Snippet: Effect of PLA2R1 knockdown on DiO and doxorubicin uptake from SSL and SPRL in PC-3 cells. PC-3 cells were treated with liposomes containing Dox and DiO for 24 to 72 h. The efficiency of DiO and drug uptake via SSL (A, B) and SPRL (C, D) was quantified using flow cytometry. Data are represented as the mean ± SEM of at least three separate experiments ( n = 3/study). *Indicates a significant difference ( p < 0.05) as compared to cells expressing scrambled shRNA.

Article Snippet: Doxorubicin (Dox) was purchased from Toronto Research Chemicals (North York, ON, Canada).

Techniques: Knockdown, Liposomes, Flow Cytometry, Expressing, shRNA

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: Cytotoxicity of amiodarone (AM) and its major metabolite desethylamiodarone (DEA) in HepG2 cells. HepG2 cells were treated with 6.25–100 μM of amiodarone or desethylamiodarone for 6 or 24 h. The cell viability was measured by a CellTiter-Glo assay at the end of treatments. Data were presented as mean±SD of three independent experiments. *P < 0.05 compared with the cell viability of amiodarone at the same concentration.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Glo Assay, Concentration Assay

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: HPLC-QDa mass spectrometry chromatograms for amiodarone (AM) and its metabolite desethylamiodarone (DEA) in an extract from a human microsomal incubation of 12.5 μM amiodarone for 60 min. The analytes were separated using a Waters e2695 HPLC System and monitored with an ACQUITY QDa detector in a positive-electrospray ionization mode. The chromatogram shows selected ion recording (SIR) channels for (a) amiodarone (m/z 646), its internal standard (b) amiodarone-d4 (m/z 650), (c) desethylamiodarone (m/z 618), and its internal standard (d) desethylamiodarone-d4 (m/z 622).

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Mass Spectrometry, Incubation

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: Desethylamiodarone formation in HepG2Cells overexpressing human CYPs. Different human CYPs overexpressed or empty vector-transduced HepG2 cells were treated with 12.5 μM of amiodarone for 6 h. At the end of treatment, cell pellets were harvested and extracted with ice-cold acetonitrile containing 100 ng/ml of deuterated internal standard desethylamiodarone-d4. Desethylamiodarone was quantified with LC–MS as described in Section 2. Data were presented as mean±SD of three independent experiments. *P < 0.05 compared with that of HepG2/empty vector.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy

Journal: Toxicology letters

Article Title: The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity ✩

doi: 10.1016/j.toxlet.2016.04.016

Figure Lengend Snippet: (A) Effects of CYP1A1 or CYP3A4 inhibition on the formation of desethylamiodarone in human microsomal incubation of amiodarone. Human microsomes were incubated with 12.5 μM of amiodarone or coincubated with α-naphthoflavone (50 μM) or ketoconazole (50 μM) in a total volume of 200 μl for 60 min. The incubations were quenched by adding 1 ml of ice-cold acetonitrile containing deuterated internal standards. The amount of amiodarone and desethylamiodarone in extractions was measured with LC–MS as described in Materials and Methods. Data are presented as mean±SD of three independent experiments. *P < 0.05 amiodarone amount compared to that of cells without CYP inhibitor addition; #P < 0.05 desethylamiodarone formation compared to that of cells without CYP inhibitor addition. (B) Effects of CYP inhibition on the formation of desethylamiodarone in HepG2 cells overexpressing CYP1A1 or CYP3A4. HepG2 cells overexpressing CYP1A1 were treated with 12.5 μM of amiodarone (AM) or co-incubated with α-naphthoflavone (10 μM) for 6 h; and HepG2 cells overexpressing CYP3A4 were treated with 12.5 μM of amiodarone or co-incubated with ketoconazole (10 μM) for 6 h. Cell pellets were harvested and extracted with ice-cold acetonitrile containing deuterated internal standards. The extractions were measured by LC–MS for the amounts of amiodarone and desethylamiodarone amounts. The bar graphs show as mean±SD of three independent experiments. *P < 0.05 versus without inhibition.

Article Snippet: Desethylamiodarone hydrochloride, amiodarone-d4 hydrochloride, and desethylamiodarone-d4 hydrochloride were obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Techniques: Inhibition, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: Oncogenic kinases ALK and SRC phosphorylate IMPDH2 at specific tyrosine residues. ( A ), Schematic of the in vitro kinase assay used to evaluate tyrosine phosphorylation of IMPDH2 by ALK and SRC kinases. ( B ), Western blot analysis of in vitro kinase reactions using ALK and SRC with recombinant IMPDH2, probed with phospho-tyrosine-specific and total IMPDH2 antibodies. ( C ), Densitometric quantification of tyrosine-phosphorylated IMPDH2 compared to total IMPDH2 levels, demonstrating enhanced phosphorylation in the presence of ALK and SRC. We compared pY peptides of IMPDH2 alone or in the presence of IMPDH2 in ALK or SRC. ( D ), Large-scale in vitro kinase assay products were resolved by NuPAGE and analyzed by LC-MS/MS phosphoproteomics to identify phosphorylation sites. ( E ), Identification of key tyrosine residues on IMPDH2 phosphorylated by ALK and SRC, as determined by mass spectrometry. ( F ), Schematic of IMPDH2 structural domains highlighting novel ALK- and SRC-mediated phosphorylation sites. All graphs represent mean ± SD from three replicates ( n = 3). Statistical significance was assessed using unpaired t -tests or one-way ANOVA: *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: In Vitro, Kinase Assay, Phospho-proteomics, Western Blot, Recombinant, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: IMPDH2 Y233 phosphorylation regulates PI3P binding . ( A ), Schematic of PIP-lipid strip assay showing membranes coated with 15 distinct phospholipid species and incubated with HA peptide as a negative control. ( B ), Recombinant human IMPDH1 (1.0 µg) incubated on PIP-lipid strips in 3% BSA/PBS overnight at 4 °C; binding detected by Western blot using IMPDH1 antibody. ( C ), Recombinant human IMPDH2 (1.0 µg) incubated under identical conditions and probed with IMPDH2 antibody. ( D ), Control binding experiment using a reduced amount (0.1 µg) of IMPDH1 and probing with IMPDH2 antibody to assess specificity. ( E ), Densitometric quantification of PI3P binding to IMPDH2 and IMPDH1, demonstrating preferential binding of PI3P to IMPDH2. ( F , G ), HA-tagged human IMPDH2 constructs expressed in HEK293T cells, with or without co-expression of SRC kinase; PI3P-bound IMPDH2-HA was detected by Western blot using anti-HA antibody. ( H ), Live-cell imaging of HEK293T cells co-transfected with IMPDH2-mCherry and EGFP-FYVE (PI3P biosensor), revealing co-localization of IMPDH2 with PI3P (Scale bar: 20 μm and 100 μm). All graphs represent mean ± SD from three biological replicates ( n = 3). Statistical significance was assessed using unpaired t -tests: **** p < 0.0001.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: Phospho-proteomics, Binding Assay, Stripping Membranes, Incubation, Negative Control, Recombinant, Western Blot, Control, Construct, Expressing, Live Cell Imaging, Transfection

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: PI3P binding inhibits IMPDH2 enzymatic activity. ( A , B ), Chemical structures of phospholipids PI3P and PIP2 used in this study. ( C ), Recombinant IMPDH2 was pre-incubated with increasing concentrations of synthetic PI3P; enzymatic activity was measured and showed dose-dependent inhibition. ( D ), Recombinant IMPDH1 was similarly pre-incubated with PI3P to assess its sensitivity to PI3P-mediated inhibition. ( E ), Comparative enzymatic activity of recombinant IMPDH1 and IMPDH2 pre-incubated with 200 µg synthetic PI3P or MPA (mycophenolic acid, a known IMPDH inhibitor) as a positive control. ( F ), Recombinant IMPDH1 was pre-incubated with increasing concentrations of synthetic PIP2; no significant inhibition of enzymatic activity was observed. ( G ), Recombinant IMPDH2 pre-incubated with synthetic PI3P confirms reproducible, dose-dependent inhibition of enzymatic activity. All graphs display mean ± SD from three biological replicates ( n = 3). Statistical significance was calculated using unpaired t -tests: * p < 0.05, ** p < 0.01, **** p < 0.0001. ns is not significant.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: Binding Assay, Activity Assay, Recombinant, Incubation, Inhibition, Positive Control

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: Structure-based virtual screening identifies a novel small-molecule inhibitor of human IMPDH2 . ( A ), Ribbon diagram of the CBS (cystathionine β-synthase) domain of human inosine monophosphate dehydrogenase 2 (IMPDH2), illustrating the secondary structural elements. ( B ), Surface representation of the GTP-binding pocket (highlighted in yellow) within the IMPDH2 structure, showing the targeted allosteric site for compound docking. ( C ), Schematic overview of the in silico screening pipeline, including structure-based virtual screening, docking, and filtering steps leading to hit identification. ( D ), Chemical structures of 15 top-ranked compounds identified from virtual screening; compound 10 (comp-10) was selected for further validation based on docking scores and predicted binding interactions. ( E ), Molecular docking pose of comp-10 within the GTP-binding pocket of human IMPDH2 (based on the PDB reference structure), revealing key interactions with the binding site residues. ( F ), In vitro enzymatic activity assay of recombinant IMPDH2 in the presence of comp-10, showing concentration-dependent inhibition and determination of the half-maximal inhibitory concentration (IC 50 ). ( G ), Enzymatic assay using the clinically established IMPDH inhibitor mycophenolic acid (MPA) as a positive control for benchmarking IC 50 and assay reproducibility. ( H – J ), Inhibition of endogenous IMPDH activity by comp-10 in human lymphoma cell lines: ( H ), anaplastic large cell lymphoma (ALCL); ( I ), mantle cell lymphoma (MCL); and ( J ), diffuse large B-cell lymphoma (DLBCL). Comp-10 treatment significantly reduced IMPDH enzymatic activity across all tested cell lines. All the graphs show mean ± SD ( n = 3 biological replicates), and all statistical analyses were conducted with unpaired t -tests: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: Binding Assay, In Silico, Biomarker Discovery, In Vitro, Enzyme Activity Assay, Recombinant, Concentration Assay, Inhibition, Enzymatic Assay, Positive Control, Activity Assay

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: Comp-10 downregulates IMPDH1/2 expression and prevents filament (rod/ring) formation in ALCL and MCL models. ( A ), Western blot analysis of IMPDH1 and IMPDH2 expression in ALK inhibitor (ALKi)-sensitive ALCL cell lines (SUPM2 and L82) following 48-h treatment with Comp-10 (300 nM) or mycophenolic acid (MPA). ( B , C ), Densitometric quantification of IMPDH1 and IMPDH2 protein bands from panel A, confirming significant downregulation following Comp-10 treatment. ( D ), Western blot of ALKi-resistant ALCL cell lines (Karpas299 and SUPM2-CR) treated with Comp-10 and MPA, showing consistent downregulation of IMPDH1/2 by Comp-10 and upregulation by MPA. ( E , F ), Densitometric analysis of protein expression in resistant ALCL lines (panel D) supports differential regulation by Comp-10 and MPA. ( G ), Quantitative RT–PCR analysis of IMPDH2 mRNA expression in MCL cell line Maver treated with Comp-10, MPA and MF. ( H ), Western blot analysis of BTK inhibitor (BTKi)-sensitive and -resistant MCL cell lines treated with Comp-10 and MPA. ( I ), Densitometric quantification of Western blots shown in panel H, confirming significant downregulation of IMPDH1/2 protein levels by Comp-10 across multiple MCL models. ( J – L ), Light microscopy of SUDHL-1 cells expressing HA–GFP–IMPDH2 shows distinct patterns of filament formation: DMSO-treated control cells. ( J ), lack filaments; MPA-treated cells. ( K ), display prominent rod/ring structures; and Comp-10-treated cells. ( L ), show no rod/ring or filament formation (scale bar: 20 μm). Densitometric values of the Western blots ( n = 2). All the graphs show mean ± SD ( n = 3 biological replicates), and all statistical analyses were conducted with unpaired t -tests: * p < 0.05, ** p < 0.01, *** p < 0.001; ns is not significant.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Light Microscopy, Control

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: Comp-10 inhibits proliferation and colony formation in ALK-positive lymphoma cells. ( A – C ), Cell viability assays of ALCL cell lines treated with comp-10 or mycophenolic acid (MPA). While both compounds similarly inhibited growth in one line. ( A ) comp-10 showed significantly stronger growth inhibition in two additional lines. ( B – F ), Comp-10 potently suppressed the growth of ALK inhibitor (ALKi)-sensitive Karpas 299 cells ( D ) and their resistant derivatives: Crizotinib-resistant ( E ) and Lorlatinib-resistant ( F ) lines. ( G – I ), Similar results were observed in parental SUP-M2 cells ( G ) and their ALKi-resistant derivatives ( H , I ), with comp-10 maintaining robust antiproliferative activity. ( J ), Representative GFP fluorescence images of colonies formed by SUDHL-1 cells stably expressing IMPDH2–HA–GFP in methylcellulose after 4 weeks of treatment with DMSO, comp-10 (100 nM), or MPA (100 nM). ( K ), Quantification of colony numbers per well. Comp-10 significantly reduced colony formation compared to both DMSO ( p < 0.0001) and MPA ( p < 0.01). ( L ), Colony numbers expressed as percentage of DMSO control. Comp-10 reduced colony formation to ~12%, while MPA-treated cells retained > 72% colony formation relative to control. ( M , N ), Cell viability assays in two mantle cell lymphoma (MCL) index lines demonstrate that comp-10 effectively inhibits cell growth than MPA. ( O ), Growth of the ovarian carcinoma cell line OVCAR3 remained unaffected by comp-10 or MPA, indicating minimal off-target toxicity in non-lymphoid malignancies. All the graphs show mean ± SD ( n = 3 biological replicates), and all statistical analyses were conducted with unpaired t -tests: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: Inhibition, Activity Assay, Fluorescence, Stable Transfection, Expressing, Control

Journal: Cancers

Article Title: A Novel Allosteric Inhibitor Targeting IMPDH at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

doi: 10.3390/cancers17203389

Figure Lengend Snippet: Schematic representation of IMPDH2 regulation and inhibition mechanisms. ( A ), In normal cells, IMPDH2 catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), a critical step in GTP biosynthesis. Elevated GTP levels act as a negative feedback regulator of IMPDH2 activity, maintaining cellular homeostasis and supporting proliferation. ( B ) Treatment with classical IMPDH inhibitors (e.g., MPA, ribavirin, or mizoribine) blocks IMPDH1/2 enzymatic activity, depleting intracellular GTP levels. This loss of feedback inhibition triggers compensatory overexpression of IMPDH1/2 and the formation of cytoplasmic rods and rings (RR) structures. ( C ), In contrast, the novel allosteric inhibitor Comp-10 targets the regulatory domain of IMPDH2, reducing both its enzymatic activity and protein abundance without inducing RR formation. This post-transcriptional mechanism represents a distinct and potentially less toxic approach to inhibiting IMPDH2.

Article Snippet: Recombinant human IMPDH1 (Catalog # 8904-DH) and IMPDH2 (catalog# Catalog # 8349-DH) proteins were obtained from R&D Systems, Minneapolis, MN, USA.

Techniques: Inhibition, Activity Assay, Over Expression, Quantitative Proteomics

Journal: Biochemistry

Article Title: Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

doi: 10.1021/acs.biochem.3c00461

Figure Lengend Snippet: Figure 1. Proteomic profiling of the 8OG-RNA interactome. (A) Structure of the 8OG or G oligo probes. Schematic for chemical proteomics workflow. (B) Volcano plot of protein enrichment ratios (8OG/G spectral counts) and p-values from label-free proteomics experiments (n = 3). To calculate enrichment ratios for proteins identified in only one of the two conditions (G or 8OG probe), we added 1 to all spectral count values. P- values were calculated based on Student’s t test. (C) Validation of protein hits from (B). Lysates were photo-cross-linked with 8OG (1) or G (2) oligo probe, and RBPs were detected by Western blotting after streptavidin enrichment. IGF2BP1 and hnRNPD were detected with corresponding antibodies against endogenous protein. Anti-FLAG M2 antibody was used to detect overexpressed epitope-tagged hnRNPDL, IGF2BP2, RBM4, and RBM4B. See Figure S1 for full Western blotting data.

Article Snippet: Plasmids encoding cDNA were obtained from Addgene: IGF2BP1 (no. 21659), IGF2BP2 (no. 91890), HNRNPD (no. 38066); purchased from Genscript: RBM4 (#OHu17642D), RBM4B (#OHu04653D); or purchased from Origene: HNRNPDL (#SC107613).

Techniques: Protein Enrichment, Biomarker Discovery, Western Blot

Journal: Biochemistry

Article Title: Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

doi: 10.1021/acs.biochem.3c00461

Figure Lengend Snippet: Figure 2. Biochemical characterization of the interaction between IGF2BP1/2 and 8OG-containing RNA oligos. (A) Electrophoretic mobility shift assay (EMSA) with IGF2BP1-KH (195−577) and fluorescein-labeled 8OG (5) or G (6) oligos. Oligos were mixed with various concentrations of IGF2BP1 and RNA−protein complexes were resolved on a TGE native gel and detected by using in-gel fluorescence. See Figure S3 for full EMSA data. Kd values were determined by fitting data to a sigmoidal dose−response curve. Values represent mean ± s.e. (n = 3). Asterisks represent statistically significant differences in binding affinity (**, p < 0.01). (B) Fluorescence anisotropy assay with IGF2BP1-KH and oligos 5 and 6. Oligos were mixed with various concentrations of protein, and binding was monitored by fluorescence polarization. Kd values were determined by fitting data to a sigmoidal dose−response curve. Values represent mean ± s.e. (n = 4). Asterisks represent statistically significant differences in binding affinity (****, p < 0.0001). (C) Photo-cross-linking of IGF2BP2 with 8OG (1) or G (2) diazirine-containing oligo probes. IGF2BP2 was mixed with various concentrations of probe and UV irradiated at 365 nm. Cross-linked RNA−protein complexes were detected by streptavidin Western blotting. See Figure S4 for full blot. EC50 values were determined by fitting data to a sigmoidal dose−response curve. Values represent mean ± s.e. (n = 4). Asterisks represent statistically significant differences in cross-linking (****, p < 0.0001). (D) Fluorescence polarization assay with IGF2BP2 and 8OG (5) or G (6) oligos. Experiment was performed as described in (B). Values represent mean ± s.e. (oligo 5, n = 4; oligo 6, n = 3;).

Article Snippet: Plasmids encoding cDNA were obtained from Addgene: IGF2BP1 (no. 21659), IGF2BP2 (no. 91890), HNRNPD (no. 38066); purchased from Genscript: RBM4 (#OHu17642D), RBM4B (#OHu04653D); or purchased from Origene: HNRNPDL (#SC107613).

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Fluorescence, Binding Assay, Irradiation, Western Blot

Journal: Journal of the American Association for Laboratory Animal Science : JAALAS

Article Title: Acrylamide Production in Autoclaved Rodent Feed

doi: 10.30802/AALAS-JAALAS-18-000011

Figure Lengend Snippet: Urine N-acetyl-S-(2-carbamoylethyl)-cysteine concentration (AAMA, μM) from mice fed NIH31 study diets. All groups represent a single pooled sample except A270P which had 2 pooled samples. Data represents mean of duplicates ± SEM. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. *P < 0.0001 compared with irradiated control group.

Article Snippet: 5 AAMA, [methyl-d3]- N -acetyl- S -(2-carbamoylethyl)-L-cysteine (AAMA-D 3 ), GAMA dicyclohexylammonium salt, and [methyl-d3]- N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (glycidamideMA-D 3 ) dicyclohexylammonium salt were purchased from Toronto Research Chemicals.

Techniques: Concentration Assay, Irradiation

Journal: Journal of the American Association for Laboratory Animal Science : JAALAS

Article Title: Acrylamide Production in Autoclaved Rodent Feed

doi: 10.30802/AALAS-JAALAS-18-000011

Figure Lengend Snippet: Urine N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine concentration (GAMA, μM) from mice fed NIH31 study diets. All groups represent a single pooled sample except A270P which had 2 pooled samples. All samples run in duplicate. Data are presented as the mean ± SEM of duplicate samples. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 0.2 ppm acrylamide; AA-high, irradiated ground fee spiked with 1.0 ppm acrylamide. *, P < 0.05; †, P < 0.01; and §, P < 0.0001 compared with the irradiated control group.

Article Snippet: 5 AAMA, [methyl-d3]- N -acetyl- S -(2-carbamoylethyl)-L-cysteine (AAMA-D 3 ), GAMA dicyclohexylammonium salt, and [methyl-d3]- N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (glycidamideMA-D 3 ) dicyclohexylammonium salt were purchased from Toronto Research Chemicals.

Techniques: Concentration Assay, Irradiation

Journal: Journal of the American Association for Laboratory Animal Science : JAALAS

Article Title: Acrylamide Production in Autoclaved Rodent Feed

doi: 10.30802/AALAS-JAALAS-18-000011

Figure Lengend Snippet: The number of hepatic glycidamide–guanine DNA adducts (N7-[2-carbamoyl-2-hydroxyethyl]-guanine or N7-glycidamide-guanine) per 108 total nucleotides in mice fed NIH31 irradiated or autoclaved diet. Data are given as mean ± SEM (n = 10) for all goups except A230 (n = 8) and A270P (n = 14). IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. †, P < 0.01; ‡, P < 0.001; and §, P < 0.0001 compared with the irradiated control group.

Article Snippet: 5 AAMA, [methyl-d3]- N -acetyl- S -(2-carbamoylethyl)-L-cysteine (AAMA-D 3 ), GAMA dicyclohexylammonium salt, and [methyl-d3]- N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (glycidamideMA-D 3 ) dicyclohexylammonium salt were purchased from Toronto Research Chemicals.

Techniques: Irradiation

Journal: Journal of the American Association for Laboratory Animal Science : JAALAS

Article Title: Acrylamide Production in Autoclaved Rodent Feed

doi: 10.30802/AALAS-JAALAS-18-000011

Figure Lengend Snippet: The number of hepatic glycidamide–adenine DNA adducts (N3-[2-carbamoyl-2-hydroxyethyl]-adenine or N3-glycidamide-adenine] per 108 total nucleotides in mice fed NIH31 irradiated or autoclaved diet. Several groups had samples in which some or all measurements were below the limit of detection (LOD) of 0.16 adducts per 108 nucleotides. Those samples were assigned a value of LOD/√2 (that is, 0.11 adducts/108 nucleotides). Data are given as mean ± SEM (n = 10) for all groups except IR (n = 14) and A270P (n = 15). The number above each column indicates the number of samples per group that were below the LOD. IR, irradiated pelleted feed (control); A230, pelleted feed autoclaved at 230 °F; A250, pelleted feed autoclaved at 250 °F; A260, pelleted feed autoclaved at 260 °F; A270P, pelleted feed autoclaved at 270 °F; A270G, ground feed autoclaved at 270 °F; AA-low, irradiated ground feed spiked with 200 ppb acrylamide; AA-high, irradiated ground fee spiked with 1000 ppb acrylamide. Statistical analysis used a 1-sample t test with hypothetical mean of 0.16. ‡, P < 0.001; §, P < 0.0001 compared with irradiated control group.

Article Snippet: 5 AAMA, [methyl-d3]- N -acetyl- S -(2-carbamoylethyl)-L-cysteine (AAMA-D 3 ), GAMA dicyclohexylammonium salt, and [methyl-d3]- N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (glycidamideMA-D 3 ) dicyclohexylammonium salt were purchased from Toronto Research Chemicals.

Techniques: Irradiation