Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 1. Chemical structure of the chromone scaffold used for the virtual screening procedure (A). Chemical structure of DB987, the compound highlighted by the screening, in which the red structure represents the part of the molecule matching query (B). Chemical structure of the reference compound, PDE9 inhibitor PF-04447943 (C).

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques:

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 2. Binding poses for PF-04447943 (A) and DB987 (B) in the predicted interaction pattern with PDE9. The residues at the interaction distance (<5 Å) within the binding pocket have been labeled.

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques: Binding Assay, Labeling

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 5. (A-B2) Immunohistochemical assessment of neuronal damage and PDE9 expression in the CA3 hippocampus of organotypic slices after treatment with KA. (A1,B1) Representative confocal images of fluorescent immunostaining of NeuN-positive neurons in area CA3 of CRL (A1) and KA (B1) slices. (A2,B2) Representative confocal images of fluorescent immunostaining of PDE9 in area CA3 of CRL (A2) and KA-treated slices (B2). (A, B) Merge of the previous images. All images were captured with a 20× objective. Scale bar: 100 μm. (C-C2) Magnifications of the framed areas of the corresponding slice shown in B, B1, and B2. (C1) Immunostaining of NeuN showing the presence of damaged neurons with an elongated, shrunk cytoplasm. (C2) Immunostaining of PDE9 showing the expression of the enzyme in many CA3 pyramidal neurons (open arrows). (C) Merge of the two previous images (open arrows indicate neurons positive for PDE9 immunostaining). Scale bar: 25 μm. (D) Quantitative analysis of PDE9 immunostaining in CA3 SP (CRL n = 10 and KA n = 9). Statistical analysis: Student’s t-test: **P < 0.01 KA vs CRL. (E-F3) Representative confocal images of triple immunofluorescent labeling of neurons (NeuN, red), PDE9 (green), and astrocytes (GFAP, blue) in CA3 of KA-treated slices captured with a 40× objective. Open arrows indicate PDE9-positive pyramidal neurons. No colocalization with astrocytes was found. Scale bars: 70 μm (E) and 15 μm (F-F3).

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Labeling

Journal: ACS chemical neuroscience

Article Title: Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

doi: 10.1021/acschemneuro.3c00431

Figure Lengend Snippet: Figure 7. Qualitative and quantitative analyses of the effects of PDE9 inhibitors in rat organotypic hippocampal slices under normal conditions or exposed to KA. (A) Hippocampal slice under normal conditions (background PI fluorescence), (B) slice exposed to 10 μM PF for 24 h, (C) slice exposed to 10 μM DB987 for 24 h, (D) slice exposed to 5 μM KA for 24 h displaying intense PI labeling in the CA3 subregion, and (E,F) CA3 damage induced by KA was attenuated by the presence of 1 μM PF and DB987. (G) PDE9 inhibitors alone did not induce side effects. (H) PDE9 inhibitors significantly attenuated CA3 damage in a dose-dependent manner. Bars represent the mean ± SEM of at least five experiments run in quadruplicate. **p < 0.01 and ***p < 0.001 vs KA (one-way ANOVA plus Dunnett’s test).

Article Snippet: To assess the inhibitory activity of DB987 against full-length recombinant human PDE9 (PDE9A, SignalChem, Richmond, Canada), the PDE-Glo phosphodiesterase assay (Promega Corp., Madison, WI, USA) was used as previously reported.29 The compound was dissolved in DMSO and mixed with the PDE-Glo reaction buffer at a v/v ratio of 1:5.

Techniques: Fluorescence, Labeling

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A, hnRNP A2, hnRNP A3, and hnRNP U are part of the same multiprotein complex. (A) Specificity of the affinity-purified peptide specific polyclonal anti-CBF-A antibody. Total protein extracts from HeLa cells were resolved by SDS-PAGE, blotted, and stained with Coomassie Blue (lane 1), immunostained with the CBF-A preimmune serum (lane 2), or with the affinity-purified anti-CBF-A antibody (lane 3) and with antibody SAK22 recognizing both CBF-A isoforms p37 and p42 (lane 4). (B) Sucrose gradient analysis of CBF-A, hnRNP A2, and hnRNP A3 from HeLa nuclear extracts. Fractions were resolved by SDS/PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2/A3. (C) Schematic representation of recombinant hnRNP A2 and A3 and CBF-A constructs. (D) Pulldown experiment using S-tagged hnRNP A2, S-tagged hnRNP A3, or GST-tagged CBF-A constructs. The beads were incubated with HeLa nuclear extracts. Bound proteins were resolved by SDS PAGE, revealed by Coomassie staining and (E) analyzed on immunoblots with antibodies to CBF-A, hnRNP A2 and A3, and hnRNP U.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Affinity Purification, SDS Page, Staining, Western Blot, Recombinant, Construct, Incubation

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A binds the MBP mRNA RTS. (A) Sequences of wild-type (wtRTS) and scrambled RTS (scrRTS) used in this study. (B) Biotinylated wtRTS and scrRTS were conjugated to streptavidin Sepharose. Beads were incubated with HeLa nuclear, cytoplasmic, and high-salt protein extracts. Bound proteins were resolved by SDS-PAGE, revealed with Coomassie, and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2 and A3. (C) RTS-binding assays using 33P-labeled wtRTS and scrRTS sequences. To perform EMSA, wtRTS and scrRTS probes were incubated with purified CBF-A and hnRNP A2 and A3 without affinity tags or (D) in the presence (+) or absence (−) of a 25-fold excess of unlabeled competitor RNA oligonucleotides as indicated. (E) Tissue distribution of CBF-A, analyzed on immunoblots, and normalized to the steady-state expression of histone H3.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Incubation, SDS Page, Western Blot, Binding Assay, Labeling, Purification, Expressing

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In cultured oligodendrocytes, CBF-A exhibits a granular cytoplasmic distribution which correlates with transported MBP mRNA. (A) Endogenous CBF-A (A–D and E–H) or (hnRNP A2 I–L and M–P) and MBP mRNA were simultaneously monitored by immuno-FISH and confocal microscopy. In D, arrows identify sites in which the distribution of CBF-A correlates with MBP RTS along processes. In E–H and M–P, oligodendrocyte processes are shown at approximately fivefold higher magnification. In H, arrowheads identify examples of CBF-A and MBP RTS-positive granules. In P, arrows point to examples of hnRNP A2- and MBP mRNA-positive granules. Scale bar, 20 μm. (B) Unbiased statistical quantification of individual CBF-A and MBP RTS-positive granules and (C) hnRNP A2 and MBP RTS-positive granules based on the immuno-FISH analysis. In both cases a linear correlation between the fluorescence intensity levels of CBF-A and RTS or hnRNP A2 and RTS is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In oli-neu cells, the distribution of endogenous CBF-A correlates with hnRNP A2. (A and E) DAPI staining, (B and F) oli-neu cells stained with a mAb to hnRNP A2. (C and G) Oli-neu cells stained with the rabbit polyclonal peptide-specific anti-CBF-A antibody and (D and H) merged images. Scale bar, 20 μm. (B) Statistical quantification of CBF-A and hnRNP A2-positive granules based on the double immunofluorescence analysis and confocal microscopy in A. A linear correlation between the fluorescence signals of CBF-A and hnRNP A2 is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Staining, Immunofluorescence, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A is associated with MBP mRNA in differentiating oligodendrocytes. (A) A complex containing CBF-A and hnRNP A2 is coprecipitated with the anti-CBF-A antibody from total protein extracts (Input) prepared from differentiating oli-neu cells in an RNA-dependent manner. Where indicated, extracts were treated with RNase A before immunoprecipitation. Bound proteins were resolved by SDS-PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2. (B) qRT-PCR was performed on reverse-transcribed cDNA derived from RNA extracts of differentiating oli-neu cells, immunoprecipitated by CBF-A. The anti-CBF-A antibody leads to enrichment of MBP mRNA, as assessed with MBP-specific primers. Mock experiments and IgG pulldowns revealed negligible RNA enrichment. Input samples were considered to be 100%; thus all samples were divided by the inputs mean value. Data are presented as average of three independent experiments. Error bars, SEM. Importantly, in each case the percentages of immunoprecipitated mRNA are relative to the total amount of each individual mRNA species (input) analyzed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Quantitative RT-PCR, Reverse Transcription, Derivative Assay

Journal: DNA repair

Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

doi: 10.1016/j.dnarep.2019.02.001

Figure Lengend Snippet: (A) For each sample, linear DNA substrate containing a single uracil or an F residue was incubated with 50 μg of POLB−/− extract, and where indicated, supplemented with 0.5 pmol of Polβ, with 0.5 pmol of Polλ−39, or (C) with 0.5 pmol of full-length Polλ. Reactions were performed and analyzed as described in Section 2. The means ± standard errors are plotted (n=3). Data points from experiment POLB−/− extract (Fig.1) are also plotted for reference. (B) A schematic showing the domain organization of Polλ.

Article Snippet: Full-length human Polλ protein and rabbit anti-Polλ polyclonal antibody were kind gifts from Drs. Thomas Kunkel and Samuel H. Wilson respectively.

Techniques: Residue, Incubation

Journal: DNA repair

Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

doi: 10.1016/j.dnarep.2019.02.001

Figure Lengend Snippet: The linear uracil and F substrates were used in a series of experiments where the indicated amounts of purified Polβ (left) or Polλ (right) were added to POLB−/− extracts. Reactions were performed with an incubation time of 30 min at 37°C and analyzed as described in Section 2.5 and 2.6. The means ± standard errors are plotted (n=3).

Article Snippet: Full-length human Polλ protein and rabbit anti-Polλ polyclonal antibody were kind gifts from Drs. Thomas Kunkel and Samuel H. Wilson respectively.

Techniques: Purification, Incubation

Journal: DNA repair

Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

doi: 10.1016/j.dnarep.2019.02.001

Figure Lengend Snippet: (A) Domain organization of Polβ showing the position of residues changed in the Polβ variants. (B) Repair of uracil and F-containing linear substrates in WT, POLB−/− MEF extracts and extracts from POLB−/− cells expressing lyase-dead (K35A/K68A/K72A) or polymerase-dead (D256A) variants of Polβ, with Polλ, or with Polλ−39. Data points from experiment with WT and POLB−/− extract (Fig.​(Fig.1)1) are also plotted for reference. (C) Repair of uracil and F-containing linear substrates in POLB−/− MEF extracts expressing D256A variant of Polβ and supplemented with 0.5 pmol of recombinant Polβ or Polλ. Data points from experiment D256A-Polβ expressing extract are also plotted for reference. The means ± standard errors are plotted (n=3).

Article Snippet: Full-length human Polλ protein and rabbit anti-Polλ polyclonal antibody were kind gifts from Drs. Thomas Kunkel and Samuel H. Wilson respectively.

Techniques: Expressing, Variant Assay, Recombinant