Journal: Nature Communications

Article Title: Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

doi: 10.1038/s41467-017-02322-5

Figure Lengend Snippet: PKD activity regulation in an in vitro model of NMDA-induced excitotoxicity. a Scheme showing activatory and autophosphorylation sites and domains in PKD1. b p-PKD(S916), p-PKD(S744/S748), PKD, p-DAPK(S308), DAPK, and Spectrin immunoblot analysis of primary mature cortical neurons stimulated with NMDA (50 μM) plus glycine (10 μM) (referred hereafter as NMDA) for various periods of time. Spectrin full-length (FL) and calpain-breakdown products (BDPs) are shown. (Right panel) Quantification of immunoblot signals of p-PKD(S916) relative to total PKD and the loading control neural-specific enolase (NSE). Each time point, p-PKD(S916) value was represented as fold increase relative to control untreated cultures ( n = 5 independent experiments). c , d PKD, p-PKD(S916), and Spectrin immunoblot analysis of neurons pretreated for 1 h with the GluN2B-specific inhibitor ifenprodil (IFN; 10 μM) or the NMDAR antagonist DL-AP5 (200 μM), and stimulated with NMDA for 5 min ( c ) or 1 h ( d ) ( n = 3 independent experiments). e Neurons were stimulated with NMDA as above for 5 min or 1 h. PKD1-Y93 phosphorylation after 5 min of NMDA treatment was detected following immunoprecipitation and immunoblotting with a novel phosphospecific monoclonal antibody. Short and long exposure images of p-PKD(S916) and PKD are included. f PKD, p-PKD(S916), and NSE (loading control) immunoblot analysis of neurons pre-incubated for 1 h with the PKC-δ inhibitor Rottlerin (5 μM) or the Src inhibitor SU6656 (5 μM) and treated with NMDA for 5 min ( n = 3 independent experiments). g Neurons were exposed for 1 h to okadaic acid (OA; 500 nM), to inhibit serine/threonine phosphatase PP1, or pervanadate (PV; 1 mM), to inhibit tyrosine phosphatases, and then stimulated with NMDA for 1 h. Levels, processing or phosphorylation of PKD, p38, STEP, or DUSP1 were determined by immunoblot analysis ( n = 3 independent experiments). h Neurons transduced with lentivirus encoding shC, shDUSP1, or shSTEP were treated with NMDA as indicated. Dusp1 or Step silencing and their effect on PKD inactivation in response to excitotoxicity was analyzed by immunoblotting. i Quantification of immunoblot signal of p-PKD(S916) higher molecular weight band in h relative to total PKD and NSE, represented as fold increase relative to untreated cultures transduced with shC is shown as mean ± s.e.m. ( n = 3 independent experiments). * P < 0.05, ** P < 0.01; n.s. not significant. two-tailed unpaired Student's t test. b – h Representative immunoblots are shown

Article Snippet: Okadaic acid (OA), 3-(4-chlorophenyl)-1-(1,1-dimethylethyl) 1-H-pyrazolo[3,4-d] pyrimidin-4-amine (PP2), DL-2-amino-5-phosphonopentanoic acid (DL-AP5), and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) were from Tocris Bioscience (Bristol, UK).

Techniques: Activity Assay, In Vitro, Western Blot, Control, Phospho-proteomics, Immunoprecipitation, Incubation, Transduction, Molecular Weight, Two Tailed Test

Journal: Neuropharmacology

Article Title: Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase.

doi: 10.1016/j.neuropharm.2022.109058

Figure Lengend Snippet: Fig. 6. DA and phosphorylation agents modulate TH activity. The decay of [3H]-DA synthesis rate with time (A) was evaluated in brain striatal minces under the effects of agents that modify TH phosphorylation state such as PD98059, a MEK/ERK pathway inhibitor; Sp-cAMP, a PKA activator; and okadaic acid (Ok), a phosphatase inhibitor. Drugs were added at the beginning of the pre-incubation time indicated in the x-axis and timeline, plus 10 min incubation with [3H]-Tyr properly used to measure [3H]-DA synthesis rate. The effects of 1 μM Ok (B) were further evaluated in combination with 0.1 and 5 μM DA. Addition of the compounds to brain striatal tissue is indicated in timelines. Data represent individual incubation values and mean ± SEM % of control [3H]-DA synthesis at 25 min (A) of N equal to 14–50 (control), 3–9 (PD98059), 4–12 (Sp-AMPc) and 4–8 (Ok) tissue incubations or (B) mean ± SEM of N equal to 16–18 (control) and 7–8 (OK) tissue in cubations. 1 incubation in (A) was excluded from the analysis after the value was considered outlier by the ROUT test. In control groups 100% corresponds to a mean ± SEM of 632,898 ± 87,744 (A) and 295,539 ± 17,413 (B) dpm [3H]-DA/mg.h. Two-way ANOVA showed in A) a significant effect of Treatment (F(1,96) = 16.4; p < 0.0001) and Time (F(2,96) = 18.4; p < 0.0001), without a significant interaction between these factors for PD98059; a significant effect of Treatment (F(1,111) = 15.9; p < 0.0001) and Time (F(2,111) = 23.7; p < 0.0001 without a significant interaction between these factors for Sp-cAMP; and a significant effect of Treatment (F (1,126) = 32.6; p < 0.0001) and Time (F(3,126) = 22.8; p < 0.0001) without a significant interaction between these factors for Ok. In B) two-way ANOVA showed a significant effect of Treatment (F(1,67) = 15.5; p < 0.0005) and DA concentration (F(2,67) = 40.8; p < 0.0001), and a significant interaction between these two factors (F(2,67) = 4.9 p < 0.05). A) *p < 0.05 vs. respective 25 min pre-incubation, ANOVA plus Dunnett’s multiple comparisons test. #p < 0.05 vs. control group, ANOVA plus Sidak’s multiple comparisons test. B) *p < 0.05vs. respective control, ANOVA plus Dunnett’s multiple comparisons test. #p < 0.05 vs. non-treated groups, ANOVA plus Sidak’s multiple comparisons test.

Article Snippet: Opti-Phase HiSafe-3 liquid scintillation cocktail (Cat. No. 1200.437) and [3, 5-3H]-L-tyrosine (3H-Tyr, 50 Ci/mmol NET127005MC) were supplied by PerkinElmer Wallac (Turku, Finland). cAMP-Sp, PD98059 (1213), TBZ (2175) and okadaic acid (1136) were obtained from Tocris Bioscience (Bristol, United Kingdom).

Techniques: Phospho-proteomics, Activity Assay, Incubation, Control, Concentration Assay

Journal: Neuropharmacology

Article Title: Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase.

doi: 10.1016/j.neuropharm.2022.109058

Figure Lengend Snippet: Fig. 7. TH phosphorylation changes in response to okadaic acid and to PD98059 at different times. A) TH phosphorylation at Ser31 and at Ser40 was determined by Western blot after different incubation times with or without 1 μM okadaic acid (Ok, added at time 0). In addition to total TH, β-actin (A) was also assessed as loading control. Mean optical density of the 55 kDa bands in A) were standardized to arbitrary units in each blot and results are expressed as pTH/TH ratio for Ser31 (B) and Ser40 (C). D) TH phosphorylation at Ser31 was determined with or without 25 μM PD98059 at different incubation times by Western blot. Dually phosphorylated ERK-1/2 in Thr202/Tyr204 was also determined as a control of PD98059 action. E) Results from D) are expressed as pS31 TH/TH ratio of standardized optical density of the 55 kDa bands. F) Graph showing phosphoERK/ERK-1/2 ratio of standardized optical density of the 42–44 kDa dimer from D). Data represent the means ± SEM of N equal to 4–5 brain striatal tissue incubations in all graphs. 0 min samples were not incubated. B–C) #p < 0.05, ##p < 0.01 vs. 0 min incubation with Ok. F) #p < 0.01 vs. 0 min incubation without PD98059 (white bars); *p < 0.0001 vs. 0 min incubation with PD98059 (black bars), ANOVA plus Sidak’s multiple comparisons test.

Article Snippet: Opti-Phase HiSafe-3 liquid scintillation cocktail (Cat. No. 1200.437) and [3, 5-3H]-L-tyrosine (3H-Tyr, 50 Ci/mmol NET127005MC) were supplied by PerkinElmer Wallac (Turku, Finland). cAMP-Sp, PD98059 (1213), TBZ (2175) and okadaic acid (1136) were obtained from Tocris Bioscience (Bristol, United Kingdom).

Techniques: Phospho-proteomics, Western Blot, Incubation, Control

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Common Markers and Small Molecule Inhibitors in Golgi Studies

doi: 10.1007/978-1-0716-2639-9_27

Figure Lengend Snippet: Small molecules

Article Snippet: Phosphatase inhibitors , Okadaic acid , general PPase inhibitor , 0.1 μM , Santa Cruz, sc-202259A , [ 90 ].

Techniques: DNA Synthesis, Activity Assay, Ubiquitin Proteomics, Protease Inhibitor, Membrane, Blocking Assay, Glycoproteomics, Inhibition, Translocation Assay

Journal: Toxins

Article Title: Combined Cytotoxicity of the Phycotoxin Okadaic Acid and Mycotoxins on Intestinal and Neuroblastoma Human Cell Models

doi: 10.3390/toxins10120526

Figure Lengend Snippet: Chemical structures of typical algal toxins (okadaic acid, saxitoxin, domoic acid), dust storm-related mycotoxins (major A. sydowii metabolites and sterigmatocystin), and shellfish-related mycotoxins (patulin, alamethicin, gliotoxin).

Article Snippet: Phycotoxin standards, saxitoxin (CRM-STX-f), domoic acid (CRM-DA-g), and okadaic acid (CRM-OA-d) were purchased from the National Research Council Canada.

Techniques:

Journal: Toxins

Article Title: Combined Cytotoxicity of the Phycotoxin Okadaic Acid and Mycotoxins on Intestinal and Neuroblastoma Human Cell Models

doi: 10.3390/toxins10120526

Figure Lengend Snippet: Summary of cytotoxicity of typical A. sydowii metabolites, dust storm/shellfish-associated mycotoxins, okadaic acid algal toxins on HT-29 and SH-SY5Y cells after 24 h exposure. Inhibitory concentration 50% (IC 50 ) values and 95% confidence interval (CI) were calculated from four replicates using the four-parameter logistic model (4PL) model.

Article Snippet: Phycotoxin standards, saxitoxin (CRM-STX-f), domoic acid (CRM-DA-g), and okadaic acid (CRM-OA-d) were purchased from the National Research Council Canada.

Techniques: Concentration Assay

Journal: Toxins

Article Title: Combined Cytotoxicity of the Phycotoxin Okadaic Acid and Mycotoxins on Intestinal and Neuroblastoma Human Cell Models

doi: 10.3390/toxins10120526

Figure Lengend Snippet: Molar combination ratio of okadaic acid and mycotoxin mixtures used in the assay.

Article Snippet: Phycotoxin standards, saxitoxin (CRM-STX-f), domoic acid (CRM-DA-g), and okadaic acid (CRM-OA-d) were purchased from the National Research Council Canada.

Techniques:

Journal: Toxins

Article Title: Combined Cytotoxicity of the Phycotoxin Okadaic Acid and Mycotoxins on Intestinal and Neuroblastoma Human Cell Models

doi: 10.3390/toxins10120526

Figure Lengend Snippet: Combination index (CI)–fraction affected ( fa , indicating fraction of cell viability affected. fa = 0.05–0.97 corresponds to 5–97% toxicity) curves for binary mixtures of okadaic acid and sydowinol, sydowinin A, alamethicin, patulin, and gliotoxin against human intestinal HT-29 cells. CI < 1, CI = 1, and CI > 1 indicate synergistic (orange rectangle), additive (red line), and antagonistic (blue rectangle) effects of binary mixtures, respectively. The error bar indicates 95% confidence intervals calculated using sequential deletion analysis (SDA).

Article Snippet: Phycotoxin standards, saxitoxin (CRM-STX-f), domoic acid (CRM-DA-g), and okadaic acid (CRM-OA-d) were purchased from the National Research Council Canada.

Techniques:

Journal: Toxins

Article Title: Combined Cytotoxicity of the Phycotoxin Okadaic Acid and Mycotoxins on Intestinal and Neuroblastoma Human Cell Models

doi: 10.3390/toxins10120526

Figure Lengend Snippet: Combination index (CI) and dose reduction index (DRI) values for okadaic acid and mycotoxin combinations in HT-29 and SH-SY5Y cells at various effect levels (IC 25 , IC 50 , IC 75 and IC 90 ). DRI values were only calculated when synergistic effects were detected. DRI implies fold of dose reduction for a given effect in a combination of toxins compared with the dose of each toxin alone.

Article Snippet: Phycotoxin standards, saxitoxin (CRM-STX-f), domoic acid (CRM-DA-g), and okadaic acid (CRM-OA-d) were purchased from the National Research Council Canada.

Techniques:

Journal: Toxins

Article Title: Combined Cytotoxicity of the Phycotoxin Okadaic Acid and Mycotoxins on Intestinal and Neuroblastoma Human Cell Models

doi: 10.3390/toxins10120526

Figure Lengend Snippet: Combination index (CI)–fraction affected ( fa , indicating fraction of cell viability affected. fa = 0.05–0.97 corresponds to 5–97% toxicity) curves for binary mixtures of okadaic acid and sydowinol, sydowinin A, alamethicin, patulin, and gliotoxin against human neuroblastoma SH-SY5Y cells. CI < 1, CI = 1, and CI > 1 indicate synergistic (orange rectangle), additive (red line), and antagonistic (blue rectangle) effects of binary mixtures, respectively. The error bar indicates 95% confidence intervals calculated using sequential deletion analysis (SDA).

Article Snippet: Phycotoxin standards, saxitoxin (CRM-STX-f), domoic acid (CRM-DA-g), and okadaic acid (CRM-OA-d) were purchased from the National Research Council Canada.

Techniques: