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Metabolic Alterations in Muscle Associated with Obesity
Published in Emmanuel Opara, NUTRITION and DIABETES, 2005
John P. Thyfault, G. Lynis Dohm
Another line of evidence that a defect in insulin signaling causes insulin resistance comes from our experiments with serine/threonine and tyrosine protein phosphatase inhibitors. These experiments were based on the fact that insulin initiates a cascade of tyrosine and serine/threonine kinase activations. Therefore, inhibiting the opposing phosphatases mimics insulin action, but without the need for the defective step in the signaling pathway. The serine/threonine phosphatase inhibitor, okadaic acid, and the tyrosine phosphatase inhibitors phenylarsine oxide and vanadate all produced robust stimulation of glucose transport in insulin-resistant muscle of obese patients.19
p21ras and Receptor Signaling
Published in Juan Carlos Lacal, Frank McCormick, The ras Superfamily of GTPases, 2017
Boudewijn M. Th. Burgering, Johannes L. Bos
Since the insulin receptor exhibits tyrosine kinase activity, most likely one of the earliest events is the phosphorylation on tyrosine of substrate molecules. However, only very few proteins have been found to become phosphorylated on tyrosine after insulin stimulation. The most abundant substrate is a 185 kDa protein found first in hepatoma cells.73 Recently, Blackshear and co-workers74 used the pharmacological compound phenylarsine oxide (PAO) and found, that upon insulin stimulation,26 proteins become rapidly phosphorylated. At least 10 of these proteins were phosphorylated on tyrosine. These proteins did not become phosphorylated after serum induction or PDGF induction in the presence of PAO, indicating some specificity for insulin. In addition, PAO was found to inhibit CD45, a tyrosine-specific phosphatase in T lymphocytes,75 and it is likely that the observed induction of insulin-induced tyrosine phosphorylation is due to the inhibition of a tyrosine-specific phosphatase. After insulin-stimulation a variety of proteins become phosphorylated on serine and threonine as well, but the kinetics are slower.74 A reasonable explanation for this delayed effect is that insulin first activates a serine/threonine kinase by tyrosine phosphorylation. For instance, MAP2 kinase has been reported to become phosphorylated on tyrosine after insulin stimulation76 and, thus, may provide the link between insulin receptor tyrosine kinase activity and the serine/threonine phosphorylation cascade as suggested by Sturgill et al.77 A large number of other serine/threonine kinases have been identified, which become activated after insulin stimulation. These include: HMG kinase; protease activated kinase II; casein kinase kinase II; glycogen synthase kinase 3; adipocyte Mn2+-dependent kinase; raf-1 kinase; and S6-kinase (for a review see Reference 78). These proteins become phosphorylated mostly on serine and, presumably, play a role more downstream in the signal transduction pathway.
Type II phosphatidylinositol 4-kinase regulates nerve terminal growth and synaptic vesicle recycling
Published in Journal of Neurogenetics, 2018
Kristyn C. Cantarutti, Jason Burgess, Julie A. Brill, Jeffrey S. Dason
PI(4,5)P2 has been shown to regulate SV exocytosis (Walter et al., 2017). Using a combination of electrophysiology and FM1-43, we show PI4KII is dispensable for SV exocytosis (Figures 2 and 3). Similarly, fwd mutants did not display any defects in neurotransmitter release (Dason et al., 2009). Pharmacological inhibition of PI4K activity by phenylarsine oxide in intact synaptosomes attenuated evoked release of glutamate (Wiedemann et al., 1998). The discrepancy between these results could be due to the other two PI4Ks compensating for loss of PI4KII or fwd in Drosophila. Functional redundancy was postulated as a reason for the lack of an effect on neurotransmitter release seen in PI4P5K or skittles mutants (Hassan et al., 1998). Alternatively, the pharmacological inhibitor, phenylarsine oxide, may have had non-specific effects on enzymes other than PI4Ks. Examining double or triple mutants of PI4Ks may help resolve these discrepancies.
Regression of prostate tumors after intravenous administration of lactoferrin-bearing polypropylenimine dendriplexes encoding TNF-α, TRAIL, and interleukin-12
Published in Drug Delivery, 2018
Najla Altwaijry, Sukrut Somani, John A. Parkinson, Rothwelle J. Tate, Patricia Keating, Monika Warzecha, Graeme R. Mackenzie, Hing Y. Leung, Christine Dufès
After removal of the medium, they were pretreated with free Lf (80 μM), phenylarsine oxide (10 µmol/L), filipin (5 µg/mL), colchicine (10 µmol/L), and poly-L-lysine (40 µg/mL) for 15 min at 37 °C. The cells were then treated with fluorescein-labeled DNA (respectively, 2.5 and 5 μg/well for qualitative and quantitative analysis) complexed to DAB-Lf (dendrimer: DNA weight ratio of 5:1) for 1 h at 37 °C, before being washed and processed for confocal microscopy and flow cytometry analysis as described above.
The Natural Alkaloid Piperlongumine Inhibits Metastatic Activity and Epithelial-to-Mesenchymal Transition of Triple-Negative Mammary Carcinoma Cells
Published in Nutrition and Cancer, 2021
Leanne M. Delaney, Nathan Farias, Javad Ghassemi Rad, Wasundara Fernando, Henry Annan, David W. Hoskin
MDA-MB-231 cells were seeded into 75 mm2 flasks at 1 × 106 cells/flask and allowed to adhere for 12 h. Medium alone, vehicle (DMSO) or piperlongumine (0.5, 1, or 2.5 µM) was then added to flasks and cells were cultured for 48 h. Following treatment, cells were harvested, washed with PBS, and resuspended in ice-cold lysis buffer (1% Nonidet P-40 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% SDS (w/v), 20 mM Tris, 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid at pH 7.5) containing a protease inhibitor cocktail (5 µg/ml leupeptin, 5 µg/ml pepstatin A, 10 µg/ml aprotinin, 100 µM sodium orthovanadate, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 µM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride, all from Sigma-Aldrich Canada) for 15 min at 4 °C. Cell lysates were cleared by centrifugation at 14,000 g for 10 min at 4 °C, supernatant was collected and total protein was quantified using a Bradford Assay Kit (Bio-Rad). Cell lysates were then diluted 1:1 in denaturing buffer (187.5 M Tris–HCl, 30% glycerol (v/v), 6% SDS (w/v), 15% β-mercaptoethanol (v/v), 0.015% bromophenol blue (w/v)) and kept at 95 °C for 5 min. Then 40 µg of protein was loaded into wells of a 10% SDS-polyacrylamide gel and electrophoresed at 120 V for 1.5 h. Proteins were transferred to PVDF membranes using the iBlot® gel transfer device (Invitrogen) and membranes were then blocked for 1 h at room temperature in 5% skim milk (w/v) in Tween-20 Tris-buffered saline (200 mM Tris, 1.5 M NaCl, pH 7.6, 0.1% Tween-20 (v/v)). Blots were probed overnight at 4 °C with primary mAb (1:1000 dilution) in blocking buffer. Membranes were washed thoroughly with Tween-20 Tris-buffered saline at room temperature and then probed for 1 h at room temperature with HRP-conjugated donkey anti-rabbit IgG Ab (1:10,000 dilution) in blocking buffer. Equal protein loading was confirmed by probing for β-actin expression. Protein bands were visualized with chemiluminescent HRP substrate LuminataTM (Millipore, Darmstadt, Germany) and Amersham high performance chemiluminescence film (GE Healthcare, Buckinghamshire, UK). Image Studio Software was used for densitometric analysis.