Cellular Responses to Adenovirus Entry
Kenneth L. Brigham in Gene Therapy for Diseases of the Lung, 2020
There is a body of literature supporting a low pH step in the adenovirus infection process (40,43,48-50). However, Perez and Carrasco (51) demonstrate that adenovirus infection is not inhibited by bafilomycin A1 (BFA), an inhibitor of endosomal acidification by the vacuolar H+-ATPase. These BFA results support the conclusion that a low pH step is not mandatory for adenovirus infection. Experiments supporting a low pH step usually demonstrate modest (less than 10-fold) declines in virus production or virus infectivity whereas one would expect a several log decline in virus production if an inhibitor is truly effective. Probably the safest conclusion is that adenovirus relies on several signals to activate endosome disruption and virus entry. A low pH will certainly facilitate capsid/membrane interactions as negatively charge residues on the surface of hexon are neutralized and the protein becomes more hydrophobic. However, in the absence of this low pH exposure, sufficient membrane perturbations can still occur to allow virus passage into the cytoplasm (51).
Mitochondrial Dysfunction in Huntington Disease
Abhai Kumar, Debasis Bagchi in Antioxidants and Functional Foods for Neurodegenerative Disorders, 2021
In 3-nitropropionic acid (3-NP)-induced rat HD model, increased formation of autophagosomes was observed in striatal cells. Biochemical analyses demonstrated active lysosomal cathepsin B and D, as well as conversion of LC3-I to LC3-II. Interestingly, 3-NP increased the expression of tumor suppressor protein 53 (p53) gene and its downstream signaling proteins, including Bax, p53-upregulated modulator of apoptosis (PUMA), and damage-regulated autophagy modulator (DRAM) (Figure 9.2). Inhibition of p53 by specific inhibitor pifithrin-α (PFT) reverses the activation of downstream molecules and 3-NP-induced striatal damage. Additionally, inhibition of autophagy using 3-methyladenine (3-MA) and bafilomycin A1 (BFA) reduces DNA fragmentation and striatal cell death (Zhang et al. 2009). In fact, postmortem analysis of brains of HD patients showed an increase in endosomal and/or lysosomal organelle, and their multivesicular bodies, which indicate enhanced macroautophagy or mitophagy in neurons (Hirano 2008). These observations recommend that autophagy, at least in part, contributes to mitochondrial dysfunction leading to neurodegeneration through p53 signaling (Zhang et al. 2009).
Pharmacology of Disogenin and Related Compounds
Amritpal Singh Saroya in Contemporary Phytomedicines, 2017
Results from 4’-6-diamidino-2-phenylindole and annexin-V/PI double-staining assay showed that caspase-3- and caspase-8-dependent, and dose-dependent apoptoses were detected after a 24-h dioscin treatment. Blockade of autophagy with bafilomycin A1 or 3-methyladenine sensitized the A549 and H1299 cells to apoptosis. Treatment of A549 and H1299 cells with dioscin caused a dose-dependent increase in ERK1/2 and JNK1/2 activity, accompanied with a decreased PI3K expression and decreased phosphorylation of Akt and mTOR (Hsieh et al. 2013).
Design, synthesis, and biological evaluation of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-Indole-2-Carbohydrazide derivatives: the methuosis inducer 12A as a Novel and selective anticancer agent
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Jun Wu, Hongyu Hu, Mingtao Ao, Zhenzhen Cui, Xiaoping Zhou, Jingbo Qin, Yafei Guo, Jingwei Chen, Yuhua Xue, Meijuan Fang
Methuosis is characterised by the accumulation of cytoplasmic vacuoles derived from macropinosomes. It has been reported that protein synthesis is needed during macropinosome formation. Bafilomycin A1 (Baf-A1), a selective inhibitor of vacuolar-type H + ATPase (V-ATPase), can inhibit nascent macropinosome formation and restore the vacuoles to normal morphology by inhibiting acid influx in cells24. Cycloheximide (CHX) is a protein synthesis inhibitor that can disrupt the process of macropinosome formation24. Accordingly, HeLa cells were treated with 12A or in combination with Baf-A1 or CHX for 8 h, and the morphological changes were then imaged. As shown in Figure 4(C), both CHX and Baf-A1 dramatically blocked the formation of vacuoles, indicating that 12A was a methuosis inducer.
The impact of assay recovery on the apparent permeability, a function of lysosomal trapping
Published in Xenobiotica, 2020
Dallas Bednarczyk, Menaka V Sanghvi
Figure 3(A) illustrates the cellular accumulation of the compounds in the presence and absence of the V-type H+-ATPase inhibitor, bafilomycin A1. Readily apparent from the figure is that compounds demonstrating significant differences between the bafilomycin A1 and vehicle condition, such as imatinib, quinacrine, and propranolol, were weak bases and compounds insensitive to bafilomycin A1, such as diclofenac, piroxicam, delavirdine, and fexofenadine, were acidic, neutral, or zwitterionic. Figure 3(B) where the bafilomycin A1 sensitive accumulation (vehicle – bafilomycin A1 condition) is shown, molecules insensitive to the presence of bafilomycin A1 show a small absolute change in the cellular accumulation of the respective molecule. By contrast, the molecules with the largest bafilomycin A1 sensitive accumulation in Figure 3(B) are weakly basic molecules, consistent with the established mechanism associated with lysosomal trapping and the inhibition of lysosomal acidification by bafilomycin A1 (Nadanaciva et al., 2011; Figure 2). Compounds that were acidic, zwitterionic, or neutral appeared to be much less sensitive to the presence of bafilomycin A1 (Figure 3(B)).
Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species
Published in mAbs, 2022
Zephania Kwong Glover, Aaron Wecksler, Baikuntha Aryal, Shrenik Mehta, Melissa Pegues, Wayman Chan, Mari Lehtimaki, Allen Luo, Alavattam Sreedhara, V. Ashutosh Rao
To measure autophagic flux, changes in LC3 protein level were measured in target cells treated with oxidized or non-oxidized antibody drugs in the presence and absence of Bafilomycin A1 (Baf). Target cells (SK-BR-3 for trastuzumab/T-DM1 and OVCAR3 for anti-NaPi2b/ anti-NaPi2b-vc-MMAE) were plated and allowed to adhere to culture dishes for 24 hours before treatment with oxidized or non-oxidized drug (20 µg/mL for trastuzumab and anti-NaPi2b; 200 ng/mL for T-DM1 and anti-NaPi2b-vc-MMAE) for 24 hours. For indicated cultures, 5 nM bafilomycin was added for the last 2 hours of culture. Cells were collected and lysed using RIPA with protease and phosphatase inhibitors. Cell lysates were then analyzed by western blotting using the primary antibodies, anti-LC3B (Novus Biologicals, NB100-2220) anti-α-tubulin (Cell Signaling, 2144), and anti-rabbit secondary antibody (LI-COR). Images were collected and densitometry analyzed using an Odyssey imager (LI-COR). Autophagic flux was calculated by dividing the LC3-II by the α-tubulin signal. For each treatment, results for are shown for treatment with and without bafilomycin. Each experiment was performed twice.
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