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Cellular Responses to Adenovirus Entry
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
Susanna Chiocca, Matthew Cotten
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).
Pharmacology of Disogenin and Related Compounds
Published in Amritpal Singh Saroya, 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).
Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sarah E. Webb, Jeffrey J. Kelu, Andrew L. Miller
In addition to the genetic manipulation of TPC2, two-pore channels have also been attenuated and inhibited pharmacologically in zebrafish (Kelu et al., 2015, 2017). Trans-ned-19 is an NAADP receptor antagonist (Naylor et al., 2009), whereas bafilomycin A1 is a vacuolar type H+ ATPase inhibitor (Bowman et al., 1988; Yoshimori et al., 1991). Although neither of these inhibitors is specific for TPC2, they do help to establish the contribution of NAADP and acidic stores to the differentiation and onset of function of SMCs and PMNs in the zebrafish trunk. By the end of epiboly (i.e., ~10 hpf), zebrafish embryos, like other teleost embryos, are somewhat impermeable to drugs due to the formation of the enveloping layer (i.e., an embryonic periderm), which completely encloses the developing blastoderm and acts as an ionic and osmotic barrier (Bruce, 2016; Collazzo et al., 1994; Keller and Trinkaus, 1987). Thus, to treat embryos at ~17–18 hpf or older with pharmacological agents, the terminal tip of the tail (i.e., ~100 µm) can be excised just before the start of the drug treatment to ensure that they can penetrate and diffuse into the muscle precursor tissue of the trunk (Brennan et al., 2005; Cheung et al., 2011; Liu and Westerfield, 1990). The exposed tail cells do heal relatively quickly forming an injury-response blastema; for example, in adult zebrafish it is reported that within 1–3 h following amputation of the caudal (tail) fin, a thin epidermis migrates to cover the wound, and then over the following 12–18 h additional epidermal layers accumulate (Azevedo et al., 2011; Poss et al., 2003). However, the experiments conducted with bafilomycin A1 or trans-ned-19 were relatively short-term such that embryos were treated with the drugs at ~17 hpf and data were either collected immediately or else within ~8 h of the start of treatment. In this way, bafilomycin A1 and trans-ned-19 were shown to have a dose-dependent effect on SMC development, with bafilomycin A1 being used at concentrations ranging between 100 nM and 5 µM, and trans-ned-19 being used at concentrations ranging between 50 µM and 500 µM (Kelu et al., 2015, 2017). Stock solutions of bafilomycin A1 and trans-ned-19 were prepared in DMSO and then diluted in Danieau’s solution just prior to incubation with embryos. Trans-ned-19 has a tendency to precipitate in aqueous solution at high concentration, but heating the drug to 65°C for 5 min (and then allowing it to cool to ~28°C) just before it was used to treat embryos helped to resolve this issue (Kelu et al., 2017). In addition, tail-excised embryos were treated with Danieau’s solution containing DMSO as negative controls.
Multi-walled carbon nanotubes trigger lysosome-dependent cell death (pyroptosis) in macrophages but not in neutrophils
Published in Nanotoxicology, 2021
Sandeep Keshavan, Govind Gupta, Sebastin Martin, Bengt Fadeel
To further address the potential involvement of autophagy, we used a murine autophagy reporter cell line (Gallud et al. 2019). RAW-Difluo™ mLC3 cells express the RFP::GFP::LC3B (microtubule-associated protein 1 light chain 3 beta) fusion protein to enable detection of autophagic flux; two fluorescent reporter proteins are deployed: RFP (acid-stable) and GFP (acid-sensitive). These cells were exposed to MWCNTs and autophagy was monitored by confocal microscopy. Bafilomycin A1 was used as a positive control (Supplemental Figure S6). We could not detect any signs of autophagy when cells were exposed to 25 µg/mL of NM400, NM401, or NM402. However, when RAW-Difluo™ mLC3 cells were exposed to 50 µg/mL of NM401, we observed predominantly red puncta indicating that autolysosomes are formed (because the GFP label is acid-sensitive and the RFP lable is acid-stable, autophagy can be monitored based on the progressive degradation of the GFP signal, and the concurrent increase of the RFP signal in autolysosomes). Thus, at the higher concentration, the long and rigid MWCNTs (NM401) evidently triggered pyroptosis with concomitant autophagic cell death.
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)).