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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).
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.
The Effects of Resveratrol on the Brain Mitochondria
Published in Shamim I. Ahmad, Handbook of Mitochondrial Dysfunction, 2019
Wu et al. (2011) have shown that RES (50 µM) pretreatment (for 24 h) inhibited the activation of caspase-3 and the cleavage of PARP in SH-SY5Y cells exposed to rotenone (a mitochondrial toxin) [87]. The inhibition of the AMPK protein significantly reduced the acetylation of histone 3 (H3, a target of SIRT1) and the levels of LC3-II in RES-treated SH-SY5Y cells. Similar effects were seen in the levels of LC3-II when the cells were treated with siRNA against SIRT1, indicating a role for the deacetylase in the activation of autophagy in the cells exposed to RES. Also, blockade of the autophagy-related signaling suppressed the RES-induced mitochondria-related cytoprotection. The utilization of siRNA targeting Beclin 1, for example, attenuated the effects of RES on the release of cytochrome c to the cytosol, as well as on the levels of cleaved PARP. Thus, RES triggered autophagy by an AMPK/SIRT1 axis-dependent manner in SH-SY5Y cells, causing neuroprotection. Beclin 1 is a protein associated with the regulation of autophagy by controlling the formation of the autophagosome [5]. The inhibition of autophagy abrogates the clearance of proteins that would be prone for aggregation, such as α-synuclein [5]. Moreover, autophagy removes dysfunctional mitochondria from the cells (also called mitophagy) [88]. In this regard, Jeong et al. (2012) reported that RES pretreatment at 2 µM for 12 h promoted mitochondrial protection and mitochondria-related anti-apoptotic effects by a mechanism associated with the triggering of autophagy in SH-SY5Y cells exposed to the amino acid residues 106–126 of the prion protein (PrP106-126) [89]. The anti-apoptotic effects induced by RES on the levels of BAX in the mitochondria and of cytochrome c in the cytosol were abrogated by the administration of 3-methyladenine (3-MA; an inhibitor of autophagy) to the cells. Furthermore, RES attenuated the loss of MMP by the same mechanism in the PrP106-126-treated cells. Thus, RES activated autophagy promoting cytoprotection by a mitochondria-associated manner. In this same line, Lin et al. (2014) have reported that the inhibition of autophagy by bafilomycin A1 (BAF) abrogated the RES-induced inhibition of caspase-3 in SH-SY5Y cells exposed to rotenone [90]. The authors have utilized RES at 20 µM in a pretreatment model for 24 h prior to the challenge with rotenone. Interestingly, the autophagy-related cytoprotective effects elicited by RES involved the activation of HO-1, since the inhibition of this enzyme by ZnPP IX (a specific inhibitor of HO-1) suppressed the RES-induced autophagy and cytoprotection. Whether there is a link between the induction of HO-1 by RES and the mitochondrial protection mediated by this polyphenol, it remains to be determined.
Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma
Published in Cancer Investigation, 2023
Sabine Wächter, Franziska Knauff, Silvia Roth, Corinna Keber, Katharina Holzer, Jerena Manoharan, Elisabeth Maurer, Detlef K. Bartsch, Pietro Di Fazio
The expression of PD-L1 confers to cancer cells the ability to resist to the extracellular death signaling by inhibiting the apoptotic transduction signal and the further caspases cleavage. A basal PD-L1 protein level was detected in all primary ATC cells included in the study (Figure 7(A)). The administration of 500 ng/ml of atezolizumab, 10 nM of panobinostat or 1 µM of sorafenib (48h) caused a significant over-expression of the protein level of PD-L1 only in the patient 1 cells. The combination of atezolizumab and panobinostat was able to induce the over-expression of PD-L1 in the patient 1 and patient 3 cells. The combination of atezolizumab and sorafenib increased PD-L1 protein level in patient 1 and decreased PD-L1 protein level in patient 3 cells. 100 pM of bafilomycin caused a significant increase of PD-L1 in patient 1 cells (Figure 7(A)).
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.
Misrouting of glucagon and stathmin-2 towards lysosomal system of α-cells in glucagon hypersecretion of diabetes
Published in Islets, 2022
Farzad Asadi, Savita Dhanvantari
αTC1-6 cells were incubated for 24 h in serum-free medium containing 16.7 mM glucose and 0.1% BSA. Cells were then washed twice with HBSS containing 16.7 mM glucose, HEPES, and 0.5% BSA and pre-incubated for 30 min in this medium. For lysosomal inhibition, cells were treated with 10 nM Bafilomycin A1 (Cat# B1793, Sigma) for 2 h. Secretion studies were done by addition of 55 mM KCl for 20 min as we have done previously18 in the presence of Bafilomycin A1. Media were then collected into microfuge tubes containing PMSF (45 mM) and Aprotinin (5 µg/mL), centrifuged at 14000 × g for 15 minutes at 4°C, and then stored at -80°C until analysis. Cells were washed in ice-cold HBSS, lysed in ice-cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100 plus 45 mM PMSF, and 5 μg/mL Aprotinin), and centrifuged at 14000 × g for 15 minutes at 4°C. The supernatant was collected and stored at -80°C until analysis. Cell protein level was measured by BCA, as mentioned above, and used for normalization of glucagon secretion.