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  • An alternative more rarely used electron microscopy based ap

    2024-02-26

    An alternative, more rarely used, electron microscopy-based approach exploits labeling of phagosomes with DAMP (3-(2,4-dinitroanilino)-30-amino-N-methyldipropylamine). DAMP is a weakly basic compound which is membrane-impermeable once it has been protonated. This way, DAMPH+ accumulates in acidic intracellular compartments. As the compound is fixable, treated cells can be analyzed in immuno-electron microscopy with a suitable monoclonal antibody. Within a reasonable range of pH and applying an extensive calibration effort the density of gold signals between compartments can reveal different pH (Clemens et al., 2004). The beauty of this approach is the proverbial visualization of pH differences at subcellular level. A more readily applicable version of the DAMP assay is the abundantly used LysoTracker™ (a trademark of Thermo Fisher Scientific) assay. Like DAMP, LysoTracker™ is a weak Benidipine HCl australia which accumulates in acidic compartments but LysoTracker™ is fluorescent. Semi-quantification of acidification of a compartment is possible by counting the number of LysoTracker™-positive phagosomes (von Bargen et al., 2009), e.g., of phagosomes with a pH below a certain threshold (in our hands around 5.8–6.0). As with DAMP, calibration is extremely difficult because the indicators would not enter the cells when added at low extracellular pH. When using LysoTracker™ it is important to not only use low nanomolar concentrations but to also keep it on cells only briefly because otherwise the compound itself can raise the pH within phagosomes. Finally, LysoTracker™ is not directly fixable due to the lack of a primary amino group and ought to be analyzed in live cells or signals become fuzzy. A recent modification of LysoTracker™ is the ‘Superior Lyso Probes’ series of fluorophores which are retained in acidic compartments for hours opposed to minutes for LysoTracker (Chen et al., 2015). Other modifications include the LysoBrite™ (AAT-Bioquest – Biomol, Germany) and pHrodo™ (ThermoFisher Scientific) stains. pHrodo™ increases fluorescence in the acidic range and can be used for pH quantification in the same way as fluorescein.
    Drugs which block V-ATPase and/or lysosome acidification Several natural and synthetic proton pump inhibitors have been developed for biomedical research into V-ATPases and the role of acidification in antimicrobial defense. Especially cancer therapy profited from a pharmacological reduction of V-ATPase-mediated acidification (Perez-Sayans et al., 2009). The commonly applied research tools bafilomycin A1 (Bowman et al., 1988) and concanamycin A (Drose et al., 1993) are first-generation V-ATPase inhibitors with IC50 values in the low nanomolar range. Later on, the benzolactone enamides e.g., salicylihamide (Erickson et al., 1997), apicularen A and B (Kunze et al., 1998) and the macrolactone archazolid (Sasse et al., 2003) were discovered as cytotoxic agents in anti-tumor or anti-virus screens and were identified as potent V-ATPase inhibitors. Pharmacological engineering of these compounds was applied to determine the common functional groups mediating V-ATPase inhibition and to synthesize optimized inhibitors for disease treatment. Manzamine A (Kallifatidis et al., 2013), diphyllin (Sorensen et al., 2007) and the pea albumin 1 subunit b (PA1b) (Chouabe et al., 2011) belong to the newest generation of V-ATPase inhibitors. Most of these drugs bind to the V-ATPase VO sector and prevent proton translocation by blocking VO rotation (Bockelmann et al., 2010, Bowman et al., 2004, Osteresch et al., 2012, Wang et al., 2005, Xie et al., 2004). Some inhibitors, e.g., N-ethylmaleimide (NEM) and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole chloride (NBD-Cl), bind to sector V1 and limit ATP hydrolysis (Forgac, 1989). It is of note, that many of these inhibitors are quite specific for V-ATPases but recent research highlighted the need for careful data interpretation whenever V-ATPase function is pharmacologically inhibited (Mauvezin and Neufeld, 2015).