Recent Applications of roGFPs

3.1. Plant Cell Jiang and co-workers have recently transformed Arabidopsis

Expression with roGFP1, targeted to both the mitochondria and cytosol

(36). Intracellularly expressed roGFP1 responded to the addition of exogenous oxidizing and reducing agents in a like manner to that reported for mammalian cells and in vitro assays (20, 22); therefore, the same calibration curves were used to convert percent oxidation of the probe to redox potentials. Parallel monitoring of the redox potential with an external redox electrode confirmed the response of roGFP1 fluorescence excitation ratios to changes in redox status. The resting redox potential of the cytoplasm, measured at three different root zones, was found to be -318 ± 13 mV, a value significantly more oxidizing than that determined from roGFP1 expressed in the mitochondria (-362 ± 10 mV). These values are nearly identical to those reported for mammalian cells (-360 mV for mitochondria in HeLa cells (20), and -315 mV reported for HeLa cell cytosol (22)). Jiang et al. also reported a more robust redox buffering capacity for the mitochondria over the cytosol, measured by comparing changes in percent oxidation of the probe in both compartments after the addition of H2O2. A comparison of the resting redox state of three root zones (root cap, meristem, and zone of elongation) revealed a somewhat-more oxidizing state in the zone of elongation compared with the meristem as well as a smaller redox buffering capacity, consistent with reports that ROS may be used as second messengers in plant cells to signal certain kinds of growth (37).

3.2. Monitoring Ischemia in neurons leads to cell-damaging oxidative stress and

Oxidative Stress in sensitivization involving depletion of glutathione levels in a process

Ischemic Neuronal that is not yet well understood. Glutathione acts as an antioxi-Cells dant buffering pool against ROS. Glutaredoxins, which derive their reducing equivalents from glutathione are, along with thioredoxins, the principle enzymes that function to maintain cytosolic thiols in their reduced state. Consequently, the depletion of glutathione pools would limit the cell's ability to respond adequately to oxidative stress. In a study of the consequences of ischemia-induced oxidative stress, Vesce and co-workers expressed roGFP2 in neuronal cells to confirm that depletion of glutathione would result in a more oxidizing thiol redox potential (38). Using monochlorobimane (mBCl), which depletes glutathione by forming a fluorescent conjugate, the researchers were able to mimic this consequence of ischemia. RoGFP2, expressed in these neurons, became significantly oxidized upon the application of mBCl. Unfortunately, fluorescence from the mBCl-glutathione conjugate interferes with measurement of roGFP emission from 400-nm excitation, thus, probe response had to be measured by comparing excitation intensities at 480 nm instead of excitation ratios.

Antibody-drug conjugate therapy is a process whereby cytotoxic compounds are specifically targeted to cancerous cells in order to maximize the effectiveness of chemotherapeutic agents while minimizing harm to the noncancerous cells of the patient. The drug is directed to tumor cells via a covalent linkage to an antibody targeting a tumor-specific antigen. Several types of covalent linkages have been used between drug and antibody, including disulfide bonds. Disulfide linkages have been shown to be effective in delivering toxic molecules to the interior of a tumor cell (39, 40), but it had been incorrectly assumed that the effect was due to a reducing environment during internalization. Scales and co-workers (4), in the process of investigating the intracel-lular cleavage of these disulfide-liked antibody-drug conjugates, found surprising evidence that endosomal compartments are not reducing but quite oxidizing, with midpoint potentials similar to those found in the ER (4).

Austin et al. (41) developed a novel intracellular cleavage assay using the drug trastuzumab attached to the fluorophore Rhodamine red (RR) via a disulfide-based linker. Trastuzumab had previously been shown to be effective against breast cancer tumors because it is an antibody specific for HER2, a member of the epidermal growth factor receptor family of tyrosine kinases

3.3. Measurement of Endosomal Redox Potential that is overexpressed in many tumor cells. HER2 is cell-surface localized but, interestingly, undergoes endocytosis at the rate of 1-2% per min, followed by recycling back to the cell surface (41). Upon endosomal cleavage of the disulfide-based trastuzumab-RR linker, there should be detectable fluorescence dequenching from the now free RR. However, because very little dequenching was observed, the researchers questioned the assumption of a reductive milieu in the endosomal compartments.

Before the development of roGFPs, no suitable redox probe was available with the pH- and proteolysis-resistance necessary to investigate conditions within endosomes. RoGFPl was therefore targeted to various endosomal compartments in order to examine their redox status. In control experiments, roGFPl was also targeted to the mitochondria and ER of the same cells. Measurements of the 380/490 nm excitation peak ratios of these constructs confirmed the suspicion that endosomal compartments are oxidizing, rather than reducing. Mitochondrial roGFP was 8.3% (±5.4%) oxidized, in agreement with that observed in mammalian cells (20, 22), whereas ER-targeted roGFP was 95.8% (±9.7%) oxidized (4). Endosomally targeted roGFPs, however, were 94-97% oxidized. Such high levels of roGFP oxidation prohibit precise determination of midpoint potentials, but Austin et al. (41) estimated that these compartments are at least as oxidizing as -240 mV, and far more oxidizing than observed for mitochondria.

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