The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tertbutyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2-and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.

The observation by Brown et al.' of a low-frequency line in the Raman spectrum of the protein a-chymotrypsin together with the subsequent report by Genzel e t aL2 of a similar vibration near 25 cm-' in the Raman spectrum of lysozyme has led to considerable speculation concerning the origin of such Among the possibilities discussed by various authors and recently reviewed by Peticolas3 are longitudinal acoustic modes (LAM), presumably localized in ordered a-helical and P-sheet segments'; internal breathing modes,' which have been modeled by treating proteins as elastic spheres6s7; intermolecular vibrations2; and motions involving the relative displacement of subunit^.^ Although Peticolas has effectively argued against an interpretation in terms of intermolecular vibrations; a more complete understanding has proved elusive. In part, this is because such low-frequency modes are extremely difficult to observe, and the reported spectra of a-chymotrypsin and lysozyme have essentially stood alone for a number of years. Fortunately, spectroscopy is still an evolving art and recent improvements in instrumentation have made the low-frequency region of the Raman spectrum more accessible. We have recently reported the observation of low-frequency modes in the Raman spectrum of bovine immunoglobulin Gs and DNA.9 The Raman spectrum of IgG was particularly intriguing in that more than one Raman line was resolved, suggesting a relationship to "hinge and elbow-bending'' modes that have been proposed for this Y-shaped molecule.'0 If we are to obtain a more specific understanding of the putative relationship between low-frequency Raman modes and the functionally important overall motions of biopolymers, a study of a wide range of proteins would seem to be necessary. Accordingly, in this communication we will consider the spectra of various proteins, chosen so as to reflect a range of molecular weights and different constituent secondary structures.The frequencies of the Raman line observed in various proteins are listed in Table I.

No abstract

Glutathione (GSH) is the major thiol-disulfide redox buffer in cells and is a critical component of antioxidant defense. Here we examined GSH redox balance in the intestinal mucosa during the annual cycle of 13-lined ground squirrels (Spermophilus tridecemlineatus). The ratio of reduced GSH to its oxidized form (glutathione disulfide, GSSG), which is an index of oxidative stress, was five-fold lower in hibernating compared with summer-active squirrels, an effect due primarily to elevated GSSG concentration in hibernators. During hibernation the total pool of GSH equivalents was lowest in squirrels undergoing arousal and highest in squirrels during interbout arousals. Hibernation decreased intestinal GSSG reductase activity by approximately 50%, but had no effect on activities of glutathione peroxidase or glucose-6-phosphate dehydrogenase. Within the hibernation season, expression of the stress protein HSP70 in intestinal mucosa was highest in squirrels entering torpor and early in a torpor bout, and lowest in squirrels arousing from torpor and during interbout euthermia. The results suggest that hibernation in ground squirrels is associated with a shift in intestinal GSH redox balance to a more oxidized state. Higher levels of HSP70 during the early phases of torpor may reflect induction of the stress response due to aberrations in protein folding or may be a mechanism to increase enterocyte tolerance to subsequent stress imposed by extended torpor or the arousal process.

Staurosporine (STP) was shown to induce cell apoptosis through formation of reactive oxygen species, but a role for cellular redox has not been defined. In this study, we report that STP (2μM) caused apoptosis (24±3% at 24h) of human colon adenocarcinoma epithelial cell line HT29 that was preceded by significant GSH and GSSG efflux (6h), but independent of changes in cellular GSH/ GSSG redox status. The blockade of GSH efflux by γ-glutamyl glutamate (γ-GG) or ophthalmic acid was associated with apoptosis attenuation; however, γ-GG administration after peak GSH efflux (8h) did not confer cytoprotection. Moreover, lowering cellular GSH through inhibition of its synthesis prevented extracellular GSH accumulation and cell apoptosis, thus validating a link between cellular GSH export and the trigger of cell apoptosis. Inhibition of γ-glutamyl transferase (GGT1 EC 2.3.2.2)-catalyzed extracellular GSH degradation with acivicin significantly blocked GSH efflux, suggesting that GSH breakdown is a driving force for GSH export. Interestingly, acivicin treatment enhanced extracellular GSSG accumulation, consistent with GSH oxidation. STP-induced HT29 cell apoptosis was associated with caspase-3 activation independent of caspase-8 or caspase-9 activity; accordingly, inhibitors of the latter caspases were without effect on STP-induced apoptosis. STP similarly induced GSH efflux and apoptosis in a nonmalignant human NCM460 colonic cell line in association with caspase-3 activation. Collectively, our results demonstrate that STP induction of apoptosis in malignant and non-malignant colonic cells is temporally linked to the export of cellular GSH and the activation of caspase-3 without caspase-8 or -9 involvement. Keywords staurosporine and cell apoptosis; GSH efflux and cell apoptosis; γ-glutamyl transferase and GSH efflux; caspase-8 and -9 independent caspase-3 activation; extracellular GSH hydrolysis and GSH efflux; GSH efflux and caspase-3 activation