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Using models of serum deprivation and 1-methyl-4-phenylpyridinium (MPP ؉ ), we investigated the mechanism by which thioredoxin (Trx) exerts its antiapoptotic protection in human neuroblastoma cells (SH-SY5Y) and preconditioning-induced neuroprotection. We showed that SH-SY5Y cells are highly sensitive to oxidative stress and responsive to both extracellularly administered and preconditioning-induced Trx. Serum deprivation and MPP ؉ produced an elevation in the hydroxyl radicals, malondialdehyde and 4-hydroxy-2,3-nonenal (HNE), causing the cells to undergo mitochondria-mediated apoptosis. Trx in the submicromolar range blocked the observed apoptosis via a multiphasic protection mechanism that includes the suppression of cytochrome c release (most likely via the induction of Bcl-2), the inhibition of procaspase-9 and procaspase-3 activation, and the elevated level of Mn-SOD. The reduced form of Trx suppresses the serum-free-induced hydroxyl radicals, lipid peroxidation, and apoptosis, indicating that Thioredoxins (Trx) 1 contain two redox-active cysteine residues in their conserved active centers that can be oxidized to form intramolecular disulfide bonds (Trx-S 2 ). Reduction of Trx-S 2 is catalyzed by Trx reductase with NADPH as the electron donor. The thiol oxidoreductase activity of Trx makes it suitable to function as an electron carrier for the catalytic actions of peroxidases and as a protector protein against unwarranted oxidant-mediated inter-or intramolecular disulfide bond formation (1-4). Trx is capable of removing H 2 O 2 (5), particularly when it is coupled with either methionine sulfoxide reductase (6) or Trx peroxidase systems (7). At a relatively high concentration, Trx can enhance the biosynthesis of Mnsuperoxide dismutase (Mn-SOD) (8). It can also selectively activate the DNA binding affinities of certain transcription factors. For example, reduced Trx enhances the NF-B binding affinity to DNA, whereas Trx-S 2 inhibits this binding (9). Similarly, Trx elevates DNA binding to transcription factors AP-1 (10) and AP-2 (11) in conjunction with the nuclear redox protein Ref-1 by reducing a specific cysteine residue in the DNA binding domain of Jun and Fos dimers.Because many oxidants or activators of cellular oxidative metabolism are inducers of apoptosis (12), oxidative stress has been implicated in the induction of apoptosis. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which metabolizes to form 1-methyl-4-phenylpyridinum (MPP ϩ ), known to generate ⅐ OH and oxidative stress in vivo (13), has consistently been shown to induce apoptosis in SH-SY5Y cells (14). Furthermore, because serum deprivation provokes apoptosis in a variety of cells, this in vitro model has been used extensively to investigate the regulation of apoptosis. The observed apoptosis has been attributed to decreased availability of cell survival factors (15) and/or increased levels of reactive oxygen species (ROS) (16). Our preliminary data (17) suggested that serum deprivation causes an increase in both nitric oxide and ⅐ OH...

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Human subjects infected with herpes or varicella-zoster viruses complain of pain, such as allodynia, in or near the region with vesicles. However, the mechanisms of the pain are unclear. We show for the first time that infection with herpes simplex virus type-1 (HSV-1) induces allodynia and hyperalgesia in mice. When HSV-1 was inoculated on the hind paw of the mouse, eruption appeared on the back on day 5 post-inoculation, and zosteriform skin lesions were developed on the inoculated side. Allodynia and hyperalgesia became apparent in the hind paw on the inoculated side on day 5 and persisted until at least day 8. HSV-1 DNA was detected in the dorsal root ganglia from days 2 to 8 post-inoculation, with a peak effect on day 5. The application of heat-inactivated HSV-1 induced no allodynia, hyperalgesia and skin lesion. When started from days 0 or 2, repeated treatment with acyclovir, anti-HSV-1 agent, inhibited the appearance of allodynia, hyperalgesia, eruption and the viral proliferation in the dorsal root ganglia. In contrast, when started from days 5 or 6, acyclovir treatment slightly inhibited the development of skin lesions and the viral proliferation, but not allodynia and hyperalgesia. These results suggest that the propagation of HSV-1 in the dorsal root ganglia produces allodynia and hyperalgesia as a result of functional abnormality of the sensory neurons in mice. This may be a useful model for studying the mechanisms of herpetic pain.

Preconditioning stress induced by a transient ischemia may increase brain tolerance to oxidative stress, and the underlying neuroprotective mechanisms are not well understood. In a series of experiments, we found that endogenous nitric oxide (NO), S-nitrosoglutathione (GSNO), and antioxidants blocked serum deprivation-induced oxidative stress and apoptosis in human neuroblastoma cells. Similar to nuclear redox factor-1 (Ref-1), mRNA of human neuronal nitric oxide synthase (hNOS1) was maximally up-regulated within 2 h after oxidative stress and down-regulated by NO/GSNO and hydroxyl radical (OH) scavenger. A brief preconditioning stress induced by serum deprivation for 2 h caused a delayed increase in the expression of hNOS1 protein and the associated formation of NO and cGMP, which in turn decreased OH generation and stress-related cell death. In addition to inhibiting caspase-3 through a dithiothreitol-sensitive S-nitrosylation process, preconditioning stress concomitantly up-regulated the expression of the anti-apoptotic bcl-2 protein and down-regulated the p66shc adaptor protein. This beneficial cytoprotective process of preconditioning stress is mediated by newly synthesized NO because it can be suppressed by the inhibition of hNOS1 and guanylyl cyclase. Therefore, the constitutive isoform of hNOS1 is dynamically redox-regulated to meet both functional and compensatory demands of NO for gene regulation, antioxidant defense, and tolerance to oxidative stress.