TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly.

Focal adhesions are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of focal adhesions is crucial for cell migration. Paxillin is a multi-adaptor protein that plays an important role in regulating focal adhesion dynamics. Here, we identify TRIM15, a member of the tripartite motif protein family, as a paxillin-interacting factor and a component of focal adhesions. TRIM15 localizes to focal contacts in a myosin-II-independent manner by an interaction between its coiled-coil domain and the LD2 motif of paxillin. Unlike other focal adhesion proteins, TRIM15 is a stable focal adhesion component with restricted mobility due to its ability to form oligomers. TRIM15-depleted cells display impaired cell migration and reduced focal adhesion disassembly rates, in addition to enlarged focal adhesions. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of focal adhesion turnover and cell migration.

Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals.

BACKGROUND: Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW: To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS: Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE: The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

The human sulfatase 2 inhibitor 2,4-disulfonylphenyl-tert-butylnitrone (OKN-007) has an antitumor effect in hepatocellular carcinoma mediated via suppression of TGFB1/SMAD2 and Hedgehog/GLI1 signaling.

Human sulfatase 2 (SULF2) functions as an oncoprotein in hepatocellular carcinoma (HCC) development by promoting tumor growth and metastasis via enhancement of fibroblast growth factor-2/extracellular signal-regulated kinase and WNT/ß-catenin signaling. Recent results implicate that SULF2 activates the transforming growth factor beta (TGFB) and Hedgehog/GLI1 pathways in HCC. OKN-007 is a novel phenyl-sulfonyl compound that inhibits the enzymatic activity of SULF2. To investigate the antitumor effect of OKN-007 in HCC, we treated Huh7 cells, which express high levels of SULF2, with OKN-007 and found that it significantly promoted tumor cell apoptosis and inhibited cell proliferation, viability, and migration. To understand the action of OKN-007 on SULF2, we used Huh7 cells which normally express SULF2 and Hep3B cells that do not normally express SULF2. Utilizing Huh7 cells transfected with short hairpin RNA targeting SULF2 and transfection of Hep3B cells with a SULF2 plasmid to enhance SULF2 expression, we showed that the antitumor activity of OKN-007 was more pronounced in cells expressing SULF2. Furthermore, in vivo experiments verified that OKN-007 repressed tumor growth significantly. These results identify SULF2 as an important target of the antitumor effect of OKN-007. To determine the molecular mechanism of the antitumor effect of OKN-007, both TGFB1/SMAD and Hedgehog/GLI1 signaling pathway activity were measured by Western blot and SMAD- or GLI-reporter luciferase assays. We found that both signaling pathways were inhibited by OKN-007. Together, these results show that OKN-007 can suppress TGFB1/SMAD and Hedgehog/GLI1 signaling via its inhibition of SULF2 enzymatic activity. We conclude that OKN-007 or more potent derivatives may be promising agents for the treatment of HCC.

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity.

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 µm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Role of volatile and non-volatile plant secondary metabolites in host tree selection by Christmas beetles.

Individual Eucalyptus trees in south-eastern Australia vary considerably in susceptibility to herbivores. On the one hand, studies with insect herbivores have suggested that variation in the concentrations of foliar monoterpenes is related to variation in susceptibility. On the other, studies with marsupial folivores have suggested that variation in the concentrations of sideroxylonals (a group of formylated phloroglucinol compounds) is responsible for variation in susceptibility. We examined relative importance of sideroxylonals and 1,8-cineole (a dominant monoterpene) in host tree selection by Christmas beetles (Anoplognathus species: Coleoptera: Scarabaeidae) by using no-choice experiments, choice/no-choice experiments, and manipulative experiments in which concentrations of sideroxylonals or 1,8-cineole were altered. We used two species of host Eucalyptus, one species of non-host Eucalyptus, and three species of non-host non-Eucalyptus trees. Leaf consumption by Christmas beetles was negatively correlated with the concentrations of sideroxylonals and 1,8-cineole. Artificial increases in the concentration of sideroxylonals or 1,8-cineole reduced leaf consumption by Christmas beetles. An artificial reduction in foliar monoterpenes had no effect on leaf consumption by the beetles when leaves contained high or very low concentrations of sideroxylonals. However, when the concentration of sideroxylonals was moderate, a reduction in the foliar monoterpenes increased leaf consumption by the beetles. Therefore, monoterpenes such as 1,8-cineole may be used as a negative cue by Christmas beetles. The pattern of food consumption on non-host Eucalyptus species and non-host non-Eucalyptus species suggest that both positive and negative cues may be used by Christmas beetles to select host trees.

Isotria medeoloides, a North American Threatened Orchid: Fungal Abundance May Be as Important as Light in Species Management.

The management of endangered or threatened plant species is difficult if protocols are not developed to propagate species for the purpose of restoration or the enhancement of existing populations. The management of endangered and threatened orchids is especially difficult because of the obligate interactions between orchids and orchid mycorrhizal fungi. Isotria medeoloides is a federally threatened forest-dwelling orchid species with a wide distribution in eastern North America. Seeds have not been successfully germinated and current management is based primarily on using subcanopy thinning to increase light in areas where monitoring demonstrates that populations are declining. We report the results of long-term monitoring efforts, canopy thinning, and orchid mycorrhizal fungus abundance studies at two locations in Virginia. The declining populations responded positively to the experimental and natural thinning of the canopy. At one site, the response was the result of understory canopy thinning. At the second site, the response was due to the natural death of a canopy tree. In light of the dramatic increase in fungal abundance following death of the canopy tree, we propose the Fungal Abundance Hypothesis as an additional approach to the management of endangered plant species. The removal of canopy trees in or adjacent to Isotria populations results in an increase in dead belowground biomass (i.e., roots of the dead canopy tree) that provides substrates for microbial growth, including orchid mycorrhizal fungi, that benefit Isotria.

Multiparametric assessment of the anti-glioma properties of OKN007 by magnetic resonance imaging.

PURPOSE: To demonstrate that OKN007, a disulfonyl derivative of phenyl-tert-butyl nitrone (PBN), has anti-glioma activity in the clinically relevant C6 rat glioma model using multi-parametric magnetic resonance imaging. MATERIALS AND METHODS: Twenty-one rats were intracerebrally implanted with C6 cells and administered OKN007 or kept as controls. Animals were monitored with MRI at 7 Tesla (T), using morphologic, diffusion-weighted and perfusion imaging, followed by histology and Western blots of angiogenesis and inflammatory markers. RESULTS: OKN007 was found to decrease tumor volumes and increase survival. The glioma tissues of OKN007-treated rats were found to have longitudinal apparent diffusion coefficients (ADC(z)) of 0.76 +/- 0.06 x 10(-3) mm(2)/s, similar to the contralateral tissue and significantly smaller than untreated gliomas (0.97 +/- 0.13 x 10(-3) mm(2)/s). They had higher perfusion rates (66 +/- 4 mL/100 g.min) than untreated gliomas (26 +/- 7 mL/100 g.min). All examined molecular markers were decreased in OKN007-treated rat gliomas, compared with elevated levels in untreated rats. CONCLUSION: MRI assessment was successfully used to monitor a decrease in tumor growth, and corresponding alterations in ADC and perfusion rates in rat C6 gliomas treated with the anti-glioma agent, OKN007.

The Prioritized Research Agenda for the Athletic Training Profession: A Report From the Strategic Alliance Research Agenda Task Force.

CONTEXT: Athletic trainers (ATs) must be equipped with evidence to inform their clinical practice. A systematic, inclusive, and continuous process for exploring research priorities is vital to the success of ATs and, more importantly, their patients' positive outcomes. OBJECTIVE: To identify research priorities and unify research with clinical practice to improve patient care and advance the profession. DESIGN: Mixed-methods study. SETTING: Focus groups and a Web-based survey. PATIENTS OR OTHER PARTICIPANTS: A total of 87 ATs (43 men [49.4%], 44 women [50.6%]; age = 40 ± 11 years; experience = 18 ± 11 years) participated in focus groups. Of the 49 332 e-mails sent, 580 were undeliverable, 5131 ATs started the survey (access rate = 10.5%), and 4514 agreed to participate (response rate = 9.3%). MAIN OUTCOME MEASURE(S): Our study consisted of 6 focus-group sessions, a content-expert review, and a Web-based survey. Themes from the focus groups were used to develop the research priorities and survey instrument. We used the 25-item validated survey to determine whether the research priorities and findings of the focus groups were generalizable. Endorsement of research priorities and recommendations was achieved when respondents indicated they agreed or strongly agreed. RESULTS: Respondents endorsed 5 research priorities: health care competency (n = 4438/4493, 98.8%), vitality of the profession (n = 4319/4455, 96.9%), health professions education (n = 3966/4419, 89.8%), health care economics (n = 4246/4425, 96.0%), and health information technology (n = 3893/4438, 87.7%). We also made the following recommendations: (1) develop funding initiatives that align with the agenda, (2) develop postdoctoral fellowships focused on clinical research, (3) facilitate collaborative relationships between clinicians and researchers, and (4) make research evidence more readily available and more applicable. CONCLUSIONS: Using a systematic and inclusive process, we developed a prioritized research agenda for the athletic training profession. The agenda was endorsed by the leaders of each Strategic Alliance organization and adopted as the Athletic Training Research Agenda.

Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model.

The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T(1)/T(2)-weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.

Effectiveness of 4-hydroxy phenyl N-tert-butylnitrone (4-OHPBN) alone and in combination with other antioxidant drugs in the treatment of acute acoustic trauma in chinchilla.

Acute acoustic trauma (AAT) results in oxidative stress to the cochlea through overproduction of cellular reactive oxygen, nitrogen, and other free radical species appearing from 1 h to 10 days after noise exposure. It has been shown that N-acetyl-L-cysteine (NAC), a glutathione prodrug, and acetyl-L-carnitine (ALCAR), a mitochondrial biogenesis agent, are effective in reducing noise-induced hearing loss. Phenyl N-tert-butylnitrone (PBN), a nitrone-based free radical trap, appears to suppress oxidative stress in a variety of disorders and several biological models. In this study, we tested whether 4-hydroxy PBN (4-OHPBN), a major metabolite of PBN, administered 4 h after noise exposure is effective in treating noise-induced hearing loss and whether a combination of antioxidant drugs (4-OHPBN plus NAC and 4-OHPBN plus NAC plus ALCAR) provides greater efficacy in attenuating AAT since each agent addresses different injury mechanisms. Chinchilla were exposed to a 105 dB octave-band noise centered at 4 kHz for 6 h. 4-OHPBN and combinations of antioxidant drugs were intraperitoneally administered beginning 4 h after noise exposure. Hearing threshold shifts in auditory brainstem responses and missing outer hair cell counts were obtained. 4-OHPBN reduced threshold shifts in a dose-dependent manner while both drug combinations showed greater effects. These results demonstrate that 4-OHPBN and combinations of antioxidants can effectively treat acute acoustic trauma and drug combinations may increase the effectiveness of treatment and decrease the required individual medication dose.

Nitrones as therapeutics.

Nitrones have the general chemical formula X-CH=NO-Y. They were first used to trap free radicals in chemical systems and then subsequently in biochemical systems. More recently several nitrones, including alpha-phenyl-tert-butylnitrone (PBN), have been shown to have potent biological activity in many experimental animal models. Many diseases of aging, including stroke, cancer development, Parkinson disease, and Alzheimer disease, are known to have enhanced levels of free radicals and oxidative stress. Some derivatives of PBN are significantly more potent than PBN and have undergone extensive commercial development for stroke. Recent research has shown that PBN-related nitrones also have anti-cancer activity in several experimental cancer models and have potential as therapeutics in some cancers. Also, in recent observations nitrones have been shown to act synergistically in combination with antioxidants in the prevention of acute acoustic-noise-induced hearing loss. The mechanistic basis of the potent biological activity of PBN-related nitrones is not known. Even though PBN-related nitrones do decrease oxidative stress and oxidative damage, their potent biological anti-inflammatory activity and their ability to alter cellular signaling processes cannot readily be explained by conventional notions of free radical trapping biochemistry. This review is focused on our studies and others in which the use of selected nitrones as novel therapeutics has been evaluated in experimental models in the context of free radical biochemical and cellular processes considered important in pathologic conditions and age-related diseases.

An Aging Interventions Testing Program: study design and interim report.

The National Institute on Aging's Interventions Testing Program (ITP) has developed a plan to evaluate agents that are considered plausible candidates for delaying rates of aging. Key features include: (i) use of genetically heterogeneous mice (a standardized four-way cross), (ii) replication at three test sites (the Jackson Laboratory, TJL; University of Michigan, UM; and University of Texas, UT), (iii) sufficient statistical power to detect 10% changes in lifespan, (iv) tests for age-dependent changes in T cell subsets and physical activity, and (v) an annual solicitation for collaborators who wish to suggest new interventions for evaluation. Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen (NFP), 4-OH-alpha-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordihydroguiaretic acid (NDGA). An interim analysis was conducted using survival data available on the date at which at least 50% of the male control mice had died at each test site. Survival of control males was significantly higher, at the interim time-point, at UM than at UT or TJL; all three sites had similar survival of control females. Males in the NDGA group had significantly improved survival (P = 0.0004), with significant effects noted at TJL (P < 0.01) and UT (P < 0.04). None of the other agents altered survival, although there was a suggestion (P = 0.07) of a beneficial effect of aspirin in males. More data will be needed to determine if any of these compounds can extend maximal lifespan, but the current data show that NDGA reduces early life mortality risks in genetically heterogeneous mice at multiple test sites.

Nitrones as therapeutics in age-related diseases.

Age-related diseases deprive individuals of a higher quality of life and therefore therapeutics for their treatment provide significant potential. An overview of the observations of nitrones as potential therapeutics in several age-related diseases is presented. Treatment of acute ischemic stroke is one condition where a nitrone (NXY-059) is in late phase 3 clinical trials now. Also presented is a summary of the most recent work we have accomplished on the anticancer activity of the nitrones in a hepatocellular carcinoma. The mechanistic basis of action of these compounds in several animal models is not yet understood at the molecular levels; however, it does appear clear that their anti-inflammatory properties are central to their action, which is based on their ability to down-regulate exacerbated signal transduction processes.

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats.

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.

HPN-07, a free radical spin trapping agent, protects against functional, cellular and electrophysiological changes in the cochlea induced by acute acoustic trauma.

Oxidative stress is considered a major cause of the structural and functional changes associated with auditory pathologies induced by exposure to acute acoustic trauma AAT). In the present study, we examined the otoprotective effects of 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07), a nitrone-based free radical trap, on the physiological and cellular changes in the auditory system of chinchilla following a six-hour exposure to 4 kHz octave band noise at 105 dB SPL. HPN-07 has been shown to suppress oxidative stress in biological models of a variety of disorders. Our results show that administration of HPN-07 beginning four hours after acoustic trauma accelerated and enhanced auditory/cochlear functional recovery, as measured by auditory brainstem responses (ABR), distortion product otoacoustic emissions (DPOAE), compound action potentials (CAP), and cochlear microphonics (CM). The normally tight correlation between the endocochlear potential (EP) and evoked potentials of CAP and CM were persistently disrupted after noise trauma in untreated animals but returned to homeostatic conditions in HPN-07 treated animals. Histological analyses revealed several therapeutic advantages associated with HPN-07 treatment following AAT, including reductions in inner and outer hair cell loss; reductions in AAT-induced loss of calretinin-positive afferent nerve fibers in the spiral lamina; and reductions in fibrocyte loss within the spiral ligament. These findings support the conclusion that early intervention with HPN-07 following an AAT efficiently blocks the propagative ototoxic effects of oxidative stress, thereby preserving the homeostatic and functional integrity of the cochlea.

Antioxidants reduce neurodegeneration and accumulation of pathologic Tau proteins in the auditory system after blast exposure.

Cochlear neurodegeneration commonly accompanies hair cell loss resulting from aging, ototoxicity, or exposures to intense noise or blast overpressures. However, the precise pathophysiological mechanisms that drive this degenerative response have not been fully elucidated. Our laboratory previously demonstrated that non-transgenic rats exposed to blast overpressures exhibited marked somatic accumulation of neurotoxic variants of the microtubule-associated protein, Tau, in the hippocampus. In the present study, we extended these analyses to examine neurodegeneration and pathologic Tau accumulation in the auditory system in response to blast exposure and evaluated the potential therapeutic efficacy of antioxidants on short-circuiting this pathological process. Blast injury induced ribbon synapse loss and retrograde neurodegeneration in the cochlea in untreated animals. An accompanying perikaryal accumulation of neurofilament light chain and pathologic Tau oligomers were observed in neurons from both the peripheral and central auditory system, spanning from the spiral ganglion to the auditory cortex. Due to its coincident accumulation pattern and well-documented neurotoxicity, our results suggest that the accumulation of pathologic Tau oligomers may actively contribute to blast-induced cochlear neurodegeneration. Therapeutic intervention with a combinatorial regimen of 2,4-disulfonyl α-phenyl tertiary butyl nitrone (HPN-07) and N-acetylcysteine (NAC) significantly reduced both pathologic Tau accumulation and indications of ongoing neurodegeneration in the cochlea and the auditory cortex. These results demonstrate that a combination of HPN-07 and NAC administrated shortly after a blast exposure can serve as a potential therapeutic strategy for preserving auditory function among military personnel or civilians with blast-induced traumatic brain injuries.

DNA cytosine methylation profile in various cancer-related genes is altered in cultured rat hepatocyte cell lines as compared with primary hepatocytes.

We determined the DNA cytosine methylation status in the promoter CpG islands of eight cancer-related genes (p16, Socs-1, Rassf1A, Hic-1, Dlc-1, Timp-1, Timp-2, and Timp-3) in five rat hepatocyte cell lines, including normal cell lines (Clone 9 and CWSV-1) and tumor cell lines (H4-II-E-C3, MH1C1, and McA-RH7777). The experimental methods used to assess the methylation profile were methylation-specific PCR (MSP) and methylation-sensitive digestion combined with PCR. The results were compared with the methylation status of rat primary hepatocytes. To evaluate methylation-mediated gene induction/silencing, the expression of gene transcripts was semi-quantitatively assessed using RT-PCR. In primary cells, the CpG islands of all genes tested were unmethylated. In contrast, there was at least one hypermethylated gene in the cultured cell lines. Three genes (p16, Socs-1 and Rassf1A) were hypermethylated in Clone 9 cells; among the other five genes, three genes (Hic-1, Timp-1 and Timp-3) were hypermethylated in the CWSV-1 cell lines and two genes (Dlc-1 and Timp-2) were hypermethylated only in the tumor cell lines. The methylation status in some of the tested genes was altered at an early stage of cell culture as compared to primary cells. It is also noteworthy that hypermethylation in Socs-1, Rassf1, Hic-1, and Timp-3 was widespread among the cell lines tested, but not in the primary cells and Clone 9 cells. This study suggests that a cautious approach is required when cell lines are utilized to study methylation-related carcinogenic, metastatic or tumoricidal mechanisms.

Ameliorative Effects of Antioxidants on the Hippocampal Accumulation of Pathologic Tau in a Rat Model of Blast-Induced Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to early onset dementia and other related neurodegenerative diseases. We previously demonstrated that damage to the central auditory pathway resulting from blast-induced TBI (bTBI) could be significantly attenuated by a combinatorial antioxidant treatment regimen. In the current study, we examined the localization patterns of normal Tau and the potential blast-induced accumulation of neurotoxic variants of this microtubule-associated protein that are believed to potentiate the neurodegenerative effects associated with synaptic dysfunction in the hippocampus following three successive blast overpressure exposures in nontransgenic rats. We observed a marked increase in the number of both hyperphosphorylated and oligomeric Tau-positive hilar mossy cells and somatic accumulation of endogenous Tau in oligodendrocytes in the hippocampus. Remarkably, a combinatorial regimen of 2,4-disulfonyl α-phenyl tertiary butyl nitrone (HPN-07) and N-acetylcysteine (NAC) resulted in striking reductions in the numbers of both mossy cells and oligodendrocytes positively labeled for these pathological Tau immunoreactivity patterns in response to bTBI. This treatment strategy represents a promising therapeutic approach for simultaneously reducing or eliminating both primary auditory injury and nonauditory changes associated with bTBI-induced hippocampal neurodegeneration.

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes.

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.

Sensitivity of protein tyrosine phosphatase activity to the redox environment, cytochrome C, and microperoxidase.

Protein tyrosine phosphatase activity depends on a catalytic thiolate group on an acidic cysteine residue that is sensitive to reactive oxygen species. Representative of this family of enzymes is protein tyrosine phosphatase 1B (PTP1B), a major target for type 2 diabetes therapy. PTP1B is sensitive to hydrogen peroxide (H2O2) in vitro and in cells. It is also sensitive to glutathionylation by glutathione disulfide (GSSG). The sensitivity of PTP1B to the redox state of its environment was partially characterized in vitro by examination of phosphatase activity in the presence of various concentrations of glutathione (GSH) and GSSG. Enzyme sensitivity to glutathionylation was dependent on the amount of available thiol groups and increased as GSH concentration was increased. The half-inhibitory concentration for H2O2 was much less than that of GSSG in the presence of low concentrations of GSH, indicating that reaction with H2O2 is much more likely than is glutathionylation by GSSG. PTP1B and a related oxidant-sensitive phosphatase, PTEN, were also sensitive to the lipid peroxidation by-product 4-hydroxynonenal. Furthermore, PTP1B was inhibited by cytochrome c and microperoxidase. Taken together, these data suggest that not only H2O2, but also a variety of redox-active metabolites and hemes can oxidatively inactivate PTPs with potentially profound implications for signal transduction.