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The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8de f and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals ([O.sub.2.sup.-]). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC ([DELTA]sodC) and a F. tularensis [DELTA]sodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB [DELTA]sodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-[gamma])-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis [DELTA]sodC and sodB [DELTA]sodC mutants showed attenuated intramacrophage survival in IFN-[gamma]-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the [DELTA]sodC mutant or inhibiting the IFN-[gamma]-dependent production of [O.sub.2.sup.-] or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The [DELTA]sodC and sodB [DELTA]sodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the [DELTA]sodC mutant was restored in ifn-[[gamma].sup.- /-], [inos.sup.-/-], and [phox.sup.-/-] mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice. doi: 10.1128/JB.00534-09

A Francisella tularensis live vaccine strain mutant ([sodB.sub.Ft]) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The [sodB.sub.Ft] mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor.

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun N[H.sub.2]-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against [H.sub.2][O.sub.2]-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 [micro]M [H.sub.2][O.sub.2], and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients. antioxidants; apoptosis; reactive oxygen species; c-Jun N[H.sub.2]-terminal kinase; c-Jun N[H.sub.2]-terminal kinase pathway

Melendez, J. Andres, James M. Vinci, John J. Jeffrey, and Brian D. Wilcox. Localization and regulation of IL-1[Alpha] in rat myometrium during late pregnancy and the postpartum period. Am J Physiol Regulatory Integrative Comp Physiol 280: R879-R888, 2001.--Interleukin-1 (IL-1) has been implicated as a participant in preterm labor that is induced by bacterial infection. Previously, we showed that serotonin-induced production of IL-1[Alpha] by myometrial smooth muscle cells in vitro is also essential for the synthesis of interstitial collagenase. It is therefore likely that IL-1[Alpha] production in uterine tissues has implications for both the normal physiology of involution and for the pathophysiological mechanisms of preterm labor. The objective of this study was to characterize the serotonin-induced production of IL-1[Alpha] by myometrial cultures in vitro and to assess the production of IL-1[Alpha] and its relationship to collagenase production in vivo during pregnancy and the postpartum period. Immunohistochemistry demonstrated IL-1[Alpha] protein in the nuclei and cytoplasm of serotonin-treated myometrial cells. IL-1[Alpha] levels were decreased by treatment with progesterone or IL-1-receptor antagonist but were unaffected by lipopolysaccharide. Western analysis of myometrium from pregnant rats showed low levels of IL-1[Alpha] during midpregnancy with increased concentrations at days 21 and 22 and postpartum. IL-1[Alpha] mRNA levels also increased from days 15 to 22. Levels of mRNA for IL-1[Beta] also increased, although to a lesser degree than IL-1[Alpha]. Both mRNAs decreased postpartum. Conversely, mRNA for interstitial collagenase was barely detectable at term but increased postpartum. Together, these data show that serotonin stimulates IL-1[Alpha] production in vitro and indicate that normal myometrium from pregnant rats is an identifiable source of IL-1 during late pregnancy. The findings are consistent with the possibility that myometrial IL-1[Alpha] participates in normal labor as well as the postpartum production of interstitial collagenase. uterus; smooth muscle; parturition; cytokine; collagenase