Disruption of nitric oxide signaling by Helicobacter pylori results in enhanced inflammation by inhibition of heme oxygenase-1.

A strong cellular cross-talk exists between the pathogen Helicobacter pylori and high-output NO production. However, how NO and H. pylori interact to signal in gastric epithelial cells and modulate the innate immune response is unknown. We show that chemical or cellular sources of NO induce the anti-inflammatory effector heme oxygenase-1 (HO-1) in gastric epithelial cells through a pathway that requires NF-κB. However, H. pylori decreases NO-induced NF-κB activation, thereby inhibiting HO-1 expression. This inhibitory effect of H. pylori results from activation of the transcription factor heat shock factor-1 by the H. pylori virulence factor CagA and by the host signaling molecules ERK1/2 and JNK. Consistent with these findings, HO-1 is downregulated in gastric epithelial cells of patients infected with cagA(+) H. pylori but not in gastric epithelial cells of patients infected with cagA(-) H. pylori. Enhancement of HO-1 activity in infected cells or in H. pylori-infected mice inhibits chemokine generation and reduces inflammation. These data define a mechanism by which H. pylori favors its own pathogenesis by inhibiting HO-1 induction through the action of CagA.

A high-throughput screening assay of ascorbate in brain samples.

Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75µM AscH(-) in the sample (0.5-10µM AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.

Normoxic versus hyperoxic resuscitation in pediatric asphyxial cardiac arrest: effects on oxidative stress.

OBJECTIVE: To determine the effects of normoxic vs. hyperoxic resuscitation on oxidative stress in a model of pediatric asphyxial cardiac arrest. DESIGN: Prospective, interventional study. SETTING: University research laboratory. SUBJECTS: Postnatal day 16-18 rats (n = 5 per group). INTERVENTIONS: Rats underwent asphyxial cardiac arrest for 9 min. Rats were randomized to receive 100% oxygen, room air, or 100% oxygen with polynitroxyl albumin (10 mL·kg⁻¹ intravenously, 0 and 30 min after resuscitation) for 1 hr from the start of cardiopulmonary resuscitation. Shams recovered in 100% oxygen or room air after surgery. MEASUREMENTS AND MAIN RESULTS: Physiological variables were recorded at baseline to 1 hr after resuscitation. At 6 hrs after asphyxial cardiac arrest, levels of reduced glutathione and protein-thiols (fluorescent assay), activities of total superoxide dismutase and mitochondrial manganese superoxide dismutase (cytochrome c reduction method), manganese superoxide dismutase expression (Western blot), and lipid peroxidation (4-hydroxynonenal Michael adducts) were evaluated in brain tissue homogenates. Hippocampal 3-nitrotyrosine levels were determined by immunohistochemistry 72 hrs after asphyxial cardiac arrest. Survival did not differ among groups. At 1 hr after resuscitation, Pao2, pH, and mean arterial pressure were decreased in room air vs. 100% oxygen rats (59 ± 3 vs. 465 ± 46 mm Hg, 7.36 ± 0.05 vs. 7.42 ± 0.03, 35 ± 4 vs. 45 ± 5 mm Hg; p < .05). Rats resuscitated with 100% oxygen had decreased hippocampal reduced glutathione levels vs. sham (15.3 ± 0.4 vs. 20.9 ± 4.1 nmol·mg protein⁻¹; p < .01). Hippocampal manganese superoxide dismutase activity was significantly increased in 100% oxygen rats vs. sham (14 ± 2.4 vs. 9.5 ± 1.6 units·mg protein⁻¹, p < .01), with no difference in protein expression of manganese superoxide dismutase. Room air and 100% oxygen plus polynitroxyl albumin groups had hippocampal reduced glutathione and manganese superoxide dismutase activity levels comparable with sham. Protein thiol levels were unchanged across groups. Compared with all other groups, rats receiving 100% oxygen had increased immunopositivity for 3-nitrotyrosine in the hippocampus and increased lipid peroxidation in the cortex. CONCLUSIONS: Resuscitation with 100% oxygen leads to increased oxidative stress in a model that mimics pediatric cardiac arrest. This may be prevented by using room air or giving an antioxidant with 100% oxygen resuscitation.

Radioprotection by short-term oxidative preconditioning: role of manganese superoxide dismutase.

Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H(2)O(2) increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.

Mitochondria-targeted (2-hydroxyamino-vinyl)-triphenyl-phosphonium releases NO(.) and protects mouse embryonic cells against irradiation-induced apoptosis.

Generation of reactive oxygen species by damaged respiratory chain followed by the formation of cytochrome c (cyt c)-cardiolipin (CL) complex with peroxidase activity are early events in apoptosis. By quenching the peroxidase activity of cyt c-CL complexes in mitochondria, nitric oxide can exert anti-apoptotic effects. Therefore, mitochondria-targeted pro-drugs capable of gradual nitric oxide radical (NO*) release are promising radioprotectants. Here we demonstrate that (2-hydroxyamino-vinyl)-triphenyl-phosphonium effectively accumulates in mitochondria, releases NO* upon mitochondrial peroxidase reaction, protects mouse embryonic cells from irradiation-induced apoptosis and increases their clonogenic survival after irradiation. We conclude that mitochondria-targeted peroxidase-activatable NO-donors represent a new interesting class of radioprotectors.