Jun Yoshitake

8-Nitroguanosine formation in viral pneumonia and its implication for pathogenesis

For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation in vivo. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virus-infected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8- Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOS-deficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS in vitro. The present data constitute a demonstration of 8-nitroguanosine formation in vivo and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.

Role of Nitric Oxide in Host Defense in Murine Salmonellosis as a Function of Its Antibacterial and Antiapoptotic Activities

ABSTRACT Host defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulent Salmonella enterica serovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD50s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 × 104 CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD50, 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD50, 6 × 102 CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute to Salmonella pathogenicity. These results indicate that NO has significant host defense functions in Salmonella infections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.

Nitrated cyclic GMP modulates guard cell signaling in Arabidopsis.

This work examines the role of the nitrated derivative of cyclic GMP (cGMP), 8-nitro-cGMP, in guard cell signaling downstream of nitric oxide (NO) and abscisic acid, finding that 8-nitro-cGMP synthesis is induced by abscisic acid, NO, and reactive oxygen species and that 8-nitro-cGMP triggers stomatal closure. Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3′,5′-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5′-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells.

Nano-scaled particles of titanium dioxide convert benign mouse fibrosarcoma cells into aggressive tumor cells.

Nanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO(2)) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO(2), either uncoated (TiO(2)-1, hydrophilic) or coated with stearic acid (TiO(2)-2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO(2)-1, but not TiO(2)-2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO(2)-1 and TiO(2)-2 treatments. However, TiO(2)-2, but not TiO(2)-1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO(2)-1 and TiO(2)-2 resulted in intracellular ROS formation, TiO(2)-2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO(2)-2, but not TiO(2)-1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO(2) toxicity acquired a tumorigenic phenotype. TiO(2)-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO(2) has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells.

Identification of and Screening for Human Helicobacter cinaedi Infections and Carriers via Nested PCR

ABSTRACT Helicobacter cinaedi is the most frequently reported enterohepatic Helicobacter species isolated from humans. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods. Here, we report a nested PCR assay that rapidly detects the cytolethal distending toxin gene (cdt) of H. cinaedi with high specificity and sensitivity. Specificity of the assay was validated by using different species of Helicobacter and Campylobacter, as well as known H. cinaedi-positive and -negative samples. The sensitivity of detection for the cdt gene in the assay was 102 CFU/ml urine or 102 CFU/105 infected RAW 264.7 cells. In an H. cinaedi-infected mouse model, the cdt gene of H. cinaedi was effectively detected via the assay with urine (6/7), stool (2/3), and blood (2/6) samples. Importantly, it detected H. cinaedi in blood, urine, and stool samples from one patient with a suspected H. cinaedi infection and three patients with known infections. The assay was further used clinically to follow up two H. cinaedi-infected patients after antibiotic treatment. Stool samples from these two patients evaluated by nested PCR after antibiotic therapy showed clearance of bacterial DNA. Finally, analysis of stool specimens from healthy volunteers showed occasional positive reactions (4/30) to H. cinaedi DNA, which suggests intestinal colonization by H. cinaedi in healthy subjects. In conclusion, this nested PCR assay may be useful for the rapid diagnosis, antimicrobial treatment evaluation, and epidemiological study of H. cinaedi infection.

Suppression of NO production and 8-nitroguanosine formation by phenol-containing endocrine-disrupting chemicals in LPS-stimulated macrophages: Involvement of estrogen receptor-dependent or -independent pathways

Since the endocrine and immune systems share portions of some intracellular signaling pathways, endocrine-disrupting chemicals (EDCs) are considered potential agents for influencing inflammatory responses. Here, we investigated the effect of EDCs on lipopolysaccharide (LPS)-induced NO production and NF-kappaB activation in the RAW264.7 mouse macrophage cell line. Five phenol-containing EDCs were investigated, namely bisphenol A (BPA), the alkyl phenols p-n-nonylphenol (NP) and p-n-octylphenol (OP), and the chlorinated phenols 2,4-dichlorophenol (DCP) and pentachlorophenol (PCP). Our results revealed that these chemicals dose-dependently suppressed LPS-induced NO production, as reflected by decreased NO(x) content. The suppressive effects of BPA, NP and OP, but not PCP or DCP, were blocked by the estrogen receptor (ER) inhibitor, ICI182780. ELISA-based quantification of the DNA-binding activity of free p65 NF-kappaB showed that LPS-induced NF-kappaB activation was significantly diminished by EDC treatment. Furthermore, immunocytochemical analysis of 8-nitroguanosine, a unique index of NO-mediated signaling, showed that 8-nitroguanosine formation increased in LPS-stimulated cells, but this increase was inhibited by the tested EDCs. These results demonstrate that EDCs suppress NO production and NF-kappaB activation in LPS-stimulated macrophages through ER-dependent (BPA, NP, OP) and -independent (PCP, DCP) pathways. The EDCs further inhibited 8-nitroguanosine formation, suggesting that they interfere with NO-mediated signaling. Thus, EDCs might play important roles in the inflammatory response and host defense system against foreign pathogens.

Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses

Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80+ foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

Nitric oxide promotes recycling of 8-nitro-cGMP, a cytoprotective mediator, into intact cGMP in cells

8-Nitro-cGMP is an endogenous nucleotide discovered under inflammation conditions as an important mediator of nitric oxide (NO) signaling. Besides cGMP-like behaviour, 8-nitro-cGMP exerts unique cytoprotective effects against oxidative stress. Although the formation of 8-nitro-cGMP from 8-nitro-GTP has previously been proposed, the mechanism by which excess or unused 8-nitro-cGMP is removed from cells remains unknown. In this study, we report a nitric oxide-dependent cellular conversion of 8-nitro-cGMP to intact cGMP in RAW 264.7 macrophage cells. In our experiments, we synthesized isotopically labeled 8-nitro-cGMP as a tool for metabolites analysis and identified 8-amino-cGMP as an initial metabolite of 8-nitro-cGMP using a LC-MS/MS technique. We also proved that endogenous 8-nitro-cGMP can be converted into 8-amino-cGMP by immunocytochemical staining with an antibody that specifically recognizes 8-amino-cGMP. Moreover, we showed that isotopically labeled 8-amino-cGMP is metabolized into cGMP under inflammation conditions. We propose that nitrosylation of 8-amino-cGMP occurs by NO formation under stress conditions and gives putative 8-diazonium-cGMP, which subsequently decomposes into cGMP. To the best of our knowledge, this study is the first to report reductive deamination of aminoguanine nucleotide at the C-8 position. The findings of this study collectively indicate that NO plays a crucial role not only in the production of 8-nitro-cGMP but also in its elimination under oxidative stress or inflammation.

Modification of Tau by 8-nitro-cGMP: Effects of Nitric Oxide-linked Chemical Modification on Tau Aggregation

Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid β peptide deposition are hallmarks of Alzheimers disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimers disease.