Kazue Satoh

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

BackgroundWe hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.MethodsUnilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.ResultsGp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.ConclusionsClassical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Preliminary evaluation of antinephritis and radical scavenging activities of glabridin from Glycyrrhiza glabra.

Antinephritis activity of glabridin, a pyranoisoflavan isolated from Glycyrrhiza glabra, was evaluated after its oral administration to mice with glomerular disease (Masugi-nephritis) by measuring the urinary protein excretion, total cholesterol, serum creatinine and blood urea nitrogen levels. Administration of glabridin for 10 days (30 mg kg(-1) day(-1)) reduced the amount of urinary protein excretion from control level (100+/-23 mg/day) to a significantly lower level (47+/-4 mg/day). ESR spectroscopy demonstrated that glabridin neither produced radical, nor affected the radical intensity of sodium ascorbate, suggesting the lack of correlation between the antinephritis activity and radical scavenging activity.

Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines — A possible mechanism

Hydrolyzable tannins showed higher cytotoxic activity against human oral squamous cell carcinoma and salivary gland tumor cell lines than against normal human gingival fibroblasts, whereas gallic acid, a component unit of tannins, showed much weaker selective cytotoxicity. The cytotoxic activity of dimeric compounds was generally higher than that of monomeric compounds. Macrocyclic ellagitannin oligomers, such as oenothein B, woodfordin C and woodfordin D showed the greatest cytotoxic activity, and their activity (per given number of molecules) was one order higher than those of gallic acid and epigallocatechin gallate, a major component of green tea. These compounds induced apoptotic cell death characterized by DNA fragmentation (as demonstrated by the TUNEL method) and cleavage of cytokeratin 18 by activated caspase(s) (as demonstrated by M30 monoclonal antibody). ESR spectroscopy revealed that these macrocyclic compounds at higher concentrations produced their own radicals and significantly enhanced the radical intensity of sodium ascorbate, possibly by their prooxidant actions. Catalase failed to eliminate their apoptosis-inducing activity, reducing the possibility of the involvement of hydrogen peroxide production in the extracellular fraction. These observations suggested that the antitumor activity of macrocyclic ellagitannin oligomers reported previously might be explained by their apoptosis-inducing activity.

Cancer prevention and therapy with kiwifruit in Chinese folklore medicine: A study of kiwifruit extracts

Kiwi gold fruits were extracted successively with hexane, acetone, methanol and 70% methanol, and further fractionated by silica gel and ODS column chromatographies for the assays of various biological activities. Five fractions H1, H2 (hexane extract), Al, A2 (acetone extract) and M2 (methanol extract) showed selective cytotoxic activity against human oral tumor cell lines, which was more sensitive than human gingival fibroblasts. More hydrophilic fractions [70M3, 70M4, 70M5] of 70% methanol extract displayed higher anti-HIV activity, radical generation and O2- scavenging activity. The antibacterial activity of 70% methanol extracts [70M0, 70M1, 70M2, 70M3, 70M4] was generally lower than that of more lipophilic fractions (hexane, acetone, methanol extracts), although each fraction did not show any specific antimicrobial action. All fractions were inactive against Helicobacter pylori. These results demonstrate that gold kiwifruit extracts contain valuable, various bioactive materials, which can be separated with each other.

Induction of cytotoxicity by chlorogenic acid in human oral tumor cell lines.

Millimolar concentrations of chlorogenic acid (CGA) showed higher cytotoxic activity against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines, as compared with that against human gingival fibroblast (HGF). The cytotoxic activity of CGA was significantly reduced by catalase or CoCl2, but not affected by FeCl3 or CuCl2. ESR spectroscopy showed that higher (millimolar) concentrations of CGA produced radicals under alkaline conditions, acting as a prooxidant, whereas lower concentrations of CGA scavenged superoxide and hydroxyl radical. CGA produced large DNA fragments (as identified by slightly faster migrating band of DNA on agarose gel electrophoresis) and nuclear condensation (as demonstrated by Hoechst (No. 33258) staining) in tumor cell lines. Activation of caspase was demonstrated by staining with M30 monoclonal antibody, which reacts with degradation products of cytokeratin 18. Contact with CGA for at least 6 h was necessary for irreversible cytotoxicity induction. Pretreatment of the cells with caspase 3 inhibitor partially inhibited the cytotoxic action of CGA. These date suggest that CGA induces cytotoxicity in oral tumor cell lines, possibly by hydrogen peroxide-mediated oxidation mechanism.

Cytotoxic Potential of Phenothiazines

Phenothiazines, a kind of sulfur-containing tricyclic compounds, have diverse biological activities including tranquilizer, antibacterial, antitumor and antihelmintic activities due to the relatively lower cytotoxicity. Phenothiazines have been used for clinical treatments as psychotropics. In contrast to the psychotropic preparations, their information of other biological activities of phenothiazines and their related compounds has been limited. This review article summarizes the interaction with DNA (using quantum calculation), antitumor activity, differentiation or apoptosis-inducing activity, tumor necrosis factor (TNF)-induction, antiproliferative activity, radical scavenging activity, antimutagenic activity, antiplasmid activity, antibacterial activity, reversal of multidrug resistance (MDR), blast transformation activity of phenothiazines, benz[c]acridines and benzo[a]phenothiazines.

Edarabone scavenges nitric oxide

Abstract Oxygen free radicals have been proposed to be major causative agents in secondary brain damage in traumatic and ischemic brain injury. Edarabone (3-methyl-1-phenyl-2-pyrazolin-5-one), a powerful antioxidative radical scavenger, is the only drug currently available in clinical practice for the treatment of cerebral infarction. There has been increasing interest in the role of nitric oxide (NO•) as a causative agent in brain injury. In the present study, we investigated the scavenging effect of Edarabone on nitric oxide (NO•), using an electron spin resonance (ESR) method. NO• was generated from 1-hydroxy-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7), and analyzed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy (carboxy-PTI) produced from the reaction between 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (carboxy-PTIO) and NO•. Edarabone directly scavenged NO• in a dose-dependent manner. These ESR studies indicate that Edarabone has a direct NO• scavenging activity and the additional possibility of novel neuroprotective activities against brain injury and focal cerebral ischemia.

Influence of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children

Although many single nucleotide polymorphism (SNP) studies have reported an association of atopy, allergic diseases and total serum immunoglobulin E (IgE) levels, almost all of these studies sought risk factors for the onset of these allergic diseases. Furthermore, many studies have analyzed a single gene and hardly any have analyzed environmental factors. In these analyses, the results could be masked and the effects of other genes and environmental factors may be decreased. Here, we described the correlation between four genes [interleukin (IL)‐4 (C‐590T), IL‐4 receptor (A1652G), FCER1B (G6842A) and STAT6 (G2964A)] in connection with IgE production; the role of IL‐10 (C‐627A) as a regulatory cytokine of allergy; and the severity of food allergy (FA) and atopic eczema (AE) in 220 Japanese allergic children. In addition to these SNPs, environmental factors, i.e., patients attitude, indoor envirmonment, and so on, were also investigated in this study.

Two Amino Acids Mutation of Ferric Uptake Regulator Determines Helicobacter pylori Resistance to Metronidazole

Metronidazole (Mtz) is a prodrug that is converted to its active form when its nitro group is reduced and superoxide radicals are generated. The superoxide radicals are directly toxic to the bacterium. On the other hand, the transcriptional regulator, ferric uptake regulator (Fur), of Helicobacter pylori is a direct suppressor of the iron-cofactored superoxide dismutase SodB, which is essential for protection against superoxide attack. Here, we demonstrate that in some Mtz-resistant strains, SodB activity is induced in a dose-dependent manner on exposure to Mtz. Further, under Mtz exposure, the generation of superoxide radicals in Mtz-resistant strains was significantly reduced as compared with that in the Mtz-susceptible strains. These Mtz-resistant strains were found to carry amino acids mutation of Fur (C78Y, P114S; mutant-type Fur). The binding affinity of the mutant-type Fur to an operator sequence on the sodB promoter (Fur-Box) was significantly reduced. Our approach demonstrated that SodB expression is derepressed by mutant-type Fur, which is associated with the development of Mtz resistance.

Free radical scavenging activity of propolis.

Abstract We investigated the radical scavenging activity of propolis by ESR spectroscopy using spin trapping method. In addition, we examined the influence of a diet of 2% propolis on mice under oxidative stress. At low concentrations, the methanolic extract of propolis exhibited strong scavenging activity in vitro towards both the superoxide anion radical, generated by the hypoxanthine-xanthine oxidase reaction, and the NO radical, generated from the mixture of NOC-7 (NO generator) and carboxy-PTIO (spin trapping agent). An inhibitory effect of propolis on lipid peroxidation in vivo was observed, as determined by measurement of thiobarbituric acid-reactive substances in mouse liver homogenate. The level of vitamin C in the brain of mice under oxidative stress significantly increased compared with control mice under atmosphere, which was not observed in the mice given 2% propolis. The level of α-tocopherol in the brain of mice given 2% propolis significantly increased compared with control mice under atmosphere, which was not observed in mice under oxidative stress. SOD activity in the brain and plasma of mice given 2% propolis significantly decreased under atmosphere and oxidative stress compared with control mice. These results suggest that propolis possesses potent antioxidant activity in vitro and in vivo.