Homologous expression and quantitative analysis of T3SS-dependent secretion of TAP-tagged XoAvrBs2 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract.

Xanthomonas oryzae pv. oryzae (Xoo) produces a putative effector, XoAvrBs2. We expressed XoAvrBs2 homologously in Xoo with a TAP-tag at the C-terminus to enable quantitative analysis of protein expression and secretion. Addition of rice leaf extracts from both Xoo-sensitive and Xoo-resistant rice cultivars to the Xoo cells induced expression of the XoAvrBs2 gene at the transcriptional and translational levels, and also stimulated a remarkable amount of XoAvrBs2 secretion into the medium. In a T3SS-defective Xoo mutant strain, secretion of the TAPtagged XoAvrBs2 was blocked. Thus, we elucidated the transcriptional and translational expressions of the XoAvrBs2 gene in Xoo was induced in vitro by the interaction with rice and the induced secretion of XoAvrBs2 was T3SSdependent. It is the first report to measure the homologous expression and secretion of XoAvrBs2 in vitro by rice leaf extract. Our system for the quantitative analysis of effector protein expression and secretion could be generally used for the study of host-pathogen interactions.

Protective effects of antioxidant supplementation on plasma lipid peroxidation in smokers.

This study was designed to investigate the possible protective effects of antioxidants (vitamin E, beta-carotene, vitamin C, and red ginseng) on lipid peroxidation in smokers (> or = 20 cigarettes/day). Male student smokers were given antioxidant supplements for 4 wk. Smokers had significantly higher plasma levels of total cholesterol, triacylglycerols, and malondialdehyde (MDA) than nonsmokers. No corresponding significant differences in lipid profiles were found between smokers and nonsmokers. Smokers had significantly lower baseline concentrations of plasma vitamin C, beta-carotene, and alpha-tocopherol. After antioxidant (200 IU vitamin E, 9 mg beta-carotene, 500 mg vitamin C, or 1.8 g red ginseng) supplementation for 4 wk, smokers had significantly higher concentrations of plasma antioxidants. After 4 wk of antioxidant supplementation with betacarotene, high-density lipoprotein (HDL) cholesterol concentrations in smokers were significantly increased. Overall, plasma MDA concentrations gradually decreased after antioxidant supplementation over the 4-wk period. Moreover, a significant reduction in plasma MDA concentrations was observed after vitamin E supplementation. The results of our study support the hypothesis that lipid peroxidation concentrations are inversely correlated with plasma antioxidant concentrations. Our data suggest that smokers have insufficient concentrations of antioxidant vitamins in plasma and that supplementation with antioxidants might protect smokers from oxidative damage.

Protective effect of green tea extract on ischemia/reperfusion-induced brain injury in Mongolian gerbils.

Free radical-induced oxidative damages of macromolecules and cell death are important factors in the pathogenesis of ischemia/reperfusion brain injury. In the present study, an investigation as to whether green tea extract reduces ischemia/reperfusion-induced brain injury in Mongolian gerbils was conducted. The effect of green tea on the ischemia/reperfusion-induced production of hydrogen peroxide, lipid peroxidation and oxidative DNA damage (formation of 8-hydroxydeoxyguanosine), and cell death in addition to locomotor activity was studied. Two doses (0.5 or 2%) of green tea extract were added into the drinking water and to be accessed by animals ad libitum for 3 weeks prior to the induction of ischemia. A global ischemia was induced by the bilateral occlusion of the common carotid arteries for 5 min. Reperfusion was achieved by releasing the occlusion and restoring blood circulation for 48 h. The infarction volumes were 112+/-31 mm(3) and 76+/-11 mm(3) in the 0.5 and 2% green tea pretreated animals compared to 189+/-12 mm(3) in the ischemia/reperfusion animals. Green tea extract also reduced the levels of ischemia/reperfusion-induced hydrogen peroxide (from 1470+/-170 to 1034+/-46 and 555+/-30 nmole/mg protein), lipid peroxidation products (from 1410+/-210 to 930+/-40 and 330+/-20 nmole/mg protein) and 8-oxodG (from 3.9+/-0.1 to 2.8+/-0.3 and1.9+/-0.3 ng/microg DNA, x10(-2)) by pretreatment of 0.5 or 2% green tea for 3 weeks, respectively. Moreover, green tea also reduced the number of ischemia/reperfusion-induced apoptotic cells (from 59+/-12 to 37+/-8, 15+/-11 apoptotic cells/high power field in the striatum region) and locomotor activity (from 15140+/-2940 to 3900+/-600 and 4100+/-1200). This study therefore suggests that green tea may be a useful agent for the prevention of cerebral ischemia damage.

Neuroprotective effect of green tea extract in experimental ischemia-reperfusion brain injury.

Eicosanoids accumulation and formation of oxygen free radicals have been implicated in the pathogenesis of ischemia/reperfusion brain injury. In the present study, we examined whether green tea extract protects against ischemia/reperfusion-induced brain injury by minimizing eicosanoid accumulation and oxygen radical-induced oxidative damage in the brain. Green tea extract (0.5%) was orally administered to Wistar rats for 3 weeks before induction of ischemia. Ischemia was induced by the occlusion of middle cerebral arteries for 60 min and reperfusion was achieved for 24 h. Infarction volume in the ipsilateral hemisphere of ischemia/reperfusion animals was 114 +/- 16 mm(3) in the 0.5% green tea pretreated animals compared to 180 +/- 54 mm(3) in left hemisphere of nontreated animals. Green tea extract (0.5%) also reduced ischemia/reperfusion-induced eicosanoid concentration: Leukotriene C(4) (from 245 +/- 51 to186 +/- 22), prostoglandin E(2) (from 306 +/- 71 to 212 +/- 43) and thromboxane A(2) (327 +/- 69 to 251 +/- 87 ng/mg protein). Ischemia/reperfusion-induced increases of hydrogen peroxide level (from 688 +/- 76 to 501 +/- 99 nmole/mg protein), lipid peroxidation products (from 1010 +/- 110 to 820 +/- 70 nmole/mg protein) and 8-oxodG formation (from 1.3 +/- 0.3 to 0.8 +/- 0.2 ng/microg DNA, x10(-2)) were also reduced. Moreover, 0.5% green tea extract also reduced the apoptotic cell number (from 44 +/- 11 to 29 +/- 1 in the striatum, and from 72 +/- 11 to 42 +/- 5 apoptotic cells/high power field in the cortex region). Green tea extract pretreatment also promoted recovery from the ischemia/reperfusion-induced inhibition of active avoidance. The present study shows that the minimizing effect of green tea extract on the eicosanoid accumulation and oxidative damage in addition to the reduction of neuronal cell death could eventually result in protective effect on the ischemia/reperfusion-induced brain injury and behavior deficit.

In vitro chemopreventive effects of plant polysaccharides (Aloe barbadensis miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor).

A plant polysaccharide, Aloe gel extract, was reported to have an inhibitory effect on benzo[a]pyrene (B[a]P)-DNA adduct formation in vitro and in vivo. Hence, chemopreventive effects of plant polysaccharides [Aloe barbadensis Miller (APS), Lentinus edodes (LPS), Ganoderma lucidum (GPS) and Coriolus versicolor (CPS)] were compared using in vitro short-term screening methods associated with both initiation and promotion processes in carcinogenesis. In B[a]P-DNA adduct formation, APS (180 micrograms/ml) was the most effective in inhibition of B[a]P binding to DNA in mouse liver cells. Oxidative DNA damage (by 8-hydroxydeoxyguanosine) was significantly decreased by APS (180 micrograms/ml) and CPS (180 micrograms/ml). In induction of glutathione S-transferase activity, GPS was found to be the most effective among plant polysaccharides. In screening anti-tumor promoting effects, APS (180 micrograms/ml) significantly inhibited phorbol myristic acetate (PMA)-induced ornithine decarboxylase activity in Balb/3T3 cells. In addition, APS significantly inhibited PMA-induced tyrosine kinase activity in human leukemic cells. APS and CPS significantly inhibited superoxide anion formation. These results suggest that some plant polysaccharides produced both anti-genotoxic and anti-tumor promoting activities in in vitro models and, therefore, might be considered as potential agents for cancer chemoprevention.

Effect of glucose, sucrose and fructose on plasma glucose and insulin responses in normal humans: comparison with white bread.

OBJECTIVE: To determine the plasma glucose and insulin responses of various doses of glucose, sucrose, fructose and white bread in normal human subjects. DESIGN: Plasma glucose and insulin were measured before and at various times after 8 subjects ate 13 different test meals in randomized order on separate days after an overnight fast. Test meals consisted of 500 ml of tea or water to which was added either nothing, 25, 50, or 100 g of glucose or sucrose, 25 or 50 g fructose, 50 g glucose plus 50 g fructose, or a 25, 50 or 100 g carbohydrate portion of white bread. The glycaemic (GI) and insulinaemic index (II) values of the sugars were calculated by expressing the incremental areas under the plasma glucose and insulin curves (AUC) after glucose, sucrose and fructose as a percentage of the respective AUC after white bread containing the same amount of carbohydrate. SETTING: University teaching hospital clinical nutrition centre. SUBJECTS: Lean, normal subjects (4 male, 4 female) 21-33 y of age. RESULTS: Plasma insulin responses increased nearly linearly as carbohydrate intake increased from 0 to 100 g, but glycaemic responses increased by only 68% and 38% as carbohydrate intake increased from 25 to 50 g and 50 to 100g, respectively. The GI and II values of glucose, 149+/-16 and 147+/-18, respectively, were significantly greater than those of bread (100; P<0.05), while the values for fructose, 16+/-4 and 22+/-3 were significantly less than those of bread (P<0.001). GI values did not differ significantly from II values. CONCLUSIONS: It is concluded that, in normal subjects, as carbohydrate intake is increased from 0 to 100 g, plasma insulin responses increase at a greater rate than plasma glucose responses. The insulinaemic responses elicited by glucose, sucrose or fructose are similar to those that would be expected from a starchy food with the same glycaemic index.

Inhibition of oxidative DNA damage, 8-OHdG, and carbonyl contents in smokers treated with antioxidants (vitamin E, vitamin C, beta-carotene and red ginseng).

The chemopreventive effects of antioxidants (vitamin E, beta-carotene, vitamin C and red ginseng) on oxidative DNA and protein (globin) damages were comparatively investigated in the peripheral blood of smokers (> or = 20 cigarettes/day). Smokers showed a lower baseline level of plasma micronutrients (vitamin C and beta-carotene) (P < 0.01) and higher baseline level of oxidative DNA or protein damage than non-smokers (N = 5; P < 0.05). During daily supplementation of antioxidants (200 IU vitamin of E, 9 mg of beta-carotene, 500 mg of vitamin C, or 1.8 g of red ginseng) for 4 weeks, smokers plasma antioxidant concentrations increased linearly, while their mean levels of 8-hydroxydeoxyguanosine (8-OHdG) and carbonyl contents decreased compared with those in smokers supplemented with a placebo (P < 0.05). Levels of urinary and plasma cotinine remained steady in smokers regardless of supplementation with antioxidants. 8-OHdG and carbonyl content decreased in a time-dependent manner (as the total intake dose increased) after supplementation with vitamin E (8-OHdG, 33.8%; carbonyl content, 43.6%) or red ginseng (8-OHdG, 31.7%; carbonyl content, 21.3%). These preliminary data suggest that supplementation with antioxidants might protect smokers from oxidative damages and could reduce cancer risk or other diseases caused by free radicals associated with smoking.

Inhibition of benzo[a]pyrene-DNA adduct formation by Aloe barbadensis Miller.

The antigenotoxic and chemopreventive effect of Aloe barbadensis Miller (polysaccharide fraction) on benzo[a]pyrene (B[a]P)-DNA adducts was investigated in vitro and in vivo. Aloe showed a time-course and dose-dependent inhibition of [3H]B[a]P-DNA adduct formation in primary rat hepatocytes (1x10(6) cells/ml) treated with [3H]B[a]P (4 nmol/ml). At concentrations of 0.4-250 microg/ml aloe, the binding of [3H]B[a]P metabolites to rat hepatocyte DNA was inhibited by 9.1-47.9%. Also, in rat hepatocytes cultured for 3-48 h with aloe (250 microg/ml) and [3H]B[a]P (4 nmol/ml), [3H]B[a]P-DNA adducts were significantly reduced by 36% compared with [3H]B[a]P alone. Aloe also inhibited cellular uptake of [3H]B[a]P in a dose-dependent manner at a concentration of 0.4-250 microg/ml by 6.3-34.1%. After a single oral administration of B[a]P to male ICR mice (10 mg/mouse), benzo[a]pyrene diol epoxide I (BPDE-I)-DNA adduct formation and persistence for 16 days following daily treatment with aloe (50 mg/mouse) were quantitated by enzyme-linked immunosorbent assay using monoclonal antibody 8E11. In this animal model, BPDE-I-DNA adduct formation was significantly inhibited in various organs (liver, kidney, forestomach and lung) (P < 0.001). When mice were pretreated with aloe for 16 days before B[a]P treatment, inhibition of BPDE-I-DNA adduct formation and persistence was enhanced. Glutathione S-transferase activity was slightly increased in the liver but cytochrome P450 content was not affected by aloe. These results suggest that the inhibitory effect of aloe on BPDE-I-DNA adduct formation might have a chemopreventive effect by inhibition of B[a]P absorption.