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.

Daidzein supplementation prevents non-alcoholic fatty liver disease through alternation of hepatic gene expression profiles and adipocyte metabolism.

INTRODUCTION: Globally, non-alcoholic fatty liver disease (NAFLD) continues to rise and isoflavones exert antisteatotic effects by the regulation of hepatic lipogenesis/insulin resistance or adiposity/a variety of adipocytokines are related to hepatic steatosis. However, there is very little information regarding the potential effects of daidzein, the secondary abundant isoflavone, on NAFLD. Here, we have assessed the hepatic global transcription profiles, adipocytokines and adiposity in mice with high fat-induced NAFLD and their alteration by daidzein supplementation. METHODS: C57BL/6J mice were fed with normal fat (16% fat of total energy), high fat (HF; 36% fat of total energy) and HF supplemented with daidzein (0.1, 0.5, 1 and 2 g per kg diet) for 12 weeks. RESULTS: Daidzein supplementation (≥ 0.5 g per kg diet) reduced hepatic lipid concentrations and alleviated hepatic steatosis. The hepatic microarray showed that daidzein supplementation (1 g per kg diet) downregulated carbohydrate responsive element binding protein, a determinant of de novo lipogenesis, its upstream gene liver X receptor ß and its target genes encoding for lipogenic enzymes, thereby preventing hepatic steatosis and insulin resistance. These results were confirmed by lower insulin and blood glucose levels as well as homeostasis model assessment insulin resistance scores. In addition, daidzein supplementation inhibited adiposity by the upregulation of genes involved in fatty acid ß-oxidation and the antiadipogeneis, and moreover augmented antisteatohepatitic leptin and adiponectin mRNA levels, whereas it reduced the mRNA or concentration of steatotic tumor necrosis factor α and ghrelin. CONCLUSIONS: These findings show that daidzein might alleviate NAFLD through the direct regulation of hepatic de novo lipogenesis and insulin signaling, and the indirect control of adiposity and adipocytokines by the alteration of adipocyte metabolism.

Combination drug therapy using edaravone and Daio-Orengedoku-to after transient focal ischemia in rats.

In this study, we investigated the effect of Daio-Orengedoku-to (DOT) on ischemic brain damage in a rat model of focal ischemia-reperfusion and attempted to identify synergistic effects for the combination of edaravone and DOT against ischemic insult. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion followed for 22 h. To determine the neuroprotective effect of DOT, it was administered orally just before reperfusion and then 2 h after reperfusion. To examine the effects of combination therapy on survival, rats were divided into groups treated with edaravone, DOT, and edaravone and DOT. Microglial activation, neutrophil infiltration and brain-derived neurotrophic factor (BDNF) expression were examined in surviving animals. Infarct volume was significantly reduced by DOT (100, 200 and 400 mg/kg; P < 0.05), and edaravone plus DOT markedly improved the survival rate after transient ischemia (P = 0.0133). Microglial activation was reduced by edaravone and DOT and their combination (P < 0.05), and neutrophil infiltration was lowered in these groups (P < 0.05). BDNF-positive cells were increased in the combination edaravone and DOT group (P < 0.05). It appears that the neuroprotective mechanisms of combined therapy involve inhibition of microglial activation, reduction of invading neutrophils and enhancement of BDNF expression.

IgE binding components in Tetranychus urticae and Panonychus ulmi-derived crude extracts and their cross-reactivity with domestic mites.

BACKGROUND: Recent investigation has revealed that spider mites such as Tetranychus urticae and Panonychus ulmi are important allergens in the development of occupational asthma among apple farmers. OBJECTIVE: To evaluate IgE binding components in T. urticae and P. ulmi-derived crude extracts and their cross-reactivity with Panonychus citri, Tyrophagus putrescentiae and Dermatophagoides pteronyssinus in apple cultivating farmers. METHODS: Thirty-one apple farmers with positive skin responses to T. urticae or P. ulmi were randomly recruited, and specific IgE levels in their sera were measured using ELISA. Cross-reactivity was evaluated by ELISA inhibition. IgE binding components were evaluated by IgE immunoblotting. RESULTS: A total of 11 IgE binding components in T. urticae and 10 in P. ulmi were found. Among them, the 17 kDa, 27 kDa, 33 kDa, 37 kDa, 41 kDa, 56 kDa, and 75 kDa allergens in T. urticae, and the 33 kDa, 41 kDa, and 51 kDa allergens in P. ulmi were identified as dominant allergens. T. urticae-specific IgE binding was completely inhibited by 100 microg/mL of T. urticae (99.7%), but only partially inhibited by P. citri (83.0%), P. ulmi (71.6%), T. putrescentiae (69.7%), and D. pternonyssinus (60.1%). P. ulmi-specific IgE binding was completely inhibited by additions of P. citri (92.3%) and P. ulmi (91.2%), but only partially inhibited by the addition of T. urticae (61.5%), T. putrescentiae (57.7%), and D. pteronyssinus (54.4%). CONCLUSION: There were seven dominant allergens found in T. urticae and three in P. ulmi. T. urticae- and P. ulmi-specific IgE bindings were partially inhibited by crude extracts derived from D. pteronyssinus and T. putrescentiae.