Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity.

UNLABELLED: The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. IMPORTANCE: Ralstonia solanacearum is the causal agent of bacterial wilt disease of plants. This pathogen injects virulence effector proteins into host cells to suppress disease resistance responses of plants. In this article, we report a biochemical activity of R. solanacearum effector protein RipAY. RipAY can degrade GSH, a tripeptide that plays important roles in the plant immune system, with its γ-glutamyl cyclotransferase activity. The high GSH degradation activity of RipAY is considered to be a good weapon for this bacterium to suppress plant immunity. However, GSH also plays important roles in bacterial tolerance to various stresses and growth. Interestingly, RipAY has an excellent safety mechanism to prevent unwanted firing of its enzyme activity in bacterial cells because RipAY is specifically activated by host eukaryotic thioredoxins. This study also reveals a novel host plant protein acting as a molecular switch for effector activation.

[Pathological evaluation of neoadjuvant chemotherapy with low-dose FP therapy for advanced gastric cancer].

In the present study, we evaluated the pathological effects of preoperative chemotherapy with low-dose CDDP and 5-FU (low-dose FP therapy) in patients with advanced gastric cancer. 50 patients diagnosed as advanced gastric cancer were administered continuous infusion of 5-FU (300 mg/m(2)/day, x 14 days) and intermittent infusion of CDDP (3 mg/m(2)/day, day 1-5 and 8-12) before surgery. The pathological effects were considered comparatively regarding the response rate of chemotherapy for gastric cancer between primary lesions and metastasis of lymph nodes and the rate of downstaging cases with low-dose FP therapy. The rates of effective cases were 26% (primary lesions) and 28% (lymph nodes). Furthermore,in the same patient,the results of low-dose FP therapy with primary tumor were more effective than those of lymph nodes (the rates of effective cases were 34% and 19%, respectively). The rate of downstaging cases with low-dose FP therapy for gastric cancer was 6%, histologically. These results indicate low-dose FP therapy for gastric cancer is promising for effective clinical management of advanced gastric cancer in preoperative treatment.

Thymidylate synthase and dihydropyrimidine dehydrogenase are related to histological effects of 5-fluorouracil and cisplatin neoadjuvant chemotherapy for primary gastric cancer patients.

UNLABELLED: Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and vascular endothelial growth factor (VEGF) are associated with the effect of 5-fluorouracil (5-FU) based adjuvant chemotherapy. However, very few studies have investigated the relationship between these factors and 5-FU neoadjuvant chemotherapy for primary gastric cancer patients. In this study, we studied the correlation between these markers and the histological chemotherapeutic effect in advanced gastric cancer with neoadjuvant chemotherapy. METHODS: Sixty-two primary advanced gastric cancer patients were recruited into the study. One cycle of continuous infusion of 5-FU (300 mg/m2/day, 14 days) plus drip infusion of cisplatin (15 mg/m2/day, Day one and Day two) was performed as neoadjuvant chemotherapy. Histological chemotherapeutic responses of the resected specimens were classified into responders and nonresponders. TS, DPD, VEGF expressions both before and after neoadjuvant chemotherapy were examined immunohistochemically. RESULTS: There was an association between the TS-low group and the responders (p < 0.05); the DPD-low group and the responders in both biopsy and surgical specimens (p < 0.01). A combination of the low-TS and low-DPD group was further associated with responders (p < 0.01). The immunoexpressions of biopsied and surgical specimens were significantly associated with each other. CONCLUSION: Neoadjuvant chemotherapy for primary gastric cancer with one cycle of 5-FU and cisplatin was associated with histological findings in patients with low baseline TS and DPD. This dual determination may predict for efficacy of neoadjuvant treatment with these drugs.

Abnormal regional cerebral blood flow in systemic lupus erythematosus patients with psychiatric symptoms.

OBJECTIVE: Single-photon emission computed tomography (SPECT) studies have demonstrated decreased regional cerebral blood flow (rCBF) in systemic lupus erythematosus (SLE) patients. However, no study has done voxel-based analysis using statistical parametric mapping (SPM) that can evaluate rCBF objectively, and the relationship between rCBF and psychiatric symptoms has not been well investigated. Using L,L-ethyl cysteinate dimer (99mTc ECD) SPECT and SPM, we aimed to clarify the association of rCBF changes with psychiatric symptoms in SLE patients whose magnetic resonance imaging (MRI) showed no morphological abnormalities. METHOD: Twenty SLE patients and 19 healthy volunteers underwent 99mTc ECD SPECT. Data were collected from August 2000 to March 2003. SLE was diagnosed according to American College of Rheumatology criteria, and psychiatric symptoms were diagnosed according to ICD-10 criteria. On the basis of the modified Carbotte, Denburg, and Denburg method, the patients were classified into 3 groups: a group with major psychiatric symptoms (hallucinosis, delusional disorder, and mood disorder), a group with minor psychiatric symptoms (anxiety disorder, dissociative disorder, and emotionally labile disorder), and a group without psychiatric symptoms. Gross organic lesions were ruled out by brain MRI. Group comparisons of rCBF were performed with analysis using SPM99. RESULTS: SLE patients without MRI lesions showed decreased rCBF in the posterior cingulate gyrus and thalamus. The reduction in rCBF was overt in patients with major psychiatric symptoms. CONCLUSION: Our study indicated that SLE patients may have dysfunction in the posterior cingulate gyrus and thalamus and that this may be associated with the severity of psychiatric symptoms.

Thymidine phosphorylase expression and efficacy of adjuvant doxifluridine in advanced colorectal cancer patients.

To clarify the correlation between the expression level of thymidine phosphorylase (TP) and efficacy of doxifluridine (5'-DFUR) and 5-fluorouracil (5-FU), samples from 177 colorectal cancer patients who underwent curative resection were evaluated by immunohistochemical staining using a newly developed monoclonal antibody 1C6-203. Patients were randomly given either oral 5'-DFUR or 5-FU as postoperative adjuvant chemotherapy. In Dukes' C staged colon cancer patients treated with 5'-DFUR, better survival was observed in the high TP patients than the low TP patients (P=0.025 by the log-rank test). The observed 5-year survival rates were 91.2 and 74.8%, respectively. No correlation between TP expression and patient prognosis was detected in the 5-FU group. In Dukes' C stage colon patients with high TP expression, the 5'-DFUR group had slightly better survival than the 5-FU group. These findings suggest that TP may be a chemosensitive marker for 5'-DFUR as postoperative adjuvant chemotherapy for advanced colon cancer patients.

Thymidine phosphorylase expression correlates with malignant potential and anti-tumor effect of doxifluridine on gastric cancer: multivariate analysis for adjuvant chemotherapy doxifluridine vs. 5-fluorouracil.

Doxifluridine (5'-DFUR) is an anticancer drug converted to 5-fluorouracil (5-FU) by thymidine phosphorylase (TP). TP is an angiogenetic and platelet-derived endothelial cell growth factor. We evaluated the relation between TP expression and chemotherapeutic efficacy and prognosis for gastric cancer. Advanced gastric cancer patients given oral adjuvant chemotherapeutics either 5'-DFUR; 163 patients or 5-FU; 162 patients were examined. TP expression was assessed with immunohistochemical staining. Multivariate analysis for influencing survival was done, employing variables such as gender, age, procedure, tumor size, location, Borrmann type, histologic factors [type, depth of invasion, lymph node metastasis (n), lymphatic invasion (ly), and venous invasion (v)], drug administered, and TP expression. In the patients with serosal invasion, 5'-DFUR in TP positive was an independent prognostic factor (risk ratio, 4.450; 95% confidence limit, 2.099-9.436), indicating significantly improved prognosis over the 5-FU group. In TP negative, n and ly were independent prognostic factors, but the survival curves of the two chemotherapeutic groups were not significantly different. TP expression was not prognostic factor in 5'-DFUR group, while, in 5-FU group, TP expression was an independent prognostic factor (2.834, 1.467-5.476). In conclusion, it was suggested that TP positive gastric cancer with serosal invasion increased malignant potential of the tumor and 5'-DFUR efficacy.

Postoperative adjuvant chemotherapy is effective in gastric cancer with serosal invasion: significance in patients chosen for multivariate analysis.

There are few reports on overall usefulness of adjuvant chemotherapy in gastric cancer patients. We tried to clarify, using multivariate analysis, usefulness of postoperative adjuvant oral chemotherapy in advanced gastric cancer patients after curative resection. Four hundred and eighty-two gastric cancer patients enrolled in a randomized controlled trial were classified into 2 groups based on postoperative chemotherapeutic regimen: oral doxifluridine (5'-DFUR, an intermediate metabolite of capecitabine) (n=245) or oral 5-fluorouracil (5-FU) (n=237). The significant prognostic factors in patients with serosal invasion were chemotherapeutics (5'-DFUR vs. 5-FU) (risk ratio 1.649; 95% CI, 1.112-2.437), lymph node metastasis (no vs. yes) (2.823; 1.422-5.604), and tumor differentiation (differentiated vs. undifferentiated) (1.727; 1.068-2.791). Significant factors influencing peritoneal recurrence time were chemotherapeutics (1.756; 1.063-2.902), serosal invasion (no vs. yes) (2.237; 1.264-3.961), lymph node metastasis (2.541; 1.267-5.095), tumor differentiation (2.656; 1.374-5.136), and tumor location (others vs. total) (3.595; 2.006-6.443). There were no differences in the overall survival between chemotherapy. However, 5'-DFUR produced a better survival time of patients with serosal invasion than 5-FU, that might be attributed to the prevention of peritoneal recurrence in this subset.