SummaryAs in many other Gram-negative phytopathogenic bacteria, the Hrp type III secretion system is essential for the pathogenicity of Ralstonia solanacearum on host plants. The expression of most of the type III effector genes previously isolated from R. solanacearum is co-regulated with those of hrp genes by an AraC-type transcriptional activator, HrpB. In order to isolate type III-related pathogenicity genes, we screened hrpB -regulated genes in R. solanacearum . Using a transposon-based system, we isolated 30 novel hpx ( hrpB -dependent expression) genes outside the hrp gene cluster. Most of the hpx genes contain a PIP (plant-inducible promoter) box-like motif in their putative promoter regions. Seven hpx genes encoded homologues of known type III effectors and type III-related proteins found in other animal and plant pathogens. Four encoded known enzymes, namely, glyoxalase I, Nudix hydrolase, spermidine synthase and transposase. Interestingly, six hpx genes encoded two types of leucine-rich repeat (LRR) protein. Products of the remaining genes did not show any significant homology to known proteins. We also identified two novel hrpB -regulated genes, hpaZ and hpaB , downstream of hrpY in the hrp cluster. The hpaB gene of R. solanacearum , but not hpaZ , was required for both the pathogenicity and ability to induce hypersensitive reaction on plants. We show that a hpaB null mutant still produces Hrp pili on the cell surface although it shows a typical Hrp-defective phenotype on plants.

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The combination therapy with sitagliptin and low dosage sulphonylureas was safe and effective for glycaemic control. Glucagon loading test indicated that 1 year administration of sitagliptin and sulphonylureas preserved insulin secretion capacity.

Currently 5-fluorouracil (5-FU) plays a central role in the chemotherapeutic regimens for colorectal cancers and thus it is important to understand the mechanisms that determine 5-FU sensitivity. The expression profiles of human colon cancer cell line DLD-1, its 5-FUresistant subclone DLD-1/FU and a futher 21 types of colon cancer cell lines were compared to identify the novel genes defining the sensitivity to 5-FU and to estimate which population of genes is responsible for 5-FU sensitivity. In the hierarchical clustering, DLD-1 and DLD-1/FU were most closely clustered despite over 100 times difference in their 50% inhibitory concentration of 5-FU. In DLD-1/FU, the population of genes differentially expressed compared to DLD-1 was limited to 3.3%, although it ranged from 4.8% to 24.0% in the other 21 cell lines, thus indicating that the difference of 5-FU sensitivity was defined by a limited number of genes. Next, the role of the cellular inhibitor of apoptosis 2 (cIAP2) gene, which was up-regulated in DLD-1/FU, was investigated for 5-FU resistance using RNA interference. The downregulation of cIAP2 efficiently enhanced 5-FU sensitivity, the activation of caspase 3/7 and apoptosis under exposure to 5-FU. The immunohistochemistry of cIAP2 in cancer and corresponding normal tissues from colorectal cancer patients in stage III revealed that cIAP2 was more frequently expressed in cancer tissues than in normal tissues, and cIAP2-positive patients had a trend toward early recurrence after fluorouracilbased chemotherapy. Although the association between drug sensitivity and the IAP family in colorectal cancer has not yet been discussed, cIAP2 may therefore play an important role as a target therapy in colorectal cancer. (Cancer Sci 2009; 100: 903-913) 5-fluorouracil (5-FU) is an anticancer drug that has been mainly used in the treatment of colorectal cancers. Recently, 5-FU has been combined with oxaliplatin or irinotecan as the first-line treatment for advanced colorectal cancers and these have significantly improved the response rates to 40-50% and prolonged overall survival.(1,2) Furthermore, novel biological agents including monoclonal antibodies such as cetuximab, which is an antibody against epidermal growth factor receptor (EGFR), and bevacizumab, which is an antibody against vascular endothelial growth factor, have been shown to provide additional clinical benefit for patients with metastatic colorectal cancers.(3-5) However, there are still a large number of patients who do not benefit from the present treatments because of anticancer drug resistance. Elucidating the mechanisms by which 5-FU resistance arises in colorectal cancer therefore remains an important issue for either overcoming or predicting such resistance.5-FU is an analog of uracil and is rapidly incorporated into the cells using the same transport system as uracil.(6) Subsequently, 5-FU is converted into active metabolites which disrupt the action of thymidylate synthetase (TS) and RNA synthesis. TS and 5-FUmetabolizing enzymes such as dihydropy...

In colorectal cancer (CRC) patients, metastasis to the regional lymph node (LN) is an important first step in the dissemination of cancers. To identify the genes possibly involved in LN metastasis of CRC, we analyzed LN metastases in an orthotopic implantation mouse model with 22 CRC cell lines using Matrigel, an extracellular matrix protein derived from mice sarcoma, and combined the data with gene expression profiles of cDNA microarray of those cell lines. With this implantation analysis, the incidence of LN metastasis was 60% in 228 orthotopically implanted mice and varied from 100% to 0% among the cell lines. KM12c and Clone A showed LN metastasis in all orthotopically implanted mice, but DLD-1, HCT-8, and SW948 did not show LN metastases at all. In contrast, the incidence of liver and lung metastasis in 22 CRC cell lines was 13% and 1%, respectively. Combining those data with cDNA microarray in vitro, we isolated 636 genes that were differentially expressed depending on the incidence of LN metastasis. T he incidence of CRC has been increasing in Japan. Approximately 94 000 CRC patients were diagnosed in 1999, (1) and approximately 40 000 patients died of CRC in 2004.(2) Metastasis to regional LN is an important first step in the dissemination of CRC, and LN metastasis and liver metastasis affect the prognoses of CRC patients. The recurrence rate of CRC is directly associated with the extent of LN metastasis. Therefore, elucidating the mechanism of LN metastasis of CRC is considered to be crucial for developing an eventual solution to this problem. It is important to establish a spontaneous LN metastasis model for analyzing the mechanisms of LN metastasis in CRC. An orthotopic animal tumor model for CRC is a complementary tool for the study of tumors in vivo.Matrigel basement membrane matrix (Becton Dickinson, Franklin Lakes, NJ, USA) is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, entactin, and nidogen.(4-9) At room temperature, Matrigel polymerizes and forms a "basement membrane" gel. The usefulness of Matrigel has been reported as an orthotopic implantation animal model of various kinds of cancers.(5,10-13) Tsutsumi et al. advocated an experimental animal model of LN metastasis using an orthotopic implantation method by intrarectal injection of human cancer cell-Matrigel solution in nude mice, but liver metastasis did not occur. (14) Takahashi et al. proposed a mouse model of rectal cancer established by intrarectal instillation of colon cancer cells following short-term induction of colitis using an irritant agent, but the metastasis to other organs such as liver and lung was not induced at significant levels.(15) Although some of the experimental models for metastasis of CRC have been developed, there is no report that addresses the relation between the metastasis of CRC and the change of gene expressions involved in the metastasis.Molecular studies have so far been ...