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Articles by T Mizutani
Total Records ( 3 ) for T Mizutani
  N Yamamichi , R Shimomura , K. i Inada , K Sakurai , T Haraguchi , Y Ozaki , S Fujita , T Mizutani , C Furukawa , M Fujishiro , M Ichinose , K Shiogama , Y Tsutsumi , M Omata and H. Iba
 

Purpose: To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH).

Experimental Design: Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21–expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps.

Results: In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation.

Conclusion: Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

  T Yazawa , Y Inaoka , R Okada , T Mizutani , Y Yamazaki , Y Usami , M Kuribayashi , M Orisaka , A Umezawa and K. Miyamoto
 

Previously, we demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) differentiate into steroidogenic cells such as Leydig and adrenocortical cells by the introduction of steroidogenic factor-1 (SF-1) and treatment with cAMP. In this study, we employed the same approach to differentiate umbilical cord blood (UCB)-derived MSCs. Despite UCB-MSCs differentiating into steroidogenic cells, they exhibited characteristics of granulosa-luteal-like cells. We found that peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) was expressed and further induced by cAMP stimulation in UCB-MSCs. Consistent with these results, tissue-specific expression of Pgc-1 was observed in rat ovarian granulosa cells. PGC-1 binds to the NR5A family [SF-1 and liver receptor homolog-1 (LRH-1)] of proteins and markedly enhances their transcriptional activities. Reporter assays revealed that PGC-1 activated the promoter activities of SF-1 and LRH-1 target genes. Infection of KGN cells (a human cell line derived from granulosa cells) with adenoviruses expressing PGC-1 resulted in the induction of steroidogenesis-related genes and stimulation of progesterone production. PGC-1 also induced SF-1 and LRH-1, with the latter induced to a greater extent. Knockdown of Pgc-1 in cultured rat granulosa cells resulted in attenuation of gene expression as well as progesterone production. Transactivation of the NR5A family by PGC-1 was repressed by Dax-1. PGC-1 binds to the activation function 2 domain of NR5A proteins via its consensus LXXLL motif. These results indicate that PGC-1 is involved in progesterone production in ovarian granulosa cells by potentiating transcriptional activities of the NR5A family proteins.

  H Tsujii , Y Eguchi , A Chenchik , T Mizutani , K Yamada and Y. Tsujimoto
 

We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding ~200,000 shRNA sequences that targeted 47,400 human transcripts were inserted into a lentivirus vector pFIV-H1-puro, and a pool of pseudovirus particles with a complexity of ~200,000 were used to infect target cells. From the cells surviving apoptogenic Fas stimulation, four candidate shRNA sequences were obtained that provided resistance to Fas-induced cell death, including two shRNAs for caspase-8, an shRNA for Bid, and an shRNA for Fas. The reconstructed shRNAs with these sequences were shown to reduce expression of the respective gene products and increase survival after Fas stimulation. When similar selection was performed for tunicamycin-induced apoptosis, no shRNA strongly inhibiting tunicamycin-induced cell death was isolated, although a few reconstructed shRNAs led to a slight increase of survival. Thus, this genome-wide shRNA library proved useful for selection of genes that are involved in cell death, but some limitation was also revealed.

 
 
 
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