Although subcellular mRNA trafficking has been demonstrated as a mechanism to control protein distribution, it is generally believed that most protein localization occurs subsequent to translation. To address this point, we developed and employed a high-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis. Surprisingly, of the 3370 genes analyzed, 71% of those expressed encode subcellularly localized mRNAs. Dozens of new and striking localization patterns were observed, implying an equivalent variety of localization mechanisms. Tight correlations between mRNA distribution and subsequent protein localization and function, indicate major roles for mRNA localization in nucleating localized cellular machineries. A searchable web resource documenting mRNA expression and localization dynamics has been established and will serve as an invaluable tool for dissecting localization mechanisms and for predicting gene functions and interactions.

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Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities. As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knockdown to investigate the function of these proteins in vivo. Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a "twisted abdomen phenotype," in which the abdomen is twisted 30 -60°, similar to the dPOMT1 mutant. Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo. We expressed dPOMTs in Sf21 cells and measured POMT activity. dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1. Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself. Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA. The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization. These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.

BACKGROUND.Circulating tumor cells (CTCs) have been shown to aid in the therapeutic management of patients. But, only a few attempts have been made at the detection of urothelial cancer cells in the blood. The purpose of this study was to test the hypothesis that CTCs are detected in patients with urothelial cancers using newly developed CellSearch Assay.METHODS.Firstly, the bladder cancer cell lines were used to evaluate the reagents for immunocytochemical detection. After, mixed with peripheral blood mononuclear cells (PBMCs) of healthy volunteers, bladder cancer cells were stained with antibodies then multiparameter flow cytometric analysis was performed for the identification of bladder cancer cells in the PBMCs. Secondary, recovery of known numbers of spiked bladder cancer cells from whole blood was examined using CellSearch Assay. Finally, blood samples from nonmetastatic and metastatic urothelial cancer patients were investigated for CTC detection using CellSearch Assay.RESULTS.1: Flow cytometric analysis revealed that it is possible to identify bladder cancer cells in PBMCs. 2: Sensitivity examination for detection of urothelial cancer cells with CellSearch Assay: Single regression analysis of the spiked number of cells vs. the recovered number of cells yielded a good correlation in this experiment. 3: Urothelial cancer cells were detected in 8 of fourteen patients (57.1%) with distant metastasis. Despite, no patient with nonmetastatic urothelial cancers showed positive result for this assay.CONCLUSION.This is the first report of attempt to detect circulating urothelial cancer cells in the peripheral blood of the patients with metastatic and nonmetastatic urothelial cancers by CellSearch Assay. Cancer 2007. © 2007 American Cancer Society.