Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.     

Attempt to simultaneously generate three chiral centers in 4-hydroxyisoleucine with microbial carbonyl reductases

A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5 mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.     

Rapid preparation of PCR-amplifiable fungal DNA by benzyl bromide extraction

For isolation of fungal DNA for PCR amplification, we compared three DNA isolation methods: enzymatic cleavage and the use of benzyl chloride or benzyl bromide. Since benzyl bromide is more reactive, its use enabled us to readily isolate the total nucleic acids as a DNA template source from various fungi, including dematiaceous hyphomycetes, for RAPD analysis.     

Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metallothionein gene (CUP1)

Cauliflower (Brassica oleracea var. botrytis) tolerates treatment with 25 µM CdCl2 for eight days, but is killed by that with a 50 µM concentration. However, even 15 µM CdCl2 is toxic in the presence of 1 mM L-buthionine sulfoximine (BSO), suggesting the presence of a Cd-inducible phytochelatin and its involvement in Cd-tolerance in cauliflower. To develop heavy metal-tolerant transgenic plants, we ligated the structural gene of yeast metallothionein gene (CUP1) downstream of CaMV35S promoter and introduced the fused gene into cauliflower. A Cd-tolerant transgenic cauliflower was selected, which grew well in the presence of 400 µM or less Cd, whereas the non-transformed cauliflower tolerated only up to 25 µM Cd. The transgenic cauliflower accumulated more Cd, especially in the upper leaves, than the non-transformed plant.In conclusion, by transfer of the yeast metallothionein gene into cauliflower increased Cd-tolerance and Cd-accumulating ability can be conferred to the plant.     

ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer

Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.     

Thyroid hormone response to thyrotrophin releasing hormone (TRH) in the sex-linked dwarf chicken

The effect of an injection of thyrotrophin releasing hormone (TRH) on plasma levels of thyroid hormones was studied in dwarf and normal Rhode Island Red chickens with similar genotypes other than for the sex-linked dwarf gene dw. The sex-linked dwarf chickens had different plasma iodothyronine levels from control normal chickens: high thyroxine (T4), low triiodothyronine (T3) and similar reverse T3 (rT3) levels. The injection of TRH (10 micrograms/kg) in 5-day- and 5-week-old normal chickens increased the plasma T4 within 30 min without a significant increase in T3, whereas the injection of TRH in 11-and 26-week-old normal chickens increased plasma T3 60 min later. In dwarfs the response of T4 to TRH was the same as that in normals but no increased T3 response was observed. The plasma level of rT3 was not influenced by the TRH injection in either strain. These results suggest that although in the sex-linked dwarfs thyroidal response to exogenous TRH is similar to that of normals, the dwarf gene dw inhibits the conversion of T4 to T3 in peripheral tissues without any inhibitory effect on rT3 production.