The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were . They also showed that histones bound to the left-handedly curved DNA, and that the TATA box was exposed out of the nucleosome core when the curved DNA was located at appropriate positions [9]. This finding prompted us to examine the effects of this left-handedly curved sequence in vivo. In this study, we investigated the in vivo effects of this sequence on plasmids when delivered in a naked form into mouse liver. We found that the position of the left-handedly curved sequence affected the transgene expression by one order of magnitude, without altering the amount of the exogenous DNAs, suggesting the different accessibility of transcriptional factors to the plasmids in vivo. Similar results were observed when plasmids complexed with cationic lipids were delivered into mouse liver. These results indicate that the left-handedly curved sequence on plasmids also determined 5 transgene expression in vivo, and that controlled interactions between the plasmid and the histones are important for the intranuclear disposition. Materials and Methods MaterialsOligodeoxyribonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) in purified forms. The pLHC4/TLN-6, pLHC4/TLN-16, pLHC4/TLN+47, and pST0/TLN-7 plasmids, containing the thymidine kinase (tk) promoter and the luciferase gene (Fig. 1) [9], were amplified in Escherichia coli strain DH5α and purified with a Qiagen (Hilden, Germany) EndoFree Plasmid Mega kit. Hydrodynamics-based injectionPlasmid DNA (20 µg in 2 ml of saline) was injected into the tail vein of male six week-old Balb/c mice within 5 sec [10,11]. The livers were harvested from the injected mice at various time points, and the luciferase activity and the amount of the exogenous DNA were measured, as described below. Luciferase activity 6Livers were minced with scissors and homogenized completely in Lysis Buffer (100 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.8). After centrifugation at 13,000 g for 10 min at 4°C, the supernatant was examined for luciferase activity, using a Luciferase Assay Systems kit (Promega, Madison, Wisconsin, USA). Isolation of nuclear DNA and Quantitative PCRLivers were homogenized in phosphate-buffered saline (PBS).After centrifugation at 2,500 g for 5 min at 4°C, the pellet was washed three times with PBS. The pellet was resuspended in DNA Lysis Buffer (100 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.5% (w/v) IGEPAL-CA630, pH 7.4) [12]. After centrifugation at 1,400 g for 5 min at 4°C, the pellet was washed three times with DNA Lysis Buffer. The intranuclear DNA was extracted with the SepaGene reagent (Sanko Jun-yaku, Tokyo, Japan).Quantitative PCR (Q-PCR) was performed using an ABI 7500 real time PCR system, and SYBR-Green...

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a lefthandedly curved sequence (20--40 repeated AKT tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo.

No abstract

A new herbicide, epyrifenacil (S-3100), inhibits protoporphyrinogen oxidase (PPO) in plants. Repeated administration of epyrifenacil in laboratory animals led to some toxicological changes related to PPO inhibition, e.g., hepatotoxicity caused by porphyrin accumulation and anemia caused by the inhibition of heme biosynthesis. In vitro studies revealed that an ester-cleaved metabolite, S-3100-CA, is predominant in mammals, exhibits PPO-inhibitory activity, and thus is the cause of epyrifenacil-induced toxicity. To assess the human risk, the effects of species differences on the dynamics (PPO inhibition) and kinetics (liver uptake) of epyrifenacil were evaluated separately. The results of in vitro assays revealed an approximately tenfold weaker inhibition of PPO by S-3100-CA in humans than in rodents and six-to thirteen-fold less hepatic uptake of S-3100-CA in humans than in mice. Finally, it was suggested that humans are less sensitive to the toxicity of epyrifenacil than are rodents, although further mechanistic research is highly anticipated.

-Apoptosis controls erythroid homeostasis by balancing survival and death of erythroid cells. The mitochondrial pathway of apoptosis involves regulation of apoptotic events caused by the Bcl-2 family proteins, including the anti-apoptotic and pro-apoptotic members. However, little has been reported on the role of the anti-apoptotic Bcl-2 family members in rat late-stage erythroblasts that are no longer erythropoietin (EPO)-dependent. In the present study, to investigate this we analyzed changes in apoptosis-related factors that occurred in vitro. EPO stimulation resulted in reduced apoptotic cell death of the late-stage erythroblasts accompanied by decreased caspase-3 and caspase-9 activities, which is indicative of the induction of apoptosis through the mitochondrial pathway. Analysis of mRNA expression of the Bcl-2 family proteins demonstrated that EPO stimulation up-regulated the Bcl-xL mRNA, resulting in decreases in the mRNA ratios of Bak, Bax, and Bad to Bcl-xL. Also, the mRNA ratios of Bak and Noxa to Mcl-1 were decreased, mainly due to up-regulation of Mcl-1 mRNA. These results showed a close association between reduced apoptotic cell death and increased mRNA levels of Bcl-xL and Mcl-1 in the presence of EPO. Thus, the present study suggests that Bcl-xL may be an important anti-apoptotic factor of rat late-stage erythroblasts as has been reported in murine erythroblasts. Moreover, the results also indicate the possibility that Mcl-1 may act on the rat late-stage erythroblasts as an anti-apoptotic factor.