Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET

Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system; a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser.     

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development.     

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356–5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.     

Synthesis and seed germination stimulating activity of some imino analogs of strigolactones

Strigolactones are germination stimulants for seeds of the root parasitic weeds, Striga and Orobanche spp. The imino analog of GR24 showed moderate germination stimulating activity against the seeds of S. hermonthica. The seed germination stimulating activity of some phenyliminoacetates and phenyliminoacetonitriles was also examined. The degree of activity of the phenyliminoacetate was less than that of the phenylacrylates. On the other hand, the degree of activity of the phenyliminoacetonitrile was comparable to that of the phenylacrylonitriles. Among the tested compounds, the 3-pyridyliminoacetonitrile showed higher activity against the seeds of O. crenata than GR24. These findings demonstrate that it is not always essential to have the Michael acceptor of the C-D ring junction moiety which has been proposed to react with nucleophilic species presented at the target site to enhance the activity.     

2-Hydroxy-2'-deoxyadenosine 5'-triphosphate enhances A.T --> C.G mutations caused by 8-hydroxy-2'-deoxyguanosine 5'-triphosphate by suppressing its degradation upon replication in a HeLa extract

The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.     

Electrostatic guarding of bookshelves for mould-free preservation of valuable library books

Old books are highly susceptible to mould infection, and an effective method for avoiding moulding is needed to safely preserve valuable books in library stack rooms. Guarding a bookshelf with an electric field screen is a physical method that prevents airborne spores from entering the space used for book preservation. In this study, insulated conductor wires (ICWs) were used as electrodes to form electric fields. The ICWs were arrayed in parallel and linked to each other and to a direct current voltage generator. The electric field screen consisted of two layers of ICWs, which were negatively and positively charged with equal voltages to make dipoles, ICW(?) and ICW(+). Both ICWs generated an attractive force that captured airborne spores of Penicillium digitatum that were blown inside the screen. The attractive force was directly proportional to the applied voltage. At ≧0.9 kV, the screen exerted sufficient force to capture all airflow-carried spores, but a few spores that were once captured were repulsed out of the electric field when subsequent spores were attracted to positions proximal to them. This phenomenon was explained by creeping discharge between spores located close to each other on the ICW surface. This spore-repulsion problem was resolved by adding an additional ICW layer to the electric field screen, namely an electric field screen with an ICW(?) layer on both sides of an ICW(+) layer. The present study demonstrated that the three-layered electric field screen remained mould-free inside a screen-guarded bookshelf, irrespective of continuous spore exposure.     

Fabrication of functionalized double-lamellar multifunctional envelope-type nanodevices using a microfluidic chip with a chaotic mixer array

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>10(5)) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency.     

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.     

Effects of target sequence and sense versus anti-sense strands on gene correction with single-stranded DNA fragments

The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.