Recrystallization and zone melting of charged colloids by thermally induced crystallization.

We examined the application of recrystallization and zone-melting crystallization methods, which have been used widely to fabricate large, high-purity crystals of atomic and molecular systems, to charged colloidal crystals. Our samples were aqueous dispersions of colloidal silica (with particle diameters of d = 108 or 121 nm and particle volume fractions of ϕ = 0.035-0.05) containing the weak base pyridine. The samples crystallized upon heating because of increases in the particle charge numbers, and they melted reversibly on cooling. During the recrystallization experiments, the polycrystalline colloids were partially melted in a Peltier cooling device and then were crystallized by stopping the cooling and allowing the system to return to ambient temperature. The zone-melting crystallization was carried out by melting a narrow zone (millimeter-sized in width) of the polycrystalline colloid samples and then moving the sample slowly over a cooling device to recrystallize the molten region. Using both methods, we fabricated a few centimeter-sized crystals, starting from millimeter-sized original polycrystals when the crystallization rates were sufficiently slow (33 µm/s). Furthermore, the optical quality of the colloidal crystals, such as the half-band widths of the diffraction peaks, was significantly improved. These methods were also useful for refining. Small amounts of impurity particles (fluorescent polystyrene particles, d = 333 nm, ϕ = 5 × 10(-5)), added to the colloidal crystals, were excluded from the crystals when the crystallization rates were sufficiently slow (∼0.1 µm/s). We expect that the present findings will be useful for fabricating large, high-purity colloidal crystals.

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro.

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.

Gel immobilization of centimeter-sized and uniform colloidal crystals formed under temperature gradient.

We report the fabrication of large and high-quality charged colloidal crystals that are incorporated in a polymer hydrogel matrix. The colloidal crystals are prepared by the thermally induced unidirectional crystallization of colloidal silica in coexistence with pyridine, whose dissociation degree increases with temperature. Their crystal structures are immobilized in the polymer hydrogel matrix by photoinduced polymerizations. The crystals are large sized (maximum dimensions: 1 x 10 x approximately 30 mm), and their lattice planes are well oriented and parallel to the gel surface. Furthermore, they have excellent spatial uniformity in the Bragg wavelengths (<0.7% in standard deviation). The present gelled colloidal crystals, which are unique in that they have large sizes as well as good optical uniformity, will be useful as photonic materials.

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.

Germination and growth inhibitors from wheat (Triticum aestivum L.) husks.

On the basis of our findings that the germination of intact wheat seeds (with husks) belonging to dormancy varieties was restrained as compared with that of the dehusked seeds (grains), the germination inhibitors in the husks were explored. The water-soluble extracts from the husks were separated by the aid of inhibition assay experiments, resulting in the characterization of 2-phenylethyl alcohol 1, 4-vinylphenol 2 and its 2-methoxy derivative 3, and dihydroactinidiolide 4, all of which showed clear inhibition of germination at 500 ppm in aqueous solution. The related compounds 1-phenylethyl alcohol 5 and tetrahydroactinidiolide 6 were as active as 1 and 4, while no noticeable difference in activity was detected among both enantiomers and the DL-form of compounds 4-6. Clear synergistic relations were observed between 4 and 1 and also 4 and 3. Since the present inhibitors have been isolated from various kinds of seed plants, they may be responsible for the general germination inhibition in the seed plants.