Atomically engineered metal-insulator transition at the TiO2/LaAlO3 heterointerface.

We demonstrate that the atomic boundary conditions of simple binary oxides can be used to impart dramatic changes of state. By changing the substrate surface termination of LaAlO3 (001) from AlO2 to LaO, the room-temperature sheet conductance of anatase TiO2 films are increased by over 3 orders of magnitude, transforming the intrinsic insulating state to a high mobility metallic state, while maintaining excellent optical transparency.

Novel ultra-cryo milling and co-grinding technique in liquid nitrogen to produce dissolution-enhanced nanoparticles for poorly water-soluble drugs.

A novel ultra-cryo milling micronization technique for pharmaceutical powders using liquid nitrogen (LN2 milling) was used to grind phenytoin, a poorly water-soluble drug, to improve its dissolution rate. LN2 milling produced particles that were much finer and more uniform in size and shape than particles produced by jet milling. However, the dissolution rate of LN2-milled phenytoin was the same as that of unground phenytoin due to agglomeration of the submicron particles. To overcome this, phenytoin was co-ground with polyvinylpyrrolidone (PVP). The dissolution rate of co-ground phenytoin was much higher than that of original phenytoin, single-ground phenytoin, a physical mixture of phenytoin and PVP, or jet-milled phenytoin. X-Ray diffraction showed that the crystalline state of mixtures co-ground by LN2 milling remained unchanged. The equivalent improvement in dissolution, whether phenytoin was co-ground or separately ground and then mixed with PVP, suggested that even when co-ground, the grinding of PVP and phenytoin occurs essentially independently. Mixing original PVP with ground phenytoin provided a slight improvement in dissolution, indicating that the particle size of PVP is important for improving dissolution. When mixed with ground phenytoin, PVP ground by LN2 milling aided the wettability and dispersion of phenytoin, enhancing utilization of the large surface area of ground phenytoin. Co-grinding phenytoin with other excipients such as Eudragit L100, hypromellose, hypromellose acetate-succinate, microcrystalline cellulose, hydroxypropylcellulose and carboxymethyl cellulose also improved the dissolution profile, indicating an ultra-cryo milling and co-grinding technique in liquid nitrogen has a broad applicability of the dissolution enhancement of phenytoin.

Development of a novel ultra cryo-milling technique for a poorly water-soluble drug using dry ice beads and liquid nitrogen.

A novel ultra cryo-milling micronization technique has been established using dry ice beads and liquid nitrogen (LN2). Drug particles were co-suspended with dry ice beads in LN2 and ground by stirring. Dry ice beads were prepared by storing dry ice pellets in LN2. A poorly water-soluble drug, phenytoin, was micronized more efficiently using either dry ice beads or zirconia beads compared to jet milling. Dry ice beads retained their granular shape without pulverizing and sublimating in LN2 as the milling operation progressed. Longer milling times produced smaller-sized phenytoin particles. The agitation speed for milling was optimized. Analysis of the glass transition temperature revealed that phenytoin particles co-ground with polyvinylpyrrolidone (PVP) by dry ice milling were crystalline, whereas a planetary ball-milled mixtures process with zirconia beads contained the amorphous form. The dissolution rate of phenytoin milled with PVP using dry ice beads or zirconia beads was significantly improved compared to jet-milled phenytoin or the physical mixture. Dry ice beads together with LN2 were spontaneously sublimated at ambient condition after milling. Thus, the yield was significantly improved by dry ice beads compared to zirconia beads since the loss arisen from adhering to the surface of dry ice beads could be completely avoided, resulting in about 85-90% of recovery. In addition, compounds milled using dry ice beads are free from abraded contaminating material originating from the beads and internal vessel wall.

One-step preparation of pharmaceutical nanocrystals using ultra cryo-milling technique in liquid nitrogen.

A novel micronization technique for pharmaceutical powders has been established using liquid nitrogen (LN2). Different from the conventional dry milling while cooling the milling pot by LN2, the materials were directly suspended in LN2 together with hard small spherical balls, called beads, and broken down by agitating intensively. The present beads milling in LN2 was named "ultra cryo-milling" in this paper. The operational conditions including the size/amount of beads made of zirconia and agitation speed were optimized to obtain the finer particles. It was found that the original crystals were effectively broken down into submicron particles, and the ultra cryo-milled particles were much finer and more uniform in size and shape than the conventional jet-milled particles. Dried powder was recovered continuously after milling process because LN2 was spontaneously evaporated at ambient temperature/pressure. Further, it was shown that this technique is applicable to the drugs with wide range of physicochemical features including heat-sensitive and water-soluble drugs. However, the resultant fine particles intrinsically tended to form the agglomerated masses. The crystalline analysis indicated that the both crystal form and crystallinity of the original bulk drugs completely remained after ultra cryo-milling process. The results demonstrated that the ultra cryo-milling would be a fundamental technique to produce the pharmaceutical nanocrystals by one step.

Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland.

Here, we investigated the expression of the claudin family of tight junction transmembrane proteins in the developing mouse submandibular gland. Data obtained by reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence microscopy showed the expression and localization of claudin-3 to -8, -10, and -11 at epithelial tight junctions. Examination of the glands taken from embryonic day (E) 14, E16, and newborn mice revealed differential expression patterns of these claudins in the developing epithelium. Claudin-3, -5, and -7 were expressed in all of the luminal epithelial cells of the ducts at all of the developmental stages examined and in those of terminal tubules at E16 and later. Claudin-4 was expressed mainly in the ducts at all the developmental stages. The expression of claudin-6 and -8 was also restricted to the ducts at E14 and E16; but after birth, the former was undetectable, whereas the latter was expressed in both the ducts and terminal tubules. Claudin-10 and -11 were detectable mainly in the terminal tubules at E16 and later. In addition to being found in the epithelium, claudin-5 was also expressed in certain mesenchymal cells, probably endothelial cells. These results will provide a valuable resource for further investigation of tubulogenesis and physiological regulation of claudin-based tight junctions.

Tissue interaction mediated by neuregulin-1 and ErbB receptors regulates epithelial morphogenesis of mouse embryonic submandibular gland.

Dimerization and activation of ErbB receptors by their ligands play crucial roles in organogenesis. Epithelial morphogenesis of embryonic mouse submandibular gland (SMG) has been shown to depend on intraepithelial signaling mediated by the epidermal growth factor (EGF) family of molecules and the EGF receptor (ErbB1). Here, we report on the neuregulin (NRG) -1 protein and its receptors ErbB2 and ErbB3 in the developing SMG. The expression of these molecules was demonstrated by reverse transcriptase-polymerase chain reaction and Western blot analysis. Immunofluorescence microscopy showed that the two ErbB receptors as well as ErbB1 were expressed mainly in the epithelium, whereas NRG-1 was exclusively found in the mesenchyme. Epithelial morphogenesis was retarded by anti-NRG-1 neutralizing antibody and promoted by recombinant NRG-1 protein. We suggest that, in the developing SMG, both mesenchyme-derived NRG molecules and epithelium-derived EGF molecules regulate ErbB signaling in the epithelium to participate in tissue morphogenesis.

Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin.

Studies with gene knockout mice have shown that Sonic hedgehog (Shh) is required for early development of hair follicles, but the role of this gene in the late stages of follicle development is not clear. By using an organ culture system of embryonic mouse skin, the role of Shh signaling in the early and late stages of follicle development was investigated. In the early stage of follicle development, the downward growth of the follicular epithelium was suppressed by cyclopamine, an inhibitor of Shh signaling, and accelerated by recombinant Shh. In addition, cyclopamine impaired dermal papilla formation, accompanied by the rearrangement of papilla cells, but not the elongation of the follicular epithelium at the later stage. These results suggest that Shh signaling is required for the proliferation of epithelial cells in the early development of hair follicles and for the morphogenetic movement of mesenchymal cells at the later stage of follicle development.

Establishment of cadherin-based intercellular junctions in the dermal papilla of the developing hair follicle.

During hair follicle development, mesenchymal cells aggregate to form the dermal papilla with hair-inducing activity. However, the cellular mechanisms underlying the aggregative behavior of dermal papilla cells are less known. The present study demonstrates that cadherin-based intercellular junctions interconnect dermal papilla cells in developing hair follicles of mice. It is shown that as mesenchymal cells aggregate to be surrounded by epithelium in developing hair follicles, cadherin-11 comes to exhibit the dotted patterns of distribution. The appearance of the dot-like distribution of the molecule is concomitant with the formation of intercellular junctions in the mesenchymal aggregate, which make a tightly packed population of cells with little extracellular space. At later stages of the development, although extracellular space reappears in the dermal papilla, the cells remain interconnected by well-developed intercellular junctions, where cadherin-11 as well as beta-catenin is localized. Taking into consideration the normal hair development in cadherin-11 mutant mice, it might be that multiple cadherins are responsible for the establishment of intercellular junctions in the dermal papilla and serve to maintain the aggregative behavior of the cells.

Branching Morphogenesis of Mouse Embryonic Submandibular Epithelia Cultured under Three Different Conditions: (mouse submandibular gland/epithelial branching/morphogenesis/collagenase/heparitinase/heparin/Matrigel).

To investigate how the mesenchyme interacts with the epithelium, we employed three different culture systems: System A, in which intact submandibular gland rudiments at the mid 13-day stage were cultured on Millipore filters; System B, in which the 13-day epithelium and mesenchyme were separated once with dispase, recombined again, and cultured on the filter; System C, in which the separated 13-day epithelium was clotted with Matrigel and cultured with the mesenchyme across the filter or in the presence of EGF instead of the mesenchyme. In Systems A and B, 13-day epithelia expanded and produced similar lobules with narrow clefts and stalk. When the 13-day epithelium was cultured in System C under the influence of the mesenchyme, it formed rather oval lobules with stalk that were superficially similar to those in System A, but narrow clefts, as seen in the intact early 13-day gland, were rarely found in System C. Furthermore, no long stalk formation was observed when EGF was introduced in place of the mesenchyme. A bacterial collagenase from Clostridium histolyticum gave a considerable inhibition of branching of the 13-day epithelium in Systems A and B, but no significant inhibition was observed in System C when the mesenchyme or EGF was employed as the source of diffusible factor(s). In contrast, although the 13-day epithelium was significantly resistant to the action of heparitinase I from Flavobacterium heparinum in Systems A and B, the enzyme almost completely inhibited the expansion and branching of the epithelium in System C. Judging from these observations, we conclude that the mechanisms of lobular formation in Systems A and B are not the same as those in System C, where the epithelium is clotted with basement membrane matrix components during tissue culture.

Removal of Heparan Sulfate Chains Halted Epithelial Branching Morphogenesis of the Developing Mouse Submandibular Gland in vitro: (mouse submandibular gland/branching morphogenesis/heparan sulfate proteoglycan/heparitinase).

The physiological function of heparan sulfate chains in the mouse embryonic submandibular gland was studied by the use of heparitinases purified from Flavobacteriu heparinum. Heparitinase I, which catalyzes the cleavage of specific glycosaminidic linkages adjacent to non-or monosulfated disaccharides of heparan sulfate chains, in the culture medium of the mid and late 12-day gland inhibited the branch-initiation and changed their round epithelial shape to elongated one, together with a concommitant reduction in lobular growth. [3 H]Thymidine incorporation experiments indicated that heparitinase I treatment blocked 24% of the DNA synthesis compared with controls. Analysis of 35 S-inorganic sulfate labeled glycosaminoglycans extracted from cultured rudiments revealed that the glands with heparitinase I contained no heparan sulfate, while in the glands without the enzyme more than 20% of total glycosaminoglycans was heparan sulfate. The heparitinase effect on morphogenesis was mimicked by the addition of heparan sulfate (1 mg ml-1 ) or heparin (75 µg ml-1 ), but not by chondroitin sulfate (1 mg ml-1 ) in the culture medium. Transmission electron microscopic study indicated that at the epithelial-mesenchymal interface close contacts between the fibroblast and epithelial cells were much fewer in heparitinase-treated glands than in controls. Immunohistochemical analysis demonstrated that the core protein of basement membrane heparan sulfate proteoglycan and type IV collagen accumulated abnormally inside the epithelial lobules of glands cultured with heparitinase I. These results strongly suggested that glycosaminoglycan chains of heparan sulfate or heparin is involved in the epithelial morphogenesis of the mouse embryonic submandibular gland.