A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing

We describe a new method with general applicability for monitoring any protein-protein interaction in vivo. The principle is based on a protein splicing system, which involves a self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to formation of a new protein in which the exteins are linked directly by a peptide bond. As the exteins, split N- and C-terminal halves of enhanced green fluorescent protein (EGFP) were used. When a single peptide consisting of an intein derived from Saccharomyces cerevisiae intervening the split EGFP was expressed in Escherichia coli, the two external regions of EGFP were ligated, thereby forming the EGFP corresponding fluorophore. Genetic alteration of the intein, which involved large deletion of the central region encoding 104 amino acids, was performed. In the expression of the residual N- and C-terminal intein fragments each fused to the split EGFP exteins, the splicing in trans did not proceed. However, upon coexpression of calmodulin and its target peptide M13, each connected to the N- and C-terminal inteins, fluorescence of EGFP was observed. These results demonstrate that interaction of calmodulin and M13 triggers the refolding of intein, which induces the protein splicing, thereby folding the ligated extein correctly for yielding the EGFP fluorophore. This method opens a new way not only to screen protein-protein interactions but also to visualize the interaction in vivo in transgenic animals.     

Development of Single‐Belt Strip Casting Simulator

Near‐net‐shape casting is one of the key technologies to improve process efficiency of steel production. Single‐belt strip casting is recognized as a promising technology for thin strip production because of the advantages like well‐controlled heat transfer rate, flexibility in production rate, compactness of equipment, and so on. In this study a newly designed simulator of the single‐belt strip casting process was developed. The simulator solidifies molten metal on the running solid metal bar with a groove for molten metal deposition pushed by a pneumatic cylinder. Capability of this simulator design was discussed by one‐dimensional numerical heat transfer analysis. It showed that a steel casting bar thicker than 40 mm was capable of casting test of 10 mm thick steel strip even if interfacial thermal resistance existed. Finally, the simulator was applied to the casting test of aluminum strip, and successfully estimated the variation of the interfacial heat flux from the solidifying strip to the casting bar.     

The Effects of Continuous and Withdrawal Voluntary Wheel Running Exercise on the Expression of Senescence-Related Genes in the Visceral Adipose Tissue of Young Mice

Obesity has become a global medical problem. The upregulation of senescence-related markers in adipose tissue may cause impairment of adipose tissue and disorders of systemic metabolism. Weight control through diet has been found to ameliorate senescence in the adipose tissue. Exercise is also important in maintaining a healthy lifestyle, however, very few researchers have examined the relationship between senescence-related markers in adipose tissue. Dietary restriction is also reported to have a legacy effect, wherein the effects are maintained for some periods after the termination of the intervention. However, very few researchers have examined the relationship between exercise and senescence-related markers in adipose tissue. Besides, there is no study on the long-term effects of exercise. Hence, we investigated whether the exercise could change the expression of senescence-related genes in the visceral adipose tissue of young mice and whether there was a legacy effect of exercise for 10 weeks after the termination of exercise. Four-week-old male ICR mice were assigned to one of the three groups: 20 weeks of sedentary condition, 20 weeks of voluntary wheel running exercise, or 10 weeks of exercise followed by 10 weeks of sedentary condition. The mice showed decreased expression in genes related to senescence and senescence-associated secretory phenotype, such as p53, p16, and IL-6, in the visceral adipose tissue in response to exercise. These effects were maintained for 10 weeks after the mice stopped exercising. Our study is the first report that exercise reduces the expression of senescence-related genes in the visceral adipose tissue of young mice, and that exercise causes the legacy effect.     

Preparation of gold nano-cones as surface-enhanced Raman scattering sensors for molecule detection

Surface-enhanced Raman spectroscopy (SERS) is a powerful novel analytical tool which integrates high levels of sensitivity for trace analysis of chemical and biomolecular species due to the massive enhancement of Raman signals by using nanometre-sized metal particles. However, SERS can be envisaged as an analytical tool only if substrates with strong, predictable and reproducible SERS enhancement can be produced. Here we have developed one simple Ar+ ions sputtering technology to prepare gold nano-cones array on silicon substrates as surface-enhanced Raman scattering (SERS)-active substrates. The tip of the gold cone-structures exhibited an extremely sharp curvature with an apex diameter of 20 nm and the interior apex angle of the nanocones was around 20 degrees. These samples were evaluated as potential SERS substrates using Rhodamine 6G molecules as molecule probe and exhibited SERS enhancement factor of greater than 10, originated from the localized electron field enhancement around the apex of cones and the surface plasmon coupling of periodic structures.     

Electrochemical Characterization of a Porous Pt Nanoparticle ＂Nanocube-Mosaic＂ Prepared by a Modified Polyol Method with HCI Addition

Porous and single crystalline platinum (Pt) nanoparticles (NPs) have been successfully synthesized by reduction of H₂PtCl₆·6H₂O and then investigated by optical spectroscopy and transmission electron microscopy. H₂PtCl₆·6H₂O was reduced using ethylene glycol in the presence of polyvinylpyrrolidone under highly acidic conditions (pH < 1) to form single crystalline Pt particles about 5 nm in size. These particles were then stacked via {100} facets, forming 50-nm length porous nanocubes with a mosaic structure. The porous Pt NPs exhibited excellent catalytic properties for methanol oxidation. In particular, the electrochemical surface area was ∼63 m²/g, five times higher than that for non-porous Pt NPs prepared using a conventional method. We suggest that the high catalytic activity of porous Pt NPs is due to a combination of the crystalline structure having exposed {100} facets and a porous morphology.