In Vitro Analyses of Spinach-Derived Opioid Peptides,Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKey^TM assay, cADDis^® cAMP assay, and PathHunter^® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKey^TM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis^® cAMP and PathHunter^® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.     

Direct numerical simulation of wall turbulent flows with microbubbles

The marker‐density‐function (MDF) method has been developed to conduct direct numerical simulation (DNS) for bubbly flows. The method is applied to turbulent bubbly channel flows to elucidate the interaction between bubbles and wall turbulence. The simulation is designed to clarify the structure of the turbulent boundary layer containing microbubbles and the mechanism of frictional drag reduction. It is deduced from the numerical tests that the interaction between bubbles and wall turbulence depends on the Weber and Froude numbers. The reduction of the frictional resistance on the wall is attained and its mechanism is explained from the modulation of the three‐dimensional structure of the turbulent flow. Copyright © 2001 John Wiley & Sons, Ltd.     

Evaluation of single-base substitution rate in DNA by affinity capillary electrophoresis

Capillary electrophoretic separation of 60 mer single-stranded DNA (ssDNA) and a single-base-substituted ssDNA was demonstrated using a size- and composition-controlled poly(ethylene glycol)-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. Under appropriate conditions, PEG-b-ODN and ssDNA with a complementary sequence formed a reversible complex via hybridization during the electrophoresis, while the copolymer did not interact with the single-base-substituted ssDNA. The copolymer's PEG length determined the electrophoretic mobility of the ssDNA; upon formation of the complex, the electrically neutral PEG added hydrodynamic friction to ssDNA. Simultaneously using two types of PEG-b-ODN copolymers whose PEG segments were of different lengths, we achieved the complete separation of the 60 mer ssDNA, its single-base-substituted ssDNA, and impurities. This method was sensitive enough to quantify a slight amount (approximately 1%) of the single-base-substituted ssDNA. The present results suggest that our approach is applicable to quantitative detection of minor genotypes.     

Synthesis and characterization of transparent silica-based aerogels using methyltrimethoxysilane precursor

Silica-based aerogels with high transparency and high bending strength were prepared using methyltrimethoxysilane and non-ionic surfactant under supercritical drying condition of CO₂. Non-ionic surfactant, ethylene oxide/propylene oxide block copolymer, was appropriate to form the three dimensional-connected thinner fibers of silica skeletons, which was extruded by soaking the wet gel in hot water at 50 °C, resulting in the formation of porous materials having small pores with narrow size distribution. The transparency of aerogels increased with decreasing the pore size, reaching to higher than 65 and 88% at 400 and 600 nm wavelength, respectively, for 10 mm thickness of sample. The formation of fiber skeletons were discussed using small angle X-ray scattering experiments.     

Study of mixed-valence dinuclear iron(II, III) complexes [Fe2(bpmp)L2](BF4)2: Asymmetrically delocalized valence states

We report the temperature dependence of Mössbauer spectra of a mixed-valence dinuclear Fe(II) Fe(III) complex, [Fe₂(bpmp)(Ph(CH₂)₂COO?)₂](BF₄)₂ 1, where Hbpmp represents 2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol. Two quadrupole doublets observed at 78 K approach each other as the temperature is increased. The two high-energy components of a doublet due to Fe(II) and of that due to Fe(III) become broad in the temperature range from 78 to 230 K and then become narrow above 260 K. The temperature dependence of the spectra was interpreted by assuming of intramolecular electron exchanges between two energetically inequivalent vibronic states Fe_A(II)Fe_B(III) and Fe_A(III)Fe_B(II) where one of them is slightly more stable than the other.     

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

The C‐terminal octapeptide of cholecystokinin (CCK8) includes some easily oxidizable amino acids. The oxidation of CCK8 by reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^?) was investigated using reversed‐phase high performance liquid chromatography (RP‐HPLC) and subsequent electrospray ionization mass spectrometry. The mechanism of oxidation of CCK8 in the H₂O₂ system differed from that of CCK8 in the Fenton system, in which OH^? are produced. In the H₂O₂ system, ²⁸Met and ³¹Met were oxidized to methionine sulfoxide, and no further oxidation or degradation/hydrolysis occurred. On the other hand, in the Fenton system, ²⁸Met and ³¹Met residues were oxidized to methionine sulfone via the formation of methionine sulfoxide. In addition, the oxidized product was observed at the Trp residue but not at the Tyr residue, and small peptide fragments from CCK8 were observed in the Fenton system. From these results, it was concluded that ²⁸Met and ³¹Met residues of CCK8 are susceptible to oxidation by ROS. Copyright © 2009 John Wiley & Sons, Ltd.     

Double Stage Flat Passband Acousto-Optic Wavelength Filter

We developed a flat passband acoustooptic filter. For the passband flattening, cascaded bandpass and notch filters with different mode converter lengths and conversion strengths were used. We attained a flat passband with flatness of less than 1 dB in the 2 nm range using the device with a second- to first-stage mode converter length ratio of 1.2.     

Results of 0.5% cold-leg small-break LOCA experiments at ROSA-IV/LSTF: Effect of break orientation

Three 0.5% cold-leg small-break loss-of-coolant accident (SBLOCA) experiment were conducted at the ROSA-IV Large Scale Test Facility (LSTF) to investigate the effects of break orientation on system thermal-hydraulic responses. In these three experiments, the break hole was located at the side, bottom, and top of the horizontal cold leg, respectively. Although the key phenomena observed in the three experiments were basically the same, the break flow rate was affected by the break orientation when phase stratification occured in the cold leg; the break flow rate was largest for the side break and smallest for the top break. The RELAP5/MOD2 code failed to predict the difference in the break flow rate observed in the experiments. Modification to the break flow calculation models, for both subcooled and two-phase flow discharge conditions, resulted in good agreement between data and predictions.