A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR

Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an “indirect/reflected” detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a “chimeric membrane protein”-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the prediction tool POODLE and 35 expression vectors were constructed. Subsequently, we obtained ¹⁵N-labeled chimeric PH0471 proteins with 25 IDPs as linkers. The NMR spectra allowed us to classify IDPs into three categories: flexible, moderately flexible, and inflexible. The inflexible IDPs contain membrane-associating or aggregation-prone sequences. This is the first attempt to use an indirect/reflected NMR method to evaluate IDPs and can verify the predictions derived from our computational tools.     

Two Triterpene Synthases from Imperata cylindrica Catalyzing the Formation of a Pair of Diastereoisomers through Boat or Chair Cyclization

Imperata cylindrica is known to produce a pair of triterpenes, isoarborinol and fernenol, that exhibit identical planar structures but possess opposite stereochemistry at six of the nine chiral centers. These differences arise from a boat or a chair cyclization of the B-ring of the substrate. Herein, we report the characterization of three OSC genes from I. cylindrica. IcOSC1 and IcOSC5 were identified as isoarborinol and fernenol synthases, respectively, while IcOSC3 was characterized as a multifunctional enzyme that produces glutinol and friedelin as its major products. Mutational studies of isoarborinol and fernenol synthases revealed that the residues surrounding the DCTAE motif partially affected the conformation of the B-ring during cyclization. Additionally, the IcOSC1-W255H mutant produced the rare triterpene boehmerol. The introduced histidine residue presumably abstracted a proton from the intermediary carbocation at C18 during the 1,2-rearrangement. Expression analysis indicated that all OSC genes were highly expressed in stems.     

A field study on serum vitellogenin levels in male Reeves' pond turtles (Chinemys reevesii) from estrogen-contaminated sites and a reference site

To ascertain whether wild male turtles were influenced by environmental estrogens, we examined serum vitellogenin (VTG) levels of male Reeves' pond turtles (Chinemys reevesii) collected from four study sites (A-D) in Kyoto, Japan. Sites A-C, which were impacted by domestic or industrial wastewater and effluents from sewage treatment plants, were chosen as contaminated sites, and site D was intended as a reference site. This contaminated/reference site characterization was confirmed by measuring estrogenic activities of the water samples collected at each site for over a year. Serum VTG levels in the turtles were quantified by an enzyme-linked immunosorbent assay established previously. Estrogenic activities of the water samples were measured using a previously validated yeast two-hybrid assay and expressed as the estradiol-17beta equivalent. Estrogenic activity was observed at high levels at sites A-C, but was almost undetected at site D throughout the sampling period: the mean and range were 0.74 (<0.07-2.1), 0.52 (0.17-1.6), 1.7 (<0.07-7.3), and 0.07 (<0.07-0.62) ng/l at sites A-D, respectively. Significant differences were found only in site D versus sites A, B, and C. Therefore, site D and sites A-C were confirmed to be a reference site and contaminated ones, respectively. Overall, 320 male turtles were captured and examined. The majority of the turtles showed normal VTG values (0.10-0.74 microg/ml). Although only five turtles from sites A-C showed unusually high VTG values (1.1-5.9 microg/ml, nearly one order of magnitude higher than normal values but much lower than values in adult females), there was no significant difference in the incidence of these high values between sites A-C and site D. Moreover, among the five turtles, one turtle was captured again 2 months later, but its VTG value dropped to the normal level. The unusually high VTG values may therefore be transient elevation caused by incidental and/or individually specific agents. Excluding the unusually high values, the mean serum VTG (accompanied with the range) was 0.22 (0.10-0.74), 0.27 (0.11-0.62), 0.27 (0.17-0.68), and 0.23 (0.10-0.57) microg/ml at sites A-D, respectively. There were no significant differences in the mean VTG values among sites A-D. Although it should be clarified how and why the unusually high VTG values occurred at sites A-C, our results suggested that wild male C. reevesii turtles would not be significantly affected by xenobiotic estrogens at environmentally relevant levels in terms of serum VTG elevation.     

A study on a porous residential building model in hot and humid regions: Part 1—the natural ventilation performance and the cooling load reduction effect of the building model

This study targets environmental load reduction in hot and humid regions. It reveals the effects that porous residential buildings have on the natural ventilation performance and, consequently, the cooling load reduction. Two residential building models, namely a model with a void ratio of 0% and a model with a void ratio of 50%, are evaluated using computational fluid dynamics (CFD) analysis and thermal and airflow network analysis. The analysis on components of the heat load indicates that improvements in the natural ventilation performance would significantly reduce the cooling load.     

Molybdenum‐Catalyzed Stereospecific Deoxygenation of Epoxides to Alkenes

Mild and simple catalytic systems consisting of molybdenum(VI) dichloride dioxide [MoO₂Cl₂] as a catalyst and a phosphine as reductant have been developed for the stereospecific deoxygenation of epoxides to alkenes. The reactions using 1,2‐bis(diphenylphosphino)ethane (dppe) and triphenylphosphine (PPh₃) proceed with retention and inversion of stereochemistry, respectively. The mild reaction tolerates the presence of various functional groups and affords stereodefined substituted olefins in good yields.

     

Oxygen release behavior of metastable tetragonal t′meta-(Ce0.5Zr0.5)2 phases prepared by reduction and successive oxidation of t′ phase

Tetragonal CaF₂-related-type t′-/Ce_0.5Zr_0.5)O₂ phase was reduced at 773 K ≤ T_red. ≤ 1073 K to prepare precursors with various oxygen compositions: Ce₂Zr₂O_712d /0.111 < δ < 0.379). Metastable tetragonal t^′_meta-(Ce_0.5Zr_0.5)O₂ phases were prepared by oxidizing in O₂ at 873 K the precursors, and subjected to evolved oxygen gas analysis by heating the samples at a constant rate. When the precursor was prepared at T_red: ? 773 K; i.e. δ ? 0:379; the oxygen release behavior of the tetragonal phase agreed approximately with that of the t^′. For δ < 0.29, distinct features of the t^′_meta-(Ce_0.5Zr_0.5)O₂ appeared in the XRD results and Raman spectra. The temperature exhibiting the maximum release rate of oxygen from the t^′_meta became lower with decreasing the d value. It could be concluded that the thermodynamic behavior of the t^′_meta is related closely to the precursor composition and becomes more unstable with decrease in the δ value. The t^′ and t^′_meta phases with disordered arrangement of Ce and Zr ions may belong to the same space group; nevertheless, difference in the random arrays of the Ce and Zr ions was suggested on the basis of the present experimental results.     

Restraint Method for Overcoming Heating Irregularity and Improving Heating Equipment Efficiency for Beverage Cans Using High‐Frequency Induction Heating

We were able to reduce heating irregularities in equipment for heating beverage cans by using the high‐frequency induction heating method and determining the resulting improvement in the heating efficiency. The heating irregularity between the top and bottom of the can was reduced to less than 3°C by improving the arrangement of the heating coils for the beverage can by thinning the winding and changing the circuit configuration. The efficiency of power conversion was improved from 93.0% to 97.4%. In this paper, the configuration of the equipment and the experimental results are reported.     

Doping effect on sinterability of polycrystalline yttria: From the viewpoint of cation diffusivity

The sintering behavior of Y₂O₃ doped with 1 mol% of a trivalent or tetravalent cation was investigated by pressureless sintering in air. Ga³⁺ or Ge⁴⁺-doped Y₂O₃ bodies exhibited higher relative densities than the undoped Y₂O₃, while the La³⁺ or Zr⁴⁺-doping suppressed the densification of Y₂O₃. An interdiffusion experiment was performed on the diffusion couples of polycrystalline Er₂O₃ and Y₂O₃ doped with Ni²⁺ or Zr⁴⁺, which are some of the most effective and least effective dopants for the improvement of the sinterability, respectively. The lattice and grain boundary diffusion coefficients of the Er³⁺ cation in Y₂O₃ were increased by the Ni²⁺-doping, but were decreased by the Zr⁴⁺-doping. High-resolution transmission electron microscopy observations and nano-probe X-ray energy dispersive spectroscopy analyses confirmed that the dopant cations segregate along the grain boundaries without forming an amorphous phase. The doping effect on the sinterability of Y₂O₃ must result from the change in the diffusivity in Y₂O₃.     

Visualization of oscillating flow in a double-inlet pulse tube refrigerator with a diaphragm inserted in a bypass-tube

The double-inlet pulse tube refrigerator that has a diaphragm inserted in a bypass-tube, which enabled it to transmit a pressure oscillation whereas to obstruct a DC gas flow, was manufactured and tested. The oscillating flow behavior inside of the refrigerator was studied by using a smoke-wire flow visualization technique. It was found that if the diaphragm was optimized, the performance would be improved more than that of the refrigerator with a bypass valve due to the increase in the P–V work of the gas and the decrease in the convective heat loss caused by a secondary flow.     

Biomechanical Evaluation of Ti-Nb-Sn Alloy Implants with a Low Young’s Modulus

Dental implants are widely used and are a predictable treatment in various edentulous cases. Occlusal overload may be causally related to implant bone loss and a loss of integration. Stress concentrations may be diminished using a mechanobiologically integrated implant with bone tissue. The purpose of this study was to investigate the biomechanical behavior, biocompatibility and bioactivity of a Ti-Nb-Sn alloy as a dental implant material. It was compared with cpTi. Cell proliferation and alkaline phosphatase (ALP) activity were quantified. To assess the degree of osseointegration, a push-in test was carried out. Cell proliferation and ALP activity in the cells grown on prepared surfaces were similar for the Ti-Nb-Sn alloy and for cpTi in all the experiments. A comparison between the Ti-Nb-Sn alloy implant and the cpTi implant revealed that no significant difference was apparent for the push-in test values. These results suggest that implants fabricated using Ti-Nb-Sn have a similar biological potential as cpTi and are capable of excellent osseointegration.