Singular value decomposition analysis of the secondary structure features contributing to the circular dichroism spectra of model proteins

Amyloid fibril formation occurs in restricted environment, such as the interface between intercellular fluids and bio-membranes. Conformational interconversion from α-helix to β-structure does not progress in fluids; however, it can occur after sedimentary aggregation during amyloid fibril formation induced by heat treatment of hen egg white lysozyme (HEWL). Secondary structures of various proteins and denatured proteins titrated with 2,2,2-trifluoroethanol (TFE) were examined using their CD spectra. Gaussian peak/trough and singular value decompositions (SVD) showed that the spectral pattern of the α-helix comprised a sharp trough at wavelength 207 nm and a broad trough at 220 nm. Conversely, we distinguished two patterns for β-sheet—a spread barrel type, corresponding to ConA, and a tightly weaved type, corresponding to the soybean trypsin inhibitor. Herein, we confirmed that the spectral/conformational interconversion of the heat-treated HEWL was not observed in the dissolved fluid.     

A Behavioral Mechanism of How Increases in Leg Strength Improve Old Adults’ Gait Speed

We examined a behavioral mechanism of how increases in leg strength improve healthy old adults’ gait speed. Leg press strength training improved maximal leg press load 40% (p = 0.001) and isometric strength in 5 group of leg muscles 32% (p = 0.001) in a randomly allocated intervention group of healthy old adults (age 74, n = 15) but not in no-exercise control group (age 74, n = 8). Gait speed increased similarly in the training (9.9%) and control (8.6%) groups (time main effect, p = 0.001). However, in the training group only, in line with the concept of biomechanical plasticity of aging gait, hip extensors and ankle plantarflexors became the only significant predictors of self-selected and maximal gait speed. The study provides the first behavioral evidence regarding a mechanism of how increases in leg strength improve healthy old adults’ gait speed.     

Transitional Reactive Oxygen Species (ROS) Production in Fertilized Egg Embryos of Devil Stinger (Inimicus japonicus), a Marine Fish Species

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.     

Role of zinc ion for catalytic activity in d-serine dehydratase from Saccharomyces cerevisiae

d-Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme catalyzing d-serine dehydration. The enzyme contains 1 mol Zn(2+) in its active site and shows a unique zinc dependence. The Zn(2+) is essential for the d-serine dehydration, but not for the α,β-elimination of β-Cl-d-alanine catalyzed as a side-reaction. The fact that dehydration of d-threonine and d-allo-threonine, also catalyzed by DsdSC, is likewise Zn(2+) dependent indicates that Zn(2+) is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of C(β) . Removal of Zn(2+) results in a less polar active site without changing the gross conformation of DsdSC. (1) H NMR determined the rates of α-hydrogen abstraction and hydroxyl group elimination of d-serine in (2) H(2) O to be 9.7 and 8.5 s(-1) , respectively, while the removal of Zn(2+) abolished both reactions. Mutation of Cys400 or His398 within the Zn(2+) binding sites to Ala endowed DsdSC with similar properties to those of the Zn(2+) -depleted wild-type enzyme: the mutants lost the reactivity toward d-serine and d-threonine but retained that toward β-Cl-d-alanine. (1) H NMR analysis also revealed that both α-hydrogen abstraction and hydroxyl group elimination from d-serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α-hydrogen abstraction and hydroxyl group elimination in a concerted fashion.     

Using Acoustics to Determine Eelgrass Bed Distribution and to Assess the Seasonal Variation of Ecosystem Service

Eelgrass beds are an important source of primary production in coastal ecosystems. Understanding seasonal variation in the abundance and distribution of eelgrass is important for conservation, and the objectives of this study were to 1) monitor seasonal variation in eelgrass beds using an acoustic monitoring method (Quantitative echo sounder) and 2) broadly quantify the carbon circulation function. We obtained acoustic data of eelgrass beds in coastal areas north and east of Ikunojima Island. Surveys were conducted nine times over the 3-year period from 2011 to 2013 in order to monitor seasonal variation. Acoustic data were obtained and used to estimate the spatial distribution of eelgrass by geostatistical methods. To determine supporting services, we determined carbon sink and carbon fixation by eelgrass beds using data from the National Research Institute of Fisheries and Environment of Inland Sea (2011). The height and distribution of eelgrass beds were at a maximum in May and at a minimum in November of each year. Distribution trends were different between the north and east areas. Supporting services showed the same patterns throughout the year. The area of distribution was considered to be coincident with the life history of eelgrass. Distribution differed by area and changed yearly due to the effects of bottom characteristics and wind direction. Quantifying the supporting services of eelgrass beds was shown to be useful for managing the conservation of coastal ecosystems.     

Discadenine Distribution in Cellular Slime Molds and Its Inhibitory Activity on Spore Germination

We constructed a hybrid plasmid to allow controlled expression of a gene and the subsequent secretion into the culture medium of the gene product in Escherichia coli. This was achieved by the use of five trp promoter-operator regions in tandem followed by the DNA fragment coding for the signal peptide and the N-terminus of the OmpF protein, and the trpR gene coding for the Trp repressor. Multiplication of the trp promoter-operator appreciably enhanced expression of the gene that followed. A single copy of the trpR gene on the chromosome was insufficient for controlling the enhanced expression. The expression was, however, completely controlled when the trpR gene was cloned onto the same plasmid. When the multiple trp promoter-operator was followed by the DNA fragment coding for the signal peptide and the N-terminus of the OmpF protein that was further followed by the gene for human β-endorphin, a β-endorphin-containing polypeptide was synthesized under the complete control of the trp promoter-operator, and secreted to the culture medium across both the cytoplasmic membrane and the outer membrane. Controlled expression of a foreign gene and subsequent secretion into the medium of the product were thus achieved.     

Structural characterization of neutral and anionic glucans from Mesorhizobium loti

The periplasmic glucans of Mesorhizobium loti were isolated and separated into fractions according to their acidity. NMR spectroscopy confirmed their backbone structure to be a cyclic beta-(1-->2)-d-glucan as in the case of other rhizobia, and revealed no non-glycosidic substituents in the neutral fraction, and glycerophosphoryl and succinyl residues as major and minor substituents, respectively, in the anionic fractions. MALDI-TOF mass spectrometry showed that the anionic glucans contain one, two, or three such substituents per molecule according to their acidity, and, in contrast, that all the anionic subfractions have a similar size distribution to that of the neutral glucans, where molecules composed of 20-24 glucosyl residues are predominant. These results clarify the periplasmic glucan composition in terms of charge-to-mass ratios in M. loti cells.     

Regulation of growth cone extension by SNARE proteins.

Recent studies have suggested that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion with the surface plasma membrane, which leads to neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. We studied this mechanism by overexpressing GFP-fusion SNAREs including VAMP-2, SNAP-25A, and syntaxin1A in PC12 cells to investigate the role of SNAREs in neurite outgrowth. When overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.     

(−)-Cercosporamide derivatives as novel antihyperglycemic agents

In our exploratory campaign for an antihyperglycemic agent with a novel mechanism of action, (?)-Cercosporamide 1, which is known as an antifungal agent, showed a potent plasma glucose lowering effect in hyperglycemic KK/Ta mice. The trouble was that it was accompanied by a decrease in food intake and a loss of body weight. We synthesized some (?)-Cercosporamide derivatives and succeeded to separate these actions. N,O-ketal type derivatives, especially compound 10, had the most potent plasma glucose lowering effect without affecting the food consumption or body weight.     

Recombinant expression,purification, and characterization of polyphenol oxidase 2 (VvPPO2) from “Shine Muscat” (Vitis labruscana Bailey × Vitis vinifera L.)

Polyphenol oxidases (PPOs) catalyze browning reactions in various plant organs, therefore controlling the reactions is important for the food industry. PPOs have been assumed to be involved in skin browning of white grape cultivars; however, the molecular mechanism underlying PPO-mediated browning process remains elusive. We have recently identified a new PPO gene named VvPPO2 from “Shine Muscat” (Vitis labruscana Bailey × V. vinifera L.), and have shown that the gene is transcribed at a higher level than the previously identified VvPPO1 in browning, physiologically disordered berry skins at the maturation stage. In this study, we expressed VvPPO2 in Escherichia coli and, using the purified preparation, revealed unique physicochemical characteristics of the enzyme. Our study opens up a way to not only understand the berry skin browning process but also to elucidate the enzymatic maturation process of grape PPOs.