Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N-and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.Sake is a Japanese traditional alcoholic beverage made from steamed rice by parallel fermentation with Aspergillus oryzae, which is a source of saccharification enzymes, and sake yeasts, which produce ethanol from glucose. Sake yeasts are classified as Saccharomyces cerevisiae and are known to produce more than 18% (vol/vol) ethanol in sake mash (16). Almost all sake yeasts form a thick foam layer on sake mash during vigorous fermentation. This foam formation has been used as an indicator of fermentation progress: the foam rises when fermentation becomes strong and it disappears when fermentation becomes weak. However, this characteristic sometimes reduces the efficiency of sake fermentation because a large part of the fermentation tank is occupied by a thick foam layer. Thus, an absence of foam formation during fermentation is a preferable property.Ouchi and Akiyama (23) developed a method to screen nonfoaming mutants from foaming sake yeast using cell affinity for bubbles, and using this method they isolated a nonfoaming mutant from an industrial sake yeast. This mutant had almost the same characteristics as the parental strain except for its nonfoaming property in fermentation. Thus, this method of screening for nonfoaming mutants was applied to various strains of sake yeasts and the resultant nonfoaming yeasts are now widely used in commercial sake brewing. Comparison of the nonfoaming mutant with its parent revealed that the cell surface of the former is less hydrophobic than that of the latter, suggesting that cell surface hydrophobicity is related to foaming ability (25). However, the detailed molecular mechanism of foaming ability of sake yeast is still unknown. In this study, we have cloned a gene from a foamin...

Digital enzyme-linked immunosorbent assay (ELISA) is a powerful analytical method for highly sensitive protein biomarker detection. The current protocol of digital ELISA requires multiple washing steps and signal amplification using an enzyme, which could be the potential drawback in in vitro diagnosis. In this study, we propose a digital immunoassay method, which we call “Digital HoNon-ELISA” (digital homogeneous non-enzymatic immunosorbent assay) for highly sensitive detection without washing and signal amplification. Target antigen molecules react with antibody-coated magnetic nanoparticles, which are then magnetically pulled into femtoliter-sized reactors. The antigens on the particles are captured by antibodies anchored on the bottom surface of the reactor via molecular tethers. Magnetic force enhances the efficiency of particle encapsulation in the reactors. Subsequent physical compartmentalization of the particles enhances the binding efficiency of antigen-carrying particles to the antibodies. The tethered particles show characteristic Brownian motion within a limited space by the molecular tethering, which is distinct from free diffusion or nonspecific binding of antigen-free particles. The number of tethered particles directly correlates with the concentration of the target antigen. Digital HoNon-ELISA was used with a prostate-specific antigen to achieve a detection of 0.093 pg/mL, which is over 9.0-fold the sensitivity of commercialized highly sensitive ELISA (0.84 pg/mL) and comparable to digital ELISA (0.055 pg/mL). This digital immunoassay strategy has sensitivity similar to digital ELISA with simplicity similar to homogeneous assay. Such similarity allows for potential application in rapid and simple digital diagnostic tests without the need for washing and enzymatic amplification.

Using rice samples derived from normal rice cultivars and endosperm starch mutant, we investigated key factors contributing to the enzyme digestibility of steamed rice grains. The chemical composition of polished rice grains, structural features of endosperm starch, and enzyme digestibility of steamed rice grains were examined. The protein content of polished rice grains was 4.6–9.1%, amylose content was 4–27%, the DPn of purified amylose was 900–1,600, the amylopectin short/long chain ratio was 1.2–5.9, and the enzyme digestibilities of steamed polished rice grains were 0.9–12.6 °Brix. Amylose content and RVA parameters (viscosity, breakdown, and setback) correlated significantly with enzyme digestibility of steamed rice grains. Multiple regression formulas were constructed to predict digestibility of steamed rice grain as a function of the molecular characteristics of the starch. When both amylose content and the short/long chain amylopectin ratio were used as predictor variables, they accounted for >80% of the observed variance in digestibility of steamed rice grains. Multiple regression revealed that the more digestible rice samples had starch with a lower amylose content and more short‐chain amylopectin. Reassociation of amylose‐lipid complex and recrystallization of amylopectin in the stored steamed rice grains was monitored by differential scanning calorimetry (DSC), and the observed retrogradation properties were related to the structural characteristics of starch and to the enzyme digestibility of steamed rice grains.

Five bitter-tasting peptides were isolated from charcoal-untreated sake, following a Sepabeads resin separation, an initial reverse-phase chromatography (RP-HPLC), a gel permeation-chromatography, and a second RP-HPLC. The isolated peptides consisted of six to thirteen amino acid residues. The N-termini were uniformly pyroglutamate residues. Based on the rice protein database, the peptides were derived from two di#erent N-termini of the rice glutelin acidic subunit. One of them was reported as a prolyl endopeptidase inhibitor. The thirteen amino acid peptides in charcoal-untreated ginjyo-type sakes were lower than that in charcoal-untreated jyunmai-type sakes. The thirteen amino acid peptides were not detected in the commercial ordinary-type sake analyzed. The concentration of analyzed peptides of nine to thirteen amino acid residues in charcoal-untreated sake exceeded their preliminary estimated sensory threshold values, suggesting that they contribute to the sensory quality of charcoal-untreated sake.

No abstract

Cereal Chem. 83(2):143-151Structural and physicochemical characteristics of endosperm starch from milled rice grains of seven Japanese cultivars used in sake production were examined. Amylose content was 15.2-20.2%, numberaverage degree of polymerization (DP n ) of amylose was 900-1,400, and the ratio of short-to-long chain amylopectin was 2.7-3.5, respectively. The degree of retrogradation of purified starch stored for seven days at 4°C after gelatinization was 20-31%. The degree of retrogradation correlated negatively with the ratio of short-to-long chain amylopectin. The effect of holding time after steaming on enzyme digestibility and starch retrogradation of steamed rice grains was investigated. The longer the holding time after steaming, the greater the extent of retrogradation, and the less the degree of enzymatic digestibility. The decreased rate of enzyme digestibility correlated with amylopectin chain length distribution. Samples with short-chain amylopectin exhibited a slow decrease in enzyme digestibility. It was determined that the structure and retrogradation properties of endosperm starch in Japanese rice cultivars affect the decreasing rate of enzyme digestibility of the steamed, milled rice grains.

Sake is a Japanese traditional alcoholic beverage made from rice and water. Recently, its export and its production in countries other than Japan have increased. In accordance, both the breeding and the cultivation of sake rice varieties have been extended to wide areas of Japan. To breed new sake rice cultivars and to cultivate rice with high suitability for sake production, an understanding of the properties of the ingredient rice grains and the factors affecting sake making is important. The influence of various rice properties, such as starch structure, protein, water absorption, and grain polishing, on sake making are being revealed in detail. In this review, the properties of rice used for sake making are summarized in terms of fermentation science and rice cultivation.