Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca²⁺-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe⁸⁵, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr¹⁸⁰, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined.     

Acetylation of loofa (Luffa cylindrica) sponge as immobilization carrier for bioprocesses involving cellulase

The feasibility of using loofa sponge for immobilization of cellulase-producing microorganisms was investigated by acetylating loofa sponge. Acetylation was achieved by autoclaving process of loofa sponge immersed in acetic anhydride at various temperatures for various times. The degree of acetylation, as inferred by the weight percentage gain (WPG), was enhanced by increasing both temperature and the duration of acetylation. The acetylation of a piece of loofa sponge in an autoclave at 120 degrees C for 20 min resulted in a WPG of about 8%, which was sufficient to protect the loofa sponge against cellulose degradation. The acetylated loofa sponge prepared under this condition was not decomposed by commercial cellulase and its structure was maintained for more than 720 h during repeated-batch treatments with commercial cellulase. A flocculating yeast (Saccharomyces cerevisiae IR-2) and a fungus (Trichoderma reesei QM9414) were successfully immobilized in the acetylated loofa sponge. In each case, the percentage of immobilized cells was as high as that obtained using nonacetylated loofa sponge. Acetylation had no adverse effects on cell growth and immobilization of T. reesei QM9414, as well as on cell growth and ethanol production by S. cerevisiae IR-2. T. reesei QM9414 immobilized on an acetylated loofa sponge was successfully used for repeated-batch cellulase production from commercial cellulose powder. Although the acetylated loofa sponge showed a slight weight loss, it was not disintegrated by activated sludge. The results obtained in this study showed that acetylated loofa sponge is suitable as an immobilization carrier for bioprocesses involving cellulase.     

PACAP-mediated sperm-cumulus cell interaction promotes fertilization

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus-oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus-oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.     

Poly[(R)-3-hydroxybutyrate] formation in Escherichia coli from glucose through an enoyl-CoA hydratase-mediated pathway

In this study, a new metabolic pathway for the synthesis of poly[(R)-3-hydroxybutyrate] [P(3HB)] was constructed in a recombinant Escherichia coli strain that utilized forward and reverse reactions catalyzed by two substrate-specific enoyl-CoA hydratases, R-hydratase (PhaJ) and S-hydratase (FadB), to epimerize (S)-3HB-CoA to (R)-3HB-CoA via a crotonyl-CoA intermediate. The R-hydratase gene (phaJ(Ac)) from Aeromonas caviae was coexpressed with the PHA synthase gene (phaC(Re)) and 3-ketothiolase gene (phaA(Re)) from Ralstonia eutropha in fadR mutant E. coli strains (CAG18497 and LS5218), which had constitutive levels of the beta-oxidation multienzyme FadB(Ec). When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5 wt% of the dry cell weight, whereas the control cells without phaJ(Ac) or fadR mutation accumulated significantly smaller amounts of P(3HB). These results suggest that PhaJ(Ac) and FadB(Ec) played an important role in supplying monomers for P(3HB) synthesis in the pathway. Furthermore, by using this pathway, a P(3HB)-concentration-dependent fluorescent staining screening technique was developed to rapidly identify cells that possess active R-hydratase.     

An Analysis of Flow in a Centrifugal Impeller by FEM with k-ε Model

IntnMluctiouIn the passage of a centrifugal impeller, there is notonly a secondary flow toward a suchon-side from aPressure-side along a casing or a hub s~e but also aleakage flow through a clearance between a casings~e and a blade hp. The~, it is imPOI'tant tO clarifysuch a passage flow with regard to the design of acentrifugal turbomashinery with high efficiency.The impeller passage nows have been inveshgatedfrom the flow measurementS with a pressure Probe, ahot-wire anemometer and a LDV…     

Solid polymerization and supramolecular chirality formation of azobenzene substituted diacetylene Langmuir–Blodgett films

An azobenzene mesogen-substituted diacetylene (NADA) monolayer has been transferred onto the solid substrate by the traditional Langmuir–Blodgett (LB) method. Solid-state polymerization of NADA LB films can be sensitized in visible light region. In situ UV–vis absorption measurement enables us to study polymerization kinetics of NADA LB films. The polymerization process can be described as a first order rate equation and polymerization rate constants are characterized in detail. Furthermore, achiral NADA molecules can form chiral LB films through overcrowded packing of the azobenzene moiety.     

The use of indoline dyes in a zinc oxide dye-sensitized solar cell

The effects of the position and the type of carboxyl anchor group in double rhodanine-type indoline dyes on the performance of a zinc oxide dye-sensitized solar cell were examined. The optimum position for the carboxymethyl group was on the inner rhodanine moiety; a carboxymethyl group gave optimum results among carboxymethyl, -ethyl, and -propyl derivatives.     

Ruthenium (II) complexes with π expanded ligand having phenylene–ethynylene moiety as sensitizers for dye-sensitized solar cells

Ruthenium(II) complexes by introducing phenylene–ethynylene subunit into a tpy (tpy=2,2′:6′,2″-terpyridine) ligand were newly synthesized, and their photophysical and photochemical properties were characterized. The molar extinction coefficients of these dyes were larger than that of black dye. The photovoltaic performances of the solar cells based on these synthesizing dyes were investigated under AM 1.5 irradiation (100 mW cm^?2). The conversion efficiencies of 1a and 1b were 5.7% and 2.4%, respectively.     

Use of multivariate analysis for the improvement in prediction accuracy of bacterial aerobic plate count by flow cytometry

Flow cytometry (FCM) and aerobic plate count (APC) by the culture method were performed on green tea samples spiked with Escherichia coli type strain NCTC9001 (ATCC11775) solutions of different concentrations. In FCM, fluorescence signals from multiple stained bacteria and other fluorophores are detected using detector channels, and recorded as events with a voltage at each channel. FCM data were analyzed in two ways: conventional and multivariate analysis. In the former, the number of events with voltages larger than the defined threshold values was regarded as the predicted APC. In the latter, voltage histograms of all channels were obtained and merged horizontally to serve as explanatory variables. Then a partial least squares regression (PLSR) model was built to predict APC from the histogram data. The coefficient of determination (R²) and the root mean square error (RMSE) between APC by the culture method and that predicted by conventional FCM were 0.916 and 1.08 cfu/ml². The APC values predicted by the PLSR model and those measured were in good agreement with R² of 0.982 and RMSE of 0.417 cfu/ml, which verified the potential of the proposed method for improving APC prediction accuracy by FCM.