An enzyme catalyzing O-prenylation of the glucose moiety of fusicoccin A, a diterpene glucoside produced by the fungus Phomopsis amygdali

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane‐bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant‐pathogenic fungus Phomopsis amygdali and has a unique O‐prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6′‐hydroxy group of the glucose moiety of FC A to yield an O‐prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K_m values were calculated as 0.49±0.037 μM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 μM for DMAPP, with a k_cat of 55.3±3.3×10^?3 s. The enzyme did not act on representative substrates of the above‐mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.     

Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties

The avidin-biotin interaction is widely employed as a universal tool in numerous biotechnological applications. In avidin-biotin technology, non-specific binding to biological macromolecules is a hindrance. The major origin of this non-specific binding is the electrical charge of the surface of biotin-binding proteins. Tamavidin 2, a fungal avidin-like protein that binds biotin with an extremely high affinity, can be produced as a soluble recombinant protein in Escherichia coli. The isoelectric point of tamavidin 2 is 7.4-7.5, lower than avidin (10.0), and slightly higher than that of streptavidin (6.0-7.5). Here, we genetically engineered charge mutants of tamavidin 2 to reduce non-specific binding. By substituting an acidic residue (glutamic acid) for basic residues (arginine and lysine), we constructed three mutant proteins (muteins) and confirmed their high-level production in soluble form in E. coli, as well as that of tamavidin 2.?We then tested these proteins for non-specific binding to salmon sperm DNA, glycoproteins (integrin and fibronectin), and IgG from human sera. The muteins showed lower non-specific binding than tamavidin 2 to these macromolecules. In particular, one mutein, tamavidin-R104EK141E, which had the lowest isoelectric point (5.8-6.2) among avidin, streptavidin and tamavidin 2, displayed the lowest non-specific binding. The affinity of this mutein to biotin was high, comparable with that of tamavidin 2. These findings indicate that tamavidin-R104EK141E has the potential to serve as a robust tool in the numerous applications of biotin-binding proteins.     

Metal release from stainless steel, Co–Cr–Mo–Ni–Fe and Ni–Ti alloys in vascular implants

A seven-day immersion test using several solutions was conducted on stainless steel, Co-based alloy, and Ni–Ti alloy, which are used for stents and stent grafts. The quantitative data on the release of each metal ion and the correlation between metal ion release rate and pH were obtained. The quantities of Fe and Ni released from stainless steel gradually decreased with increasing solution pH (pH 2–7.5). For Co–Cr–Mo–Ni–Fe alloy, the quantity of Cr released steadily increased as pH decreased (pH ≤ 6) and reached nearly zero at pHs higher than 6 (pH 6–7.5). Co release was slightly affected by a variation in pH. The quantities of Ni and Ti released from Ni–Ti alloy markedly increased with decreasing pH (pH ≤ 4) and they leveled off from pH 4 (pH 4–7.5). Although the rapid increases were observed at approximately pH 2, the quantities were even higher than that of Co released from the Co–Cr–Mo and Co–Cr–Mo–Ni–Fe alloys. For further investigation of the rapid increase in the quantities of metals released at pH 2, an anodic polarization test was employed to study the passive and transpassive behaviours of Ni–Ti alloy. The critical current density for the passivation of Ni–Ti alloy markedly increased as pH decreased (pH ≤ 4) and was low (1.4 μA/cm²) at pH higher than 4 (pH 4–7.5). The potential at a current density of 10 μA/cm², by contrast, markedly rose with decreasing pH (pH ≤ 2), and was 1.2 V from pH 2 (pH 2–7.5).     

Ultra-high-precision time control system over any long time delay for laser pump and synchrotron x-ray probe experiment

An ultra-high-precision clock system for long time delay has been developed for picosecond time-resolved x-ray diffraction measurements using synchrotron radiation (SR) pulses and synchronized femtosecond laser pulses. The time delay control between pump laser pulse and the probe SR pulse was achieved by combining an in-phase quadrature modulator and a synchronous counter. This method allowed us to change the delay time by a nearly infinite amount while maintaining the precision of +/-8.40 ps. Time-resolved diffraction measurements using the delay control system were demonstrated for precise measurement of an acoustic velocity in a single crystal of gallium arsenide.     

Application of β-sheet proteins as a compatibilizer in injection molding of kenaf core/polypropylene composites

ABSTRACT

Injection-molding was performed using ethylene–propylene copolymer as the matrix by varying the physicochemical treatment of the reinforcement, i.e., core portion of kenaf stem that was dry-distilled and coated with or without β-sheet protein derivatives. Although dry distillation reduced the moisture content, the Charpy impact strength of the molded composites was low. When molding was performed using the fillers coated with β-sheet protein derivatives, especially coated with liquid silk (LS), a satisfactory Charpy impact strength of the molded composites was markedly gained. Thus, the β-sheet protein derivatives had potential to work as a compatibilizer. Coating the fillers with β-sheet proteins was also effective in suppressing the generation of silver streaks on the surface of composites and in preventing the occurrence of defective injection molding.     

Reaction products of α-tocopherol with a free radical initiator, 2,2′-azobis(2,4-dimethylvaleronitrile)

α-Tocopherol wss reacted with an alkylperoxyl radical at 37°C in ethanol. 2,2′-Azobis(2,4-dimethylvaleronitrile) was used to generate the alkylperoxyl radicals. The reaction products of α-tocopherol were isolated by reverse-phase and normal-phase high performance liquid chromatography, and their structures were characterized by infrared, ultraviolet,¹H and¹³C nuclear magnetic resonances and mass spectrometry. They were 8aS-hydroperoxy-α-tocopherone, 8aR-hydroperoxy-α-tocopherone, a mixure of 7,8-epoxy-8aS-ethoxy-α-tocopherone, and 7,8-epoxy-8aR-ethoxy-α-tocopherone, 8aS-(1R-cyano-1,3-dimethyl)butylperoxy-α-tocopherone, 8aS-(1S-cyano-1,3-dimethyl)butylperoxy-α-tocopherone, a mixture of 8aR-(1S-cyano-1,3-dimethyl)butylperoxy-α-tocopherone and 8aR-(1S-cyano-1,3-dimethyl)butylperoxy-α-tocopherone, 4aR,5S-epoxy-8aS-ethoxy-α-tocopherone, 4aS,5R-epoxy-8aS-ethoxy-α-tocopherone and 4aR,5S-epoxy-8aR-ethoxy-α-tocopherone.     

Solvent extraction of metal ions using a new extractant,biuret(C8)

A new extractant biuret(C8) is synthesized and tested for solvent extraction of hard acid metals, for example, actinides, and soft acid metals. This compound has a central frame similar to that of malonamide but with an additional amino functional group introduced into the central framework; in this case, both the amidic oxygen atoms and the amino nitrogen atom may bond with metals. We found that both hard and soft acid metals can be extracted from nitric or perchloric acids to n-dodecane using biuret(C8). It is clear that D(Pd, Pu, U) values of approximately 100 can be obtained using 0.1 M biuret(C8)/n-dodecane.     

EFFECT OF DISTRIBUTOR ON BUBBLE SIZE AND BUBBLE RISE VELOCITY IN THE SLUGGING REGIME OF A SEMI-CYLINDRICAL GAS-SOLID FLUIDIZED BED†

A semi-cylindrical fluidized bed of 15 cm internal diameter, equipped with a transparent flat glass plate for the front wall, was employed to visually observe bubbles in the bubbling to slugging transition regime and in the slugging regime. Five kinds of perforated distributors were used to investigate the effect of distributor type on the bubble size and the bubble rise velocity. The average bubble size was not affected by distributor type in these flow regimes, and could be predicted by Darton et al's correlation (1974)of hole number 22. In other words, this comes from the inapplicability of the correlation to the slugging regime. The bubble rise velocity agreed well with Allahwala et al's correlation (1979) and was not affected by the type of distributor.     

Efficient intraliposomal entrapment of hydrophilic platinum oligonuclear complexes

A simple method for intraliposomal entrapment of platinum complexes is presented, where hydrophilic platinum oligonuclear complexes, 1-methyluracil green (MeUG), uridine green (UdG) and uridine blue (UdB), are included inside liposomes and allowed to react with bilayer lipids. The liposomes prepared in this method exhibit higher entrapment efficiency and higher distribution to organs (liver, kidney, spleen, lung) and blood (but not B16 cancer cells) than those prepared from mononuclear Pt complexes [cis-diamminedichloroplatinum, cis-diammine-1,1′-dicarboxylatocyclobutaneplatinum, and cis-dichloro-cis-dihydroxy-trans-bis(isopropylamine)platinum)].