Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.     

Postnatal changes in activation of polyunsaturated fatty acid,acylation of lysophosphoglyceride,and phosphorylation of diacylglycerol in cortical gray matter of rat cerebrum

Activation of polyunsaturated fatty acid, acylation of lysophosphoglyceride, and phosphorylation of diacylglycerol in postnatal developing rat cerebrum were studiedin vitro, using cortical gray matter homogenates. The rate of n-3 and n-6 fatty acid activation was maximal at ten days after birth and minimal at adulthood. Acylation of lysophosphoglyceride was most active at the neonatal stage and gradually decreased with age. In addition, the amount of phosphatidic acid formed from 1, 2-dioleoyl-glycerol was also maximum at the neonatal stage, and then gradually decreased. These results suggest that the specific activity of glycerolipid synthesis in cortical gray matter declines during postnatal development.     

Strictly Conserved Residues in Euphorbia tirucalli β‐Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation–π Interaction and CH–π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.     

A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB

The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d -Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d -Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB.     

EXPERIMENT AND SIMULATION FOR THICK-PLATE BENDING BY HIGH FREQUENCY INDUCTOR

1.IntroductionSo-called"line--heating"technologyhasbeenemployedintheproductionofcurvedthickshells,whichareutilizedintheouterbodyinships[1].Oneoftheadvalltageofthisbendingprocessingisthattherequiredshapecanbeachievedwithoutdie,sothatwedonotneedanylargescalemachiningequipment.Actuallyitisimpossibletouseapunchpressforsuchlargeplateswithmorethan10mminthickness.Themechanismofthistechniqueisbasedonthethermalstressesarisingfromthedifferenceintemperaturebetweenbothsidesoftheplate.Agasburnerisusuallye…     

The present status of dental titanium casting

Experimentation in all aspects of titanium casting at universities and industries throughout the world for the last 20 years has made titanium and titanium-alloy casting nearly feasible for fabricating sound cast dental prostheses, including crowns, inlays, and partial and complete dentures. Titanium casting in dentistry has now almost reached the stage where it can seriously be considered as a new method to compete with dental casting using conventional noble and base-metal alloys. More than anything else, the strength of titanium’s appeal lies in its excellent biocompatibility, coupled with its comparatively low price and abundant supply. Research efforts to overcome some problems associated with this method, including studies on the development of new titanium alloys suitable for dental use, will continue at many research sites internationally. Toru Okabe earned his Ph.D. in metallurgical and materials engineering at the University of Florida in 1969. He is currently professor and chair of biomaterials science at Baylor College of Dentistry. Dr. Okabe is a member of TMS. Chikahiro Ohkubo earned his Ph.D. in removable prosthodontics and his D.M.D. in dentistry at Tsurumi University in 1989 and 1985, respectively. He is currently an instructor at the School of Dental Medicine at Tsurumi University in Yokohama, Japan. Ikuya Watanabe earned his Ph.D. in dental science at Nagasaki University in 1995. He is currently working at Nagasaki University, Japan. Osamu Okuno earned his Ph.D. in engineering at Waseda University in 1976. He is currently a professor and chair in the Department of Dental Materials Science, School of Dentistry, Tohoku University, Japan. Yukyo Takada earned his Ph.D. in engineering at Waseda University in 1991. He is currently assistant professor in the Department of Dental Materials Science, School of Dentistry, Tohoku University, Japan.     

Effects of diffuser shape and back pressure on the performance of disk MHD generators

The effects of diffuser shape and back pressure on the performance of closed‐cycle disk MHD generators are investigated with two‐dimensional numerical simulations taking account of the boundary layer. The enthalpy extraction ratio decreases and fluctuates periodically with increase in the back pressure. The amplitude of power output fluctuation and the period depend on the volume of the inverse flow region. For the reduced height diffuser, the inverse flow region is suppressed and the fluctuation becomes smaller than that for the constant height diffuser. When operated with a back pressure lower than an optimum value, the diffuser does not work because of reacceleration in the diffuser. For the higher back pressure, on the contrary, the inverse flow region propagates into the MHD channel, then the enthalpy extraction ratio is reduced and the Mach number at the exit of the MHD channel becomes lower than unity. For the optimum back pressure, high enthalpy extraction ratio is achieved and the adiabatic efficiency at the diffuser exit is decreased by several percent from that evaluated at the MHD channel exit. © 2000 Scripta Technica, Electr Eng Jpn, 133(2): 11–19, 2000     

Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of CU2+ ion

An affinity membrane was prepared from a porous cellulose membrane, and adsorption and recovery of serum proteins were investigated from the viewpoint that affinity membranes are efficacious against separation and purification of biomaterials. Into the cellulose membrane, iminodiacetate (IDA) group that acts as a ligand to metal ions was introduced (Cell–IDA membrane), and then Cu²⁺ ion was immobilized (Cell–IDA–Cu membrane). Bovine serum albumin (BSA) and γ-globulin (BγG), which are the major proteins in blood, were adopted as model proteins to be separated. The Cell–IDA–Cu membrane had large adsorption capacity for these proteins despite the low degree of modification. The amounts of proteins adsorbed on the Cell–IDA–Cu membrane increased with increasing pH, and BγG was adsorbed more than BSA. High protein recoveries from the Cell–IDA–Cu membrane were obtained. The separation of these proteins was also conducted under the optimum conditions of adsorption and recovery, and BγG was concentrated more than BSA although the initial concentration of BγG was lower than that of BSA. © 1996 John Wiley & Sons, Inc.     

Hydrogen isotope permeation from cooling water through various metal piping

In order to investigate the behavior of hydrogen isotope on the water–metal boundary, deuterium permeation experiments from heavy water vessel through various metal piping, such as pure iron (Fe), nickel (Ni), stainless steel (SS304), and pure iron with 10 μm gold plating, were performed at 573 K and at 15 MPa. During the experiment, surfaces of metal piping except gold plating one were oxidized at the heavy water boundary and then deuterium would generate by the oxidation reactions. This deuterium could be detected by mass spectrometer, which monitored the inside gases of the piping under continuous evacuation. The result showed clearly that the deuterium permeated through Fe, Ni, and SS304 piping was detected as mainly deuterium gas (D₂) under continuous evacuation, though that through gold plating one could not be detected effectively. The D₂ permeation rate through Fe, Ni, and SS304 piping reached equilibrium conditions with oxide generation at D₂O–metal boundary, although concluding the transfer mechanism will require further testing and modeling activities.     

Influence of tungsten–carbon mixed layer and irradiation defects on deuterium retention behavior in tungsten

The D₂⁺ fluence dependence on deuterium (D) retention was studied to clarify the D retention mechanism in tungsten. The additional D desorption stage was observed around 660 K in the TDS spectrum for a sample implanted with D₂⁺ up to the fluence of 10²³ D⁺ m^?2, which desorption stage was not observed the D₂⁺ implanted sample with the fluence less than 10²² D⁺ m^?2. The TEM observation showed that the highly dense voids were formed in tungsten by D₂⁺ implantation with the fluence of 10²³ D⁺ m^?2, considering that the D would be trapped by voids. To understand the D trapping by voids in C⁺ implanted tungsten, C⁺–D₂⁺ sequential implantation experiments at various C⁺ implantation temperatures were performed. It was found that the amount of D desorbed around 560 K was increased by increasing the C⁺ implantation temperature. The formation of the voids was observed with increasing the C⁺ implantation temperature by TEM, indicating that the increase of D desorption around 560 K was caused by the formation of voids. However, the desorption temperature of D trapped by voids in C⁺ implanted sample was lower than that in D₂⁺ implanted one. TEM observation and XPS measurement indicated that this difference was caused by the increase of void size and/or the presence of implanted carbon.