Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system

Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.     

Effect of Interlayers in Ceramic-Metal Joints with Thermal Expansion Mismatches

The internal stress resulting from the thermal expansion mismatch between alumina and steel with and without an interlayer was evaluated. On the basis of the calculation, alumina and steel were bonded using the interlayer method and hot isostatic pressing at 1273 to 1573 K under 100 MPa for 30 min. When a laminated interlayer (niobiumlmolybdenum) was used, a joint with high bonding strength was obtained.     

Temperature Dependence of Young's Modulus and Internal Friction in Alumina, Silicon Nitride, and Partially Stabilized Zirconia Ceramics

The temperature dependence of Young's modulus and internal friction (Q⁻¹)in alumina, silicon nitride, and partially stabilized zirconia (Y-PSZ) ceramics was studied. Little change in Q⁻¹ was found for alumina, whereas Q⁻¹ for silicon nitride ceramics increased above 700°C. The Q⁻¹ of Y-PSZ increased markedly with increasing temperature up to a peak at ∼200°C.     

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.     

Mating-induced mating-type cassette conversion in Saccharomyces cerevisiae

We have previously reported that the HMRa-bearing restriction fragment of a rho degrees sir4-11 strain (HMLalpha-MATalpha-HMRa), which acts as an alpha-mater because of being rho degrees , changes its electrophoretic mobility when the strain mates with a certain group of a-mating strains (HMLalpha-MATa-HMRa). In this study, we found that the sir4-11 strain being rho degrees was not essential for this phenomenon and also that the altered form of the fragment contained HMRalpha in place of HMRa. Furthermore, we observed conversion of HMLa to HMLalpha in the cross in which a sir4-11 HMLa-MATalpha-HMRalpha strain was mated with a representative of the above-mentioned a-mating strain. In addition, when this a-mating strain was mated with a SIR(+) HMLalpha-MATa-HMRalpha strain, the resultant diploid was found to be HMLalpha/HMLalpha MATa/MATalphaHMRa/HMRalpha, indicating that conversion of MATa to MATalpha had taken place in the course of mating. From all these observations, we conclude that there is a group of S. cerevisiae strains that carries factor(s) that induces conversion of a mating-type cassette of the mating partner to alpha mating-type cassette and that this mating type cassette conversion takes place in all three mating type loci, HML, MAT and HMR, if the loci are in the non-silenced condition.     

Efficient synthesis of nonnatural mutants in Escherichia coli S30 in vitro protein synthesizing system

Factors that affect the efficiency of in vitro synthesis of mutant proteins that contain nonnatural amino acids were investigated. The process of the nonnatural mutagenesis consists of chemical aminoacylation of a tRNA that contains a 4-base anticodon, followed by in vitro synthesis in the presence of an mRNA that contains the corresponding 4-base codon. Detailed studies on the time courses of the synthesis revealed two major factors that suppress the yield of nonnatural mutants compared with the wild-type protein. First, a cyclic tRNA that exists as a by-product of the chemical aminoacylation inhibits the protein synthesis. Second, the very short lifetime of a tRNA aminoacylated with a nonnatural amino acid limits the protein yield. As a simple and practical way of surmounting these factors, aminoacyl tRNA was added into the in vitro system at 5 min after the start of the synthesis. The addition increased the protein yield up to the level of conventional proteins in the in vitro system.     

Lithium intercalation and alloying effects on electronic structures of spinel lithium manganese oxides

Employing the DV-Xα molecular orbital method, lithium intercalation effect and alloying effect have been investigated on the electronic structures of spinel M_xMn_2−xO₄ and LiM_xMn_2−xO₄ (M=Ti, V, Cr, Mn, Fe, Co, Ni and Cu). It is found that the lithium intercalation induces the electron transfer from Li to the surrounding O ions, and enlarges the Mn-3d/O-2p band gap greatly. Such a modification of the oxygen electronic state is probably related to the electrochemical cell voltage. This lithium intercalation effect still holds even in the alloyed systems. However, the amount of transferred charges from M to O ions changes depending on the alloying element, M. So the alloying addition modifies the oxygen electronic state and influences the lithium intercalation process indirectly.     

Microscopic implication of rapid shrinking of comb-type grafted poly(N-isopropylacrylamide) hydrogels

The shrinking mechanism of comb-type grafted poly(N-isopropylacrylamide) gel was investigated by small-angle X-ray scattering (SAXS). The SAXS reveals that the microdomain structure with characteristic spacing of 460 Å is developed in the comb-type grafted poly(N-isopropylacrylamide) gel during the shrinking process. These observations suggest that the freely mobile characteristics of the grafted chains are expected to show the rapid dehydration to make tightly packed globules with temperature, followed by the subsequent hydrophobic intermolecular aggregation of the dehydrated graft chains. The dehydrated grafted chains created the hydrophobic cores, which enhance the hydrophobic aggregation of the networks. These aggregations of the NIPA chains contribute to an increase in void volume, which allow the gel having a pathway of water molecules by the phase separation.     

Relationship Between Structure and Coordination Strength of N and N,O-Hybrid Donor Ligands with Trivalent Lanthanides

ABSTRACT

To effectively separate lanthanides (Ln(III)) from actinides (An(III)), symmetrical 2,2′-bipyridyl (Bpy), 1,10-phenanthroline (Phen), and unsymmetrical N-methyl-N-tolyl-1,10-phenanthroline-2-carboxamide (MeTol-PTA) were investigated. According to the crystal structures and EXAFS experiments, the decreasing ionic radius from light to heavy Ln led to decreases in the Ln–N (Bpy) and Ln–N (Phen) distances, while log β simply increased due to the electrostatic interaction and the order of Ln–O (MeTol-PTA) < Ln–N (Bpy, Phen) < Ln–N (MeTol-PTA) was obtained. This indicated that the bulky phenanthroline moiety of MeTol-PTA may not allow N (MeTol-PTA) to come close to Ln. Consequently, the log β of MeTol-PTA exhibited a local maximum (around Nd).     

Passivation effects of deuterium exposure on boron-doped CVD homoepitaxial diamond

The deuterium (hydrogen) passivation effect on acceptors in boron-doped CVD homoepitaxial diamond was studied by electrical (Hall-effect) and secondary ion mass spectroscopy (SIMS) measurements. Deuterium was incorporated into the samples using microwave (MW) deuterium plasma at 673 K for 2–24 h. We observed the progress of acceptor passivation with p-type conduction, which finally resulted in a highly resistive state.