High cell-density expression system: a novel method for extracellular production of difficult-to-express proteins

Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.     

Glycine Protects Against Strong Protein-Denaturing Activity of Oleuropein, a Phenolic Compound in Privet Leaves

In previous studies, we reported the existence of a high concentration of free glycine in the digestive juice of several Lepidoptera larvae, particularly in the digestive juice of species that feed on the privet tree, Ligustrum oblusifolium. The water extract of privet leaves showed very strong protein-denaturing activity and lysine-decreasing activity, which closely resembled activity of oxidized polyphenolics. Addition of 1% glycine to the extract could completely inhibit these activities. Free glycine may be secreted into the digestive juice by larvae as an adaptive mechanism for chemical defense against its host plants. The protein-denaturing compound in privet leaves is present in the cytosol or in the vacuoles of the leaf cells. The compound does not show protein-denaturing activity without oxidation, but when mixed with intact organelles under low osmotic conditions to give an osmotic shock, a very high protein-denaturing activity is produced. Our results suggest that the privet tree is endowed with a defense mechanism in which a stable compound in the cytosol or in the vacuoles is activated into a chemically active denaturant by an enzyme present in the organelles (including chloroplasts) after the leaves are eaten by insects and the organelles are broken by osmotic shock or by digestive mechanisms. Based upon HPLC and NMR data, we conclude that the denaturing compound is oleuropein, an o-dihydroxyphenolic compound. This compound makes up 3% of the wet weight of privet leaves. The protein-denaturing activity of purified oleuropein activated by the leaf enzyme is high enough to account for all the denaturing activity in a water extract of privet leaves. The denaturing reaction is completely inhibited by free glycine. Our results suggest that the protein-denaturing activity and lysine-decreasing activity of privet leaves are caused by oxidized polyphenolics, and that some insects secrete free glycine to counter the denaturing activity of oxidized phenolics. The chemical mechanism of counteraction by glycine is also considered. Free glycine in the midgut of insects probably protects proteins from denaturation by competing with proteins for oxidized phenolics.     

Thin inter-polyoxide films for flash memories grown at lowtemperature (400°C) by oxygen radicals

Thin polyoxide films grown at 400°C on n⁺-poly-Si films, used as gate insulators in MOS capacitors, are shown to exhibit superior performance, with respect to conventional, thermally grown polyoxide films at 900°C, i.e., they possess lower leakage currents and can sustain higher electrical fields. They are also shown to be superior to, either polyoxide films grown at 980°C on substrates treated by chemical mechanical polishing (CMP) or polyoxide films grown by electron cyclotron resonance (ECR) nitrous oxide plasma at 400°C. The present film growth techniques utilize oxygen radicals (O*), rather than oxygen molecules (O₂), which are used in conventional processing. The oxygen radicals are generated by microwave (2.45 GHz) excited high-density plasma of Kr/O₂ gas mixture     

Delayless subband adaptive filtering using the Hadamard transform

In this correspondence, an analysis of a delayless critically decimated subband adaptive filter structure is presented. In this structure, adaptive weights in each subband are computed by the LMS algorithm and then transformed into those in fullband by the Hadamard transform. It is shown that a stationary point of the proposed algorithm corresponds to the fullband Wiener filter. Some numerical results are also presented to show the performance of this scheme     

Rho/ROCK Pathway and Noncoding RNAs: Implications in Ischemic Stroke and Spinal Cord Injury

Ischemic strokes (IS) and spinal cord injuries (SCI) are major causes of disability. RhoA is a small GTPase protein that activates a downstream effector, ROCK. The up-regulation of the RhoA/ROCK pathway contributes to neuronal apoptosis, neuroinflammation, blood-brain barrier dysfunction, astrogliosis, and axon growth inhibition in IS and SCI. Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), were previously considered to be non-functional. However, they have attracted much attention because they play an essential role in regulating gene expression in physiological and pathological conditions. There is growing evidence that ROCK inhibitors, such as fasudil and VX-210, can reduce injury in IS and SCI in animal models and clinical trials. Recently, it has been reported that miRNAs are decreased in IS and SCI, while lncRNAs are increased. Inhibiting the Rho/ROCK pathway with miRNAs alleviates apoptosis, neuroinflammation, oxidative stress, and axon growth inhibition in IS and SCI. Further studies are required to explore the significance of ncRNAs in IS and SCI and to establish new strategies for preventing and treating these devastating diseases.     

Endo‐α‐Mannosidase‐Catalyzed Transglycosylation

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo ‐ α‐mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo‐α‐mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α‐glycosyl‐fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1– 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high‐mannose‐type undecasaccharide (Glc₁Man₉GlcNAc₂).     

Counter-Rotating Type Tidal Stream Power Unit Mounted on a Mono-pile

The authors have invented a unique counter-rotating type tidal stream power unit, which is composed of tandem propellers and a double rotational armature type generator without a stator. The front and the rear propellers drive, as for an upstream type, the inner and the outer rotational armatures in the counter-rotating directions respectively, which keep the rotational torques counter-balanced between both propellers and armatures. This paper investigates experimentally the output and forces acting on a pile in a water channel, to get design materials of the mono-pile type tidal stream power unit. The output is maximal at the moderate rotational speed, as the same as a wind turbine. The force acting on the pile is affected by the drag, the Karman vortex and the dynamic balances of the tandem propellers, and has dominant frequencies due to not only the individual but also the interacting rotation of the front and the rear propellers.     

On the so-called “semi-ionic” C–F bond character in fluorine–GIC

The short-range structures of fluorine–graphite intercalation compounds with stage-1 structures, C_xF (x = 2.47, 2.84, 3.61), were analyzed by means of neutron diffraction. It has been shown that the so-called “semi-ionic” C–F bond character in C_xF is essentially covalent with the bond length of 0.140 nm, and the original planar graphene sheets are buckled at the sp³-hybridized carbon atoms bound to fluorine atoms. Conjugated C–C double bonds with the bond length of 0.142 nm are preserved between the carbon atoms unbound to fluorine atoms in C_xF, while other C–C bonds are single bonds with the bond lengths of 0.153 nm. The C–F bond order in C_xF is slightly lower than those in poly(carbon monofluoride) ((CF)_n) and poly(dicarbon monofluoride) ((C₂F)_n), which is explained by the hyperconjugation involving the C–C bonds on the carbon sheets and C–F bond.