In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.     

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases

Spectroscopic characterization of Ag^I-ion-mediated C-Ag^I-A and C-Ag^I-T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by Ag^I ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of Ag^I ions is in the order C-A≈C-T>C-C, the stabilizing effect of Ag^I ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to Ag^I-mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.     

‘Lipase and protease production of dairy Penicillium sp. on milk‐protein‐based solid substrates’

The lipolytic and proteolytic activity of Penicillium camemberti PC TT033 and Penicillium roqueforti PR G3, cultured on the whey solids or simulated cheese media, were compared under several pH reaction conditions. Lipolytic activity was higher when both strains had been cultured on the whey medium than on the simulated cheese medium, whereas proteolytic activity was less influenced by the culture medium. The relationship between the reaction pH and these enzyme activities was dependent on the culture medium, which suggested that the expression level and balance of isozyme rely on the culture substrate.     

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.     

Processing of Ultrafine-Grained Materials through the Application of Severe Plastic Deformation

Processing through the application of severe plastic deformation (SPD) has become an absorbing tool because it provides the potential for refining the grain size of polycrystalline bulk metals to the submicrometer or even the nanometer level. Several SPD methods are now available, but the more promising procedures seem to be equal-channel angular pressing (ECAP) and high-pressure torsion (HPT). This report examines these procedures with an emphasis on the principles of grain refinement and the mechanical properties developed at high temperatures in materials after processing by ECAP and the hardness homogeneity and microstructural evolution in materials processed by HPT. Moreover, recent observations on the ECAP and HPT processing also are discussed.     

Achieving homogeneity in a Cu–Zr alloy processed by high-pressure torsion

A copper alloy, Cu–0.1 %Zr, was subjected to severe plastic deformation at room temperature using quasi-constrained high-pressure torsion. Disks were strained through different numbers of revolutions up to a maximum of ten turns under an applied pressure of 6.0 GPa and then examined to evaluate the evolution in the Vickers microhardness, Hv, and the microstructure. The results show lower values of Hv in the center regions of the disks in the early stages of processing but a gradual evolution to a high degree of hardness homogeneity after five and ten turns. Under conditions of hardness homogeneity, the distributions of the grain boundary misorientations are essentially identical at the center and the periphery of the sample. Homogeneity was further confirmed by conducting tensile testing at elevated temperatures where similar stress–strain curves and similar elongations to failure were recorded after processing through five and ten turns of HPT.