Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis

We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA. In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities. We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli. To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution. We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies. Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region. Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis. Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5. These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region.     

An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4R,6R)‐Actinol in a One‐Pot Reduction System

(4R,6R)‐Actinol can be stereo‐selectively synthesized from ketoisophorone by a two‐step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)‐levodione reductase. However, (4S)‐phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p‐HBA, and introduced point mutations into the substrate‐recognition loop. The most effective mutant (P295G) showed two‐ and 12‐fold higher catalytic activities toward ketoisophorone and (4S)‐phorenol, respectively, than the wild‐type, and improved the yield of the two‐step conversion from 67.2 to 90.1 %. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate‐recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.     

Influence of pH on the synthesis and properties of polypyrrole on copper from phytic acid solution for corrosion protection

Polypyrrole (PPy) films were deposited on copper from “green” inhibitor of phytic acid solution for corrosion protection of copper. The corrosion protection property of the PPy layer was studied by an immersion test in a NaCl aqueous solution. The polymerization process of PPy on copper changed with the pH values of phytic acid solution and current density applied. When one oxidized bare copper in phytic acid solution at various pHs containing pyrrole monomer, a thin layer consisting of complex compound of Cu-phytate was firstly formed, followed by the formation of the PPy layer doped with phytate anion on the complex compound layer. The complex compound layer passivated the copper surface and its thickness increased with the lower pH value of the solution and the lower current density applied. It was found that the PPy coating prepared in the phytic acid solution at pH 4 exhibit the most protective property against copper corrosion.     

Deposition of Metal Oxide Films from Metal–EDTA Complexes by Flame Spray Technique

R₂O₃ (R = Y, Eu, Er) metal oxides were synthesized from metal–ethylenediaminetetraacetic acid (EDTA) complexes using a flame spray technique. As this technique enables high deposition rates, films with thickness of several tens of micrometers were obtained. Films of yttria, europia, and erbia phase were synthesized on stainless-steel substrates with reaction assistance by H₂–O₂ combustion gas. The oxide films consisted of the desired crystalline phase with micropores. The porosity of the films was in the range of 6–15%, varying with the metal used. These results suggest that the true density of the metal oxide obtained from metal–EDTA powder through the thermal reaction process plays an important role in achieving film with the desired porosity.     

Investigation of the cause of peculiar irradiation behavior of 9Cr-ODS steel in BOR-60 irradiation tests

Four experimental fuel assemblies (EFAs) containing 9Cr-ODS steel cladding fuel pins were previously irradiated in the BOR-60 to demonstrate the in-reactor performance of 9Cr-ODS steel for use as fuel cladding tubes. One of the EFAs achieved the best data, a peak burn-up of 11.9at% and a neutron dose of 51 dpa, without any microstructure instability or any fuel pin rupture. On the other hand, in another EFA (peak burn-up, 10.5at%; peak neutron dose, 44 dpa), peculiar irradiation behaviors, such as microstructure instability and fuel pin rupture, occurred. Investigations of the cause of these peculiar irradiation behaviors were carried out. The detection sensitivity in an ultrasonic inspection test was shown to be low for the metallic Cr and metallic Fe inclusions. The peculiar microstructure change reappeared with high-temperature thermal-aging of the 9Cr-ODS steel containing metallic Cr inclusions. The strength and ductility of the defective part containing metallic Cr inclusions were appreciably lower than those of a standard part without the inclusions. The combined effects of matrix Cr heterogeneity (presence of metallic Cr inclusions) and high-temperature irradiation were concluded to be the main cause of the peculiar microstructure change in 9Cr-ODS steel cladding tubes in the BOR-60 irradiation tests. They contributed to the fuel pin rupture.     

Novel Polarimetric Technique Exploring Spatiotemporal Birefringent Response of an Anti-ferroelectric Liquid Crystal Cell

Abstract

This paper describes a novel polarimetric technique for mapping the spatiotemporal birefringent parameters characterizing an anti-ferroelectric liquid crystal cell. A linearly polarized beam of light transmitted by the liquid crystal cell turns into an elliptically polarized beam. Some birefringent irregularities existing over the cell make state of polarization of the elliptically polarized beam of light be spatiotemporal. This beam works as a signal beam to interfere with a reference beam consisting of two orthogonal linearly polarized components. The resultant interference pattern is taken by a charge-coupled device video camera and recorded in a computer. The computer gives the information required for determining the spatiotemporal birefringent parameters that characterize the liquid crystal cell. The major advantage is that the two-dimensional distribution of the birefringent axes as well as the two principal refractive indices can be determined simultaneously, and no use of any optical components for polarization alignment makes it possible to follow a rapid change in birefringent parameters within the maximum frame rate of the video camera. A change in time-dependent birefringent parameter is measured at every 0·1 ms for the demonstration experiment.     

Intra-arterial infusion chemotherapy for advanced endometrial cancer before surgical treatment

Nine cases of advanced uterine body cancer (stage III: 8, stage IVa: 1) were treated by intra-arterial infusion chemotherapy before curative operation. This treatment produced primary tumors smaller than half sizes in eight cases. Necrotic changes were found in over two-thirds of the lesions in six cases. This chemotherapy enabled us to operate curatively in seven cases. After the operations, we performed various types of treatment including intra-arterial infusion chemotherapy. No evidences of disease have been found in four cases, but there is no significant difference between the groups receiving and not receiving the intra-arterial infusion chemotherapy (n = 16) in terms of survival rate of stage III. Further study of the prognosis is necessary.     

The role of surface lysines in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody

The roles of four VH surface lysine side-chains in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. Three of the four lysines (at positions H62, H64 and H66) are strongly conserved; however, the presence of lysine at position H68 was highly unusual. Each lysine was replaced individually by alanine; in addition, a quadruple mutant was also created. Studies of the binding kinetics revealed that these lysine residues mainly influenced the association phase. Our results suggest that these side chains help to guide the DNA polyanion to the antigen binding pocket by electrostatic effects.     

Effect of Oxygen Segregation at Grain Boundaries on Deformation of B, C-Doped Silicon Carbides at Elevated Temperatures

The effect of oxygen segregation at grain boundaries on the deformation of 1 wt% boron (B)- and carbon (C)-doped β-silicon carbide (B, C-doped SiC) was investigated by compression testing at 2073 K. We studied the deformation of sinter-forged B, C-doped SiC (sinter-forged SiC), which contained the minimum amount (0.07 wt%) of oxygen as an impurity, and that of hot isostatically pressed B, C-doped SiC (HIPed SiC), which contained 1 wt% oxygen. Oxygen was detected at grain boundary in HIPed SiC by energy-dispersive X-ray spectroscopy, but it was not detected in sinter-forged SiC. The strain rate of sinter-forged SiC was one order of magnitude lower than that of HIPed SiC at the same grain size. The grain growth rate of sinter-forged SiC was lower than that of HIPed SiC also. These results suggest that the oxygen segregation at grain boundaries, together with boron segregation, promoted the grain-boundary diffusion in B, C-doped SiC. But, the oxygen segregation without boron was less effective in promoting deformation than the boron segregation without oxygen.