N-terminal specific fluorescence labeling of proteins through incorporation of fluorescent hydroxy acid and subsequent ester cleavage

We have developed a novel method to attach a fluorescent label at the N terminus of proteins through a four-base codon-mediated incorporation of a fluorescent hydroxy acid and subsequent cleavage of the ester bond in a cell-free translation system. We found that a fluorescent-labeled p-amino-L-phenyllactic acid was successfully incorporated downstream of N-terminal tag peptides in response to a CGGG codon, and the tag peptides could be removed through ester cleavage to leave the fluorescent hydroxy acid at the N terminus of the proteins. Immunoprecipitation analysis revealed that ester cleavage occurred spontaneously during the translation reaction. The efficiency of the ester cleavage and the yield of the labeled proteins were dependent on the peptide tag sequence. We demonstrate that the insertion of an asparagine residue between the N-terminal T7 tag and the fluorescent hydroxy acid achieved both quantitative ester cleavage and efficient expression of the labeled proteins. The present method is a potential tool for N-terminal specific labeling of proteins with various compounds.     

Vapor-Phase Dehydration of 1,3-butanediol over CeO2–ZrO2 Catalysts

The dehydration of 1,3-butanediol was investigated over CeO₂–ZrO₂ catalysts prepared by impregnation at temperatures of 325–375 °C. Pure CeO₂ selectively catalyzed the dehydration of 1,3-butanediol to form 3-buten-2-ol and 2-buten-1-ol, while pure ZrO₂, which was less active than pure CeO₂, catalyzed the dehydration to 3-buten-1-ol. In the CeO₂/ZrO₂ catalyst in which CeO₂ was supported on zirconia, the presence of a small amount of CeO₂ suppressed the formation of 3-buten-1-ol and induced the dehydration of 1,3-butanediol to form 3-buten-2-ol and 2-buten-1-ol and the subsequent dehydrogenation of 3-buten-2-ol to form 3-buten-2-one and butanone. The activity would be related to the redox features of CeO₂. The monoclinic phase of zirconia support decreased while the cubic CeO₂ phase increased as CeO₂ content was increased. In contrast, in the ZrO₂/CeO₂ catalyst in which ZrO₂ was supported on cubic CeO₂, only the cubic CeO₂ phase was observed and ZrO₂ species appeared in the form of a solid solution of CeO₂–ZrO₂ with fluorite structure. Regardless of zirconia loading, ZrO₂ species did not affect the catalytic activity of ZrO₂/CeO₂, which was controlled by CeO₂ species.     

Towards Tuneable Retaining Glycosidase‐Inhibiting Peptides by Mimicry of a Plant Flavonol Warhead

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in‐depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α‐amylase (HPA). Notably, incorporation of l ‐DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (K_i=480 pm ) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural‐product‐derived motif, already specific for the catalytic side‐chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.     

Analysis of the tilt angle of the moiré fringes produced by two inclined gratings in talbot interferometry

Abstract

Features of the moiré fringes produced by the inclined grating planes in Talbot interferometry are studied under the illumination of a plane wave. Inclinations of the two grating planes are introduced by rotating the beam-splitter and the detector gratings around the axes parallel to the line directions of each grating, when the line directions of the two grating are different. Theoretical analyses indicate that the tilt angle of the moiré fringes is sensitive to the inclination angle, and the analyses are supported by experimental results. Some simple and practical methods for judging and removing the inclinations are also discussed.     

Energy‐saving Technologies for Automobiles

Since entering the 21st century, we have been facing a globally urgent need for all nations, without exception, to deal with environmental concerns such as global warming and energy issues arising from the depletion of fossil fuels. As a countermeasure, next‐generation vehicles for a recycling‐based society that are energy‐efficient, compact, light, low‐cost and reliable need to spread in the society. In this study, we will explain the basic philosophy of how to approach next‐generation energy‐saving technologies for automobiles, the current status and technological issues of internal combustion engines (ICE), hybrid electric vehicles (HEVs), electric vehicles (EVs), and fuel cell vehicles (FCVs) with the latest examples. Copyright © 2008 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.     

Automatic FeO activity determinator for slag control

An automatic facility for rapid determination of the activities of FeO in metallurgical slags has been developed, by employing an electrochemical technique incorporating stabilized zirconia as solid electrolyte, a mixture of Mo + MoO₂ as reference electrode and an Mo rod as an electrical contact. With this equipment, one datum is obtainable within 5 minutes. In the present article, discussions were held on potential applications of this system to steelmaking process control. A particular emphasis is given to the control of FeO levels in slags used for secondary steelmaking and chromium levels in stainless steelmaking slags.     

Charge trapping characteristics of Al2O3/Al-rich Al2O3/SiO2 stacked films fabricated by radio-frequency magnetron co-sputtering

A thin-film structure comprising Al₂O₃/Al-rich Al₂O₃/SiO₂ was fabricated on Si substrate. We used radio-frequency magnetron co-sputtering with Al metal plates set on an Al₂O₃ target to fabricate the Al-rich Al₂O₃ thin film, which is used as a charge storage layer for nonvolatile Al₂O₃ memory. We investigated the charge trapping characteristics of the film. When the applied voltage between the gate and the substrate is increased, the hysteresis window of capacitance-voltage (C-V) characteristics becomes larger, which is caused by the charge trapping in the film. For a fabricated Al-O capacitor structure, we clarified experimentally that the maximum capacitance in the C-V hysteresis agrees well with the series capacitance of insulators and that the minimum capacitance agrees well with the series capacitance of the semiconductor depletion layer and stacked insulator. When the Al content in the Al-rich Al₂O₃ is increased, a large charge trap density is obtained. When the Al content in the Al-O is changed from 40 to 58%, the charge trap density increases from 0 to 18 × 10¹⁸ cm^− 3, which is 2.6 times larger than that of the trap memory using SiN as the charge storage layer. The device structure would be promising for low-cost nonvolatile memory.     

Corrosion products of tin in humid air containing sulfur dioxide and nitrogen dioxide at room temperature

To obtain a fundamental understanding of the corrosion behavior of tin in corrosive gas environments, in situ infrared reflection absorption spectroscopy measurements were carried out on tin in humid air containing SO₂ and NO₂ at room temperature. A series of time-resolved in situ IR spectra in air of 90% relative humidity (RH) containing 10-22 ppm SO₂ suggested that the oxide films on tin specimens had a protective effect and that no significant corrosion occurred. The corrosion products in air of 80-90% RH containing 10-22 ppm NO₂ were SnO₂, SnO, nitrate and hyponitrite. The synergistic effect of SO₂ and NO₂ on corrosion of tin was not observed in humid air (RH of 90%) containing 0.84 ppm SO₂ and 1.8 ppm NO₂.     

Occurrence and production of chloramines in the chlorination of creatinine in aqueous solution

Occurrence and production of stable chloramines in the chlorination of creatinine, a constituent of perspiration and urine, in aqueous media were studied. Creatinine (5 x 10(-5)M) was treated with free chlorine in aqueous solutions at molar ratios of 0.5-8 (chlorine/creatinine) at pH 7.0 at room temperature for several days. At lower ratios of chlorine, two stable N-chlorocreatinine derivatives, which were determined as dichloramine fractions by the DPD method, were isolated by HPLC and identified by EI-MS and (1)H-NMR. One was 2-chloroamino-1-methylimidazolin-4-one (creatinine chloramine) and the other was 2-chloroamino-5-hydroxy-1-methylimidazolin-4-one (hydroxycreatinine chloramine). In addition, the formation of methylamine was identified by GC-MS analyses of its imine derivative formed with pentafluorobenzaldehyde. Methylamine forms stable chloramines, which might be determined as mono- and/or di-chloramine fractions together with free chlorine by the DPD method in the reaction mixtures at higher molar ratios of chlorine. In practice, small amounts of methylamine (ca. 19 microg/L) were detected in water samples collected from several swimming pools. Hence, methylamine may be an origin of elusive organic chloramine formed in the chlorination of swimming pools. A probable mechanism of the occurrence and processing of chlorination products of creatinine is suggested.     

Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF1: enzymatic analysis related to C1 metabolism

Formaldehyde is a highly toxic compound to most living organisms. We have isolated a bacterial strain that is able to efficiently degrade formaldehyde and use it as a sole carbon source. The isolated strain was identified as Methylobacterium sp. MF1, which could grow on formaldehyde and methanol. Methylobacterium sp. MF1 was grown in batch culture using 1.2 g/l formaldehyde as a sole carbon source, which was all consumed within 200 h. In order to decompose formaldehyde more efficiently, formaldehyde-limited chemostat cultivation of Methylobacterium sp. MF1 was investigated. Formaldehyde was consumed at 1.7 g/l/d when the dilution rate was 0.012 h(-1). Under these conditions, the cell turbidity (OD610) reached 2.0. Furthermore, when the initial turbidity was adjusted to 3.0 using methanol-grown cells, continuous cultivation could be started at an initial dilution rate of 0.008 h(-1). Using these conditions, consumption of formaldehyde could be continued for at least 600 h. The enzyme activities of cells growing as a chemostat culture, using methanol or formaldehyde as a sole carbon source, were compared to that of C1 metabolism. No difference was detected in the enzyme activities for the oxidation and assimilation of C1 compounds between the two cell-free extracts. Furthermore, methanol dehydrogenase activity was detected at the same level when formaldehyde was used as a sole carbon source. These results suggest that the resistance to the toxic effects of formaldehyde exhibited by Methylobacterium sp. MF1 is related to factors other than C1 metabolism.