Engineering Plant Alternative Oxidase Function in Mammalian Cells: Substitution of the Motif-like Sequence ENV for QDT Diminishes Catalytic Activity of Arum concinnatum AOX1a Expressed in HeLa Cells

Alternative oxidase (AOX) is a nonproton motive quinol–oxygen oxidoreductase which is a component of the mitochondrial respiratory chain in higher plants. In this study, we have characterized the catalytic activity and regulatory behaviors of Arum concinnatum AOX isoforms, namely AcoAOX1a and AcoAOX1b, and their artificial mutants in HeLa cells. We demonstrated that substitution of the motif-like sequence ENV on the C-terminal half of AcoAOX1a for QDT diminishes its activity and proposed that the innate inactivity of AcoAOX1b in HeLa cells is, at least in part, attributable to its QDT motif. Furthermore, we show that introduction of F130L in the hydrophilic N-terminal extension of AcoAOX1a resulted in greater activity in the presence of pyruvate. This result indicates that functional significance of the N-terminal extension is not particular to the conventional regulatory cysteine. On the basis of these findings, we discuss new insights into the structural integrity of AOX in HeLa cells and the applicability of mammalian cells for functional analysis of this enzyme.     

Synthesis of a “molecular rope curtain”: Preparation and characterization of a sliding graft copolymer with grafted poly(ethylene glycol) side chains by the “grafting onto” strategy

A sliding graft copolymer (SGC) with poly(ethylene glycol) (PEG) side chains was prepared by ester formation between terminal carboxyl groups of oxidized PEG methyl ether with molecular weight of 2000 (mPEG2000‐COOH) and hydroxyl groups of a polyrotaxane consisting of PEG and cyclodextrins (CDs). Formation of the SGC structure was confirmed by ¹H NMR, attenuated total reflectance Fourier‐transformed infrared, and gel permeation chromatography. The SGC was soluble in good solvents of PEG and insoluble in poor solvents of PEG. Estimation of the number of grafted mPEG chains suggested a “rope‐curtain” like structure, in which an mPEG chain is connected to each CD ring. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Reinvestigation of palladium-catalyzed allylation of the monoacetate of 4-cyclopentene-1,3-diol and synthesis of the coronafacic acid ethyl ester

A larger quantity of a β-keto ester that is 1.5–1.7 equiv more than the base (t-BuOK, NaH) was found to be essential in securing sufficient yields of the products in the palladium-catalyzed allylic substitution of the monoacetate of 4-cyclopentene-1,3-diol with β-keto esters. This requirement also works well for substitutions with the TBS ether of the monoacetate and the monoacetate of 2-cyclohexene-1,4-diol. As an application, the coronafacic acid ethyl ester was synthesized as an optically active form.     

High magnetic field solid‐state NMR analyses by combining MAS,MQ‐MAS,homo‐nuclear and hetero‐nuclear correlation experiments

A general strategy of structural analysis of alumina silicate by combining various solid‐state NMR measurements such as single pulse, multi‐quantum magic angle spinning, double‐quantum homo‐nuclear correlation under magic angle spinning (DQ‐MAS), and cross‐polarization hetero‐nuclear correlation (CP‐HETCOR) was evaluated with the aid of high magnetic field NMR (800 MHz for ¹H Larmor frequency) by using anorthite as a model material. The high magnetic field greatly enhanced resolution of ²⁷Al in single pulse, DQ‐MAS, and even in triple‐quantum magic angle spinning NMR spectra. The spatial proximities through dipolar couplings were probed by the DQ‐MAS methods for homo‐nuclear correlations between both ²⁷Al–²⁷Al and ²⁹Si–²⁹Si and by CP‐HETCOR for hetero‐nuclear correlations between ²⁷Al–²⁹Si in the anorthite framework. By combining various NMR methodologies, we elucidated detailed spatial correlations among various aluminum and silicon species in anorthite that was hard to be determined using conventional analytical methods at low magnetic field. Moreover, the presented approach is applicable to analyze other alumina‐silicate minerals. Copyright © 2012 John Wiley & Sons, Ltd.     

Cyclic sample pooling using two‐dimensional liquid chromatography system enhances coverage in shotgun proteomics

We report a cyclic sample pooling technique devised in two‐dimensional liquid chromatography–electrospray ionization mass spectrometry (LC‐ESI‐MS) shotgun proteomics that renders deeper proteome coverage; we combined low pH reversed‐phase (RP) LC in trifluoroacetic acid in the first dimension, followed by cyclic sample pooling of the eluate and low‐pH RP‐LC in formic acid in the second dimension. The new protocol has a significantly higher resolving power suitable for LC‐ESI‐MS/MS shotgun proteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Combinatorial and approximative analyses in a spatially random division process

For a spatial characteristic, there exist commonly fat-tail frequency distributions of fragment-size and -mass of glass, areas enclosed by city roads, and pore size/volume in random packings. In order to give a new analytical approach for the distributions, we consider a simple model which constructs a fractal-like hierarchical network based on random divisions of rectangles. The stochastic process makes a Markov chain and corresponds to directional random walks with splitting into four particles. We derive a combinatorial analytical form and its continuous approximation for the distribution of rectangle areas, and numerically show a good fitting with the actual distribution in the averaging behavior of the divisions.     

Adsorption of ammonia and water on functionalized edge-rich carbon nanofibers

The preparation, characterization and ammonia and water adsorption properties of edge-rich carbon nanofibers (CNFs) were studied, including platelet CNFs (PCNFs) and cup-stacked CNFs (CSCNFs). Since PCNFs and CSCNFs have many chemically active exposed edges, functionalization by oxidizing the edges was carried out by ozone stream and by nitric acid. Transmission electron microscopy, N₂ adsorption isotherms and temperature-programmed desorption analysis showed that the nitric acid treatment partly destroyed the graphite structure of the PCNFs and created acid functional groups and micropores, whereas the ozone treatment created functional groups without damaging the structure. Ammonia adsorption isotherms clarified that NH₃ adsorption on PCNFs and CSCNFs occurred mainly on oxygen-containing groups, whereas the adsorption on activated carbon fibers (ACFs) occurred on both oxygen-containing groups and the carbon surface without the functional groups, and the CSCNFs showed larger amounts of adsorbed ammonia compared to the PCNFs. Especially at a relatively low pressure range (<0.2 atm), the PCNFs/CSCNFs/ACFs showed the same ammonia adsorption mechanism; that is, the one-to-one interaction between oxygen atoms in the functional groups and hydrogen atoms in ammonia molecules. In addition, the adsorption on the ACFs appeared to occur mainly by interaction with the carbon surface at relatively high pressure (0.3–1.0 atm). Our experimental results and previous findings suggest that NH₃ adsorption on PCNFs is due mainly to NH…O hydrogen bonding between oxygen-containing groups and ammonia rather than to chemical bonding.