Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.     

Characteristics of listener sensitivity to talker-specific phonetic detail

Previous research shows that listeners are sensitive to talker differences in phonetic properties of speech, including voice-onset-time (VOT) in word-initial voiceless stop consonants, and that learning how a talker produces one voiceless stop transfers to another word with the same voiceless stop [Allen, J. S., and Miller, J. L. (2004). J. Acoust. Soc. Am. 115, 3171-3183]. The present experiments examined whether transfer extends to words that begin with different voiceless stops. During training, listeners heard two talkers produce a given voiceless-initial word (e.g., pain). VOTs were manipulated such that one talker produced the voiceless stop with relatively short VOTs and the other with relatively long VOTs. At test, listeners heard a short- and long-VOT variant of the same word (e.g., pain) or a word beginning with a different voiceless stop (e.g., cane or coal), and were asked to select which of the two VOT variants was most representative of a given talker. In all conditions, which variant was selected at test was in line with listeners' exposure during training, and the effect was equally strong for the novel word and the training word. These findings suggest that accommodating talker-specific phonetic detail does not require exposure to each individual phonetic segment.     

Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586

The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.     

Catalytic C-H amination with aromatic amines

Aniline joins the club: A β-diketiminato copper(I) catalyst enables C-H amination of anilines employing low catalyst loadings to preclude oxidation to the diazene ArN=NAr. Electron-poor anilines are particularly resistant towards diazene formation and participate in the amination of strong and unactivated C-H bonds. N-alkyl anilines also take part in C-H amination.     

Investigation into omacetaxine solution stability for in vitro study

Omacetaxine is a natural product extract originating from Chinese medicine and finding therapeutic use as a potent myelosuppressive agent in leukemia. When planning in vitro cell biology experiments to assess omacetaxine activity against primary leukemic stem cells, it became apparent that the literature rarely describes the in vitro stability of the molecule, although accessible chromatographic methods have been published. Clearly whole organisms vs their component cells will differ in the way in which they handle xenobiotics, with the latter more dependent on physiochemical parameters such as pH and temperature in the absence of active metabolism or excretion. This could impact on the cells' experience of drug in culture. We therefore report here on examination of a modified, high‐performance liquid chromatography (HPLC) method with assessment of degradant production from a 72 h solution stability study, clearly demonstrating that omacetaxine is highly stable in representative cell culture conditions (37 °C, neutral pH) and persists for many days in marked contrast to its short‐half life in vivo. Copyright © 2011 John Wiley & Sons, Ltd.     

Magnetic discontinuities in magnetohydrodynamic turbulence and in the solar wind

Recent measurements of solar wind turbulence report the presence of intermittent, exponentially distributed angular discontinuities in the magnetic field. In this Letter, we study whether such discontinuities can be produced by magnetohydrodynamic (MHD) turbulence. We detect the discontinuities by measuring the fluctuations of the magnetic field direction, Δθ, across fixed spatial increments Δx in direct numerical simulations of MHD turbulence with an imposed uniform guide field B(0). A large region of the probability density function (pdf) for Δθ is found to follow an exponential decay, proportional to exp(-Δθ/θ(*)), with characteristic angle θ(*)≈(14°)(b(rms)/B(0))(0.65) for a broad range of guide-field strengths. We find that discontinuities observed in the solar wind can be reproduced by MHD turbulence with reasonable ratios of b(rms)/B(0). We also observe an excess of small angular discontinuities when Δx becomes small, possibly indicating an increasing statistical significance of dissipation-scale structures. The structure of the pdf in this case closely resembles the two-population pdf seen in the solar wind. We thus propose that strong discontinuities are associated with inertial-range MHD turbulence, while weak discontinuities emerge from dissipation-range turbulence. In addition, we find that the structure functions of the magnetic field direction exhibit anomalous scaling exponents, which indicates the existence of intermittent structures.     

Atom-scale ptychographic electron diffractive imaging of boron nitride cones

Ptychographic coherent diffractive imaging (CDI) has been extensively applied using both x rays and electrons. The extension to atomic resolution has been elusive. This Letter demonstrates ptychographic electron diffractive imaging at atomic resolution, permitting identification of structure in a boron nitride helical cone at a resolution of order 1 ?, beyond that of comparative Z-contrast images. A scanning transmission electron microscope is used to create a diverging illumination in a defocused Fresnel CDI geometry, providing a robust strategy leading to a unique solution.     

A new application of scanning electrochemical microscopy for the label-free interrogation of antibody–antigen interactions: Part 2

Within this paper we describe the use of scanning electrochemical microscopy (SECM) to fabricate a dotted array of biotinylated polyethyleneimine which was then used to immobilise first neutravidin and then a biotinylated antibody towards a relevant antigen of interest (PSA, NTx, ciprofloxacin). These antigens were selected both for their clinical relevance but also since they display a broad range of molecular weights, to determine whether the size of the antigen used effects the sensitivity of this approach. The SECM was then used to image the binding of both complementary and non-complementary antigens in a label-free assay. Imaging of the arrays before and following exposure to various concentrations of antigen in buffer showed clear evidence for specific binding of the complementary antigens to the antibody functionalised dots. Non-specific binding was also quantified by control experiments with other antigens. This demonstrated non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen of interest at the surface of the dots. The binding of ciprofloxacin was investigated both in simple buffer solution and in a more complex media, bovine milk.     

The role of C-H and C-C stretching modes in the intrinsic non-radiative decay of triplet states in a Pt-containing conjugated phenylene ethynylene

The intrinsic non-radiative decay (internal conversion) from the triplet excited state in phosphorescent dyes can be described by a multi-phonon emission process. Since non-radiative decay of triplet excitons can be a significant process in organic light-emitting diodes, a detailed understanding of this decay mechanism is important if the overall device efficiency is to be controlled. We compare a deuterated Pt(II)-containing phenylene ethynylene with its non-deuterated counterpart in order to investigate which phonon modes control to the non-radiative decay path. We observe that deuteration does not decrease the non-radiative decay rate. A Franck-Condon analysis of the phosphorescence spectra shows that the electronic excitation is coupled strongly to the breathing mode of the phenyl ring and the C≡C carbon stretching modes, while high-energy C-H or C-D stretching modes play an insignificant role. We, therefore, associate the internal conversion process with the carbon-carbon stretching vibrations.