Characterization of a Fungal Thioesterase Having Claisen Cyclase and Deacetylase Activities in Melanin Biosynthesis

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Highlights? Revised C. lagenarium Pks1 coding sequence enabling in vitro analysis ? Clarified third fungal route to dihydroxynaphthalene melanin ? Revealed thioesterase-catalyzed tandem Claisen cyclization and deacetylation ? Lid swap chimera illuminate features necessary for C-C bond making and breaking     

The role of molecular chaperonins in warm ischemia and reperfusion injury in the steatotic liver: A proteomic study

ABSTRACT: BACKGROUND: The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software. RESULTS: The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R. CONCLUSION: Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.     

Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B(1)

The biosynthesis of the potent environmental carcinogen aflatoxin B(1) is initiated by norsolorinic acid synthase (NorS), a complex of an iterative type I polyketide synthase and a specialized yeast-like pair of fatty acid synthases. NorS has been partially purified from Aspergillus parasiticus, has been found to have a mass of approximately 1.4 x 10(6) Da, and carries out the synthesis of norsolorinic acid in the presence of acetylCoA, malonylCoA, and NADPH where hexanoylCoA is not a free intermediate. The N-acetylcysteamine thioester of hexanoic acid can substitute for the catalytic functions of HexA/B to initiate norsolorinic acid synthesis by the complex in the presence of only malonylCoA. An alpha(2)beta(2)gamma(2) stoichiometry is proposed for NorS in keeping with its estimated mass and the observed dimeric or higher-order quarternary structures of PKS and FAS enzymes.     

Luminescence detection of phase transitions,local environment and nanoparticle inclusions

Luminescence data offer delicate probes of changes in structure and local environment in insulating materials. Therefore they have long been employed in studies of imperfections and characterisation of lattice distortions. Luminescence techniques are inherently very sensitive so respond to small concentrations of intrinsic defects and impurities, and intentionally added probe ions, such as rare earth or chromium ions, are particularly effective at displaying responsive changes linked to modified structural environments. Parameters of interest are variations in luminescence efficiency, the details of the emission spectra, polarisation, temperature dependence and changes in excited state lifetimes. These are suitable properties to monitor variations in both short and long range lattice structure, composition, pressure and temperature. For modern photonic materials such probes are powerful tools to follow changes introduced by processing to make waveguides, surface layers and new materials. The use of different excitation conditions for the luminescence can resolve differences between near surface and bulk features. A less common approach is to use luminescence as a route to detect phase transitions. This is particularly valuable for rapid survey studies of new materials which are of relevance to modern optics. One of the more surprising results is the observation that phase changes of very small inclusions, such as phase precipitates or impurity nanoparticles, can totally dominate the luminescence response of the bulk material. Specific examples include the frequent observation of trapped nanoparticles of water, N₂, O₂ and CO₂. Further, the excitation techniques, and ion implantation or surface stresses can all induce surface relaxations which, in materials such as SrTiO₃ or ZnO, result in phase changes propagating throughout the entire crystal. This review rapidly recalls the methods used in the older studies of imperfections and then uses a range of examples to show how luminescence studies can be effective in detecting and confirming the presence of phase transitions. Interestingly the examples include not only transparent insulating materials but also fullerenes and superconductors.     

Interrogation of global active site occupancy of a fungal iterative polyketide synthase reveals strategies for maintaining biosynthetic fidelity

Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using the norsolorinic acid anthrone-producing polyketide synthase, PksA, from the aflatoxin biosynthetic pathway in Aspergillus parasiticus. Here we report the simultaneous observation of covalent intermediates from all catalytic domains of PksA from in vitro reconstitution reactions. The data provide snapshots of iterative catalysis and reveal an underappreciated editing function for the C-terminal thioesterase domain beyond its recently established synthetic role in Claisen/Dieckmann cyclization and product release. The specificity of thioesterase catalyzed hydrolysis was explored using biosynthetically relevant protein-bound and small molecule acyl substrates and demonstrated activity against hexanoyl and acetyl, but not malonyl. Processivity of polyketide extension was supported by the inability of a nonhydrolyzable malonyl analog to trap products of intermediate chain lengths and by the detection of only fully extended species observed covalently bound to, and as the predominant products released by, PksA. High occupancy of the malonyl transacylase domain and fast relative rate of malonyl transfer compared to starter unit transfer indicate that rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation.     

Nuclear data for space radiation

Human space flight requires protecting astronauts from the harmful effects of space radiation. The availability of measured nuclear cross-section data needed for these studies is reviewed in the present paper. The energy range of interest for radiation protection is approximately 100 MeV/n–10 GeV/n. The majority of data are for projectile fragmentation partial and total cross-sections, including both charge changing and isotopic cross-sections. The cross-section data are organized into categories which include charge changing, elemental, isotopic for total, single and double differential with respect to momentum, energy and angle. Gaps in the data relevant to space radiation protection are discussed and recommendations for future experiments are made.     

Anyons from strings

The Nambu-Goto string in a three-dimensional (3D) Minkowski spacetime is quantized preserving Lorentz invariance and parity. The spectrum of massive states contains anyons. An ambiguity in the ground state energy is resolved by the 3D N=1 Green-Schwarz superstring, which has massless ground states describing a dilaton and dilatino, and first-excited states of spin 1/4.     

Performance of a quarter-wavelength particle concentrator

A series of devices have been investigated which use acoustic radiation forces to concentrate micron sized particles. These multi-layered resonators use a quarter-wavelength resonance in order to position an acoustic pressure node close to the top surface of a fluid layer such that particles migrate towards this surface. As flow-through devices, it is then possible to collect a concentrate of particulates by drawing off the particle stream and separating it from the clarified fluid and so can operate continuously as opposed to batch processes such as centrifugation. The methods of construction are described which include a micro-fabricated, wet-etched device and a modular device fabricated using a micro-mill. These use silicon and macor, a machinable glass ceramic, as a carrier layer between the transducer and fluid channel, respectively. Simulations using an acoustic impedance transfer model are used to determine the influence of various design parameters on the acoustic energy density within the fluid layer and the nodal position. Concentration tests have shown up to 4.4-, 6.0- and 3.2-fold increases in concentration for 9, 3 and 1 microm diameter polystyrene particles, respectively. The effect of voltage and fluid flow rates on concentration performance is investigated and helps demonstrate the various factors which determine the increase in concentration possible.     

Phase matching considerations in second harmonic generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology

We present a heuristic treatment which relates SHG image intensities, signal directionality, and observed morphology to the physical structure of collagen and cellulose fibrillar tissues. The SHG creation model is based upon relaxed phase matching conditions which account for dispersion, randomness, and axial momentum contributions from the media, and includes a mathematical treatment which relates SHG conversion efficiency to fibril diameter and packing through the inclusion of potential intensity amplification resultant from quasi-phase matching (QPM). A direct consequence of this theory is that SHG in biological tissues is not strictly a coherent process, and that the forward directed SHG has a longer coherence length than the backward component, Through this treatment, we show that the emission directionality and also conversion efficiency do not arise solely from the fibril size but also depend on packing density and order of the inter-fibril structure. We demonstrate these principles in comparing the SHG response in normal and Osteogenesis Imperfecta (OI) skin. We show that the observed directionality and decreased relative intensity in the diseased state is consistent with phase matching conditions arising from the decreased fibril size and more random assembly. We further use this theory to explain the differences in morphology seen in forward and backward collected SHG in fibrillar tissues (e.g., collagenous and cellulosic). Specifically, we attribute segmented appearance to destructive interference between small fibrils separated by less than the coherence length. We suggest the approach based on relaxed phasematching conditions is general in predicting the SHG response in tissues and may be broadly applicable in interpreting the SHG contrast for diagnostic applications.     

Landau levels and Riemann zeros

The number N(E) of complex zeros of the Riemann zeta function with positive imaginary part less than E is the sum of a "smooth" function N[over ](E) and a "fluctuation." Berry and Keating have shown that the asymptotic expansion of N[over ](E) counts states of positive energy less than E in a "regularized" semiclassical model with classical Hamiltonian H=xp. For a different regularization, Connes has shown that it counts states "missing" from a continuum. Here we show how the "absorption spectrum" model of Connes emerges as the lowest Landau level limit of a specific quantum-mechanical model for a charged particle on a planar surface in an electric potential and uniform magnetic field. We suggest a role for the higher Landau levels in the fluctuation part of N(E).