Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of 8–12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.

On subspace lattices I. Closedness type properties and tensor products

We study properties of subspace lattices related to the continuity of the map Lat and the notion of reflexivity. We characterize various “closedness” properties in different ways and give the hierarchy between them. We investigate several properties related to tensor products of subspace lattices and show that the tensor product of the projection lattices of two von Neumann algebras, one of which is injective, is reflexive.

The complex array of bradykinin-related peptides (BRPs) in the peptidome of pickerel frog (Rana palustris) skin secretion is the product of transcriptional economy.

Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.

Antioxidant properties of kilned and roasted malts

Compounds possessing antioxidant activity play a crucial role in delaying or preventing lipid oxidation in foods and beverages during processing and storage. Such reactions lead to loss of product quality, especially as a consequence of off-flavor formation. The aim of this study was to determine the antioxidant activity of kilned (standard) and roasted (speciality) malts in relation to phenolic compounds, sugars, amino acids, and color [assessed as European Brewing Convention units (degrees EBC) and absorbance at 420 nm]. The concentrations of sugars and amino acids decreased with the intensity of the applied heat treatment, and this was attributed to the extent of the Maillard reaction, as well as sugar caramelization, in the highly roasted malts. Proline, followed by glutamine, was the most abundant free amino/imino acid in the malt samples, except those that were highly roasted, and maltose was the most abundant sugar in all malts. Levels of total phenolic compounds decreased with heat treatment. Catechin and ferulic acid were the most abundant phenolic compounds in the majority of the malts, and amounts were highest in the kilned samples. In highly roasted malts, degradation products of ferulic acid were identified. Antioxidant activity increased with the intensity of heating, in parallel with color formation, and was significantly higher for roasted malts compared to kilned malts. In kilned malts, phenolic compounds were the main identified contributors to antioxidant activity, with Maillard reaction products also playing a role. In roasted malts, Maillard reaction products were responsible for the majority of the antioxidant activity.

Fermentation of heated gluten systems by gut microflora

The Maillard reaction causes changes to protein structure and occurs in foods mainly during thermal treatment. Melanoidins, the final products of the Maillard reaction, may enter the gastrointestinal tract, which is populated by different species of bacteria. In this study, melanoidins were prepared from gluten and glucose. Their effect on the growth of faecal bacteria was determined in culture with genotype and phenotype probes to identify the different species involved. Analysis of peptic and tryptic digests showed that low molecular mass products are formed from the degradation of melanoidins. Results showed a change in the growth of bacteria. This in vitro study demonstrated that melanoidins, prepared from gluten and glucose, affect the growth of the gut microflora.

Analysis of hop acids by capillary electrophoresis

Micellar electrokinetic. chromatography (MEKC) was used to separate components of hop extracts. The separation of a sample of iso-alpha -acids by MEKC was better and faster than by an established HPLC method, giving <0.8 % RSD on migration times and 5-10% RSD on peak areas. MEKC was also successfully used to separate the oxidation products of the - and beta -acids and thus to monitor the stability of hop products containing them. Furthermore, MEKC distinguished among samples of reduced iso-alpha -acids (rho-, tetrahydro- and hexahydro- derivatives). (C) 2001 Elsevier Science Ltd. All rights reserved.

Gas-phase photocatalytic oxidation of dichlorobutenes

Gas-phase photocatalysis of 1,4-dichlorobut-2-enes and 3,4-dichlorobut-1-ene (DCB) has been studied using TiO2 and 3%WO3/TiO2 supported on SiO2. DCB was found to oxidize efficiently over these catalysts; however, only low rates of CO2 formation were observed. With these chlorinated hydrocarbons, the catalysts were found to deactivate over time, probably via the formation of aldol condensation products of chloroacetaldehyde, which is the predominant intermediate observed. The variation in rate and selectivity of the oxidation reactions with O-2 concentration is reported and a mechanism is proposed. Using isotope ratio mass spectrometry, the initial step for the DCB removal has been shown not to be a carbon bond cleavage but is likely to be hydroxyl radical addition to the carbon-carbon double bond.

Determination of absolute configuration of conformationally flexible cis-dihydrodiol metabolites: Effect of diene substitution pattern on the circular dichroism spectra and optical rotations

Absolute configurations of a number of cis-dihydrodiols (cis-1,2-dihydroxy-3,5-cyclohexadienes), synthetically useful products of TDO-catalyzed dihydroxylations of 1,2- and 1,3-disubstituted benzene derivatives, have been determined by a comparison of calculated and experimental CD spectra and optical rotations and by methods involving X-ray crystallography, H-1 NMR spectra of diastereoisomeric derivatives, and by stereochemical correlations. The computations disclosed a significant effect of the substituents on conformational equilibria of cis-dihydrodiols and chiroptical properties of individual conformers. The assigned absolute configurations of cis-dihydrodiols have allowed the validity of a simple predictive model for TDO-catalyzed arene dihydroxylations to be extended.

Non-trivial stably free modules over crossed products

We consider the class of crossed products of noetherian domains with universal enveloping algebras of Lie algebras. For algebras from this class we give a sufficient condition for the existence of projective non-free modules. This class includes Weyl algebras and universal envelopings of Lie algebras, for which this question, known as noncommutative Serre's problem, was extensively studied before. It turns out that the method of lifting of non-trivial stably free modules from simple Ore extensions can be applied to crossed products after an appropriate choice of filtration. The motivating examples of crossed products are provided by the class of RIT algebras, originating in non-equilibrium physics.

An array-based study of reactivity under solvent-free mechanochemical conditions-insights and trends

An array-based approach is put forward to obtain insight into reactivity under mechanochemical solvent-free conditions. We describe a survey of sixty potential reactions between twelve metal salts MX2 {(M = Cu, X-2 = (OAc)(2), (HCO2)(2), (F3CCO2)(2), (acac)(2), (F(6)acac)(2), (NO3)(2), SO4; M = Ni, X-2 = (OAc)(2), (NO3)(2), SO4; M = Zn, X-2 (OAc)(2), (NO3)(2)} and five bridging organic ligands {isonicotinic acid (HINA), 1,4-benzenedicarboxylic acid (H2BDC), acetylenedicarboxylic acid (H(2)ADC), 1,3,5-benzenetricarboxylic acid (H3BTC), 4,4'-bipyridyl (BIPY). Reaction conditions involved a ball mill, applied for 15 min at 30 Hz, without external heating. When examined by XRPD, forty of the combinations gave detectable reactions, thirty-eight with crystalline products. Of these, twenty-nine reactions were quantitative (consuming all of at least one reactant). Comparison of XRPD patterns with patterns simulated from single crystal X-ray diffraction data in the Cambridge Structural Database allowed structural identification of six products. Of particular interest are the microporous framework materials [Cu(INA)(2)] and [Cu-3(BTC)(2)] (HKUST-1) obtained by reaction of the corresponding carboxylic acids with copper acetate. Other non-porous polymers with 3-dimensional connectivity, [Ni(ADC)(H2O)(4)], or 1-dimensional connectivity, [Cu(acac)(2)(BIPY)] and [Cu(F6acac)(BIPY)] were also obtained. Reaction between zinc acetate and H2ADC gave a new product which had not previously been characterised by single-crystal X-ray crystallography, but whose XRPD pattern suggests that it is isostructural with the known nickel polymer [Ni(ADC)(H2O)(4)]. Two further isostructural nickel and zinc products were obtained in reactions between HINA and nickel nitrate and zinc nitrate. Trends observed within the array are discussed. Copper acetate and copper formate were the most effective starting materials for reaction with carboxylic acids, potentially related to the basicity of their anions and the solvating effects of the formic and acetic acid byproducts. Amongst the ligands there was a general negative corelation between melting point and reactivity. The issue of pore templating in microporous phases and the generation of new structures is also discussed in relation to the Cu(INA)(2), Cu-3(BTC)(2) and nickel nitrate-BIPY systems. Overall, the study suggests that mechanochemical reactivity between metal salts and organic ligands under solvent free conditions is remarkably general. Use of array-based approaches as demonstrated here is advocated a useful way to reveal underlying trends in reactivity under solvent free mechanochemical conditions and to highlight particular cases for more detailed study.

Assessment of Self-Compacting Concrete Immersed in Acidic Solutions

Ettringite and thaumasite can be found among the deterioration products of cementitious materials exposed to sulfate and hydrochloric attack. The results of a test program to investigate the acid resistance of self-compacting concrete (SCC) and conventional concrete (CC), immersed up to 18 weeks at 20°C in sulfuric and hydrochloric acid solutions, are described. The SCC was prepared with 47% carboniferous limestone powder, as a replacement for cement, and an ordinary portland cement. The CC was prepared with portland cement only. The water-binder ratios of the SCC and CC were 0.36 and 0.46, respectively. The parameter investigated was the time, in weeks, taken to cause 10% mass loss of fully immersed concrete specimens in a 1% solution of sulfuric acid and the same amount of loss in a 1% solution of hydrochloric acid. The investigation indicated that the SCC performed better than the CC in sulfuric solution but was slightly more vulnerable to hydrochloric acid attack compared to CC. The mode of attack between the two solutions was different.

Formation of NH4[Hg-3(NH)(2)](NO3)(3) and transformation to [Hg2N](NO3)

NH4[Hg-3(NH)(2)](NO3)(3) (1) and [Hg2N](NO3) (2) are obtained from cone. aqueous ammonia solutions of Hg(NO3)(2) at ambient temperature and under hydrothermal conditions at 180 degreesC, respectively, as colourless and dark yellow to light brown single crystals. The crystal structures {NH4[Hg-3(NH)(2)](NO3)(3): cubic, P4(I)32, a = 1030.4(2) pm, Z = 4, R-all = 0.028; [Hg2N](NO3): tetragonal, P4(3)2(1)2, a = 1540.4(1), c = 909.8(1) pm, Z = 4, R-all = 0.054} have been determined from single crystal data. Both exhibit network type structures in which [HNHg3] and [NHg4] tetrahedra of the partial structures of 1 and 2 are connected via three and four vertices, respectively. 1 transforms at about 270 degreesC in a straightforward reaction to 2 whereby the decomposition products of NH4NO3 are set free. 2 decomposes at about 380 degreesC forming yellow HgO. Most certainly, I is identical with a mineral previously analyzed as

Evidence of natural occurrence of the banned antibiotic chloramphenicol in herbs and grass

Chloramphenicol (CAP), a broad-spectrum antibiotic, was detected in several herb and grass samples from different geographic origins. Due to its suspected carcino-genicity and linkages with the development of aplastic anemia in humans, CAP is banned for use in food-producing animals in the European Union (EU) and many other countries. However, products of animal origin originating from Asian countries entering the European market are still found noncompliant (containing CAP) on a regular basis, even when there is no history of chloramphenicol use in these countries. A possible explanation for the continued detection of these residues is the natural occurrence of CAP in plant material which is used as animal feed, with the consequent transfer of the substance to the animal tissues. Approximately 110 samples were analyzed using liquid chromatography coupled with mass spectrometric detection. In 26 samples, the presence of CAP was confirmed using the criteria for banned substances defined by the EU. Among other plant materials, samples of the Artemisia family retrieved from Mongolia and from Utah, USA, and a therapeutic herb mixture obtained from local stores in the Netherlands proved to contain CAP at levels ranging from 0.1 to 450 mu g/kg. These findings may have a major impact in relation to international trade and safety to the consumer. The results of this study demonstrate that noncompliant findings in animal-derived food products may in part be due to the natural occurrence of chloramphenicol in plant material. This has implications for the application of current EU, USA, and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane(1). Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses(1). The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class I-A Pi3ks (ref. 2). Mice lacking only the p85a isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants(3). Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites, We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class I-A Pi3k catalytic subunits: nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

The Type Ic SN 2007gr: a census of the ejecta from late-time optical-infrared spectra

Nebular spectra of supernovae (SNe) offer an unimpeded view of the inner region of the ejecta, where most nucleosynthesis takes place. Optical spectra cover most, but not all, of the emitting elements and therefore offer only a partial view of the products of the explosion. Simultaneous optical-infrared spectra, on the other hand, contain emission lines of all important elements, from C and O through to the intermediate mass elements (IME) Mg, Si, S, Ca and to Fe and Ni. In particular, Si and S are best seen in the IR. The availability of IR data makes it possible to explore in greater detail the results of the explosion. SN 2007gr is the first Type Ic SN for which such data are available. Modelling the spectra with a non-local thermodynamic equilibrium (NLTE) code reveals that the inner ejecta contain similar to 1M(circle dot) of material within a velocity of approximate to 4500 km s(-1). The same mass of Ni-56 derived from the light-curve peak (0.076M(circle dot)) was used to power the spectrum, yielding consistent results. Oxygen is the dominant element, contributing similar to 0.8M(circle dot). The C/O ratio is