Crystal structure of a monomeric form of SARS-CoV endonuclease nsp15 suggests a role for hexamerization as an allosteric switch

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn2+-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 A. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by approximately 120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

Making ecological science policy relevant: issues of scale and disciplinary integration

In this paper, we ask why so much ecological scientific research does not have a greater policy impact in the UK. We argue that there are two potentially important and related reasons for this failing. First, much current ecological science is not being conducted at a scale that is readily meaningful to policy-makers. Second, to make much of this research policy-relevant requires collaborative interdisciplinary research between ecologists and social scientists. However, the challenge of undertaking useful interdisciplinary research only re-emphasises the problems of scale: ecologists and social scientists traditionally frame their research questions at different scales and consider different facets of natural resource management, setting different objectives and using different language. We argue that if applied ecological research is to have greater impact in informing environmental policy, much greater attention needs to be given to the scale of the research efforts as well as to the interaction with social scientists. Such an approach requires an adjustment in existing research and funding infrastructures.

Assembling spatially explicit landscape models of pollen and spore dispersal by wind for risk assessment

Models of windblown pollen or spore movement are required to predict gene flow from genetically modified (GM) crops and the spread of fungal diseases. We suggest a simple form for a function describing the distance moved by a pollen grain or fungal spore, for use in generic models of dispersal. The function has power-law behaviour over sub-continental distances. We show that air-borne dispersal of rapeseed pollen in two experiments was inconsistent with an exponential model, but was fitted by power-law models, implying a large contribution from distant fields to the catches observed. After allowance for this 'background' by applying Fourier transforms to deconvolve the mixture of distant and local sources, the data were best fit by power-laws with exponents between 1.5 and 2. We also demonstrate that for a simple model of area sources, the median dispersal distance is a function of field radius and that measurement from the source edge can be misleading. Using an inverse-square dispersal distribution deduced from the experimental data and the distribution of rapeseed fields deduced by remote sensing, we successfully predict observed rapeseed pollen density in the city centres of Derby and Leicester (UK).

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

1 Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). 2 Ligand binding studies were used to determine dissociation constants for agonist binding to the 5HT(1A) receptor: (a) K-i values for agonists were determined in competition versus the binding of the agonist [H-3]-8-OH DPAT. Competition data were all fitted best by a one-binding site model. (b) K-i values for agonists were also determined in competition versus the binding of the antagonist [H-3]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (K-h) and lower affinity (K-1) sites were determined. 3 The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [S-35]-GTPgammaS binding. Maximal effects of agonists (E-max) and their potencies (EC50) were determined from concentration/response curves for stimulation of [S-35]-GTPgammaS binding. 4 K-1/K-h determined from ligand binding assays correlated with the relative efficacy (relative Em) of agonists determined in [S-35]-GTPgammaS binding assays. There was also a correlation between K-1/K-h and K-1/EC50 for agonists determined from ligand binding and [S-35]-GTPgammaS binding assays. 5 Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of K-1/K-h for predicting the relative efficacy of agonists. British Journal of Pharmacology (2003) 138, 1129-1139. doi: 10. 1038/sj.bjp.705085.

Direct detection of dimethylstannylene and tetramethyldistannene in solution and the gas phase by laser flash photolysis of 1,1-dimethylstannacyclopent-3-enes

The photochemistry of 1,1-dimethyl- and 1,1,3,4-tetramethylstannacyclopent-3-ene (4a and 4b,respectively) has been studied in the gas phase and in hexane solution by steady-state and 193-nm laser flash photolysis methods. Photolysis of the two compounds results in the formation of 1,3-butadiene (from 4a) and 2,3-dimethyl-1,3-butadiene (from 4b) as the major products, suggesting that cycloreversion to yield dimethylstannylene (SnMe2) is the main photodecomposition pathway of these molecules. Indeed, the stannylene has been trapped as the Sn-H insertion product upon photolysis of 4a in hexane containing trimethylstannane. Flash photolysis of 4a in the gas phase affords a transient absorbing in the 450-520nm range that is assigned to SnMe2 by comparison of its spectrum and reactivity to those previously reported from other precursors. Flash photolysis of 4b in hexane solution affords results consistent with the initial formation of SnMe2 (lambda(max) approximate to 500 nm), which decays over similar to 10 mu s to form tetramethyldistannene (5b; lambda(max) approximate to 470 nm). The distannene decays over the next ca. 50 mu s to form at least two other longer-lived species, which are assigned to higher SnMe2 oligomers. Time-dependent DFT calculations support the spectral assignments for SnMe2 and Sn2Me4, and calculations examining the variation in bond dissociation energy with substituent (H, Me, and Ph) in disilenes, digermenes, and distannenes rule out the possibility that dimerization of SnMe2 proceeds reversibly. Addition of methanol leads to reversible reaction with SnMe2 to form a transient absorbing at lambda(max) approximate to 360 nm, which is assigned to the Lewis acid-base complex between SnMe2 and the alcohol.

Copper(II) complexes of mono-anionic glutamate: anionic influence in the variations of molecular and supramolecular structures

Three new polynuclear copper(II) complexes of singly deprotonated L-glutamic acid (L-glu), {[Cu(bipy)(2)][Cu(bipy)(L-glu)H2O](2)(BF4)(4)center dot(H2O)(3)}(n) (1), {[Cu(bipy)(L-glu)H2O][Cu(bipy)(L-glu)(ClO4)]( ClO4)center dot(H2O)(2)}(n) ((2)) and [Cu(phen)(L-glu)H2O](2)(NO3)(2)center dot(H2O)(4) (3) (bipy = 2,2-bipyridine, phen = 1,10-phenanthroline), were synthesized in acidic pH (ca. 2.5) and characterized structurally. In all the complexes, L-glutamic acid acts as a bidentate chelating ligand, leaving the protonated carboxylic acid free. Both in 1 and 2, two different types of species [Cu(bipy)(2)](BF4)(2) and [Cu(bipy)(L-glu)H2O] BF4 for 1 and [Cu(bipy)(L-glu)H2O]ClO4 and [Cu(bipy)(L-glu)(ClO4)] for 2 coexist in the solid state. In complex 1, the [C( bipy)(L-glu)H2O]+ units are joined together by syn-anti carboxylate bridges to form an enantiopure (M) helical chain and the [Cu(bipy)(2)](2+) presents a very rare example of the four-coordinate distorted tetrahedral geometry of Cu(II). In complex 2, the [Cu(bipy)(L gluClO(4))] units are joined together by weakly coordinating perchlorate ions to form a 1D polymeric chain while the [Cu(bipy)(L-glu)H2O]+ units remain as mononuclear species. The different coordinating ability of the two counter anions along with their involvement in the H-bonding network seems likely to be responsible for the difference in the final polymeric structures in the two compounds. Variable-temperature (2-300 K) magnetic susceptibility measurements show negligible coupling for both the complexes. The structure of 3 consists of two independent monomeric [Cu(phen)(L-glu)H2O]+ cations, two nitrate anions and four water molecules. The copper atom occupies a five-coordinate square pyramidal environment with a water molecule in the axial position.

Cyclopropylidenes, bicyclopropylidenes, and vinylcarbenes - some modes of formation and preparative applications

The routes of transformation of the simplest bicyclopropylidene into the derivatives of the second and third generations and synthesis of perspirocyclopropanated [3]rotane and linear spiral [4]- and [5]triangulanes are discussed.

Supramolecular helix and beta-sheet through self-assembly of two isomeric tetrapeptides in crystals and formation of filaments and ribbons in the solid state

Single crystal X-ray diffraction studies show that the beta-turn structure of tetrapeptide I, Boc-Gly-Phe-Aib-Leu-OMe (Aib: alpha-amino isobutyric acid) self-assembles to a supramolecular helix through intermolecular hydrogen bonding along the crystallographic a axis. By contrast the beta-turn structure of an isomeric tetrapeptide II, Boc-Gly-Leu-Aib-Phe-OMe self-assembles to a supramolecular beta-sheet-like structure via a two-dimensional (a, b axis) intermolecular hydrogen bonding network and pi-pi interactions. FT-IR studies of the peptides revealed that both of them form intermolecularly hydrogen bonded supramolecular structures in the solid state. Field emission scanning electron micrographs (FE-SEM) of the dried fibrous materials of the peptides show different morphologies, non-twisted filaments in case of peptide I and non-twisted filaments and ribbon-like structures in case of peptide II.

Solvent-induced dynamic single-crystal-to-single-crystal transformation of a synthetic peptide-based cyclic compound

Solvent induced single-crystal-to-single-crystal transformation has been demonstrated indicating the dynamic behavior of one dimensional arrays obtained from a self-assembled new synthetic cyclic peptide.

Unusual transformation of trialkylamines mediated by platinum

An interesting chemical transformation of trialkylamines has taken place during the reaction of 2-(2', 6'-dimethylphenylazo)- 4-methylphenol ( 1) with K-2[ PtCl4] in refluxing methanol in the presence of trialkylamines, leading to the formation of organoplatinum complexes ( 2 and 3), where ligand 1 is coordinated as a bidentate N, O donor and the transformed trialkylamines are coordinated as bidentate C, N donors.

Gas-solid reactions of single crystals: a study of reactions of NH3 and NO2 with single crystalline organic substrates by infrared microspectroscopy

Reaction of single crystals of benzoic and trans-cinnamic acids with 200 Torr pressure of ammonia gas in a sealed glass bulb at 20 degrees C generates the corresponding ammonium salts; there is no sign of any 1:2 adduct as has been reported previously for related systems. Isotopic substitution using ND3 has been used to aid identification of the products. Adipic acid likewise reacts with NH3 gas to form a product in which ammonium salts are formed at both carboxylic acid groups. Reaction of 0.5 Torr pressure of NO2 gas with single crystals of 9-methylanthracene and 9-anthracenemethanol in a flow system generates nitrated products where the nitro group appears to be attached at the 10-position, i.e. the position trans to the methyl or methoxy substituent on the central ring. Isotopic substitution using (NO2)-N-15 has been used to confirm the identity of the bands arising from the coordinated NO2 group. The products formed when single crystals of hydantoin are reacted with NO2 gas under similar conditions depend on the temperature of the reaction. At 20 degrees C, a nitrated product is formed, but at 65 degrees C this gives way to a product containing no nitro groups. The findings show the general applicability of infrared microspectroscopy to a study of gas-solid reactions of organic single crystals. (c) 2005 Elsevier B.V. All rights reserved.

Educational participation post-16: a longitudinal analysis of intentions and outcomes

The issue of levels of participation in post-compulsory education has been emphasised by the current policy initiatives to increase the age to which some form of participation is compulsory. One of the acknowledged weaknesses of research in the field of children's intentions with regard to participation is the lack of longitudinal data. This paper offers a longitudinal analysis using the Youth Survey from the British Household Panel Survey. The results show that most children can express intentions with regard to future participation very early in their secondary school careers and that these intentions are good predictors of actual behaviour five years later. Intentions to stay on are more consistent than intentions to leave and most children who finally leave at 16 have at some point said they want to remain in education post-16. The strongest association with participation levels is attainment at GCSE. However, there are also influences of gender and parental background and these remain, even after attainment is held constant. The results show the value of focusing on intentions for participation at a very early stage of children's school careers and also the importance of current attempts to reform curriculum and assessment for the 14-19 age group.

The role of drama in the teaching of speaking and listening as the basis for social capital

This paper explores how the concept of 'social capital' relates to the teaching of speaking and listening. The argument draws on Bourdieu's notion that a common language is an illusion but posits that an understanding of the grammar of speech can be productive in the development of both an understanding of what constitutes effective speech and the development of competence in speaking. It is argued that applying structuralist notions of written grammar is an inadequate approach to understanding speech acts or enhancing the creative use of speech. An analysis is made of how typical features of speech relate to dramatic dialogue and how the meaning of what is said is contingent upon aural and visual signifiers. On this basis a competent speaker is seen as being one who produces expressions appropriate for a range of situations by intentionally employing such signifiers. The paper draws on research into the way drama teachers make explicit reference to and use of semiotics and dramatic effectiveness in order to improve students' performance and by so doing empower them to increase their social capital. Ultimately, it is concluded that helping students identify, analyse and employ the aural, visual and verbal grammar of spoken English is not an adjunct to the subject of drama, but an intrinsic part of understanding the art form. What is called for is a re-appraisal by drama teachers of their own understanding of concepts relating to speech acts in order to enhance this area of their work.

Effects of enhanced consumption of fruit and vegetables on plasma antioxidant status and oxidative resistance of LDL in smokers supplemented with fish oil

Objective: To determine whether consumption of five portions of fruit and vegetables per day reduces the enhancement of oxidative stress induced by consumption of fish oil. Subjects: A total of 18 free-living healthy smoking volunteers, aged 18-63 y, were recruited by posters and e-mail in The University of Reading, and by leaflets in local shops. Design: A prospective study. Setting: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights PO Box 226, Reading RG6 6AP, UK. Intervention: All subjects consumed a daily supplement of 4 x 1 g fish oil capsules for 9 weeks. After 3 weeks, they consumed an additional five portions of fruits and vegetables per day, and then they returned to their normal diet for the last 3 weeks of the study. Fasting blood samples were taken at the ends of weeks 0, 3, 6 and 9. Results: The plasma concentrations of ascorbic acid, lutein, beta-cryptoxanthin, alpha-carotene and beta-carotene all significantly increased when fruit and vegetable intake was enhanced (P<0.05). Plasma concentrations of α-tocopherol, retinol and uric acid did not change significantly during the period of increased fruit and vegetable consumption. Plasma oxidative stability, assessed by the oxygen radical absorbance capacity (ORAC) assay, also increased from weeks 3-6 (P<0.001) but not in association with increases in measured antioxidants. Lag phase before oxidation of low-density lipoprotein (LDL) significantly decreased in the first 3 weeks of the study, reflecting the incorporation of EPA and DHA into LDL (P<0.0001). Subsequent enhanced fruit and vegetable consumption significantly reduced the susceptibility of LDL to oxidation (P<0.005). Conclusion: Fish oil reduced the oxidative stability of plasma and LDL, but the effects were partially offset by the increased consumption of fruit and vegetables.