Amino acid pairing preferences in parallel beta-sheets in proteins

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http://www.rubic.rdg.ac.uk/betapairprefsparallel/).

Amino acid pairing preferences in parallel beta-sheets in proteins

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favourability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB) versus Thr(HB)-Asn(nHB). Significantly (p <= 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi(1) rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi(1) conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi(1) and chi(2) conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favourability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http:// www.rubic.rdg.ac.uk/betapairprefsparallel/). (c) 2005 Elsevier Ltd. All rights reserved.

A phyloclimatic study of Cyclamen

Background: The impact of global climate change on plant distribution, speciation and extinction is of current concern. Examining species climatic preferences via bioclimatic niche modelling is a key tool to study this impact. There is an established link between bioclimatic niche models and phylogenetic diversification. A next step is to examine future distribution predictions from a phylogenetic perspective. We present such a study using Cyclamen (Myrsinaceae), a group which demonstrates morphological and phenological adaptations to its seasonal Mediterranean-type climate. How will the predicted climate change affect future distribution of this popular genus of garden plants? Results: We demonstrate phylogenetic structure for some climatic characteristics, and show that most Cyclamen have distinct climatic niches, with the exception of several wide-ranging, geographically expansive, species. We reconstruct climate preferences for hypothetical ancestral Cyclamen. The ancestral Cyclamen lineage has a preference for the seasonal Mediterranean climate characteristic of dry summers and wet winters. Future bioclimatic niches, based on BIOCLIM and Maxent models, are examined with reference to a future climate scenario for the 2050s. Over the next 50 years we predict a northward shift in the area of climatic suitability, with many areas of current distribution becoming climatically unsuitable. The area of climatic suitability for every Cyclamen species is predicted to decrease. For many species, there may be no areas with a suitable climate regardless of dispersal ability, these species are considered to be at high risk of extinction. This risk is examined from a phylogenetic perspective. Conclusion: Examining bioclimatic niches from a phylogenetic perspective permits novel interpretations of these models. In particular, reconstruction of ancestral niches can provide testable hypothesis about the historical development of lineages. In the future we can expect a northwards shift in climatic suitability for the genus Cyclamen. If this proves to be the case then dispersal is the best chance of survival, which seems highly unlikely for ant-dispersed Cyclamen. Human-assisted establishment of Cyclamen species well outside their native ranges offers hope and could provide the only means of dispersal to potentially suitable future environments. Even without human intervention the phylogenetic perspective demonstrates that major lineages could survive climate change even if many species are lost.

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations

Objectives and methods: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. Results and conclusions: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.

Structure of the snake-venom toxin convulxin

Snake venoms contain a number of proteins that interact with components of the haemostatic system that promote or inhibit events leading to blood- clot formation. The snake- venom protein convulxin ( Cvx) binds glycoprotein ( GP) VI, the platelet receptor for collagen, and triggers signal transduction. Here, the 2.7 Angstrom resolution crystal structure of Cvx is presented. In common with other members of this snake-venom protein family, Cvx is an alphabeta- heterodimer and conforms to the C- type lectin- fold topology. Comparison with other family members allows a set of Cvx residues that form a concave surface to be putatively implicated in GPVI binding. Unlike other family members, with the exception of flavocetin- A ( FL- A), Cvx forms an (alphabeta)(4) tetramer. This oligomeric structure is consistent with Cvx clustering GPVI molecules on the surface of platelets and as a result promoting signal transduction activity. The Cvx structure and the location of the putative binding sites suggest a model for this multimeric signalling assembly.

Ecology of sponge assemblages (Porifera) in the Wakatobi region, south-east Sulawesi, Indonesia: richness and abundance

The influence of sedimentation, depth and substratum angle on sponge assemblages in the Wakatobi region, south-eastern Sulawesi, Indonesia was considered. Sponge assemblages were sampled from two reef localities. The first reef (Sampela) was highly impacted by high sedimentation rates with fine sediment particles that settle slowly, while the second (Hoga) experienced only fast settling coarse sediment with lower overall sedimentation rates. Sponge assemblages were sampled (area occupied and numbers) on the reef fiat (0 m) and at 5 (reef crest), 10 and 15 m (15 m at Hoga only). Some significant (P < 0.001) differences were observed in the area occupied and the number of sponge patches between surface angles and sites. Significantly lower (t > 4.61, df = 9, P < 0.001) sponge numbers, percentage cover and richness were associated with the reef flat at both sites compared with all other depths at each site, with the exception of abundance of sponges on the reef flat at Sampela, which was much greater than at any other depth sampled. Species richness increased with depth at both sites but differences between surface angles were only recorded at Sampela, with higher species richness being found on vertical, inclined and horizontal surfaces respectively A total of 100 sponge species (total area sampled 52.5 m(2)) was reported from the two sites, with 58 species found at Sampela and 71 species at Hoga (41% of species shared). Multi-dimensional scaling (MDS) indicated differences in assemblage structure between sites and most depth intervals, but not substratum angles. A number of biological (e.g. competition and predation) and physical (e.g. sedimentation and aerial exposure) factors were considered to control sponge abundance and richness. Unexpectedly a significant (F-1,F-169 = 148.98, P < 0.001) positive linear relationship was found between sponge density and area occupied. In areas of high sponge coverage, the number of patches was also high, possibly due to fragmentation of large sponges produced as a result of predation and physical disturbance. The MDS results were also the same whether sponge numbers or percentage cover estimates were used, suggesting that although these different approaches yield different sorts of information, the same assemblage structure can be identified.

Study of tyrosine kinases and protein tyrosine phosphorylation

In recent years, our increased understanding of the complex signal transduction mechanisms that regulate cellular function has fueled huge advances in all aspects of biomedical science and cell biology. Platelet and megakaryocyte function is no exception to this. In the last 10 yr our understanding of the receptor biochemistry and the systems that they control has been pivotal in the development of new strategies to inhibit platelet function and thereby prevent thrombosis. Experimental techniques have become more and more elegant, however; the basic toolbox that a researcher requires to study signaling in platelets and megakaryoctes is described in this and several subsequent chapters.

Investigation of the mechanism of agonist and inverse agonist action at D-2 dopamine receptors

This study investigated, for the D-2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. K-i values for agonists were determined in competition, versus the binding of the antagonist [H-3]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (K-h) (G protein-coupled) and lower affinity (K-l) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [H-3]N-propylnorapomorphine (NPA) to provide a second estimate of K-h,. Maximal agonist effects (E-max) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[S-32] thiotriphosphate) ([S-35]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (K-l/K-h, determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative E-max, Kl/EC50) of agonists determined in [S-35]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed For a subset of compounds, however, there was a relation between K-l/K-h and E-max.. Competition-binding data versus [H-3]spiperone and [H-3]NPA for a range of inverse agonists were fitted best by a one-binding site model. K-i values for the inverse agonists tested were slightly lower in competition versus [H-3]NPA compared to [H-3]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor. (C) 2004 Elsevier Inc. All rights reserved.

Temporal and spatial variability of mobile fauna on a submarine cliff and boulder scree complex: a community in flux

The mobile component of a community inhabiting a submarine boulder scree/cliff was investigated at Lough Hyne, Ireland at dawn, midday, dusk and night over a 1-week period. Line transects (50 m) were placed in the infralittoral (6 m) and circumlittoral (18 m) zones and also the interface between these two zones (12 m). The dominant mobile fauna of this cliff consisted of echinoderms (6 species), crustaceans (10 species) and fish (23 species). A different component community was identified at each time/depth interval using Multi-Dimensional Scaling (MDS) even though both species diversity (Shannon-Wiener indices) and richness (number of species) remained constant. These changes in community composition provided indirect evidence for migration by these mobile organisms. However, little evidence was found for migration between different zones with the exception of the several wrasse species. These species were observed to spend the daytime foraging in the deeper zone, but returned to the upper zone at night presumably for protection from predators. For the majority of species, migration was considered to occur to cryptic habitats such as holes and crevices. The number of organisms declined during the night, although crustacean numbers peaked, while fish and echinoderms were most abundant during day, possibly due to predator-prey interactions. This submarine community is in a state of flux, whereby, community characteristics, including trophic and energetic relationships, varied over small temporal (daily) and spatial (m) scales.

Conservation and sustainable use of wild species as sources of new ornamentals

While only about 1-200 species are used intensively in commercial floriculture (e.g. carnations, chrysanthemums, gerbera, narcissus, orchids, tulips, lilies, roses, pansies and violas, saintpaulias, etc.) and 4-500 as house plants, several thousand species of herbs, shrubs and trees are traded commercially by nurseries and garden centres as ornamentals or amenity species. Most of these have been introduced from the wild with little selection or breeding. In Europe alone, 12 000 species are found in cultivation in general garden collections (i.e. excluding specialist collections and botanic gardens). In addition, specialist collections (often very large) of many other species and/or cultivars of groups such as orchids, bromeliads, cacti and succulents, primulas, rhododendrons, conifers and cycads are maintained in several centres such as botanic gardens and specialist nurseries, as are 'national collections' of cultivated species and cultivars in some countries. Specialist growers, both professional and amateur, also maintain collections of plants for cultivation, including, increasingly, native plants. The trade in ornamental and amenity horticulture cannot be fully estimated but runs into many billions of dollars annually and there is considerable potential for further development and the introduction of many new species into the trade. Despite this, most of the collections are ad hoc and no co-ordinated efforts have been made to ensure that adequate germplasm samples of these species are maintained for conservation purposes and few of them are represented at all adequately in seed banks. Few countries have paid much attention to germplasm needs of ornamentals and the Ornamental Plant Germplasm Center in conjunction with the USDA National Plant Germplasm System at The Ohio State University is an exception. Generally there is a serious gap in national and international germplasm strategies, which have tended to focus primarily on food plants and some forage and industrial crops. Adequate arrangements need to be put in place to ensure the long- and medium-term conservation of representative samples of the genetic diversity of ornamental species. The problems of achieving this will be discussed. In addition, a policy for the conservation of old cultivars or 'heritage' varieties of ornamentals needs to be formulated. The considerable potential for introduction of new ornamental species needs to be assessed. Consideration needs to be given to setting up a co-ordinating structure with overall responsibility for the conservation of germplasm of ornamental and amenity plants.

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 nun but increased - 50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (&LE; 9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (&SIM; 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only. detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 nun; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (&SIM;6-fold higher in F4; P<0.0001); levels then fell &SIM;2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose &SIM;3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell &SIM;4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

Pyrolysis of polymer-derived carbons in the formation of graphitizing carbons and nanoparticles of zirconia

Carbons have been prepared by the low-temperature pyrolysis, under argon, of a number of long-chain polymers. We have found that the resistivity (Omega cm(-1)) varies considerably with the temperature of pyrolysis; thus, for ammonium polyacrylate, the resistivity of that pyrolyzed at 600 degrees C is 9.7 x 10(4) Omega cm(-1) whereas that pyrolyzed at 1000 degrees C is ca. 3 Omega cm(-1). A similar situation arises for the other polymers studied (including radiolyzed cross-linked polyacrylamide). All those pyrolyzed at 600 degrees C had a resistivity of > 1 x 10(6) Omega cm(-1), whereas those pyrolyzed at 1000 degrees C had a resistivity of ca. 3-5 Omega cm(-1). A notable exception was that of unirradiated polyacrylamide, where the resistivity remained at > 1 x 10(6) Omega cm(-1) over the range of temperatures studied. The decrease of resistivity with increase of temperature of pyrolysis has been related to the formation of glassy carbon. Nanoparticles (4 nm) of tetragonal zirconia were formed when zirconium polyacrylate was pyrolyzed under similar conditions.

New dioxadiaza-, trioxadiaza- and hexaaza-macrocycles containing dibenzofuran units

New dioxadiaza-, trioxadiaza-, and hexaaza-macrocycles containing rigid dibenzofuran groups (DBF) were prepared by a convenient synthetic route in high yields. The structures of the macrocycles were unequivocally established by electrospray mass spectrometry (ESIMS) studies together with NMR spectroscopy, with the exception of [14](DBF)N-3. The structures of the copper complex of [14](DBF)N-3 and of the diprotonated form of [22](DBF)N2O3 were determined by single crystal X-ray diffraction. Conformational analyses on the free macrocycles [14](DBF)N-3 and [22](DBF)N2O3 as well as on their larger counterparts containing two DBF units were undertaken in order to understand the synthetic findings. (c) 2006 Elsevier Ltd. All rights reserved.

Oxovanadium(V) complexes of bis(phenolate) ligands with acetylacetone as co-ligand: synthesis, crystal structure, electrochemical and kinetics studies on the oxidation of ascorbic acid

Two vanadium(V) complexes, [VO(L-1)]acac)] (1) and [VO(L-2)(acac)] (2), where H2L1 = N,N-bis(2-hydroxy-3-5-di-tert-butyl-benzyl)propylamine and H2L2 = 2,2'-selenobis(4,6-di-tert-butylphenol), have been synthesized and characterized by elemental analyses, IR, V-51 NMR, both in the solid and in solution, and cyclic voltammetric studies. Single crystal X-ray studies reveal that in complex 1 the vanadium atom is octahedrally coordinated with an O5N donor environment, where the oxygen atom of the V-V=O moiety and the N atom of the ONO ligand occupy the axial sites while two oxygen atoms (O1 and O2) from the bisphenolate ligand and two oxygen atoms (O3 and O4) from the acac ligand occupy the equatorial plane. A similar bonding pattern has also been encountered for 2 with the exception that a Se atom instead of N is involved in weak bonding to the metal center. Both complexes showed reversible cyclic voltammeric responses and E-1/2 appears at -0.18 and 0.10 V versus NHE for complexes 1 and 2, respectively. The kinetics of oxidation of ascorbic acid by complex 1 were carried out in 50% MeCN-50% HO (v/v) at 25 degrees C. The high formation constant value, Q = 63 +/- 7 M-1, reveals that the reaction proceeds through the rapid formation of a H-bonded intermediate. The low k(2)Q(2)/k(1)Q(1) ratio (13.4) for 1 points out that there is extensive H-bonding between the oxygen atom of the V-V=O group and the OH group of ascorbic acid. (c) 2007 Published by Elsevier Ltd.

Transition metal-directed self-assembly of calix[4]arene based dithiocarbamate ligands

The transition metal-directed self-assembly of dithiocarbamate ligand functionalised upper and lower rim calix[4]arenes affords novel dimeric bimetallic bis(calix[4]arene) species as determined by a combination of analytical methods including X-ray crystallography. An exception is a zinc(II) dithiocarbamate upper rim calix[4]arene assembly which is monomeric in nature. Electrochemical investigations reveal the bimetallic copper(II) bis(calix[4]arene) systems can electrochemically sense dihydrogen phosphate and carboxylate anions via significant cathodic perturbations of the respective copper(II)/(III) dithiocarbamate oxidation wave.