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Sample records for neonicotinoids structural insights

  1. Diverse actions and target-site selectivity of neonicotinoids: structural insights.

    PubMed

    Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B

    2009-07-01

    The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO(2) group of imidacloprid and 2) neonicotinoid-unique stacking and CH-pi bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-alpha nAChR subunits.

  2. Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

    PubMed Central

    Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.

    2009-01-01

    The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits. PMID:19321668

  3. Studies on an acetylcholine binding protein identify a basic residue in loop G on the β1 strand as a new structural determinant of neonicotinoid actions.

    PubMed

    Ihara, Makoto; Okajima, Toshihide; Yamashita, Atsuko; Oda, Takuma; Asano, Takuya; Matsui, Mikana; Sattelle, David B; Matsuda, Kazuhiko

    2014-12-01

    Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the β1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the α7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.

  4. Quantitative structure-activity relationship study using refractotopological state atom index on some neonicotinoid insecticides.

    PubMed

    Debnath, Bikash; Gayen, Shovanlal; Basu, Anindya; Ghosh, Balaram; Srikanth, Kolluru; Jha, Tarun

    2004-12-01

    Importance of atom-level topological descriptors like electrotopological state atom (E-state) index in QSAR study is increasing. These descriptors help to relate structure and activity at atomic/fragmental level. In view of the earlier success of E-state index on some azidopyridinyl neonicotinoid insecticides, a relatively new atom-level topological descriptor; refractotopological state atom (R-state) index was used in this work. This was used to identify the important atoms/fragments related to dispersive/van der Waals interactions of neonicotinoids with the nicotinic acetylcholine receptor (nAChR). This study showed the structural requirements for the mammal alpha(4)beta(2) and Drosophila nAChR agonistic activity. It also revealed that substituted imine, nitromethylene at X-position were selective to the insecticidal activity. Azido substitution at pyridine ring of neonicotinoids disfavored the binding with the receptors. This study confirmed the validity of the R-state index as a new tool for quantitative structure-activity relationships. It has the ability to find out the required structural features as well as to predict the activity of the neonicotinoids.

  5. Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

    PubMed Central

    Ihara, Makoto; Okajima, Toshihide; Yamashita, Atsuko; Oda, Takuma; Hirata, Koichi; Nishiwaki, Hisashi; Morimoto, Takako; Akamatsu, Miki; Ashikawa, Yuji; Kuroda, Shun’ichi; Mega, Ryosuke; Kuramitsu, Seiki; Sattelle, David B.

    2008-01-01

    Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs. PMID:18338186

  6. Insight into the mechanism of reproductive dysfunction caused by neonicotinoid pesticides.

    PubMed

    Hoshi, Nobuhiko; Hirano, Tetsushi; Omotehara, Takuya; Tokumoto, Junko; Umemura, Yuria; Mantani, Youhei; Tanida, Takashi; Warita, Katsuhiko; Tabuchi, Yoshiaki; Yokoyama, Toshifumi; Kitagawa, Hiroshi

    2014-01-01

    Neonicotinoids, which were developed in the 1990 s as an insecticide having selective toxicity, were later found to cause reproductive abnormalities in experimental animals. In Japan there is an attempt to preserve endangered animals, including the Japanese crested ibis, and there is a question of whether neonicotinoids affect the reproduction of this bird, since they are used in its habitat. Hence, we investigated whether the daily oral administration of the neonicotinoid clothianidin (CTD) has any deleterious effects on the reproductive function of mature male only or both young male and female quails as experimental animals. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, as well as the number and size of vacuoles in hepatocytes, increased dose-dependently. The ovaries showed abnormal histology in the granulosa cells, which produce progesterone. There were significant differences in egg-laying rates and embryo weights between the groups. Glutathione Peroxidase 4 (GPx4) and Manganese Superoxide Dismutase (Mn-SOD), which protect the organism from oxidative damage, showed a dose-dependent decrease. Thus, it is possible neonicotinoids affect the bird's reproductive system through oxidative stress, reflecting an imbalance between the production of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Responding to our study, Sado Island has since succeeded in breeding Japanese crested ibis in the wild without the use of neonicotinoids.

  7. Seven-membered azabridged neonicotinoids: synthesis, crystal structure, insecticidal assay, and molecular docking studies.

    PubMed

    Xu, Renbo; Luo, Ming; Xia, Rui; Meng, Xiaoqing; Xu, Xiaoyong; Xu, Zhiping; Cheng, Jiagao; Shao, Xusheng; Li, Houju; Li, Zhong

    2014-11-19

    To study the influence of the ring sizes, 37 novel seven-membered azabridged neonicotinoid analogues were synthesized by reactions of nitromethylene analogues, succinaldehyde, and aniline hydrochlorides. Most of the title compounds presented higher insecticidal activities than that of imidacloprid (IMI), cycloxaprid (CYC), and eight-membered compounds against cowpea aphid (Aphis craccivora), armyworm (Pseudaletia separata Walker), and brown planthopper (Nilaparvata lugens), which indicated that introducing the structure of a seven-membered azabridge could significantly improve the insecticidal activities of neonicotinoid analogues. Docking study and binding mode analysis also revealed that introducing methyl group into position 2 of phenyl ring could increase the hydrophobic interactions with receptor, which implied that position 2 might be the key site to get high insecticidal compounds.

  8. Advanced research on cis-Neonicotinoids.

    PubMed

    Shao, Xusheng; Ye, Zhejun; Bao, Haibo; Liu, Zewen; Xu, Xiaoyong; Li, Zhong; Qian, Xuhong

    2011-01-01

    cis-Neonicotinoids are a type of neonicotinoid, in which the nitro or the cyano group are in cis-configuration relative to heteroaromatic moiety, which show excellent activities against a range of insect species. This review covers cis-neonicotinoids with commercialization perspectives, structural optimization (phenylazoneonicotinoids and chlorothiazolyl analogues of Paichongding), modes of action studies, radiao-synthesis of Paichongding and Cycloxaprid, and photostability of neonicotinoids.

  9. Divalent and oxabridged neonicotinoids constructed by dialdehydes and nitromethylene analogues of imidacloprid: design, synthesis, crystal structure, and insecticidal activities.

    PubMed

    Shao, Xusheng; Fu, Hua; Xu, Xiaoyong; Xu, Xinglei; Liu, Zewen; Li, Zhong; Qian, Xuhong

    2010-03-10

    A series of divalent and oxabridged neonicotinoids were synthesized by reactions of nitromethylene analogues of imidacloprid and dialdehydes, and their structures were confirmed by (1)H NMR, (13)C NMR, high-resolution mass spectroscopy, and X-ray diffraction analysis. The bioassays indicated that some of them were endowed with excellent insecticidal activities against cowpea aphid ( Aphis craccivora ), armyworm ( Pseudaletia separata Walker), and brown planthopper ( Nilaparvata lugens ). Divalent neonicotinoid 6 and oxabridged 8a had higher activities than imidacloprid against cowpea aphids and armyworm; furthermore, the activity of 8a was 40.4-fold higher than that of imidacloprid against imidacloprid-resistant brown planthopper.

  10. Structural features of phenoxycarbonylimino neonicotinoids acting at the insect nicotinic receptor.

    PubMed

    Ohno, Ikuya; Tomizawa, Motohiro; Miyazu, Nozomi; Kushibiki, Gohito; Noda, Kumiko; Hasebe, Yasunori; Durkin, Kathleen A; Miyake, Taiji; Kagabu, Shinzo

    2010-10-01

    Substituted-phenoxycarbonylimino neonicotinoid ligands with an electron-donating group showed significantly higher affinity to the insect nicotinic receptor relative to that of the analogue with an electron-withdrawing substituent, thereby establishing in silico binding site interaction model featuring that the phenoxy ring of neonicotinoids and the receptor loop D tryptophan indole plane form a face-to-edge aromatic interaction.

  11. Imidacloprid and thiacloprid neonicotinoids bind more favourably to cockroach than to honeybee α6 nicotinic acetylcholine receptor: insights from computational studies.

    PubMed

    Selvam, Balaji; Graton, Jérôme; Laurent, Adèle D; Alamiddine, Zakaria; Mathé-Allainmat, Monique; Lebreton, Jacques; Coqueret, Olivier; Olivier, Christophe; Thany, Steeve H; Le Questel, Jean-Yves

    2015-02-01

    The binding interactions of two neonicotinoids, imidacloprid (IMI) and thiacloprid (THI) with the extracellular domains of cockroach and honeybee α6 nicotinic acetylcholine receptor (nAChR) subunits in an homomeric receptor have been studied through docking and molecular dynamics (MD) simulations. The binding mode predicted for the two neonicotinoids is validated through the good agreement observed between the theoretical results with the crystal structures of the corresponding complexes with Ac-AChBP, the recognized structural surrogate for insects nAChR extracellular ligand binding domain. The binding site of the two insect α6 receptors differs by only one residue of loop D, a serine residue (Ser83) in cockroach being replaced by a lysine residue (Lys108) in honeybee. The docking results show very close interactions for the two neonicotinoids with both α6 nAChR models, in correspondence to the trends observed in the experimental neonicotinoid-Ac-AChBP complexes. However, the docking parameters (scores and energies) are not significantly different between the two insect α6 nAChRs to draw clear conclusions. The MD results bring distinct trends. The analysis of the average interaction energies in the two insects α6 nAChRs shows indeed better affinity of neonicotinoids bound to α6 cockroach compared to honeybee nAChR. This preference is explained by tighter contacts with aromatic residues (Trp and Tyr) of the binding pocket. Interestingly, the non-conserved residue Lys108 of loop D of α6 honeybee nAChR interacts through van der Waals contacts with neonicotinoids, which appear more favourable than the direct or water mediated hydrogen-bond interaction between the OH group of Ser83 of α6 cockroach nAChR and the electronegative terminal group of the two neonicotinoids (nitro in IMI and cyano in THI). Finally, in both insects nAChRs, THI is consistently found to bind more favourably than IMI. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Comparative analysis of neonicotinoid binding to insect membranes: I. A structure-activity study of the mode of [3H]imidacloprid displacement in Myzus persicae and Aphis craccivora.

    PubMed

    Kayser, Hartmut; Lee, Connie; Decock, Arnaud; Baur, Markus; Haettenschwiler, Joerg; Maienfisch, Peter

    2004-10-01

    Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.

  13. Neonicotinoids-from zero to hero in insecticide chemistry.

    PubMed

    Jeschke, Peter; Nauen, Ralf

    2008-11-01

    In recent years, neonicotinoids have been the fastest-growing class of insecticides in modern crop protection, with widespread use against a broad spectrum of sucking and certain chewing pests. As potent agonists, they act selectively on insect nicotinic acetylcholine receptors, their molecular target site. The discovery of neonicotinoids can be considered as a milestone in insecticide research and facilitates greatly the understanding of the functional properties of insect nicotinic acetylcholine receptors. Because of the relatively low risk for non-target organisms and environment, the high target specificity of neonicotinoid insecticides and their versatility in application methods, this important class has to be maintained globally for integrated pest management strategies and insect resistance management programmes. This review comprehensively describes particularly the origin, structure and bonding as well as associated properties of neonicotinoid insecticides.

  14. Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

    SciTech Connect

    Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radi, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

    2008-07-28

    Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 {angstrom} in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.

  15. Cross-resistance relationships between neonicotinoids and pymetrozine in Bemisia tabaci (Hemiptera: Aleyrodidae).

    PubMed

    Gorman, Kevin; Slater, Russell; Blande, James D; Clarke, Alison; Wren, Jodie; McCaffery, Alan; Denholm, Ian

    2010-11-01

    Although cross-resistance between compounds in the same insecticide group is a frequently observed phenomenon, cross-resistance between groups that differ in structural and functional characteristics can be extremely unpredictable. In the case of controlling the whitefly, Bemisia tabaci Gennadius, neonicotinoids and the pyridine azomethine antifeedant pymetrozine represent independent lines of discovery that should be suited for alternation to avoid prolonged selection for the same resistance mechanism. Reports of an association between responses to neonicotinoids and pymetrozine were investigated by resistance profiling of seven B. tabaci strains and complementary reciprocal selection experiments. All strains demonstrated a consistent correlation between responses to three neonicotinoid compounds: thiamethoxam, imidacloprid and acetamiprid. Responses to neonicotinoids for six field strains clearly correlated with responses to pymetrozine. Reciprocal selection experiments confirmed an unexpected case of intergroup cross-resistance. A seventh strain exhibited a so far unique phenotype of strong resistance to pymetrozine but full susceptibility to neonicotinoids. Selection experiments confirmed that in this strain the mechanism of pymetrozine resistance is specific and has no implications for neonicotinoids. Cross-resistance between neonicotinoids and pymetrozine in B. tabaci probably reflects the overexpression of a cytochrome-P450-dependent monooxygenase capable of metabolising both types of compound in spite of their apparent structural dissimilarity. Given the predominance of this mechanism in B. tabaci, both can contribute to resistance management but should be placed within the same treatment 'window'. Copyright © 2010 Society of Chemical Industry.

  16. N-haloacetylimino neonicotinoids: potency and molecular recognition at the insect nicotinic receptor.

    PubMed

    Tomizawa, Motohiro; Durkin, Kathleen A; Ohno, Ikuya; Nagura, Kyoko; Manabe, Mio; Kumazawa, Satoru; Kagabu, Shinzo

    2011-06-15

    This structure-activity relationship study for neonicotinoids with an N-haloacetylimino pharmacophore identifies several candidate compounds showing outstanding insecticidal potency and consequently leads to establishing their molecular recognition at an insect nicotinic receptor structural model, wherein the neonicotinoid halogen atoms (fluorine, chlorine, bromine, and iodine) variously interact with the receptor loops C-D interfacial niche via H-bonding and/or hydrophobic interactions.

  17. Occurrence of neonicotinoid insecticides in finished drinking water and fate during drinking water treatment

    USGS Publications Warehouse

    Klarich, Kathryn L.; Pflug, Nicholas C.; DeWald, Eden M.; Hladik, Michelle; Kolpin, Dana W.; Cwiertny, David M.; LeFevre, Gergory H.

    2017-01-01

    Neonicotinoid insecticides are widespread in surface waters across the agriculturally-intensive Midwestern US. We report for the first time the presence of three neonicotinoids in finished drinking water and demonstrate their general persistence during conventional water treatment. Periodic tap water grab samples were collected at the University of Iowa over seven weeks in 2016 (May-July) after maize/soy planting. Clothianidin, imidacloprid, and thiamethoxam were ubiquitously detected in finished water samples and ranged from 0.24-57.3 ng/L. Samples collected along the University of Iowa treatment train indicate no apparent removal of clothianidin and imidacloprid, with modest thiamethoxam removal (~50%). In contrast, the concentrations of all neonicotinoids were substantially lower in the Iowa City treatment facility finished water using granular activated carbon (GAC) filtration. Batch experiments investigated potential losses. Thiamethoxam losses are due to base-catalyzed hydrolysis at high pH conditions during lime softening. GAC rapidly and nearly completely removed all three neonicotinoids. Clothianidin is susceptible to reaction with free chlorine and may undergo at least partial transformation during chlorination. Our work provides new insights into the persistence of neonicotinoids and their potential for transformation during water treatment and distribution, while also identifying GAC as an effective management tool to lower neonicotinoid concentrations in finished drinking water.

  18. Overview of the status and global strategy for neonicotinoids.

    PubMed

    Jeschke, Peter; Nauen, Ralf; Schindler, Michael; Elbert, Alfred

    2011-04-13

    In recent years, neonicotinoid insecticides have been the fastest growing class of insecticides in modern crop protection, with widespread use against a broad spectrum of sucking and certain chewing pests. As potent agonists, they act selectively on insect nicotinic acetylcholine receptors (nAChRs), their molecular target site. The discovery of neonicotinoids can be considered as a milestone in insecticide research and greatly facilitates the understanding of functional properties of the insect nAChRs. In this context, the crystal structure of the acetylcholine-binding proteins provides the theoretical foundation for designing homology models of the corresponding receptor ligand binding domains within the nAChRs, a useful basis for virtual screening of chemical libraries and rational design of novel insecticides acting on these practically relevant channels. Because of the relatively low risk for nontarget organisms and the environment, the high target specificity of neonicotinoid insecticides, and their versatility in application methods, this important class has to be maintained globally for integrated pest management strategies and insect resistance management programs. Innovative concepts for life-cycle management, jointly with the introduction of generic products, have made neonicotinoids the most important chemical class for the insecticide market.

  19. Neonicotinoids interfere with specific components of navigation in honeybees.

    PubMed

    Fischer, Johannes; Müller, Teresa; Spatz, Anne-Kathrin; Greggers, Uwe; Grünewald, Bernd; Menzel, Randolf

    2014-01-01

    Three neonicotinoids, imidacloprid, clothianidin and thiacloprid, agonists of the nicotinic acetylcholine receptor in the central brain of insects, were applied at non-lethal doses in order to test their effects on honeybee navigation. A catch-and-release experimental design was applied in which feeder trained bees were caught when arriving at the feeder, treated with one of the neonicotinoids, and released 1.5 hours later at a remote site. The flight paths of individual bees were tracked with harmonic radar. The initial flight phase controlled by the recently acquired navigation memory (vector memory) was less compromised than the second phase that leads the animal back to the hive (homing flight). The rate of successful return was significantly lower in treated bees, the probability of a correct turn at a salient landscape structure was reduced, and less directed flights during homing flights were performed. Since the homing phase in catch-and-release experiments documents the ability of a foraging honeybee to activate a remote memory acquired during its exploratory orientation flights, we conclude that non-lethal doses of the three neonicotinoids tested either block the retrieval of exploratory navigation memory or alter this form of navigation memory. These findings are discussed in the context of the application of neonicotinoids in plant protection.

  20. Neonicotinoids Interfere with Specific Components of Navigation in Honeybees

    PubMed Central

    Fischer, Johannes; Müller, Teresa; Spatz, Anne-Kathrin; Greggers, Uwe; Grünewald, Bernd; Menzel, Randolf

    2014-01-01

    Three neonicotinoids, imidacloprid, clothianidin and thiacloprid, agonists of the nicotinic acetylcholine receptor in the central brain of insects, were applied at non-lethal doses in order to test their effects on honeybee navigation. A catch-and-release experimental design was applied in which feeder trained bees were caught when arriving at the feeder, treated with one of the neonicotinoids, and released 1.5 hours later at a remote site. The flight paths of individual bees were tracked with harmonic radar. The initial flight phase controlled by the recently acquired navigation memory (vector memory) was less compromised than the second phase that leads the animal back to the hive (homing flight). The rate of successful return was significantly lower in treated bees, the probability of a correct turn at a salient landscape structure was reduced, and less directed flights during homing flights were performed. Since the homing phase in catch-and-release experiments documents the ability of a foraging honeybee to activate a remote memory acquired during its exploratory orientation flights, we conclude that non-lethal doses of the three neonicotinoids tested either block the retrieval of exploratory navigation memory or alter this form of navigation memory. These findings are discussed in the context of the application of neonicotinoids in plant protection. PMID:24646521

  1. Structural insights into transcription complexes.

    PubMed

    Berger, Imre; Blanco, Alexandre G; Boelens, Rolf; Cavarelli, Jean; Coll, Miquel; Folkers, Gert E; Nie, Yan; Pogenberg, Vivian; Schultz, Patrick; Wilmanns, Matthias; Moras, Dino; Poterszman, Arnaud

    2011-08-01

    Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of structural proteomics on our understanding of the molecular basis of gene expression. While most atomic structures were obtained by X-ray crystallography, the impact of solution NMR and cryo-electron microscopy is far from being negligible. Here, we summarize some highlights and illustrate the importance of specific technologies on the structural biology of protein-protein or protein/DNA transcription complexes: structure/function analysis of components the eukaryotic basal and activated transcription machinery with focus on the TFIID and TFIIH multi-subunit complexes as well as transcription regulators such as members of the nuclear hormone receptor families. We also discuss molecular aspects of promoter recognition and epigenetic control of gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Neonicotinoids show selective and diverse actions on their nicotinic receptor targets: electrophysiology, molecular biology, and receptor modeling studies.

    PubMed

    Matsuda, Kazuhiko; Shimomura, Masaru; Ihara, Makoto; Akamatsu, Miki; Sattelle, David B

    2005-08-01

    Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.

  3. [Current insights into chromatin structure organization].

    PubMed

    Ilatovskiĭ, A V; Lebedev, D V; Filatov, M V; Petukhov, M G; Isaev-Ivanov, V V

    2012-01-01

    This review summarizes current insights into organization of chromatin structure at different levels of DNA compaction. Analysis of available experimental data allowed concluding that only nucleosomal level of structural organization was sufficiently investigated, whereas structure of a 30-nm chromatin fiber remains an open issue. The data on the chromatin structure obtained at the level of the nucleus speak in favor of a biphasic fractal organization of chromatin.

  4. Structural insights into Elongator function.

    PubMed

    Glatt, Sebastian; Müller, Christoph W

    2013-04-01

    The eukaryotic Elongator complex was initially identified in yeast as a RNA polymerase II (Pol II) associated transcription elongation factor, although there is accumulating evidence that its main cellular function is the specific modification of uridines at the wobble base position of tRNAs. Elongator complex is built up by six highly conserved subunits and was shown to be involved in a variety of different cellular activities. Here, we summarize structural and functional information on individual Elongator subunits or subcomplexes. On the basis of homology models of the Elp1, Elp2 and Elp3 subunits and the crystal structure of the Elp456 subcomplex, the role of each subunit in Elongator complex assembly and catalytic activity is discussed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Structural insights into RNA interference.

    PubMed

    Sashital, Dipali G; Doudna, Jennifer A

    2010-02-01

    Virtually all animals and plants utilize small RNA molecules to control protein expression during different developmental stages and in response to viral infection. Structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small RNA biogenesis, formation of RNA-induced silencing complexes (RISCs), and targeting of complementary mRNAs. Here we review exciting recent progress in understanding how regulatory RNAs are produced and how they trigger specific destruction of mRNAs during RNA interference (RNAi).

  6. Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

    PubMed Central

    Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radić, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

    2008-01-01

    Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 Å in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids. PMID:18477694

  7. Structural insights on complement activation.

    PubMed

    Alcorlo, Martín; López-Perrote, Andrés; Delgado, Sandra; Yébenes, Hugo; Subías, Marta; Rodríguez-Gallego, César; Rodríguez de Córdoba, Santiago; Llorca, Oscar

    2015-10-01

    The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio-ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio-ester-containing domain (TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3-convertase of the alternative pathway (AP). In addition, the displacement of the TED and its interaction with the macroglobulin 1 (MG1) domain generates an extended surface in C3b where the complement regulators factor H (FH), decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1) can bind, mediating accelerated decay of the AP C3-convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED can display a collection of conformations around the MG ring, as well as a few specialized positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation by affecting the interaction with regulators.

  8. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  9. Sorption, desorption and degradation of neonicotinoids in four agricultural soils and their effects on soil microorganisms.

    PubMed

    Zhang, Peng; Ren, Chao; Sun, Hongwen; Min, Lujuan

    2017-09-29

    In this study, the sorption, desorption and degradation of three neonicotinoids, imidacloprid (IMI), clothianidin (CLO) and thiacloprid (THI), and their effects on microorganisms in four different agricultural soils were systematically evaluated. The sorption of neonicotinoids on the soils was generally low with distribution coefficients (Kd) up to 16.2L/kg at Ce of 0.05mg/L following the order THI>IMI≈CLO, and the sorption were mainly influenced by the soil organic carbon content. The percentage degradation rates of the pesticides in different soils ranged from 25.4% to 80.9%, all following the order THI>IMI≈CLO. All the three neonicotinoids degraded much faster under non-sterilized conditions than sterilized conditions, indicating considerable contribution of biodegradation. The total degradation or biodegradation of neonicotinoids was the fastest in the soil with the highest organic carbon content, and the neonicotinoids' bioavailability was not the primary influencing factor due to their weak sorption. The chemical degradation was mainly affected by pH and cation exchange capacity. The degradation of neonicotinoids occurred mainly via nitrate reduction, cyano hydrolysis and chloropyridinyl dechlorination. High-throughput sequencing data showed that the microbial community structure and abundance changed greatly in neonicotinoid-spiked soils as compared to the control, which might influence their degradation pathways. Some microbe families associated with the biodegradation of neoniconoids were found, which were all belonging to Proteobacteria and Actinobacteria. The degradation of neoniconoids influenced the soil nitrifying process. The present study provides valuable information for comprehensively understanding the fate of neonicotinoids in soils. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Biological Monitoring of Human Exposure to Neonicotinoids Using Urine Samples, and Neonicotinoid Excretion Kinetics

    PubMed Central

    Harada, Kouji H.; Tanaka, Keiko; Sakamoto, Hiroko; Imanaka, Mie; Niisoe, Tamon; Hitomi, Toshiaki; Kobayashi, Hatasu; Okuda, Hiroko; Inoue, Sumiko; Kusakawa, Koichi; Oshima, Masayo; Watanabe, Kiyohiko; Yasojima, Makoto; Takasuga, Takumi; Koizumi, Akio

    2016-01-01

    Background Neonicotinoids, which are novel pesticides, have entered into usage around the world because they are selectively toxic to arthropods and relatively non-toxic to vertebrates. It has been suggested that several neonicotinoids cause neurodevelopmental toxicity in mammals. The aim was to establish the relationship between oral intake and urinary excretion of neonicotinoids by humans to facilitate biological monitoring, and to estimate dietary neonicotinoid intakes by Japanese adults. Methodology/Principal Findings Deuterium-labeled neonicotinoid (acetamiprid, clothianidin, dinotefuran, and imidacloprid) microdoses were orally ingested by nine healthy adults, and 24 h pooled urine samples were collected for 4 consecutive days after dosing. The excretion kinetics were modeled using one- and two-compartment models, then validated in a non-deuterium-labeled neonicotinoid microdose study involving 12 healthy adults. Increased urinary concentrations of labeled neonicotinoids were observed after dosing. Clothianidin was recovered unchanged within 3 days, and most dinotefuran was recovered unchanged within 1 day. Around 10% of the imidacloprid dose was excreted unchanged. Most of the acetamiprid was metabolized to desmethyl-acetamiprid. Spot urine samples from 373 Japanese adults were analyzed for neonicotinoids, and daily intakes were estimated. The estimated average daily intake of these neonicotinoids was 0.53–3.66 μg/day. The highest intake of any of the neonicotinoids in the study population was 64.5 μg/day for dinotefuran, and this was <1% of the acceptable daily intake. PMID:26731104

  11. Biological Monitoring of Human Exposure to Neonicotinoids Using Urine Samples, and Neonicotinoid Excretion Kinetics.

    PubMed

    Harada, Kouji H; Tanaka, Keiko; Sakamoto, Hiroko; Imanaka, Mie; Niisoe, Tamon; Hitomi, Toshiaki; Kobayashi, Hatasu; Okuda, Hiroko; Inoue, Sumiko; Kusakawa, Koichi; Oshima, Masayo; Watanabe, Kiyohiko; Yasojima, Makoto; Takasuga, Takumi; Koizumi, Akio

    2016-01-01

    Neonicotinoids, which are novel pesticides, have entered into usage around the world because they are selectively toxic to arthropods and relatively non-toxic to vertebrates. It has been suggested that several neonicotinoids cause neurodevelopmental toxicity in mammals. The aim was to establish the relationship between oral intake and urinary excretion of neonicotinoids by humans to facilitate biological monitoring, and to estimate dietary neonicotinoid intakes by Japanese adults. Deuterium-labeled neonicotinoid (acetamiprid, clothianidin, dinotefuran, and imidacloprid) microdoses were orally ingested by nine healthy adults, and 24 h pooled urine samples were collected for 4 consecutive days after dosing. The excretion kinetics were modeled using one- and two-compartment models, then validated in a non-deuterium-labeled neonicotinoid microdose study involving 12 healthy adults. Increased urinary concentrations of labeled neonicotinoids were observed after dosing. Clothianidin was recovered unchanged within 3 days, and most dinotefuran was recovered unchanged within 1 day. Around 10% of the imidacloprid dose was excreted unchanged. Most of the acetamiprid was metabolized to desmethyl-acetamiprid. Spot urine samples from 373 Japanese adults were analyzed for neonicotinoids, and daily intakes were estimated. The estimated average daily intake of these neonicotinoids was 0.53-3.66 μg/day. The highest intake of any of the neonicotinoids in the study population was 64.5 μg/day for dinotefuran, and this was <1% of the acceptable daily intake.

  12. Neonicotinoid insecticides: highlights of a symposium on strategic molecular designs.

    PubMed

    Tomizawa, Motohiro; Casida, John E

    2011-04-13

    Neonicotinoids are the newest of the five major classes of insecticides (the others are chlorinated hydrocarbons, organophosphorus compounds, methylcarbamates, and pyrethroids), and they make up approximately one-fourth of the world insecticide market. Nithiazine was the lead compound from Shell Development Co. in California later optimized by Shinzo Kagabu of Nihon Tokushu Noyaku Seizo to increase the potency and photostability, resulting in imidacloprid and thiacloprid. These discoveries are the basis for the International Award for Research in Agrochemicals of the American Chemical Society presented in 2010 to Professor Shinzo Kagabu. Five other neonicotinoids were added by others for the current set of seven commercial compounds. This symposium considers the progress in discovery and development of novel chemotype nicotinic insecticides with enhanced effectiveness, unique biological properties, and maximal safety. Chemorational approaches considered include physicochemical properties, metabolic activation and detoxification, and chemical and structural biology aspects potentially facilitating receptor structure-guided insecticide design.

  13. cis-Nitromethylene neonicotinoids as new nicotinic family: synthesis, structural diversity, and insecticidal evaluation of hexahydroimidazo[1,2-alpha]pyridine.

    PubMed

    Shao, Xusheng; Zhang, Wenwen; Peng, Yanqing; Li, Zhong; Tian, Zhongzhen; Qian, Xuhong

    2008-12-15

    A series of neonicotinoids analogues of hexahydroimidazo[1,2-alpha]pyridine were modified at 5-, 6-, and 7-positions, and their insecticidal activities were evaluated. Introducing a methyl or ethyl at 7-position increased the insecticidal activities, while other substituents decreased activities. When alkyl substituents were introduced to 7-position, the insecticidal activities against Pea aphids decreased in the order methyl (7a)>ethyl (7b)>n-butyl (7e)>phenyl (7f)>n-propyl (7c)>iso-propyl (7d), p-NO(2)-phenyl (7g). Modifications at 5-, 6- or both at 6- and 7-positions with methyl or ethyl were unfavorable to activities. Interestingly, introducing methyl to 7-position not only increased insecticidal activities against pea aphids, but also show higher insecticidal activities than imidacloprid against imidacloprid-resistant brown planthopper.

  14. Monitoring of neonicotinoid pesticides in beekeeping.

    PubMed

    Cicero, Nicola; Naccari, Clara; Cammilleri, Gaetano; Giangrosso, Giuseppe; Cicero, Antonello; Gervasi, Teresa; Tropea, Alessia; Albergamo, Ambrogina; Ferrantelli, Vincenzo

    2017-06-01

    The decline of pollinating species is correlated to the extensive use of neonicotinoids against pest insects for crop protection. In this study, the concentrations of neonicotinoid insecticides were determined in honeybees, honeycomb and honey samples, collected in Spring 2015 (blooming period) from different areas in Sicily (IT), to carry out an evaluation of bees products' safety and an overview of neonicotinoid contamination in beekeeping. The results obtained showed only the presence of clothianidin in bee samples and these concentrations don't represent a risk for bees' vitality and safety. The absence of residue in all honey samples, instead, showed the quality of bee products.

  15. Orthobunyaviruses: recent genetic and structural insights.

    PubMed

    Elliott, Richard M

    2014-10-01

    Orthobunyaviruses, which have small, tripartite, negative-sense RNA genomes and structurally simple virions composed of just four proteins, can have devastating effects on human health and well-being, either by causing disease in humans or by causing disease in livestock and crops. In this Review, I describe the recent genetic and structural advances that have revealed important insights into the composition of orthobunyavirus virions, viral transcription and replication and viral interactions with the host innate immune response. Lastly, I highlight outstanding questions and areas of future research.

  16. Structural insights into microtubule doublet interactions inaxonemes

    SciTech Connect

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

    Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of the axoneme. Recent structural studies of the axoneme have used cryo-electron tomography to reveal new details of the interactions among some of the multitude of proteins that form the axoneme and regulate its movement. Connections among the several sets of dyneins, in particular, suggest ways in which their actions may be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding the doublet's mechanical properties that are related to the bending of the axoneme, and has also offered insight into its potential role in the mechanism of dynein activity regulation.

  17. Recent structural insights into transcription preinitiation complexes.

    PubMed

    Nogales, E

    2000-12-01

    Our understanding of the elaborate mechanism of gene transcription initiation in eukaryotes has been widened by recent structural information on some of the key components of the complex preinitiation transcriptional machinery. The high-resolution structures of both bacterial and eukaryotic polymerases are technical landmarks of great biological significance that have given us the first molecular insight into the mechanism of this large enzyme. While new atomic structures of different domains of general transcription factors, such as the double bromodomain of TAF250, have become available by means of X-ray crystallography and NMR studies, more global pictures of multisubunit transcription complexes, such as TFIID, TFIIH or the yeast mediator, have now been obtained by electron microscopy and image-reconstruction techniques. A combination of methodologies may prove essential for a complete structural description of the initial steps in the expression of eukaryotic genes.

  18. Neonicotinoid insecticide toxicology: mechanisms of selective action.

    PubMed

    Tomizawa, Motohiro; Casida, John E

    2005-01-01

    The neonicotinoids, the newest major class of insecticides, have outstanding potency and systemic action for crop protection against piercing-sucking pests, and they are highly effective for flea control on cats and dogs. Their common names are acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam. They generally have low toxicity to mammals (acute and chronic), birds, and fish. Biotransformations involve some activation reactions but largely detoxification mechanisms. In contrast to nicotine, epibatidine, and other ammonium or iminium nicotinoids, which are mostly protonated at physiological pH, the neonicotinoids are not protonated and have an electronegative nitro or cyano pharmacophore. Agonist recognition by the nicotinic receptor involves cation-pi interaction for nicotinoids in mammals and possibly a cationic subsite for interaction with the nitro or cyano substituent of neonicotinoids in insects. The low affinity of neonicotinoids for vertebrate relative to insect nicotinic receptors is a major factor in their favorable toxicological profile.

  19. Synthesis, insecticidal, and antibacterial activities of novel neonicotinoid analogs with dihydropyridine.

    PubMed

    He, Yinju; Hu, Deyu; Lv, Mingming; Jin, Linhong; Wu, Jian; Zeng, Song; Yang, Song; Song, Baoan

    2013-04-26

    Nilaparvata lugens, a major pest in rice-growing areas, is extremely difficult to manage. Neonicotinoids have increasingly been used in crop protection and animal health care against N. lugens. To discover new bioactive molecules and pesticides, we combined the active structure of cyanoacrylates, aromatic aldehydes, and substituted pyridyl (thiazolyl) methyl-2-substituted-methylidene-imidazolidine derivatives for the design and synthesis of a series of novel neonicotinoid analogs with dihydropyridine. A series of neonicotinoid analogs with dihydropyridine were synthesized. Their structures were characterized by IR, 1H NMR, 13C NMR, and elemental analysis and their insecticidal and antibacterial activities were assessed. Preliminary biological activity tests showed that all of the title compounds feature insecticidal activities against N. lugens at 500 mg/L. Moreover, some compounds showed promising antibacterial activities against Pseudomonas solanacearum (e.g., Tobacco bacterial wilt and Tomato bacterial wilt) at a dose of 200 mg/L. A synthetic route to obtain neonicotinoid analogs with dihydropyridine by the reaction of intermediates 2 (pyridyl (thiazolyl) methyl-2-substituted-methyl-ideneimidazolidine) and intermediates 1 (cyanoacrylates) and different aromatic aldehydes in acetonitrile under reflux conditions is presented. The effects of different solvents, bases, and reaction time on the reaction of 3a were investigated. The results of this study suggest that neonicotinoid analogs with dihydropyridine could cause N. lugens death and restrain P. solanacearum growth.

  20. Latest insights on adenovirus structure and assembly.

    PubMed

    San Martín, Carmen

    2012-05-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

  1. Latest Insights on Adenovirus Structure and Assembly

    PubMed Central

    San Martín, Carmen

    2012-01-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies. PMID:22754652

  2. Review of crop pests targeted by neonicotinoid seed treatments

    USDA-ARS?s Scientific Manuscript database

    Seed treatment with neonicotinoid insecticides is an increasingly popular crop protection practice, intended to reduce damage due to early season pests. A large proportion of major U.S. crops are planted with neonicotinoid-treated seed. Use of the three most popular neonicotinoids (imidacloprid, thi...

  3. Synthesis and biological evaluation of nitromethylene neonicotinoids based on the enhanced conjugation.

    PubMed

    Lu, Siyuan; Zhuang, Yingying; Wu, Ningbo; Feng, Yue; Cheng, Jiagao; Li, Zhong; Chen, Jie; Yuan, Jing; Xu, Xiaoyong

    2013-11-20

    The neonicotinoids with a nitroconjugated system had excellent bioactivity, which could rival imidacloprid, and has been previously reported. However, the photodegradation and hydrolysis of this series of neonicotinoids was very quick according to our further investigation, which cannot be developed as a pesticide further. The approach to further enhance the conjugation was tried not only to increase the bioactivities but also to improve the stability in water and in the sun. A substituted phenyl group was introduced into the furan ring of compound 3. A total of 13 novel neonicotinoid analogues with a higher conjugation system were designed and synthesized. The target molecular structures have been confirmed on the basis of satisfactory analytical and spectral data. All compounds presented significant insecticidal activities on cowpea aphid ( Aphis craccivora ), cotton aphid ( Aphis gossypii ), and brown planthopper ( Nilaparvata lugens ). The stability test exhibited that the stability of novel analogues in water and under the mercury lamp has been improved significantly in comparison to compound 3.

  4. Understanding cochleate formation: insights into structural development.

    PubMed

    Nagarsekar, Kalpa; Ashtikar, Mukul; Steiniger, Frank; Thamm, Jana; Schacher, Felix; Fahr, Alfred

    2016-04-20

    Understanding the structure and the self-assembly process of cochleates has become increasingly necessary considering the advances of this drug delivery system towards the pharmaceutical industry. It is well known that the addition of cations like calcium to a dispersion of anionic lipids such as phosphatidylserines results in stable, multilamellar cochleates through a spontaneous assembly. In the current investigation we have studied the intermediate structures generated during this self-assembly of cochleates. To achieve this, we have varied the process temperature for altering the rate of cochleate formation. Our findings from electron microscopy studies showed the formation of ribbonlike structures, which with proceeding interaction associate to form lipid stacks, networks and eventually cochleates. We also observed that the variation in lipid acyl chains did not make a remarkable difference to the type of structure evolved during the formation of cochleates. More generally, our observations provide a new insight into the self-assembly process of cochleates based on which we have proposed a pathway for cochleate formation from phosphatidylserine and calcium. This knowledge could be employed in using cochleates for a variety of possible biomedical applications in the future.

  5. Bees prefer foods containing neonicotinoid pesticides

    NASA Astrophysics Data System (ADS)

    Kessler, Sébastien C.; Tiedeken, Erin Jo; Simcock, Kerry L.; Derveau, Sophie; Mitchell, Jessica; Softley, Samantha; Stout, Jane C.; Wright, Geraldine A.

    2015-05-01

    The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

  6. Bees prefer foods containing neonicotinoid pesticides

    PubMed Central

    Simcock, Kerry L.; Derveau, Sophie; Mitchell, Jessica; Softley, Samantha; Stout, Jane C.; Wright, Geraldine A.

    2015-01-01

    The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies1-3. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants4. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure4,5. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly-used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO) in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX, and CLO neither elicited spiking responses from gustatory neurons in the bees’ mouthparts nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a significant hazard to foraging bees. PMID:25901684

  7. Bees prefer foods containing neonicotinoid pesticides.

    PubMed

    Kessler, Sébastien C; Tiedeken, Erin Jo; Simcock, Kerry L; Derveau, Sophie; Mitchell, Jessica; Softley, Samantha; Radcliffe, Amy; Stout, Jane C; Wright, Geraldine A

    2015-05-07

    The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

  8. Structural insight into Slit-Robo signalling.

    PubMed

    Hohenester, Erhard

    2008-04-01

    Drosophila Slit and its vertebrate orthologues Slit1-Slit3 are secreted glycoproteins that play important roles in the development of the nervous system and other organs. Human Slits are also involved in a number of pathological situations, such as cancer and inflammation. Slits exert their effects by activating receptors of the Robo (Roundabout) family, which resemble cell adhesion molecules in their ectodomains and have large, mainly unstructured cytosolic domains. HS (heparan sulfate) is required for Slit-Robo signalling. The hallmark of Slit proteins is a tandem of four LRR (leucine-rich repeat) domains, which mediate binding to the IG (immunoglobulin-like) domains of Robos. A major question is how Slit binding is translated into the recruitment of effector molecules to the cytosolic domain of Robo. Detailed structure-function studies have shown that the second LRR domain of Slit (D2) binds to the first two IG domains of Robo, and that HS serves to stabilize the Slit-Robo interaction and is required for biological activity of Slit D2. Very recently, the crystal structure of a minimal Slit-Robo complex revealed that the IG1 domain of Robo is bound by the concave face of Slit D2, confirming earlier mutagenesis data. To define the mechanism of Robo transmembrane signalling, these structural insights will have to be complemented by new cell biology and microscopy approaches.

  9. Structural insights into calicivirus attachment and uncoating.

    PubMed

    Bhella, David; Gatherer, Derek; Chaudhry, Yasmin; Pink, Rebecca; Goodfellow, Ian G

    2008-08-01

    The Caliciviridae family comprises positive-sense RNA viruses of medical and veterinary significance. In humans, caliciviruses are a major cause of acute gastroenteritis, while in animals respiratory illness, conjunctivitis, stomatitis, and hemorrhagic disease are documented. Investigation of virus-host interactions is limited by a lack of culture systems for many viruses in this family. Feline calicivirus (FCV), a member of the Vesivirus genus, provides a tractable model, since it may be propagated in cell culture. Feline junctional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for FCV. We have analyzed the structure of this virus-receptor complex by cryo-electron microscopy and three-dimensional image reconstruction, combined with fitting of homology modeled high-resolution coordinates. We show that domain 1 of fJAM-1 binds to the outer face of the P2 domain of the FCV capsid protein VP1, inducing conformational changes in the viral capsid. This study provides the first structural view of a native calicivirus-protein receptor complex and insights into the mechanisms of virus attachment and uncoating.

  10. Target-site resistance to neonicotinoids.

    PubMed

    Crossthwaite, Andrew J; Rendine, Stefano; Stenta, Marco; Slater, Russell

    2014-10-01

    Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

  11. Agonist actions of neonicotinoids on nicotinic acetylcholine receptors expressed by cockroach neurons.

    PubMed

    Tan, Jianguo; Galligan, James J; Hollingworth, Robert M

    2007-07-01

    indicated by maximal current (I(max)). Agonist efficacy, but not affinity, was positively correlated with insecticidal activity. These findings indicate that: (1) agonist affinity and efficacy vary independently with neonicotinoid structure; (2) high agonist efficacy is dependent on the presence of an acyclic electronegative pharmacophore group; (3) agonist efficacy is a significant factor in the insecticidal activity of neonicotinoids to cockroaches; (4) lower efficacy compounds cause excitatory symptoms (Type A), while high efficacy compounds cause depressive/paralytic symptoms (Type B).

  12. Mechanism of Neonicotinoid Toxicity: Impact on Oxidative Stress and Metabolism.

    PubMed

    Wang, Xu; Anadón, Arturo; Qinghua, Wu; Qiao, Fang; Ares, Irma; Martínez-Larrañaga, María-Rosa; Yuan, Zonghui; Martínez, María-Aránzazu

    2017-10-02

    Thousands of tons of neonicotinoids are widely used around the world as broad-spectrum systemic insecticides and veterinary drugs. Researchers originally thought that neonicotinoids exhibited low mammalian toxicity. However, following their widespread use, it became increasingly evident that neonicotinoids could have various toxic effects on vertebrates and invertebrates. The primary focus of this review is to summarize the research progress associated with oxidative stress as a plausible mechanism for neonicotinoid-induced toxicity as well as neonicotinoid metabolism. This review summarizes the research conducted over the past decade into the production of reactive oxygen species, reactive nitrogen species, and oxidative stress as result of neonicotinoid treatments, along with their correlation with the toxicity and metabolism of neonicotinoids. The metabolism of neonicotinoids and protection of various compounds against neonicotinoid-induced toxicity based on their antioxidative effects is also discussed. This review sheds new light on the critical roles of oxidative stress in neonicotinoid-induced toxicity to nontarget species. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 58 is January 6, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  13. Schedule for Review of Neonicotinoid Pesticides

    EPA Pesticide Factsheets

    The dockets for all the neonicotinoid pesticides, which pose exposure risk to honey bees and other pollinators, have been opened. Our goal is to review the pesticides in this class in the same timeframe so we can ensure consistency across the class.

  14. A worldwide survey of neonicotinoids in honey.

    PubMed

    Mitchell, E A D; Mulhauser, B; Mulot, M; Mutabazi, A; Glauser, G; Aebi, A

    2017-10-06

    Growing evidence for global pollinator decline is causing concern for biodiversity conservation and ecosystem services maintenance. Neonicotinoid pesticides have been identified or suspected as a key factor responsible for this decline. We assessed the global exposure of pollinators to neonicotinoids by analyzing 198 honey samples from across the world. We found at least one of five tested compounds (acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam) in 75% of all samples, 45% of samples contained two or more of these compounds, and 10% contained four or five. Our results confirm the exposure of bees to neonicotinoids in their food throughout the world. The coexistence of neonicotinoids and other pesticides may increase harm to pollinators. However, the concentrations detected are below the maximum residue level authorized for human consumption (average ± standard error for positive samples: 1.8 ± 0.56 nanograms per gram). Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  15. Neonicotinoids in the Canadian aquatic environment: a literature review on current use products with a focus on fate, exposure, and biological effects.

    PubMed

    Anderson, J C; Dubetz, C; Palace, V P

    2015-02-01

    Developed to replace organophosphate and carbamate insecticides, neonicotinoids are structurally similar to nicotine. The three main neonicotinoid insecticides, imidacloprid, clothianidin, and thiamethoxam, are being re-evaluated by Health Canada's Pest Management Regulatory Agency (PMRA). An important aspect of the re-evaluation is the potential for effects in non-target organisms, including aquatic organisms. Leaching into surface waters is one of the major concerns surrounding extensive use of neonicotinoids, especially in close proximity to water bodies. The PMRA has classified IMI as 'persistent' with a 'high' leaching potential. Globally, neonicotinoids have been detected in a variety of water bodies, typically at concentrations in the low μg/L range. While IMI has been included in some monitoring exercises, there are currently very few published data for the presence of CLO and THM in Canadian water bodies. The majority of neonicotinoid toxicity studies have been conducted with IMI due to its longer presence on the market and high prevalence of use. Aquatic insects are particularly vulnerable to neonicotinoids and chronic toxicity has been observed at concentrations of IMI below 1 μg/L. Acute toxicity has been reported at concentrations below 20 μg/L for the most sensitive species, including Hyalella azteca, ostracods, and Chironomus riparius. Fish, algae, amphibians, and molluscs are relatively insensitive to IMI. However, the biological effects of THM and CLO have not been as well explored. The Canadian interim water quality guideline for IMI is 0.23 μg/L, but there is currently insufficient use, fate, and toxicological information available to establish guidelines for CLO and THM. Based on concentrations of neonicotinoids reported in surface waters in Canada and globally, there is potential for aquatic invertebrates to be negatively impacted by neonicotinoids. Therefore, it is necessary to address knowledge gaps to inform decisions around guidelines

  16. Binary mixtures of neonicotinoids show different transcriptional changes than single neonicotinoids in honeybees (Apis mellifera).

    PubMed

    Christen, Verena; Bachofer, Sara; Fent, Karl

    2017-01-01

    Among the many factors responsible for the decline of bee populations are plant protection products such as neonicotinoids. In general, bees are exposed to not only one but mixtures of such chemicals. At environmental realistic concentrations neonicotinoids may display negative effects on the immune system, foraging activity, learning and memory formation of bees. Neonicotinoids induce alterations of gene transcripts such as nicotinic acetylcholine receptor (nAChR) subunits, vitellogenin, genes of the immune system and genes linked to memory formation. While previous studies focused on individual compounds, the effect of neonicotinoid mixtures in bees is poorly known. Here we investigated the effects of neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam as single compounds, and binary mixtures thereof in honeybees. We determined transcriptional changes of nAChR subunits and vitellogenin in the brain of experimentally exposed honeybees after exposure up to 72 h. Exposure concentrations were selected on the basis of lowest effect concentrations of the single compounds. Transcriptional induction of nAChRs and vitellogenin was strongest for thiamethoxam, and weakest for acetamiprid. To a large extent, binary mixtures did not show additive transcriptional inductions but they were less than additive. Our data suggest that the joint transcriptional activity of neonicotinoids cannot be explained by concentration addition. The in vivo effects are not only governed by agonistic interaction with nAChRs alone, but are more complex as a result of interactions with other pathways as well. Further studies are needed to investigate the physiological joint effects of mixtures of neonicotinoids and other plant protection products on bees to better understand their joint effects.

  17. Bacterial biodegradation of neonicotinoid pesticides in soil and water systems.

    PubMed

    Hussain, Sarfraz; Hartley, Carol J; Shettigar, Madhura; Pandey, Gunjan

    2016-12-01

    Neonicotinoids are neurotoxic systemic insecticides used in plant protection worldwide. Unfortunately, application of neonicotinoids affects both beneficial and target insects indiscriminately. Being water soluble and persistent, these pesticides are capable of disrupting both food chains and biogeochemical cycles. This review focuses on the biodegradation of neonicotinoids in soil and water systems by the bacterial community. Several bacterial strains have been isolated and identified as capable of transforming neonicotinoids in the presence of an additional carbon source. Environmental parameters have been established for accelerated transformation in some of these strains. Studies have also indicated that enhanced biotransformation of these pesticides can be accomplished by mixed microbial populations under optimised environmental conditions. Substantial research into the identification of neonicotinoid-mineralising bacterial strains and identification of the genes and enzymes responsible for neonicotinoid degradation is still required to complete the understanding of microbial biodegradation pathways, and advance bioremediation efforts.

  18. Solid-phase purification and extraction for the determination of trace neonicotinoid pesticides in tea infusion.

    PubMed

    Zhang, Minglu; Chen, Hongping; Zhu, Li; Wang, Chuanpi; Ma, Guicen; Liu, Xin

    2016-03-01

    An analytical protocol that includes solid-phase purification and extraction is successfully developed for the determination of trace neonicotinoid pesticides in tea infusion. The method consists of a purification on amino-functionalized mesoporous silica SBA-15 followed by a solid-phase extraction based on graphene oxide before ultra high performance liquid chromatography with tandem mass spectrometry analysis. Parameters that significantly affected the extraction of the neonicotinoids onto graphene oxide, such as the amount of adsorbent, extraction time, pH, elution solvent, etc. were optimized. The amino-functionalized mesoporous silica SBA-15 has been proved to be an efficient adsorbent for removal of polyphenols especially catechins from tea infusion. Graphene oxide exhibits a very rapid adsorption rate (within 10 min) and high adsorption capacities for neonicotinoids at low initial concentration (0.01-0.5 mg/L). The analysis method gave a good determination coefficient (r(2) > 0.99) for each pesticide and high recoveries in the range of 72.2-95.0%. Powder X-ray diffraction, Raman spectroscopy, transmission electron microscopy, and UV-vis spectroscopy were utilized to identify the structure and morphology of graphene oxide. The adsorption driving force of neonicotinoids on graphene oxide mainly depends on π-π electron donor-acceptor interaction and electrostatic interaction. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

    PubMed

    Tomizawa, M; Latli, B; Casida, J E

    1996-10-01

    Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

  20. Neonicotinoid pesticides severely affect honey bee queens

    PubMed Central

    Williams, Geoffrey R.; Troxler, Aline; Retschnig, Gina; Roth, Kaspar; Yañez, Orlando; Shutler, Dave; Neumann, Peter; Gauthier, Laurent

    2015-01-01

    Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances. PMID:26459072

  1. Neonicotinoid pesticides severely affect honey bee queens.

    PubMed

    Williams, Geoffrey R; Troxler, Aline; Retschnig, Gina; Roth, Kaspar; Yañez, Orlando; Shutler, Dave; Neumann, Peter; Gauthier, Laurent

    2015-10-13

    Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances.

  2. Risks of neonicotinoid insecticides to honeybees

    PubMed Central

    Fairbrother, Anne; Purdy, John; Anderson, Troy; Fell, Richard

    2014-01-01

    The European honeybee, Apis mellifera, is an important pollinator of agricultural crops. Since 2006, when unexpectedly high colony losses were first reported, articles have proliferated in the popular press suggesting a range of possible causes and raising alarm over the general decline of bees. Suggested causes include pesticides, genetically modified crops, habitat fragmentation, and introduced diseases and parasites. Scientists have concluded that multiple factors in various combinations—including mites, fungi, viruses, and pesticides, as well as other factors such as reduction in forage, poor nutrition, and queen failure—are the most probable cause of elevated colony loss rates. Investigators and regulators continue to focus on the possible role that insecticides, particularly the neonicotinoids, may play in honeybee health. Neonicotinoid insecticides are insect neurotoxicants with desirable features such as broad-spectrum activity, low application rates, low mammalian toxicity, upward systemic movement in plants, and versatile application methods. Their distribution throughout the plant, including pollen, nectar, and guttation fluids, poses particular concern for exposure to pollinators. The authors describe how neonicotinoids interact with the nervous system of honeybees and affect individual honeybees in laboratory situations. Because honeybees are social insects, colony effects in semifield and field studies are discussed. The authors conclude with a review of current and proposed guidance in the United States and Europe for assessing the risks of pesticides to honeybees. PMID:24692231

  3. Risks of neonicotinoid insecticides to honeybees.

    PubMed

    Fairbrother, Anne; Purdy, John; Anderson, Troy; Fell, Richard

    2014-04-01

    The European honeybee, Apis mellifera, is an important pollinator of agricultural crops. Since 2006, when unexpectedly high colony losses were first reported, articles have proliferated in the popular press suggesting a range of possible causes and raising alarm over the general decline of bees. Suggested causes include pesticides, genetically modified crops, habitat fragmentation, and introduced diseases and parasites. Scientists have concluded that multiple factors in various combinations-including mites, fungi, viruses, and pesticides, as well as other factors such as reduction in forage, poor nutrition, and queen failure-are the most probable cause of elevated colony loss rates. Investigators and regulators continue to focus on the possible role that insecticides, particularly the neonicotinoids, may play in honeybee health. Neonicotinoid insecticides are insect neurotoxicants with desirable features such as broad-spectrum activity, low application rates, low mammalian toxicity, upward systemic movement in plants, and versatile application methods. Their distribution throughout the plant, including pollen, nectar, and guttation fluids, poses particular concern for exposure to pollinators. The authors describe how neonicotinoids interact with the nervous system of honeybees and affect individual honeybees in laboratory situations. Because honeybees are social insects, colony effects in semifield and field studies are discussed. The authors conclude with a review of current and proposed guidance in the United States and Europe for assessing the risks of pesticides to honeybees. © 2014 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc.

  4. Potential exposure of pollinators to neonicotinoid insecticides from the use of insecticide seed treatments in the mid-southern United States.

    PubMed

    Stewart, Scott D; Lorenz, Gus M; Catchot, Angus L; Gore, Jeff; Cook, Don; Skinner, John; Mueller, Thomas C; Johnson, Donald R; Zawislak, Jon; Barber, Jonathan

    2014-08-19

    Research was done during 2012 to evaluate the potential exposure of pollinators to neonicotinoid insecticides used as seed treatments on corn, cotton, and soybean. Samples were collected from small plot evaluations of seed treatments and from commercial fields in agricultural production areas in Arkansas, Mississippi, and Tennessee. In total, 560 samples were analyzed for concentrations of clothianidin, imidacloprid, thiamethoxam, and their metabolites. These included pollen from corn and cotton, nectar from cotton, flowers from soybean, honey bees, Apis mellifera L., and pollen carried by foragers returning to hives, preplanting and in-season soil samples, and wild flowers adjacent to recently planted fields. Neonicotinoid insecticides were detected at a level of 1 ng/g or above in 23% of wild flower samples around recently planted fields, with an average detection level of about 10 ng/g. We detected neonicotinoid insecticides in the soil of production fields prior to planting at an average concentration of about 10 ng/g, and over 80% of the samples having some insecticide present. Only 5% of foraging honey bees tested positive for the presence of neonicotinoid insecticides, and there was only one trace detection (< 1 ng/g) in pollen being carried by those bees. Soybean flowers, cotton pollen, and cotton nectar contained little or no neonicotinoids resulting from insecticide seed treatments. Average levels of neonicotinoid insecticides in corn pollen ranged from less than 1 to 6 ng/g. The highest neonicotinoid concentrations were found in soil collected during early flowering from insecticide seed treatment trials. However, these levels were generally not well correlated with neonicotinoid concentrations in flowers, pollen, or nectar. Concentrations in flowering structures were well below defined levels of concern thought to cause acute mortality in honey bees. The potential implications of our findings are discussed.

  5. Environmental fate and exposure; neonicotinoids and fipronil.

    PubMed

    Bonmatin, J-M; Giorio, C; Girolami, V; Goulson, D; Kreutzweiser, D P; Krupke, C; Liess, M; Long, E; Marzaro, M; Mitchell, E A D; Noome, D A; Simon-Delso, N; Tapparo, A

    2015-01-01

    Systemic insecticides are applied to plants using a wide variety of methods, ranging from foliar sprays to seed treatments and soil drenches. Neonicotinoids and fipronil are among the most widely used pesticides in the world. Their popularity is largely due to their high toxicity to invertebrates, the ease and flexibility with which they can be applied, their long persistence, and their systemic nature, which ensures that they spread to all parts of the target crop. However, these properties also increase the probability of environmental contamination and exposure of nontarget organisms. Environmental contamination occurs via a number of routes including dust generated during drilling of dressed seeds, contamination and accumulation in arable soils and soil water, runoff into waterways, and uptake of pesticides by nontarget plants via their roots or dust deposition on leaves. Persistence in soils, waterways, and nontarget plants is variable but can be prolonged; for example, the half-lives of neonicotinoids in soils can exceed 1,000 days, so they can accumulate when used repeatedly. Similarly, they can persist in woody plants for periods exceeding 1 year. Breakdown results in toxic metabolites, though concentrations of these in the environment are rarely measured. Overall, there is strong evidence that soils, waterways, and plants in agricultural environments and neighboring areas are contaminated with variable levels of neonicotinoids or fipronil mixtures and their metabolites (soil, parts per billion (ppb)-parts per million (ppm) range; water, parts per trillion (ppt)-ppb range; and plants, ppb-ppm range). This provides multiple routes for chronic (and acute in some cases) exposure of nontarget animals. For example, pollinators are exposed through direct contact with dust during drilling; consumption of pollen, nectar, or guttation drops from seed-treated crops, water, and consumption of contaminated pollen and nectar from wild flowers and trees growing near

  6. Structural insights into ABC transporter mechanism

    SciTech Connect

    Oldham, Michael L.; Davidson, Amy L.; Chen, Jue

    2010-07-27

    ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

  7. The discovery of thiamethoxam: a second-generation neonicotinoid.

    PubMed

    Maienfisch, P; Huerlimann, H; Rindlisbacher, A; Gsell, L; Dettwiler, H; Haettenschwiler, J; Sieger, E; Walti, M

    2001-02-01

    Neonicotinoids represent a novel and distinct chemical class of insecticides with remarkable chemical and biological properties. In 1985, a research programme was started in this field, in which novel nitroimino heterocycles were designed, prepared and assayed for insecticidal activity. The methodology for the synthesis of 2-nitroimino-hexahydro-1,3,5-triazines, 4-nitroimino-1,3,5-oxadiazinanes and 4-nitroimino-1,3,5-thiadiazinanes is outlined. Bioassays demonstrated that 3-(6-chloropyridin-3-ylmethyl)-4-nitroimino-1,3,5-oxadiazinane exhibited better insecticidal activity than the corresponding 2-nitroimino-hexahydro-1,3,5-triazine and 4-nitroimino-1,3,5-thiadiazinane. In most tests, this compound was equally or only slightly less active than imidacloprid. A series of structural modifications on this lead structure revealed that replacement of the 6-chloro-3-pyridyl group by a 2-chloro-5-thiazolyl moiety resulted in a strong increase of activity against chewing insects, whereas the introduction of a methyl group as pharmacophore substituent increased activity against sucking pests. The combination of these two favourable modifications led to thiamethoxam (CGA 293 343). Thiamethoxam is the first commercially available second-generation neonicotinoid and belongs to the thianicotinyl sub-class. It is marketed under the trademarks Actara for foliar and soil treatment and Cruiser for seed treatment. The compound has broad-spectrum insecticidal activity and offers excellent control of a wide variety of commercially important pests in many crops. Low use rates, flexible application methods, excellent efficacy and the favourable safety profile make this new insecticide well-suited for modern integrated pest management programmes in many cropping systems.

  8. Structural insights into the translational infidelity mechanism

    NASA Astrophysics Data System (ADS)

    Rozov, Alexey; Demeshkina, Natalia; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

    2015-06-01

    The decoding of mRNA on the ribosome is the least accurate process during genetic information transfer. Here we propose a unified decoding mechanism based on 11 high-resolution X-ray structures of the 70S ribosome that explains the occurrence of missense errors during translation. We determined ribosome structures in rare states where incorrect tRNAs were incorporated into the peptidyl-tRNA-binding site. These structures show that in the codon-anticodon duplex, a G.U mismatch adopts the Watson-Crick geometry, indicating a shift in the tautomeric equilibrium or ionization of the nucleobase. Additional structures with mismatches in the 70S decoding centre show that the binding of any tRNA induces identical rearrangements in the centre, which favours either isosteric or close to the Watson-Crick geometry codon-anticodon pairs. Overall, the results suggest that a mismatch escapes discrimination by preserving the shape of a Watson-Crick pair and indicate that geometric selection via tautomerism or ionization dominates the translational infidelity mechanism.

  9. Design, synthesis, and particular biological behaviors of chain-opening nitromethylene neonicotinoids with cis configuration.

    PubMed

    Lu, Siyuan; Shao, Xusheng; Li, Zhong; Xu, Zhiping; Zhao, Shishuai; Wu, Yinli; Xu, Xiaoyong

    2012-01-11

    On the basis of the structure of heterocyclic-fused cis configuration derivatives and chain-opening neonicotinoids, two series of novel chain-opening tetrahydropyridine analogues were designed and synthesized. The preliminary bioassay tests were determined on cowpea aphid (Aphis craccivora) and armyworm (Pseudaletia separata Walker). The results showed that some of the target compounds exhibited repellent effects, whereas others showed good insecticidal activities.

  10. Structural insights into a circadian oscillator.

    PubMed

    Johnson, Carl Hirschie; Egli, Martin; Stewart, Phoebe L

    2008-10-31

    An endogenous circadian system in cyanobacteria exerts pervasive control over cellular processes, including global gene expression. Indeed, the entire chromosome undergoes daily cycles of topological changes and compaction. The biochemical machinery underlying a circadian oscillator can be reconstituted in vitro with just three cyanobacterial proteins, KaiA, KaiB, and KaiC. These proteins interact to promote conformational changes and phosphorylation events that determine the phase of the in vitro oscillation. The high-resolution structures of these proteins suggest a ratcheting mechanism by which the KaiABC oscillator ticks unidirectionally. This posttranslational oscillator may interact with transcriptional and translational feedback loops to generate the emergent circadian behavior in vivo. The conjunction of structural, biophysical, and biochemical approaches to this system reveals molecular mechanisms of biological timekeeping.

  11. Insight into Amyloid Structure Using Chemical Probes

    PubMed Central

    Reinke, Ashley A.; Gestwicki, Jason E.

    2011-01-01

    Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloids in the brain. One prominent form of amyloid is composed of repeating units of the amyloid-β (Aβ) peptide. Over the past decade, it has become clear that these Aβ amyloids are not homogeneous; rather, they are composed of a series of structures varying in their overall size and shape and the number of Aβ peptides they contain. Recent theories suggest that these different amyloid conformations may play distinct roles in disease, although their relative contributions are still being discovered. Here, we review how chemical probes, such as congo red, thioflavin T and their derivatives, have been powerful tools for better understanding amyloid structure and function. Moreover, we discuss how design and deployment of conformationally selective probes might be used to test emerging models of AD. PMID:21457473

  12. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  13. Structural and functional insights into Mimivirus ORFans

    PubMed Central

    Saini, Harpreet Kaur; Fischer, Daniel

    2007-01-01

    Background Mimivirus isolated from A. polyphaga is the largest virus discovered so far. It is unique among all the viruses in having genes related to translation, DNA repair and replication which bear close homology to eukaryotic genes. Nevertheless, only a small fraction of the proteins (33%) encoded in this genome has been assigned a function. Furthermore, a large fraction of the unassigned protein sequences bear no sequence similarity to proteins from other genomes. These sequences are referred to as ORFans. Because of their lack of sequence similarity to other proteins, they can not be assigned putative functions using standard sequence comparison methods. As part of our genome-wide computational efforts aimed at characterizing Mimivirus ORFans, we have applied fold-recognition methods to predict the structure of these ORFans and further functions were derived based on conservation of functionally important residues in sequence-template alignments. Results Using fold recognition, we have identified highly confident computational 3D structural assignments for 21 Mimivirus ORFans. In addition, highly confident functional predictions for 6 of these ORFans were derived by analyzing the conservation of functional motifs between the predicted structures and proteins of known function. This analysis allowed us to classify these 6 previously unannotated ORFans into their specific protein families: carboxylesterase/thioesterase, metal-dependent deacetylase, P-loop kinases, 3-methyladenine DNA glycosylase, BTB domain and eukaryotic translation initiation factor eIF4E. Conclusion Using stringent fold recognition criteria we have assigned three-dimensional structures for 21 of the ORFans encoded in the Mimivirus genome. Further, based on the 3D models and an analysis of the conservation of functionally important residues and motifs, we were able to derive functional attributes for 6 of the ORFans. Our computational identification of important functional sites in these

  14. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  15. Structural insights into sulfite oxidase deficiency.

    PubMed

    Karakas, Erkan; Wilson, Heather L; Graf, Tyler N; Xiang, Song; Jaramillo-Busquets, Sandra; Rajagopalan, K V; Kisker, Caroline

    2005-09-30

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  16. New insights into prion structure and toxicity.

    PubMed

    Harris, David A; True, Heather L

    2006-05-04

    Prion diseases in humans and animals are due to conformational conversion of PrP(C), a cellular glycoprotein of unknown function, into PrP(Sc), an isoform that appears to be infectious in the absence of nucleic acids. Proteins that behave as prions are also found in yeast and filamentous fungi. Although there is now strong experimental support for the hypothesis that prions are infectious proteins, two subjects have remained poorly understood: the structure of prions, and the mechanisms by which they kill neurons. In this review, we will highlight recent studies that shed new light on these important issues.

  17. A beekeeper's perspective on the neonicotinoid ban.

    PubMed

    Carreck, Norman L

    2017-07-01

    Bees and agrochemicals have a long history. For example, the first volume of IBRA's journal Bee World in 1919 contains mention of poisoning of bees by spraying an orchard with lead arsenate. Bees being insects, it is self-evident that the use of insecticides to control crop pests poses a risk to them. Bee poisoning incidents became a very serious problem in the 1960s and 1970s with spraying of, in particular, oilseed rape with organophosphorus compounds. The introduction of carbamates and then especially synthetic pyrethroids reduced these problems. Data from the Wildlife Incident Investigation Scheme show that in recent years there have been very few poisoning incidents in the United Kingdom that can be attributed to agricultural insecticides. The introduction of neonicotinoid insecticides has, however, been very controversial. Almost as soon as they were introduced in the 1990s, French beekeepers blamed colony losses on imidacloprid used on sunflowers and maize, but restrictions on its use did not lead to a reduction in losses or to a reduction in beekeepers' concerns. Acute pesticide poisoning incidents by neonicotinoids in Germany and Italy in 2008 further sealed their reputation. Despite laboratory evidence showing their harm, field experience remains equivocal, and many commercial beekeepers continue to move their colonies to oilseed rape crops for honey production. The neonicotinoid moratorium has undoubtedly led to the increased use of older insecticides, and the effect of this on bee populations is unknown and unquantified. Many beekeepers are currently confused by the conflicting evidence. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  18. Are neonicotinoid insecticides driving declines of widespread butterflies?

    PubMed Central

    Bunnefeld, Nils; Wilson, John McVean; Botham, Marc S.; Brereton, Tom M.; Fox, Richard; Goulson, Dave

    2015-01-01

    There has been widespread concern that neonicotinoid pesticides may be adversely impacting wild and managed bees for some years, but recently attention has shifted to examining broader effects they may be having on biodiversity. For example in the Netherlands, declines in insectivorous birds are positively associated with levels of neonicotinoid pollution in surface water. In England, the total abundance of widespread butterfly species declined by 58% on farmed land between 2000 and 2009 despite both a doubling in conservation spending in the UK, and predictions that climate change should benefit most species. Here we build models of the UK population indices from 1985 to 2012 for 17 widespread butterfly species that commonly occur at farmland sites. Of the factors we tested, three correlated significantly with butterfly populations. Summer temperature and the index for a species the previous year are both positively associated with butterfly indices. By contrast, the number of hectares of farmland where neonicotinoid pesticides are used is negatively associated with butterfly indices. Indices for 15 of the 17 species show negative associations with neonicotinoid usage. The declines in butterflies have largely occurred in England, where neonicotinoid usage is at its highest. In Scotland, where neonicotinoid usage is comparatively low, butterfly numbers are stable. Further research is needed urgently to show whether there is a causal link between neonicotinoid usage and the decline of widespread butterflies or whether it simply represents a proxy for other environmental factors associated with intensive agriculture. PMID:26623186

  19. Are neonicotinoid insecticides driving declines of widespread butterflies?

    PubMed

    Gilburn, Andre S; Bunnefeld, Nils; Wilson, John McVean; Botham, Marc S; Brereton, Tom M; Fox, Richard; Goulson, Dave

    2015-01-01

    There has been widespread concern that neonicotinoid pesticides may be adversely impacting wild and managed bees for some years, but recently attention has shifted to examining broader effects they may be having on biodiversity. For example in the Netherlands, declines in insectivorous birds are positively associated with levels of neonicotinoid pollution in surface water. In England, the total abundance of widespread butterfly species declined by 58% on farmed land between 2000 and 2009 despite both a doubling in conservation spending in the UK, and predictions that climate change should benefit most species. Here we build models of the UK population indices from 1985 to 2012 for 17 widespread butterfly species that commonly occur at farmland sites. Of the factors we tested, three correlated significantly with butterfly populations. Summer temperature and the index for a species the previous year are both positively associated with butterfly indices. By contrast, the number of hectares of farmland where neonicotinoid pesticides are used is negatively associated with butterfly indices. Indices for 15 of the 17 species show negative associations with neonicotinoid usage. The declines in butterflies have largely occurred in England, where neonicotinoid usage is at its highest. In Scotland, where neonicotinoid usage is comparatively low, butterfly numbers are stable. Further research is needed urgently to show whether there is a causal link between neonicotinoid usage and the decline of widespread butterflies or whether it simply represents a proxy for other environmental factors associated with intensive agriculture.

  20. Theoretical Insight into Polymer Structure near Interfaces

    NASA Astrophysics Data System (ADS)

    Curro, John G.

    2000-03-01

    A molecule near a surface experiences an anisotropic environment in contrast to a molecule in a bulk liquid. As a consequence, the packing of macromolecules is different in the vicinity of a surface than in a bulk polymer melt or solution, and, in addition, must be treated with a theoretical formalism appropriate for inhomogeneous systems. Not surprisingly, this interface structure plays an important role in determining many properties of interest including adhesion, surface tension, wetting, and polymer compatibility. This work is concerned with modeling the structure and packing of macromolecules near a surface. Our approach, like many other previous studies, is a self-consistent field theory. In this theory the many chain problem near a surface is mapped onto a single chain problem in the presence of an external field. The effects of the multiple chain correlations are incorporated into a self-consistently determined, external field. Our approach differs from previous work in that we use a form of this external field that allows us to account for atomistic details in the calculation. We employ the classical density functional theory (DFT) of Chandler, McCoy, and Singer in which the excess free energy of the inhomogeneous system is expanded to second order about the uniform system. This leads to a nonlocal external field involving the direct correlation function C(r) of the homogeneous, bulk polymer liquid. C(r) can be determined from polymer reference interaction site (PRISM) theory, or simulation. The self-consistent calculation of the density profile can be computed numerically for chain models that are Markovian. For more realistic chain models, however, a single chain Monte Carlo simulation in an external field must be performed. Applications of this DFT method will be discussed and comparisons will be made between theory and full, many chain simulations.

  1. Specific Synergist for Neonicotinoid Insecticides: IPPA08, a cis-Neonicotinoid Compound with a Unique Oxabridged Substructure.

    PubMed

    Bao, Haibo; Shao, Xusheng; Zhang, Yixi; Deng, Yayun; Xu, Xiaoyong; Liu, Zewen; Li, Zhong

    2016-06-29

    Insecticide synergists are key components to increase the control efficacy and reduce active ingredient use. Here, we describe a novel insecticide synergist with activity specific for insecticidal neonicotinoids. The synergist IPPA08, a cis configuration neonicotinoid compound with a unique oxabridged substructure, could increase the toxicity of most neonicotinoid insecticides belonging to the Insecticide Resistance Action Committee (IRAC) 4A subgroup against a range of insect species, although IPPA08 itself was almost inactive to insects at synergistic concentrations. Unfortunately, similar effects were observed on the honey bee (Apis mellifera) and the brown planthopper (Nilaparvata lugens), resistant to imidacloprid. IPPA08 did not show any effects on toxicity of insecticides with different targets, which made us define it as a neonicotinoid-specific synergist. Unlike most insecticide synergists, by inhibition of activities of detoxification enzymes, IPPA08 showed no effects on enzyme activities. The results revealed that IPPA08 worked as a synergist through a distinct way. Although the modulating insect nicotinic acetylcholine receptors (nAChRs, targets of neonicotinoid insecticides) were supposed as a possible mode of action for IPPA08 as a neonicotinoid-specific synergist, direct evidence is needed in further studies. In insect pest control, IPPA08 acts as a target synergist to increase neonicotinoid toxicity and reduce the amount of neonicotinoid used. Combinations of IPPA08 and insecticidal neonicotinoids may be developed into new insecticide formulations. In summary, combining an active ingredient with a "custom" synergist appears to be a very promising approach for the development of effective new insecticide products.

  2. Benefits of Neonicotinoid Seed Treatments to Soybean Production

    EPA Pesticide Factsheets

    Read about EPA’s analysis of use of the neonicotinoid seed treatments for insect control in U.S. soybean production. EPA concludes that these seed treatments provide little or no overall benefits to soybean production in most situations.

  3. The effects of neonicotinoid exposure on embryonic development and organ mass in northern bobwhite quail (Colinus virginianus).

    PubMed

    Gobeli, Amanda; Crossley, Dane; Johnson, Jeff; Reyna, Kelly

    2017-05-01

    Since their emergence in the early 1990s, neonicotinoid use has increased exponentially to make them the world's most prevalent insecticides. Although there has been considerable research concerning the lethality of neonicotinoids, their sub-lethal and developmental effects are still being explored, especially with regard to non-mammalian species. The goal of this research was to investigate the effects of the neonicotinoid imidacloprid on the morphological and physiological development of northern bobwhite quail (Colinus virginianus). Bobwhite eggs (n=390) were injected with imidacloprid concentrations of 0 (sham), 10, 50, 100, and 150mg/kg of egg mass, which was administered at day 0 (pre-incubation), 3, 6, 9, or 12 of growth. Embryos were dissected, weighed, staged, and examined for any overt structural deformities after 19days of incubation. The mass of the embryonic heart, liver, lungs and kidneys was also recorded. The majority of treatments produced no discernible differences in embryo morphology; however, in some instances, embryos were subject to increased frequency of anatomical deformity and altered organ masses. Some impacts were more pronounced in specific dosing periods, implying that there may be critical windows of development when embryos are more susceptible to neonicotinoid exposure. This investigation suggests that imidacloprid has the potential to impact bobwhite quail embryonic development and chick survival. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Insights into the structural biology of Gaucher disease.

    PubMed

    Smith, Laura; Mullin, Stephen; Schapira, Anthony H V

    2017-09-18

    Gaucher disease, the most common lysosomal storage disorder, is caused by mutations in the gene encoding the acid-β-glucosidase lysosomal hydrolase enzyme that cleaves glucocerebroside into glucose and ceramide. Reduced enzyme activity and impaired structural stability arise due to >300 known disease-causing mutations. Several of these mutations have also been associated with an increased risk of Parkinson disease (PD). Since the discovery of the acid-β-glucosidase X-ray structure, there have been major advances in our understanding of the structural properties of the protein. Analysis of specific residues has provided insight into their functional and structural importance and provided insight into the pathogenesis of Gaucher disease and the contribution to PD. Disease-causing mutations are positioned throughout the acid-β-glucosidase structure, with many located far from the active site and thus retaining some enzymatic activity however, thus far no clear relationship between mutation location and disease severity has been established. Here, we review the crystal structure of acid-β-glucosidase, while highlighting important structural aspects of the protein in detail. This review discusses the structural stability of acid-β-glucosidase, which can be altered by pH and glycosylation, and explores the relationship between known Gaucher disease and PD mutations, structural stability and disease severity. Copyright © 2017. Published by Elsevier Inc.

  5. Seed coating with a neonicotinoid insecticide negatively affects wild bees.

    PubMed

    Rundlöf, Maj; Andersson, Georg K S; Bommarco, Riccardo; Fries, Ingemar; Hederström, Veronica; Herbertsson, Lina; Jonsson, Ove; Klatt, Björn K; Pedersen, Thorsten R; Yourstone, Johanna; Smith, Henrik G

    2015-05-07

    Understanding the effects of neonicotinoid insecticides on bees is vital because of reported declines in bee diversity and distribution and the crucial role bees have as pollinators in ecosystems and agriculture. Neonicotinoids are suspected to pose an unacceptable risk to bees, partly because of their systemic uptake in plants, and the European Union has therefore introduced a moratorium on three neonicotinoids as seed coatings in flowering crops that attract bees. The moratorium has been criticized for being based on weak evidence, particularly because effects have mostly been measured on bees that have been artificially fed neonicotinoids. Thus, the key question is how neonicotinoids influence bees, and wild bees in particular, in real-world agricultural landscapes. Here we show that a commonly used insecticide seed coating in a flowering crop can have serious consequences for wild bees. In a study with replicated and matched landscapes, we found that seed coating with Elado, an insecticide containing a combination of the neonicotinoid clothianidin and the non-systemic pyrethroid β-cyfluthrin, applied to oilseed rape seeds, reduced wild bee density, solitary bee nesting, and bumblebee colony growth and reproduction under field conditions. Hence, such insecticidal use can pose a substantial risk to wild bees in agricultural landscapes, and the contribution of pesticides to the global decline of wild bees may have been underestimated. The lack of a significant response in honeybee colonies suggests that reported pesticide effects on honeybees cannot always be extrapolated to wild bees.

  6. Translocation of the neonicotinoid seed treatment clothianidin in maize

    PubMed Central

    Krupke, Christian H.

    2017-01-01

    Neonicotinoid seed treatments, typically clothianidin or thiamethoxam, are routinely applied to >80% of maize (corn) seed grown in North America where they are marketed as a targeted pesticide delivery system. Despite this widespread use, the amount of compound translocated into plant tissue from the initial seed treatment to provide protection has not been reported. Our two year field study compared concentrations of clothianidin seed treatments in maize to that of maize without neonicotinoid seed treatments and found neonicotinoids present in root tissues up to 34 days post planting. Plant-bound clothianidin concentrations followed an exponential decay pattern with initially high values followed by a rapid decrease within the first ~20 days post planting. A maximum of 1.34% of the initial seed treatment was successfully recovered from plant tissues in both study years and a maximum of 0.26% was recovered from root tissue. Our findings show neonicotinoid seed treatments may provide protection from some early season secondary maize pests. However, the proportion of the neonicotinoid seed treatment clothianidin translocated into plant tissues throughout the growing season is low overall and this observation may provide a mechanism to explain reports of inconsistent efficacy of this pest management approach and increasing detections of environmental neonicotinoids. PMID:28282441

  7. Translocation of the neonicotinoid seed treatment clothianidin in maize.

    PubMed

    Alford, Adam; Krupke, Christian H

    2017-01-01

    Neonicotinoid seed treatments, typically clothianidin or thiamethoxam, are routinely applied to >80% of maize (corn) seed grown in North America where they are marketed as a targeted pesticide delivery system. Despite this widespread use, the amount of compound translocated into plant tissue from the initial seed treatment to provide protection has not been reported. Our two year field study compared concentrations of clothianidin seed treatments in maize to that of maize without neonicotinoid seed treatments and found neonicotinoids present in root tissues up to 34 days post planting. Plant-bound clothianidin concentrations followed an exponential decay pattern with initially high values followed by a rapid decrease within the first ~20 days post planting. A maximum of 1.34% of the initial seed treatment was successfully recovered from plant tissues in both study years and a maximum of 0.26% was recovered from root tissue. Our findings show neonicotinoid seed treatments may provide protection from some early season secondary maize pests. However, the proportion of the neonicotinoid seed treatment clothianidin translocated into plant tissues throughout the growing season is low overall and this observation may provide a mechanism to explain reports of inconsistent efficacy of this pest management approach and increasing detections of environmental neonicotinoids.

  8. Molecular Effects of Neonicotinoids in Honey Bees (Apis mellifera).

    PubMed

    Christen, Verena; Mittner, Fabian; Fent, Karl

    2016-04-05

    Neonicotinoids are implicated in the decline of bee populations. As agonists of nicotinic acetylcholine receptors, they disturb acetylcholine receptor signaling leading to neurotoxicity. Several behavioral studies showed the link between neonicotinoid exposure and adverse effects on foraging activity and reproduction. However, molecular effects underlying these effects are poorly understood. Here we elucidated molecular effects at environmental realistic levels of three neonicotinoids and nicotine, and compared laboratory studies to field exposures with acetamiprid. We assessed transcriptional alterations of eight selected genes in caged honey bees exposed to different concentrations of the neonicotinoids acetamiprid, clothianidin, imidacloporid, and thiamethoxam, as well as nicotine. We determined transcripts of several targets, including nicotinic acetylcholine receptor α 1 and α 2 subunit, the multifunctional gene vitellogenin, immune system genes apidaecin and defensin-1, stress-related gene catalase and two genes linked to memory formation, pka and creb. Vitellogenin showed a strong increase upon neonicotinoid exposures in the laboratory and field, while creb and pka transcripts were down-regulated. The induction of vitellogenin suggests adverse effects on foraging activity, whereas creb and pka down-regulation may be implicated in decreased long-term memory formation. Transcriptional alterations occurred at environmental concentrations and provide an explanation for the molecular basis of observed adverse effects of neonicotinoids to bees.

  9. The global status of insect resistance to neonicotinoid insecticides.

    PubMed

    Bass, Chris; Denholm, Ian; Williamson, Martin S; Nauen, Ralf

    2015-06-01

    The first neonicotinoid insecticide, imidacloprid, was launched in 1991. Today this class of insecticides comprises at least seven major compounds with a market share of more than 25% of total global insecticide sales. Neonicotinoid insecticides are highly selective agonists of insect nicotinic acetylcholine receptors and provide farmers with invaluable, highly effective tools against some of the world's most destructive crop pests. These include sucking pests such as aphids, whiteflies, and planthoppers, and also some coleopteran, dipteran and lepidopteran species. Although many insect species are still successfully controlled by neonicotinoids, their popularity has imposed a mounting selection pressure for resistance, and in several species resistance has now reached levels that compromise the efficacy of these insecticides. Research to understand the molecular basis of neonicotinoid resistance has revealed both target-site and metabolic mechanisms conferring resistance. For target-site resistance, field-evolved mutations have only been characterized in two aphid species. Metabolic resistance appears much more common, with the enhanced expression of one or more cytochrome P450s frequently reported in resistant strains. Despite the current scale of resistance, neonicotinoids remain a major component of many pest control programmes, and resistance management strategies, based on mode of action rotation, are of crucial importance in preventing resistance becoming more widespread. In this review we summarize the current status of neonicotinoid resistance, the biochemical and molecular mechanisms involved, and the implications for resistance management.

  10. Honey bees, neonicotinoids and bee incident reports: the Canadian situation.

    PubMed

    Cutler, G Christopher; Scott-Dupree, Cynthia D; Drexler, David M

    2014-05-01

    Neonicotinoid insecticides have been the target of much scrutiny as possible causes of recent declines observed in pollinator populations. Although neonicotinoids have been implicated in honey bee pesticide incidents, there has been little examination of incident report data. Here we summarize honey bee incident report data obtained from the Canadian Pest Management Regulatory Agency (PMRA). In Canada, there were very few honey bee incidents reported in 2007-2011 and data were not collected prior to 2007. In 2012, a significant number of incidents were reported in the province of Ontario, where exposure to neonicotinoid dust during planting of corn was suspected to have caused the incident in up to 70% of cases. Most of these incidents were classified as 'minor' by the PMRA, and only six cases were considered 'moderate' or 'major'. In that same year, there were over three times as many moderate or major incidents due to older non-neonicotinoid pesticides, involving numbers of hives or bees far greater than the number of moderate or major incidents suspected to be due to neonicotinoid poisoning. These data emphasize that, while exposure of honey bees to neonicotinoid-contaminated dust during corn planting needs to be mitigated, other pesticides also pose a risk. © 2013 Society of Chemical Industry.

  11. Visualization of elusive structures using intracardiac echocardiography: Insights from electrophysiology

    PubMed Central

    Szili-Torok, T; McFadden, EP; Jordaens, LJ; Roelandt, JRTC

    2004-01-01

    Electrophysiological mapping and ablation techniques are increasingly used to diagnose and treat many types of supraventricular and ventricular tachycardias. These procedures require an intimate knowledge of intracardiac anatomy and their use has led to a renewed interest in visualization of specific structures. This has required collaborative efforts from imaging as well as electrophysiology experts. Classical imaging techniques may be unable to visualize structures involved in arrhythmia mechanisms and therapy. Novel methods, such as intracardiac echocardiography and three-dimensional echocardiography, have been refined and these technological improvements have opened new perspectives for more effective and accurate imaging during electrophysiology procedures. Concurrently, visualization of these structures noticeably improved our ability to identify intracardiac structures. The aim of this review is to provide electrophysiologists with an overview of recent insights into the structure of the heart obtained with intracardiac echocardiography and to indicate to the echo-specialist which structures are potentially important for the electrophysiologist. PMID:15253772

  12. Managing resistance is critical to future use of Pyrethroids and Neonicotinoids

    USDA-ARS?s Scientific Manuscript database

    Synthetic pyrethroids and neonicotinoids are the most readily available alternatives to the organophosphate and carbamate insecticides. Pyrethroids have become widely used in California, and problems with insecticide resistance and non-target impacts have already been identified. Neonicotinoids are ...

  13. Monitoring changes in bemisia tabaci susceptibility to neonicotinoid insecticides in Arizona and California

    USDA-ARS?s Scientific Manuscript database

    Laboratory bioassays were carried out on field-collected and laboratory strains of Bemisia tabaci to evaluate relative toxicities of four neonicotinoid insecticides: acetamiprid, dinotefuran, imidacloprid and thiamethoxam. Susceptibility to all four neonicotinoids in leaf-uptake bioassays varied co...

  14. Signal Transduction in Histidine Kinases: Insights from New Structures

    PubMed Central

    Bhate, Manasi P.; Molnar, Kathleen S.; Goulian, Mark; DeGrado, William F.

    2015-01-01

    Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last five years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric-to-symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering and thermodynamics paves the way for a deeper molecular understanding of histidine kinase signal transduction. PMID:25982528

  15. Opioid receptors: Structural and mechanistic insights into pharmacology and signaling

    PubMed Central

    Shang, Yi; Filizola, Marta

    2015-01-01

    Opioid receptors are important drug targets for pain management, addiction, and mood disorders. Although substantial research on these important subtypes of G protein-coupled receptors has been conducted over the past two decades to discover ligands with higher specificity and diminished side effects, currently used opioid therapeutics remain suboptimal. Luckily, recent advances in structural biology of opioid receptors provide unprecedented insights into opioid receptor pharmacology and signaling. We review here a few recent studies that have used the crystal structures of opioid receptors as a basis for revealing mechanistic details of signal transduction mediated by these receptors, and for the purpose of drug discovery. PMID:25981301

  16. Opioid receptors: Structural and mechanistic insights into pharmacology and signaling.

    PubMed

    Shang, Yi; Filizola, Marta

    2015-09-15

    Opioid receptors are important drug targets for pain management, addiction, and mood disorders. Although substantial research on these important subtypes of G protein-coupled receptors has been conducted over the past two decades to discover ligands with higher specificity and diminished side effects, currently used opioid therapeutics remain suboptimal. Luckily, recent advances in structural biology of opioid receptors provide unprecedented insights into opioid receptor pharmacology and signaling. We review here a few recent studies that have used the crystal structures of opioid receptors as a basis for revealing mechanistic details of signal transduction mediated by these receptors, and for the purpose of drug discovery.

  17. Structural insights into the transport of small molecules across membranes

    PubMed Central

    Noinaj, Nicholas; Buchanan, Susan K.

    2014-01-01

    While hydrophobic small molecules often can freely permeate a lipid bilayer, ions and other polar molecules cannot and require transporters to mediate their transport. Recently, a number of important structures have been reported which have advanced our understanding of how membrane protein transporters function to transport small molecules. Structures of TbpA/B and HmuUV provided new insight into iron uptake by pathogenic bacteria while the structures of NarK, ASBT, and VcINDY revealed molecular details about the transport of nitrate, bile acids and dicarboxylates, respectively. The structure of the folate ECF transporter indicated that the S component likely undergoes a large conformational shift to mediate folate transport, while the cellulose synthase/transporter contains an elongated translocation pore for passage through the inner membrane. PMID:24681594

  18. Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

    PubMed

    Nestl, Bettina M; Geinitz, Christopher; Popa, Stephanie; Rizek, Sari; Haselbeck, Robert J; Stephen, Rosary; Noble, Michael A; Fischer, Max-Philipp; Ralph, Erik C; Hau, Hoi Ting; Man, Henry; Omar, Muhiadin; Turkenburg, Johan P; van Dien, Stephen; Culler, Stephanie J; Grogan, Gideon; Hauer, Bernhard

    2017-03-01

    The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

  19. Neonicotinoid-induced impairment of odour coding in the honeybee.

    PubMed

    Andrione, Mara; Vallortigara, Giorgio; Antolini, Renzo; Haase, Albrecht

    2016-12-01

    Exposure to neonicotinoid pesticides is considered one of the possible causes of honeybee (Apis mellifera) population decline. At sublethal doses, these chemicals have been shown to negatively affect a number of behaviours, including performance of olfactory learning and memory, due to their interference with acetylcholine signalling in the mushroom bodies. Here we provide evidence that neonicotinoids can affect odour coding upstream of the mushroom bodies, in the first odour processing centres of the honeybee brain, i.e. the antennal lobes (ALs). In particular, we investigated the effects of imidacloprid, the most common neonicotinoid, in the AL glomeruli via in vivo two-photon calcium imaging combined with pulsed odour stimulation. Following acute imidacloprid treatment, odour-evoked calcium response amplitude in single glomeruli decreases, and at the network level the representations of different odours are no longer separated. This demonstrates that, under neonicotinoid influence, olfactory information might reach the mushroom bodies in a form that is already incorrect. Thus, some of the impairments in olfactory learning and memory caused by neonicotinoids could, in fact, arise from the disruption in odor coding and olfactory discrimination ability of the honey bees.

  20. Neonicotinoid-induced impairment of odour coding in the honeybee

    PubMed Central

    Andrione, Mara; Vallortigara, Giorgio; Antolini, Renzo; Haase, Albrecht

    2016-01-01

    Exposure to neonicotinoid pesticides is considered one of the possible causes of honeybee (Apis mellifera) population decline. At sublethal doses, these chemicals have been shown to negatively affect a number of behaviours, including performance of olfactory learning and memory, due to their interference with acetylcholine signalling in the mushroom bodies. Here we provide evidence that neonicotinoids can affect odour coding upstream of the mushroom bodies, in the first odour processing centres of the honeybee brain, i.e. the antennal lobes (ALs). In particular, we investigated the effects of imidacloprid, the most common neonicotinoid, in the AL glomeruli via in vivo two-photon calcium imaging combined with pulsed odour stimulation. Following acute imidacloprid treatment, odour-evoked calcium response amplitude in single glomeruli decreases, and at the network level the representations of different odours are no longer separated. This demonstrates that, under neonicotinoid influence, olfactory information might reach the mushroom bodies in a form that is already incorrect. Thus, some of the impairments in olfactory learning and memory caused by neonicotinoids could, in fact, arise from the disruption in odor coding and olfactory discrimination ability of the honey bees. PMID:27905515

  1. Neonicotinoid metabolism: compounds, substituents, pathways, enzymes, organisms, and relevance.

    PubMed

    Casida, John E

    2011-04-13

    Neonicotinoids are one of the three principal insecticide chemotypes. The seven major commercial neonicotinoids are readily biodegraded by metabolic attack at their N-heterocyclylmethyl moiety, heterocyclic or acyclic spacer, and N-nitroimine, nitromethylene, or N-cyanoimine tip. Phase I metabolism is largely dependent on microsomal CYP450 isozymes with situ selectivity in hydroxylation, desaturation, dealkylation, sulfoxidation, and nitro reduction. Cytosolic aldehyde oxidase is a nitroreductase for some neonicotinoids. Phase II metabolism involves methylation, acetylation, and formation of glucuronide, glucoside, amino acid, and sulfate- and glutathione-derived conjugates. Some neonicotinoids act as proinsecticides with metabolism to more potent nicotinic agonists. Pest resistance is more commonly due to synergist-reversible CYP450 detoxification than to nAChR mutants or variants. Metabolites in some cases contribute to mammalian hepatotoxicity and carcinogenesis and in others to enhanced plant vigor and stress shields. These relationships explain much of neonicotinoid comparative toxicology and provide the basis for continued and improved safety and effectiveness of this chemotype.

  2. Neonicotinoid insecticides can serve as inadvertent insect contraceptives

    PubMed Central

    Villamar-Bouza, Laura; Bruckner, Selina; Chantawannakul, Panuwan; Gauthier, Laurent; Khongphinitbunjong, Kitiphong; Retschnig, Gina; Troxler, Aline; Vidondo, Beatriz; Neumann, Peter; Williams, Geoffrey R.

    2016-01-01

    There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones), Apis mellifera. Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts. PMID:27466446

  3. Neonicotinoid insecticides can serve as inadvertent insect contraceptives.

    PubMed

    Straub, Lars; Villamar-Bouza, Laura; Bruckner, Selina; Chantawannakul, Panuwan; Gauthier, Laurent; Khongphinitbunjong, Kitiphong; Retschnig, Gina; Troxler, Aline; Vidondo, Beatriz; Neumann, Peter; Williams, Geoffrey R

    2016-07-27

    There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones), Apis mellifera Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts.

  4. New insight in magnetic saturation behavior of nickel hierarchical structures

    NASA Astrophysics Data System (ADS)

    Ma, Ji; Zhang, Jianxing; Liu, Chunting; Chen, Kezheng

    2017-09-01

    It is unanimously accepted that non-ferromagnetic inclusions in a ferromagnetic system will lower down total saturation magnetization in unit of emu/g. In this study, ;lattice strain; was found to be another key factor to have critical impact on magnetic saturation behavior of the system. The lattice strain determined assembling patterns of primary nanoparticles in hierarchical structures and was intimately related with the formation process of these architectures. Therefore, flower-necklace-like and cauliflower-like nickel hierarchical structures were used as prototype systems to evidence the relationship between assembling patterns of primary nanoparticles and magnetic saturation behaviors of these architectures. It was found that the influence of lattice strain on saturation magnetization outperformed that of non-ferromagnetic inclusions in these hierarchical structures. This will enable new insights into fundamental understanding of related magnetic effects.

  5. Emerging structural insights into the function of ionotropic glutamate receptors

    PubMed Central

    Karakas, Erkan; Regan, Michael C.; Furukawa, Hiro

    2015-01-01

    Summary Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission crucial for brain development and function including learning and memory formation. Recently a wealth of structural studies on iGluRs, including AMPA receptors (AMPARs), kainate receptors, and NMDA receptors (NMDARs) became available.. These studies showed structures of non-NMDARs including AMPAR and kainate receptor in various functional states, thereby providing the first visual sense of how non-NMDAR iGluRs may function in the context of homotetramers. Furthermore, they provided the first view of heterotetrameric NMDAR ion channels, which illuminated the similarities with and differences from non-NMDARs, thus raising a mechanistic distinction between the two groups of iGluRs. Here we review mechanistic insights into iGluR functions gained through structural studies of multiple groups. PMID:25941168

  6. Structural and mechanistic insights into hepatitis C viral translation initiation.

    PubMed

    Fraser, Christopher S; Doudna, Jennifer A

    2007-01-01

    Hepatitis C virus uses an internal ribosome entry site (IRES) to control viral protein synthesis by directly recruiting ribosomes to the translation-start site in the viral mRNA. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of translation-initiation factors by binding and inducing conformational changes in the 40S ribosomal subunit. Direct interactions of the IRES with initiation factor eIF3 are also crucial for efficient translation initiation, providing clues to the role of eIF3 in protein synthesis.

  7. Structural Insights into the Mechanism of PEPCK Catalysis

    SciTech Connect

    Holyoak,T.; Sullivan, S.; Nowak, T.

    2006-01-01

    Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the {gamma}-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn{sup 2+}, Mn{sup 2+}-PEP, or Mn{sup 2+}-malonate-Mn{sup 2+}GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn{sup 2+}. Upon formation of the PEPCK-Mn{sup 2+}-PEP or PEPCK-Mn{sup 2+}-malonate-Mn{sup 2+}GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn{sup 2+}-malonate-Mn{sup 2+}GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn{sup 2+}-malonate-Mn{sup 2+}GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.

  8. Neonicotinoids as seed potato treatments to control wireworms.

    PubMed

    Huiting, H F; Ester, A

    2009-01-01

    A series of field trials were carried out from 2000 to 2003. Neonicotinoid insecticides applied as seed potato treatments at planting were tested to control wireworms in potato crops. Compounds were applied as drench or spray. Neonicotinoids tested were imidacloprid at rates of 35, 70, 88, and 175 g a.i.; thiamethoxam at 17.5, 35, 50, 70, and 140 g a.i.; and thiacloprid at 72 and 144 g a.i. per metric ton seed potatoes. Treatment with imidacloprid at 70 g/ton seed and thiamethoxam at 50 g/ton seed showed significant control of wireworms at harvest but thiacloprid showed insufficient protection. No phytotoxicity was recorded at harvest. Prospects and benefits of seed potato treatments with neonicotinoids are discussed, including lowering of the amount of insecticide needed for adequate protection.

  9. Acute poisoning with neonicotinoid insecticides: a case report and literature review.

    PubMed

    Lin, Pei-Chen; Lin, Hung-Jung; Liao, Yu-Ying; Guo, How-Ran; Chen, Kuo-Tai

    2013-04-01

    Neonicotinoids are a new class of insecticides widely applied for crop protection. These insecticides act as agonists at nicotinic acetylcholine receptors, which cause insect paralysis and death. The high specificity for receptors in insects was considered to possess highly selective toxicity to insects and relative sparing of mammals. However, an increasing number of cases of acute neonicotinoid poisoning have been reported in recent years. We reported a man who developed respiratory failure and shock after ingestion of neonicotinoid insecticide. A detailed literature review found that respiratory, cardiovascular and certain neurological presentations are warning signs of severe neonicotinoid intoxication. The amounts of ingested neonicotinoid insecticide and the plasma neonicotinoid concentration are not useful guides for the management of intoxicated patients. Supportive treatment and decontamination are the practical methods for the management of all neonicotinoid-poisoned patients. © 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society.

  10. Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

    PubMed Central

    Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

    2009-01-01

    Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

  11. Enzymes and inhibitors in neonicotinoid insecticide metabolism.

    PubMed

    Shi, Xueyan; Dick, Ryan A; Ford, Kevin A; Casida, John E

    2009-06-10

    Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19, and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19, and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than it does that of TMX. Imidacloprid (IMI), CLO, and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI, and 4-hydroxythiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid, and CLO metabolism in vivo in mice, elevating brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites.

  12. Insights into structural mechanisms of gating induced regulation of aquaporins.

    PubMed

    Sachdeva, Ruchi; Singh, Balvinder

    2014-04-01

    Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation.

  13. Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins

    PubMed Central

    Xu, Chengchen; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming

    2014-01-01

    Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively. PMID:25229189

  14. Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins.

    PubMed

    Xu, Chengchen; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming

    2014-09-16

    Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.

  15. Structure-function insights into prokaryotic and eukaryotic translation initiation.

    PubMed

    Myasnikov, Alexander G; Simonetti, Angelita; Marzi, Stefano; Klaholz, Bruno P

    2009-06-01

    Translation initiation is the rate-limiting and most complexly regulated step of protein synthesis in prokaryotes and eukaryotes. In the last few years, cryo-electron microscopy has provided several novel insights into the universal process of translation initiation. Structures of prokaryotic 30S and 70S ribosomal initiation complexes with initiator transfer RNA (tRNA), messenger RNA (mRNA), and initiation factors have recently revealed the mechanism of initiator tRNA recruitment to the assembling ribosomal machinery, involving molecular rearrangements of the ribosome and associated factors. First three-dimensional pictures of the particularly complex eukaryotic translation initiation machinery have been obtained, revealing how initiation factors tune the ribosome for recruiting the mRNA. A comparison of the available prokaryotic and eukaryotic structures shows that--besides significant differences--some key ribosomal features are universally conserved.

  16. Structural insight into CIDE domains: the Janus face of CIDEs.

    PubMed

    Park, Hyun Ho

    2015-02-01

    Cell-death inducing DFF45-like effect domain (CIDE domain) is a protein interaction module that was initially found in DNA fragmentation factor (DFF) proteins DFF40 and DFF45. Several other CIDE-containing proteins, CIDE-A, CIDE-B, and CIDE-3, have since been identified in humans. Although the main function of these proteins is associated with apoptosis, recent studies have identified roles of CIDE-containing proteins in energy metabolism, especially involvement in control of the size of lipid droplets. Because CIDE-containing proteins perform critical tasks in apoptosis and energy metabolism and have been linked to many human diseases including cancer and obesity, studies of CIDE domains and CIDE-containing proteins are of great biological importance. This review summarizes the structural insight into CIDE and the CIDE-CIDE complex and speculates on a generalized strategy for the CIDE-CIDE interaction based on the available CIDE structures and molecular modelling.

  17. Hydroxyl radicals in ice: insights into local structure and dynamics.

    PubMed

    Codorniu-Hernández, Edelsys; Kusalik, Peter G

    2012-09-07

    The hydroxyl radical and its reactivity within ice environments are crucial to many important atmospheric reactions. The associated molecular mechanisms are largely unknown due to challenges posed by direct experimental measurements and computational studies of this transient species. Here we report insights into the local structure and behaviour of the hydroxyl radical in bulk ice through an extensive study utilizing Car-Parrinello molecular dynamics simulations. Interstitial and in-lattice hydroxyl radicals in hexagonal ice were investigated at primarily 190 K. Our findings, utilizing both HCTH/120 and BLYP functionals, show that OH* can exhibit greater mobility than other ice defects (the trapping energy estimated to be only 0.09 eV). We observe the formation of a two-center three-electron hemibond structure between the hydroxyl radical and an in-lattice water molecule; while controversial, such a structure in ice may be amenable to experimental detection due to its relative stability. Our results show that interstitial water molecules can strongly influence the mobility of the hydroxyl radical in bulk ice through the displacement of the radical to an interstitial location. We also demonstrate that the H-transfer reaction from an interstitial water to the radical is a rare event in ice. Together, these results predict that the radical can be a reactive species in bulk ice, as both interstitial and in-lattice OH* can be available for reactions with other species. These microscopic insights should contribute to our understanding of the reactivity of OH* in ice and its implications to atmospheric reactions.

  18. Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

    PubMed Central

    MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

    2008-01-01

    Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

  19. Structure Prediction: New Insights into Decrypting Long Noncoding RNAs

    PubMed Central

    Yan, Kun; Arfat, Yasir; Li, Dijie; Zhao, Fan; Chen, Zhihao; Yin, Chong; Sun, Yulong; Hu, Lifang; Yang, Tuanmin; Qian, Airong

    2016-01-01

    Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological functions such as dosage compensation, chromatin regulation, genomic imprinting, and nuclear organization. However, understanding the wide range of functions of lncRNAs related to various processes of cellular networks remains a great experimental challenge. Structural versatility is critical for RNAs to perform various functions and provides new insights into probing the functions of lncRNAs. In recent years, the computational method of RNA structure prediction has been developed to analyze the structure of lncRNAs. This novel methodology has provided basic but indispensable information for the rapid, large-scale and in-depth research of lncRNAs. This review focuses on mainstream RNA structure prediction methods at the secondary and tertiary levels to offer an additional approach to investigating the functions of lncRNAs. PMID:26805815

  20. Insights from the Sea: Structural Biology of Marine Polyketide Synthases

    PubMed Central

    Akey, David L.; Gehret, Jennifer J.; Khare, Dheeraj; Smith, Janet L.

    2013-01-01

    The world’s oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe2+/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for β-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown. PMID:22498975

  1. Insights from the sea: structural biology of marine polyketide synthases.

    PubMed

    Akey, David L; Gehret, Jennifer J; Khare, Dheeraj; Smith, Janet L

    2012-10-01

    The world's oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe(2+)/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for β-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown.

  2. Structural insights into substrate traffic and inhibition in acetylcholinesterase

    PubMed Central

    Colletier, Jacques-Philippe; Fournier, Didier; Greenblatt, Harry M; Stojan, Jure; Sussman, Joel L; Zaccai, Giuseppe; Silman, Israel; Weik, Martin

    2006-01-01

    Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge. PMID:16763558

  3. Structural insights into SAM domain-mediated tankyrase oligomerization.

    PubMed

    DaRosa, Paul A; Ovchinnikov, Sergey; Xu, Wenqing; Klevit, Rachel E

    2016-09-01

    Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP-ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain-mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head-to-tail polymer that facilitates TNKS self-association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM-TNKS2 SAM) hetero-oligomeric structures mediated by their SAM domains. Though wild-type tankyrase proteins have very low solubility, model-based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP-ribosyl)ation (PARylation) and PARylation-dependent ubiquitylation. © 2016 The Protein Society.

  4. Structural insights into SAM domain‐mediated tankyrase oligomerization

    PubMed Central

    DaRosa, Paul A.; Ovchinnikov, Sergey

    2016-01-01

    Abstract Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP‐ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain‐mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head‐to‐tail polymer that facilitates TNKS self‐association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM‐TNKS2 SAM) hetero‐oligomeric structures mediated by their SAM domains. Though wild‐type tankyrase proteins have very low solubility, model‐based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP‐ribosyl)ation (PARylation) and PARylation‐dependent ubiquitylation. PMID:27328430

  5. Transmembrane signaling by GPCRs: insight from rhodopsin and opsin structures.

    PubMed

    Choe, Hui-Woog; Park, Jung Hee; Kim, Yong Ju; Ernst, Oliver P

    2011-01-01

    G-protein-coupled receptors (GPCRs), also known as seven-transmembrane (7TM) receptors, are the largest family of membrane proteins in the human genome. As versatile signaling molecules, they mediate cellular responses to extracellular signals. Diffusible ligands like hormones and neurotransmitters bind to GPCRs to modulate GPCR activity. An extraordinary and highly specialized GPCR is the photoreceptor rhodopsin which contains the chromophore retinal as its covalently bound ligand. For receptor activation the configuration of retinal is altered by photon absorption. To date, rhodopsin is the only GPCR for which crystal structures of inactive, active and ligand-free conformations are known. Although the photochemical activation is unique to rhodopsin, many mechanistic insights from this receptor can be generalized for GPCRs. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Magnetic apatite for structural insights on the plasma membrane.

    PubMed

    Stanca, Sarmiza E; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

    2015-01-21

    The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

  7. Magnetic apatite for structural insights on the plasma membrane

    NASA Astrophysics Data System (ADS)

    Stanca, Sarmiza E.; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

    2015-01-01

    The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

  8. Substrate specificity of rabbit aldehyde oxidase for nitroguanidine and nitromethylene neonicotinoid insecticides.

    PubMed

    Dick, Ryan A; Kanne, David B; Casida, John E

    2006-01-01

    of 0.80, 1.24, and 0.79 nmol/min/mg protein for clothianidin, imidacloprid, and dinotefuran, respectively. These in vitro observations show large structural differences in the rates of AOX-catalyzed reduction and help to interpret the extensive studies on in vivo metabolism of neonicotinoid insecticides.

  9. Recent Structural and Mechanistic Insights into Endplate Acetylcholine Receptors

    PubMed Central

    Sine, Steven M.; Gao, Fan; Lee, Won Yong; Mukhtasimova, Nuriya; Wang, Hai-Long; Engel, Andrew G.

    2012-01-01

    Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize themuscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms. PMID:18567853

  10. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    PubMed Central

    Wang, Tao; Li, Hua; Lin, Gang; Tang, Chunyan; Li, Dongyang; Nathan, Carl; Darwin, K. Heran; Li, Huilin

    2009-01-01

    Summary Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPγS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved inter-domain showed a five-stranded double β-barrel structure containing a Greek key motif. The structure and mutagenesis indicate a major role of the inter-domain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome. PMID:19836337

  11. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  12. Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees

    PubMed Central

    Henry, Mickaël; Cerrutti, Nicolas; Aupinel, Pierrick; Decourtye, Axel; Gayrard, Mélanie; Odoux, Jean-François; Pissard, Aurélien; Rüger, Charlotte; Bretagnolle, Vincent

    2015-01-01

    European governments have banned the use of three common neonicotinoid pesticides due to insufficiently identified risks to bees. This policy decision is controversial given the absence of clear consistency between toxicity assessments of those substances in the laboratory and in the field. Although laboratory trials report deleterious effects in honeybees at trace levels, field surveys reveal no decrease in the performance of honeybee colonies in the vicinity of treated fields. Here we provide the missing link, showing that individual honeybees near thiamethoxam-treated fields do indeed disappear at a faster rate, but the impact of this is buffered by the colonies' demographic regulation response. Although we could ascertain the exposure pathway of thiamethoxam residues from treated flowers to honeybee dietary nectar, we uncovered an unexpected pervasive co-occurrence of similar concentrations of imidacloprid, another neonicotinoid normally restricted to non-entomophilous crops in the study country. Thus, its origin and transfer pathways through the succession of annual crops need be elucidated to conveniently appraise the risks of combined neonicotinoid exposures. This study reconciles the conflicting laboratory and field toxicity assessments of neonicotinoids on honeybees and further highlights the difficulty in actually detecting non-intentional effects on the field through conventional risk assessment methods. PMID:26582026

  13. Effective extraction method for determination of neonicotinoid residues in tea.

    PubMed

    Hou, Ru-Yan; Jiao, Wei-Ting; Qian, Xiao-San; Wang, Xiao-Hui; Xiao, Yu; Wan, Xiao-Chun

    2013-12-26

    Sample preparation using an absorbent for removal of polyphenols and a solid-phase extraction (SPE) cartridge for cleanup followed by high-performance liquid chromatography (HPLC) has been investigated for the simultaneous determination of eight neonicotinoid insecticides (dinotefuran, nitenpyram, thiamethoxam, imidacloprid, clothianidin, imidaclothiz, acetamiprid, and thiacloprid). After tea samples were soaked with water and extracted with acetonitrile, sample extracts were treated with an appropriate amount of polyvinylpolypyrrolidone (PVPP) to effectively remove polyphenols. The treated extract was cleaned up with a Carb-PSA cartridge. Neonicotinoid insecticides were eluted with acetonitrile from the cartridge and dried. The extract was redissolved with methanol/water (1:9, v/v) and analyzed by conventional HPLC coupled with an ultraviolet detector. The recoveries of eight neonicotinoid insecticides in tea samples were 71.4-106.6% at 0.1-1.0 mg kg(-1) spiked levels. Relative standard deviations were <10% for all of the recovery tests. The established method was simple, effective, and accurate and could be used for monitoring neonicotinoid insecticides in tea.

  14. Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees.

    PubMed

    Henry, Mickaël; Cerrutti, Nicolas; Aupinel, Pierrick; Decourtye, Axel; Gayrard, Mélanie; Odoux, Jean-François; Pissard, Aurélien; Rüger, Charlotte; Bretagnolle, Vincent

    2015-11-22

    European governments have banned the use of three common neonicotinoid pesticides due to insufficiently identified risks to bees. This policy decision is controversial given the absence of clear consistency between toxicity assessments of those substances in the laboratory and in the field. Although laboratory trials report deleterious effects in honeybees at trace levels, field surveys reveal no decrease in the performance of honeybee colonies in the vicinity of treated fields. Here we provide the missing link, showing that individual honeybees near thiamethoxam-treated fields do indeed disappear at a faster rate, but the impact of this is buffered by the colonies' demographic regulation response. Although we could ascertain the exposure pathway of thiamethoxam residues from treated flowers to honeybee dietary nectar, we uncovered an unexpected pervasive co-occurrence of similar concentrations of imidacloprid, another neonicotinoid normally restricted to non-entomophilous crops in the study country. Thus, its origin and transfer pathways through the succession of annual crops need be elucidated to conveniently appraise the risks of combined neonicotinoid exposures. This study reconciles the conflicting laboratory and field toxicity assessments of neonicotinoids on honeybees and further highlights the difficulty in actually detecting non-intentional effects on the field through conventional risk assessment methods. © 2015 The Author(s).

  15. A common neonicotinoid pesticide, thiamethoxam, impairs honey bee flight ability.

    PubMed

    Tosi, Simone; Burgio, Giovanni; Nieh, James C

    2017-04-26

    Pesticides can pose environmental risks, and a common neonicotinoid pesticide, thiamethoxam, decreases homing success in honey bees. Neonicotinoids can alter bee navigation, but we present the first evidence that neonicotinoid exposure alone can impair the physical ability of bees to fly. We tested the effects of acute or chronic exposure to thiamethoxam on the flight ability of foragers in flight mills. Within 1 h of consuming a single sublethal dose (1.34 ng/bee), foragers showed excitation and significantly increased flight duration (+78%) and distance (+72%). Chronic exposure significantly decreased flight duration (-54%), distance (-56%), and average velocity (-7%) after either one or two days of continuous exposure that resulted in bees ingesting field-relevant thiamethoxam doses of 1.96-2.90 ng/bee/day. These results provide the first demonstration that acute or chronic exposure to a neonicotinoid alone can significantly alter bee flight. Such exposure may impair foraging and homing, which are vital to normal colony function and ecosystem services.

  16. Effects of neonicotinoids and fipronil on non-target invertebrates.

    PubMed

    Pisa, L W; Amaral-Rogers, V; Belzunces, L P; Bonmatin, J M; Downs, C A; Goulson, D; Kreutzweiser, D P; Krupke, C; Liess, M; McField, M; Morrissey, C A; Noome, D A; Settele, J; Simon-Delso, N; Stark, J D; Van der Sluijs, J P; Van Dyck, H; Wiemers, M

    2015-01-01

    We assessed the state of knowledge regarding the effects of large-scale pollution with neonicotinoid insecticides and fipronil on non-target invertebrate species of terrestrial, freshwater and marine environments. A large section of the assessment is dedicated to the state of knowledge on sublethal effects on honeybees (Apis mellifera) because this important pollinator is the most studied non-target invertebrate species. Lepidoptera (butterflies and moths), Lumbricidae (earthworms), Apoidae sensu lato (bumblebees, solitary bees) and the section "other invertebrates" review available studies on the other terrestrial species. The sections on freshwater and marine species are rather short as little is known so far about the impact of neonicotinoid insecticides and fipronil on the diverse invertebrate fauna of these widely exposed habitats. For terrestrial and aquatic invertebrate species, the known effects of neonicotinoid pesticides and fipronil are described ranging from organismal toxicology and behavioural effects to population-level effects. For earthworms, freshwater and marine species, the relation of findings to regulatory risk assessment is described. Neonicotinoid insecticides exhibit very high toxicity to a wide range of invertebrates, particularly insects, and field-realistic exposure is likely to result in both lethal and a broad range of important sublethal impacts. There is a major knowledge gap regarding impacts on the grand majority of invertebrates, many of which perform essential roles enabling healthy ecosystem functioning. The data on the few non-target species on which field tests have been performed are limited by major flaws in the outdated test protocols. Despite large knowledge gaps and uncertainties, enough knowledge exists to conclude that existing levels of pollution with neonicotinoids and fipronil resulting from presently authorized uses frequently exceed the lowest observed adverse effect concentrations and are thus likely to have large

  17. Neonicotinoid insecticide exposures reported to six poison centers in Texas.

    PubMed

    Forrester, M B

    2014-06-01

    Neonicotinoids are a relatively newer class of insecticide. Used primarily in agriculture, neonicotinoids are also used for flea control in domestic animals. Information on human exposures to neonicotinoids is limited. Neonicotinoid exposures reported to Texas poison centers during 2000-2012 were identified and the distribution by selected factors examined. Of 1,142 total exposures, most products contained imidacloprid (77%) or dinotefuran (17%). The exposures were seasonal with half reported during May-August. The most common routes of exposure were ingestion (51%), dermal (44%), and ocular (11%). The distribution by patient age was 5 years or less (28%), 6-19 years (9%), 20 years or more (61%), and unknown (2%); and 64% of the patients were female. Of all, 97% of the exposures were unintentional and 97% occurred at the patient's own residence. The management site was on-site (92%), already at/en route to a health care facility (6%), and referred to a health care facility (2%). The medical outcomes included no effect (22%), minor effect (11%), moderate effect (1%), not followed judged nontoxic (14%), not followed minimal effects (46%), unable to follow potentially toxic (1%), and unrelated effect (4%). The most commonly reported adverse clinical effects were ocular irritation (6%), dermal irritation (5%), nausea (3%), vomiting (2%), oral irritation (2%), erythema (2%), and red eye (2%). The most frequently reported treatments were dilution/wash (85%) and food (6%). In summary, these data suggest that the majority of neonicotinoid exposures reported to the poison centers may be managed outside of health care facilities with few clinical effects expected. © The Author(s) 2014.

  18. Dietary traces of neonicotinoid pesticides as a cause of population declines in honey bees: an evaluation by Hill's epidemiological criteria.

    PubMed

    Cresswell, James E; Desneux, Nicolas; vanEngelsdorp, Dennis

    2012-06-01

    Honey bees are important pollinators of both crops and wild plants. Pesticide regimes that threaten their sustainability should therefore be assessed. As an example, evidence that the agricultural use of neonicotinoid pesticides is a cause of the recently observed declines in honey bees is examined. The aim is to define exacting demographic conditions for a detrimental factor to precipitate a population decline, and Hill's epidemiological 'causality criteria' are employed as a structured process for making an expert judgement about the proposition that trace dietary neonicotinoids in nectar and pollen cause population declines in honey bees. In spite of the absence of decisive experimental results, the analysis shows that, while the proposition is a substantially justified conjecture in the context of current knowledge, it is also substantially contraindicated by a wide variety of circumstantial epidemiological evidence. It is concluded that dietary neonicotinoids cannot be implicated in honey bee declines, but this position is provisional because important gaps remain in current knowledge. Avenues for further investigations to resolve this longstanding uncertainty are therefore identified. Copyright © 2012 Society of Chemical Industry.

  19. Nanoscale insights on one- and two-dimensional material structures

    NASA Astrophysics Data System (ADS)

    Floresca, Herman Carlo

    The race for smaller, faster and more efficient devices has led researchers to explore the possibilities of utilizing nanostructures for scaling. These one-dimensional and two-dimensional materials have properties that are attractive for this purpose but are still not well controlled. Control comes with a complete understanding of the materials' electrical, thermal, optical and structural characteristics but is difficult to obtain due to their small scale. This work is intended to help researchers overcome the difficulty in studying nanostructures by providing techniques for analysis and insights of nanostructures that have not been previously available. Two nanostructures were studied: silicon nanowires and graphene. The nanowires were prepared for cross-section transmission electron microscopy (TEM) to discover the effects that controlled oxidation has on the dimensions and shape of the nanowires. Since cross-section TEM is not able to provide information about surface structure, a method for manipulating the wires with orientation control was developed. With this ability, all three orthogonal views of the nanowire were compiled for a comprehensive study on its structure in terms of shape and surface roughness. Graphene was used for a two-dimensional analytical technique that took advantage of customized computer programs for data acquisition, measurement and display. With the information provided, distinctions between grain boundary types in polycrystalline graphene were made and supported by statistical information from the software's output. It was also applied to a growth series of graphene samples in conjunction with scanning electron microscopy (SEM) images and electron backscatter diffraction (EBSD) maps. The results help point to origins of graphene's polycrystalline nature. This dissertation concludes with a thought towards the future by highlighting a method that can help analyze nanostructures, which may become incorporated into the structures of large

  20. Amyloid-beta fibrillogenesis: structural insight and therapeutic intervention.

    PubMed

    Dasilva, Kevin A; Shaw, James E; McLaurin, Joanne

    2010-06-01

    Structural insight into the conformational changes associated with aggregation and assembly of fibrils has provided a number of targets for therapeutic intervention. Solid-state NMR, hydrogen/deuterium exchange and mutagenesis strategies have been used to probe the secondary and tertiary structure of amyloid fibrils and key intermediates. Rational design of peptide inhibitors directed against key residues important for aggregation and stabilization of fibrils has demonstrated effectiveness at inhibiting fibrillogenesis. Studies on the interaction between Abeta and cell membranes led to the discovery that inositol, the head group of phosphatidylinositol, inhibits fibrillogenesis. As a result, scyllo-inositol is currently in clinical trials for the treatment of AD. Additional small-molecule inhibitors, including polyphenolic compounds such as curcumin, (-)-epigallocatechin gallate (EGCG), and grape seed extract have been shown to attenuate Abeta aggregation through distinct mechanisms, and have shown effectiveness at reducing amyloid levels when administered to transgenic mouse models of AD. Although the results of ongoing clinical trials remain to be seen, these compounds represent the first generation of amyloid-based therapeutics, with the potential to alter the progression of AD and, when used prophylactically, alleviate the deposition of Abeta.

  1. Insights from the structural analysis of protein heterodimer interfaces

    PubMed Central

    Sowmya, Gopichandran; Anita, Sathyanarayanan; Kangueane, Pandjassarame

    2011-01-01

    Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions. PMID:21572879

  2. Insight into Structural Phase Transitions from Density Functional Theory

    NASA Astrophysics Data System (ADS)

    Ruzsinszky, Adrienn

    2014-03-01

    Structural phase transitions caused by high pressure or temperature are very relevant in materials science. The high pressure transitions are essential to understand the interior of planets. Pressure or temperature induced phase transitions can be relevant to understand other phase transitions in strongly correlated systems or molecular crystals.Phase transitions are important also from the aspect of method development. Lower level density functionals, LSDA and GGAs all fail to predict the lattice parameters of different polymorphs and the phase transition parameters at the same time. At this time only nonlocal density functionals like HSE and RPA have been proved to resolve the geometry-energy dilemma to some extent in structural phase transitions. In this talk I will report new results from the MGGA_MS family of meta-GGAs and give an insight why this type of meta-GGAs can give a systematic improvement of the geometry and phase transition parameters together. I will also present results from the RPA and show a possible way to improve beyond RPA.

  3. Insights from the structural analysis of protein heterodimer interfaces.

    PubMed

    Sowmya, Gopichandran; Anita, Sathyanarayanan; Kangueane, Pandjassarame

    2011-05-07

    Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.

  4. Structural Insights into Calicivirus Attachment and Uncoating▿ †

    PubMed Central

    Bhella, David; Gatherer, Derek; Chaudhry, Yasmin; Pink, Rebecca; Goodfellow, Ian G.

    2008-01-01

    The Caliciviridae family comprises positive-sense RNA viruses of medical and veterinary significance. In humans, caliciviruses are a major cause of acute gastroenteritis, while in animals respiratory illness, conjunctivitis, stomatitis, and hemorrhagic disease are documented. Investigation of virus-host interactions is limited by a lack of culture systems for many viruses in this family. Feline calicivirus (FCV), a member of the Vesivirus genus, provides a tractable model, since it may be propagated in cell culture. Feline junctional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for FCV. We have analyzed the structure of this virus-receptor complex by cryo-electron microscopy and three-dimensional image reconstruction, combined with fitting of homology modeled high-resolution coordinates. We show that domain 1 of fJAM-1 binds to the outer face of the P2 domain of the FCV capsid protein VP1, inducing conformational changes in the viral capsid. This study provides the first structural view of a native calicivirus-protein receptor complex and insights into the mechanisms of virus attachment and uncoating. PMID:18550656

  5. Impacts of neonicotinoid use on long-term population changes in wild bees in England

    NASA Astrophysics Data System (ADS)

    Woodcock, Ben A.; Isaac, Nicholas J. B.; Bullock, James M.; Roy, David B.; Garthwaite, David G.; Crowe, Andrew; Pywell, Richard F.

    2016-08-01

    Wild bee declines have been ascribed in part to neonicotinoid insecticides. While short-term laboratory studies on commercially bred species (principally honeybees and bumblebees) have identified sub-lethal effects, there is no strong evidence linking these insecticides to losses of the majority of wild bee species. We relate 18 years of UK national wild bee distribution data for 62 species to amounts of neonicotinoid use in oilseed rape. Using a multi-species dynamic Bayesian occupancy analysis, we find evidence of increased population extinction rates in response to neonicotinoid seed treatment use on oilseed rape. Species foraging on oilseed rape benefit from the cover of this crop, but were on average three times more negatively affected by exposure to neonicotinoids than non-crop foragers. Our results suggest that sub-lethal effects of neonicotinoids could scale up to cause losses of bee biodiversity. Restrictions on neonicotinoid use may reduce population declines.

  6. Impacts of neonicotinoid use on long-term population changes in wild bees in England.

    PubMed

    Woodcock, Ben A; Isaac, Nicholas J B; Bullock, James M; Roy, David B; Garthwaite, David G; Crowe, Andrew; Pywell, Richard F

    2016-08-16

    Wild bee declines have been ascribed in part to neonicotinoid insecticides. While short-term laboratory studies on commercially bred species (principally honeybees and bumblebees) have identified sub-lethal effects, there is no strong evidence linking these insecticides to losses of the majority of wild bee species. We relate 18 years of UK national wild bee distribution data for 62 species to amounts of neonicotinoid use in oilseed rape. Using a multi-species dynamic Bayesian occupancy analysis, we find evidence of increased population extinction rates in response to neonicotinoid seed treatment use on oilseed rape. Species foraging on oilseed rape benefit from the cover of this crop, but were on average three times more negatively affected by exposure to neonicotinoids than non-crop foragers. Our results suggest that sub-lethal effects of neonicotinoids could scale up to cause losses of bee biodiversity. Restrictions on neonicotinoid use may reduce population declines.

  7. Impacts of neonicotinoid use on long-term population changes in wild bees in England

    PubMed Central

    Woodcock, Ben A.; Isaac, Nicholas J. B.; Bullock, James M.; Roy, David B.; Garthwaite, David G.; Crowe, Andrew; Pywell, Richard F.

    2016-01-01

    Wild bee declines have been ascribed in part to neonicotinoid insecticides. While short-term laboratory studies on commercially bred species (principally honeybees and bumblebees) have identified sub-lethal effects, there is no strong evidence linking these insecticides to losses of the majority of wild bee species. We relate 18 years of UK national wild bee distribution data for 62 species to amounts of neonicotinoid use in oilseed rape. Using a multi-species dynamic Bayesian occupancy analysis, we find evidence of increased population extinction rates in response to neonicotinoid seed treatment use on oilseed rape. Species foraging on oilseed rape benefit from the cover of this crop, but were on average three times more negatively affected by exposure to neonicotinoids than non-crop foragers. Our results suggest that sub-lethal effects of neonicotinoids could scale up to cause losses of bee biodiversity. Restrictions on neonicotinoid use may reduce population declines. PMID:27529661

  8. Quantitative analysis of neonicotinoid insecticide residues in foods: implication for dietary exposures.

    PubMed

    Chen, Mei; Tao, Lin; McLean, John; Lu, Chensheng

    2014-07-02

    This study quantitatively measured neonicotinoids in various foods that are common to human consumption. All fruit and vegetable samples (except nectarine and tomato) and 90% of honey samples were detected positive for at least one neonicotinoid; 72% of fruits, 45% of vegetables, and 50% of honey samples contained at least two different neonicotinoids in one sample, with imidacloprid having the highest detection rate among all samples. All pollen samples from New Zealand contained multiple neonicotinoids, and five of seven pollens from Massachusetts detected positive for imidacloprid. These results show the prevalence of low-level neonicotinoid residues in fruits, vegetables, and honey that are readily available in the market for human consumption and in the environment where honeybees forage. In light of new reports of toxicological effects in mammals, the results strengthen the importance of assessing dietary neonicotinoid intakes and the potential human health effects.

  9. Quantitative Analysis of Neonicotinoid Insecticide Residues in Foods: Implication for Dietary Exposures

    PubMed Central

    2015-01-01

    This study quantitatively measured neonicotinoids in various foods that are common to human consumption. All fruit and vegetable samples (except nectarine and tomato) and 90% of honey samples were detected positive for at least one neonicotinoid; 72% of fruits, 45% of vegetables, and 50% of honey samples contained at least two different neonicotinoids in one sample, with imidacloprid having the highest detection rate among all samples. All pollen samples from New Zealand contained multiple neonicotinoids, and five of seven pollens from Massachusetts detected positive for imidacloprid. These results show the prevalence of low-level neonicotinoid residues in fruits, vegetables, and honey that are readily available in the market for human consumption and in the environment where honeybees forage. In light of new reports of toxicological effects in mammals, the results strengthen the importance of assessing dietary neonicotinoid intakes and the potential human health effects. PMID:24933495

  10. Determination of toxicity of neonicotinoids on the genome level using chemogenomics in yeast.

    PubMed

    Mattiazzi Ušaj, Mojca; Kaferle, Petra; Toplak, Alenka; Trebše, Polonca; Petrovič, Uroš

    2014-06-01

    Neonicotinoid insecticides are an important contribution to plant protection products. At the same time, their environmental impact on non-target organisms is often problematic. It has been shown that the toxicity of formulations of neonicotinoid insecticides can originate from non-neonicotinoid additives. In the present study we used chemogenomics to analyse side effects of purified neonicotinoids, additives and formulations on the genome-wide scale. We show that the additives in formulations have more pronounced effects than the active components, and that these effects could explain previously observed negative effects of neonicotinoid insecticides on spermatogenesis in animals. We also demonstrate that cell wall organization and biogenesis in yeast is negatively affected by neonicotinoid substances.

  11. Structural Insights into Regioselectivity in the Enzymatic Chlorination of Tryptophan

    PubMed Central

    Zhu, Xiaofeng; De Laurentis, Walter; Leang, Khim; Herrmann, Julia; Ihlefeld, Katja; van Pée, Karl-Heinz; Naismith, James H.

    2009-01-01

    The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom. Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family. These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position. We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl-, O2, and 1,5-dihydroflavin adenine dinucleotide. We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module. The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role. This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily. PMID:19501593

  12. Structural and functional insights into alphavirus polyprotein processing and pathogenesis

    PubMed Central

    Shin, Gyehwa; Yost, Samantha A.; Miller, Matthew T.; Elrod, Elizabeth J.; Grakoui, Arash; Marcotrigiano, Joseph

    2012-01-01

    Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1–4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus. PMID:23010928

  13. Structural and functional insights into alphavirus polyprotein processing and pathogenesis.

    PubMed

    Shin, Gyehwa; Yost, Samantha A; Miller, Matthew T; Elrod, Elizabeth J; Grakoui, Arash; Marcotrigiano, Joseph

    2012-10-09

    Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1-4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.

  14. Neonicotinoid formaldehyde generators: possible mechanism of mouse-specific hepatotoxicity/hepatocarcinogenicity of thiamethoxam.

    PubMed

    Swenson, Tami L; Casida, John E

    2013-02-04

    Thiamethoxam (TMX), an important insecticide, is hepatotoxic and hepatocarcinogenic in mice but not rats. Studies of Syngenta Central Toxicology Laboratory on species specificity in metabolism established that TMX is a much better substrate for mouse liver microsomal CYPs than the corresponding rat or human enzymes in forming desmethyl-TMX (dm-TMX), which is also hepatotoxic, and clothianidin (CLO), which is not hepatotoxic or hepatocarcinogenic. They proposed that TMX hepatotoxicity/hepatocarcinogencity is due to dm-TMX and a further metabolite desmethyl-CLO (dm-CLO) (structurally analogous to a standard inducible nitric oxide synthase inhibitor) acting synergistically. The present study considers formation of formaldehyde (HCHO) and N-methylol intermediates as an alternative mechanism of TMX hepatotoxicity/hepatocarcinogenicity. Comparison of neonicotinoid metabolism by mouse, rat and human microsomes with NADPH showed two important points. First, TMX and dm-TMX yield more HCHO than any other commercial neonicotinoid. Second, mouse microsomes give much higher conversion than rat or human microsomes. These observations provide an alternative hypothesis of HCHO and N-methylol intermediates from CYP-mediated oxidative oxadiazinane ring cleavage as the bioactivated hepatotoxicants. However, the proposed mono-N-methylol CYP metabolites are not observed, possibly further reacting in situ. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

    PubMed Central

    Uchigashima, Mikiko; Watanabe, Eiki; Ito, Shigekazu; Iwasa, Seiji; Miyake, Shiro

    2012-01-01

    Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops. PMID:23202236

  16. Insights into structural variations and genome rearrangements in prokaryotic genomes.

    PubMed

    Periwal, Vinita; Scaria, Vinod

    2015-01-01

    Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Insights.

    ERIC Educational Resources Information Center

    Bogner, Donna, Ed.

    1988-01-01

    Describes two methods to teach radioactive decay to secondary students with wide ranging abilities. Activities are designed to follow classroom discussions of atomic structure, transmutation, half life, and nuclear decay. Includes "The Tasmanian Empire: A Radioactive Dating Activity" and an exercise to teach concepts of half life without…

  18. Insights.

    ERIC Educational Resources Information Center

    Bogner, Donna, Ed.

    1988-01-01

    Describes two methods to teach radioactive decay to secondary students with wide ranging abilities. Activities are designed to follow classroom discussions of atomic structure, transmutation, half life, and nuclear decay. Includes "The Tasmanian Empire: A Radioactive Dating Activity" and an exercise to teach concepts of half life without…

  19. Survey of neonicotinoids and fipronil in corn seeds for agriculture.

    PubMed

    Sabatino, Leonardo; Scordino, Monica; Pantò, Valentina; Chiappara, Elena; Traulo, Pasqualino; Gagliano, Giacomo

    2013-01-01

    Recently, legislative decisions withdrew or temporarily suspended the use of neonicotinoids and fipronil as seeds tanning in many countries because of their endocrine-disrupting activity imputable to the bees' toxicity. In this study, the occurrence of acetamiprid, fipronil, clothianidin, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam was detected in 66 samples of commercial treated corn seeds, collected in the Italian market in the frame of ministerial institutional quality control activity. Because of the lack of a validated analytical protocol for neonicotinoid detection in seeds, a routinely suitable liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analytical method was developed and statistically validated on fortified corn seeds. Survey results demonstrated that 88% of the investigated seed samples showed the presence of residues of clothianidin, fipronil, thiamethoxam and thiacloprid, either individually or simultaneously, with values that ranged from about 0.002 to 20 mg kg(-1), which evidenced the alarming illicit use of these pesticides in seed treatments.

  20. Neonicotinoid insecticides induce salicylate-associated plant defense responses

    PubMed Central

    Ford, Kevin A.; Casida, John E.; Chandran, Divya; Gulevich, Alexander G.; Okrent, Rachel A.; Durkin, Kathleen A.; Sarpong, Richmond; Bunnelle, Eric M.; Wildermuth, Mary C.

    2010-01-01

    Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl- and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl- and chlorothiazolyl-neonicotinoids induce SA responses associated with enhanced stress tolerance. PMID:20876120

  1. Improving Mitochondrial Function Protects Bumblebees from Neonicotinoid Pesticides.

    PubMed

    Powner, Michael B; Salt, Thomas E; Hogg, Chris; Jeffery, Glen

    2016-01-01

    Global pollination is threatened by declining insect pollinator populations that may be linked to neonicotinoid pesticide use. Neonicotinoids over stimulate neurons and depolarize their mitochondria, producing immobility and death. However, mitochondrial function can be improved by near infrared light absorbed by cytochrome c oxidase in mitochondrial respiration. In flies, daily exposure to 670nm light throughout life increases average lifespan and aged mobility, and reduces systemic inflammation. Here we treat bumble bees with Imidacloprid a common neonicotinoid. This undermined ATP and rapidly induced immobility and reduced visual function and survival. Bees exposed to insecticide and daily to 670nm light showed corrected ATP levels and significantly improved mobility allowing them to feed. Physiological recordings from eyes revealed that light exposure corrected deficits induced by the pesticide. Overall, death rates in bees exposed to insecticide but also given 670nm light were indistinguishable from controls. When Imidacloprid and light exposure were withdrawn, survival was maintained. Bees and insects generally cannot see deep red light so it does not disturb their behaviour. Hence, we show that deep red light exposure that improves mitochondrial function, reverses the sensory and motor deficits induced by Imidacloprid. These results may have important implications as light delivery is economic and can be placed in hives/colonies.

  2. Biological response of earthworm, Eisenia fetida, to five neonicotinoid insecticides.

    PubMed

    Wang, Kai; Pang, Sen; Mu, Xiyan; Qi, Suzhen; Li, Dongzhi; Cui, Feng; Wang, Chengju

    2015-08-01

    Earthworms (Eisenia fetida) are one of the most abundant terrestrial species, and play an important role in maintaining the ecological function of soil. Neonicotinoids are some of the most widely used insecticides applied to crops. Studies on the effect of neonicotinoids on E. fetida are limited. In the present work, we evaluated the effects of five neonicotinoid insecticides on reproduction, cellulase activity and the tissues of E. fetida. The results showed that, the LC50 of imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid was 3.05, 2.69, 4.34, 0.93 and 2.68mgkg(-1), respectively. They also could seriously affect the reproduction of E. fetida, reducing the fecundity by 84.0%, 39.5%, 54.3%, 45.7% and 39.5% at the sub-lethal concentrations of 2.0, 1.5, 0.80, 2.0 and 1.5mgkg(-1), respectively. The cellulase activity of E. fetida was most sensitive to clothianidin. Significant disruption of the epidermal and midgut tissue was observed after 14d exposure. In summary, we demonstrate that imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid have high toxic to earthworm, and can significantly inhibited fecundity and cellulase activity of E. fetida, and they also damage the epidermal and midgut cells of earthworm. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Improving Mitochondrial Function Protects Bumblebees from Neonicotinoid Pesticides

    PubMed Central

    Powner, Michael B.; Salt, Thomas E.; Hogg, Chris; Jeffery, Glen

    2016-01-01

    Global pollination is threatened by declining insect pollinator populations that may be linked to neonicotinoid pesticide use. Neonicotinoids over stimulate neurons and depolarize their mitochondria, producing immobility and death. However, mitochondrial function can be improved by near infrared light absorbed by cytochrome c oxidase in mitochondrial respiration. In flies, daily exposure to 670nm light throughout life increases average lifespan and aged mobility, and reduces systemic inflammation. Here we treat bumble bees with Imidacloprid a common neonicotinoid. This undermined ATP and rapidly induced immobility and reduced visual function and survival. Bees exposed to insecticide and daily to 670nm light showed corrected ATP levels and significantly improved mobility allowing them to feed. Physiological recordings from eyes revealed that light exposure corrected deficits induced by the pesticide. Overall, death rates in bees exposed to insecticide but also given 670nm light were indistinguishable from controls. When Imidacloprid and light exposure were withdrawn, survival was maintained. Bees and insects generally cannot see deep red light so it does not disturb their behaviour. Hence, we show that deep red light exposure that improves mitochondrial function, reverses the sensory and motor deficits induced by Imidacloprid. These results may have important implications as light delivery is economic and can be placed in hives/colonies. PMID:27846310

  4. Nitromethylene neonicotinoids analogues with tetrahydropyrimidine fixed cis-configuration: synthesis, insecticidal activities, and molecular docking studies.

    PubMed

    Sun, Chuanwen; Yang, Dingrong; Xing, Jiahua; Wang, Haifeng; Jin, Jia; Zhu, Jun

    2010-03-24

    Two series of new nitromethylene neonicotinoid analogues (2a-2h and 3a-3h) were designed and prepared, with the cis-configuration confirmed by X-ray diffraction. Preliminary bioassays showed that most analogues exhibited excellent insecticidal activities at 500 mg/L, and analogues with optical activity (2c-2g) were highly potent at 100 mg/L, while compound 2d had >90% mortality at 20 mg/L, which suggested that it could be used as a lead for future insecticides development. Modeling the ligand-receptor complexes by molecular docking study explained the structure-activity relationships observed in vitro and revealed an intriguing molecular binding mode at the active site of the nAChR model, thereby possibly providing some useful information for future receptor structure-based designs of novel insecticidal compounds.

  5. Environmental Fate of Soil Applied Neonicotinoid Insecticides in an Irrigated Potato Agroecosystem

    PubMed Central

    Huseth, Anders S.; Groves, Russell L.

    2014-01-01

    Since 1995, neonicotinoid insecticides have been a critical component of arthropod management in potato, Solanum tuberosum L. Recent detections of neonicotinoids in groundwater have generated questions about the sources of these contaminants and the relative contribution from commodities in U.S. agriculture. Delivery of neonicotinoids to crops typically occurs as a seed or in-furrow treatment to manage early season insect herbivores. Applied in this way, these insecticides become systemically mobile in the plant and provide control of key pest species. An outcome of this project links these soil insecticide application strategies in crop plants with neonicotinoid contamination of water leaching from the application zone. In 2011 and 2012, our objectives were to document the temporal patterns of neonicotinoid leachate below the planting furrow following common insecticide delivery methods in potato. Leaching loss of thiamethoxam from potato was measured using pan lysimeters from three at-plant treatments and one foliar application treatment. Insecticide concentration in leachate was assessed for six consecutive months using liquid chromatography-tandem mass spectrometry. Findings from this study suggest leaching of neonicotinoids from potato may be greater following crop harvest in comparison to other times during the growing season. Furthermore, this study documented recycling of neonicotinoid insecticides from contaminated groundwater back onto the crop via high capacity irrigation wells. These results document interactions between cultivated potato, different neonicotinoid delivery methods, and the potential for subsurface water contamination via leaching. PMID:24823765

  6. Environmental fate of soil applied neonicotinoid insecticides in an irrigated potato agroecosystem.

    PubMed

    Huseth, Anders S; Groves, Russell L

    2014-01-01

    Since 1995, neonicotinoid insecticides have been a critical component of arthropod management in potato, Solanum tuberosum L. Recent detections of neonicotinoids in groundwater have generated questions about the sources of these contaminants and the relative contribution from commodities in U.S. agriculture. Delivery of neonicotinoids to crops typically occurs as a seed or in-furrow treatment to manage early season insect herbivores. Applied in this way, these insecticides become systemically mobile in the plant and provide control of key pest species. An outcome of this project links these soil insecticide application strategies in crop plants with neonicotinoid contamination of water leaching from the application zone. In 2011 and 2012, our objectives were to document the temporal patterns of neonicotinoid leachate below the planting furrow following common insecticide delivery methods in potato. Leaching loss of thiamethoxam from potato was measured using pan lysimeters from three at-plant treatments and one foliar application treatment. Insecticide concentration in leachate was assessed for six consecutive months using liquid chromatography-tandem mass spectrometry. Findings from this study suggest leaching of neonicotinoids from potato may be greater following crop harvest in comparison to other times during the growing season. Furthermore, this study documented recycling of neonicotinoid insecticides from contaminated groundwater back onto the crop via high capacity irrigation wells. These results document interactions between cultivated potato, different neonicotinoid delivery methods, and the potential for subsurface water contamination via leaching.

  7. Determination of neonicotinoids in Estonian honey by liquid chromatography-electrospray mass spectrometry.

    PubMed

    Laaniste, Asko; Leito, Ivo; Rebane, Riin; Lõhmus, Rünno; Lõhmus, Ants; Punga, Fredrik; Kruve, Anneli

    2016-07-02

    The aim of the study was to provide a comprehensive overview of neonicotinoid pesticide residues in honey samples for a single country and compare the results with the import data for neonicotinoid pesticides. The levels of four neonicotinoid pesticides, namely thiamethoxam, imidacloprid, acetamiprid, and thiacloprid, were determined in 294 honey samples harvested from 2005 to 2013 from more than 200 locations in Estonia. For the analyzed honey samples, 27% contained thiacloprid, and its levels in all cases were below the maximum residue level set by the European Union. The other neonicotinoids were not detected. The proportion of thiacloprid-positive samples for different years correlates well with the data on thiacloprid imports into Estonia, indicating that honey contamination with neonicotinoids can be estimated based on the import data.

  8. Country-specific effects of neonicotinoid pesticides on honey bees and wild bees.

    PubMed

    Woodcock, B A; Bullock, J M; Shore, R F; Heard, M S; Pereira, M G; Redhead, J; Ridding, L; Dean, H; Sleep, D; Henrys, P; Peyton, J; Hulmes, S; Hulmes, L; Sárospataki, M; Saure, C; Edwards, M; Genersch, E; Knäbe, S; Pywell, R F

    2017-06-30

    Neonicotinoid seed dressings have caused concern world-wide. We use large field experiments to assess the effects of neonicotinoid-treated crops on three bee species across three countries (Hungary, Germany, and the United Kingdom). Winter-sown oilseed rape was grown commercially with either seed coatings containing neonicotinoids (clothianidin or thiamethoxam) or no seed treatment (control). For honey bees, we found both negative (Hungary and United Kingdom) and positive (Germany) effects during crop flowering. In Hungary, negative effects on honey bees (associated with clothianidin) persisted over winter and resulted in smaller colonies in the following spring (24% declines). In wild bees (Bombus terrestris and Osmia bicornis), reproduction was negatively correlated with neonicotinoid residues. These findings point to neonicotinoids causing a reduced capacity of bee species to establish new populations in the year following exposure. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. Impact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure

    PubMed Central

    Sandrock, Christoph; Tanadini, Matteo; Tanadini, Lorenzo G.; Fauser-Misslin, Aline; Potts, Simon G.; Neumann, Peter

    2014-01-01

    Background Honeybees provide economically and ecologically vital pollination services to crops and wild plants. During the last decade elevated colony losses have been documented in Europe and North America. Despite growing consensus on the involvement of multiple causal factors, the underlying interactions impacting on honeybee health and colony failure are not fully resolved. Parasites and pathogens are among the main candidates, but sublethal exposure to widespread agricultural pesticides may also affect bees. Methodology/Principal Findings To investigate effects of sublethal dietary neonicotinoid exposure on honeybee colony performance, a fully crossed experimental design was implemented using 24 colonies, including sister-queens from two different strains, and experimental in-hive pollen feeding with or without environmentally relevant concentrations of thiamethoxam and clothianidin. Honeybee colonies chronically exposed to both neonicotinoids over two brood cycles exhibited decreased performance in the short-term resulting in declining numbers of adult bees (−28%) and brood (−13%), as well as a reduction in honey production (−29%) and pollen collections (−19%), but colonies recovered in the medium-term and overwintered successfully. However, significantly decelerated growth of neonicotinoid-exposed colonies during the following spring was associated with queen failure, revealing previously undocumented long-term impacts of neonicotinoids: queen supersedure was observed for 60% of the neonicotinoid-exposed colonies within a one year period, but not for control colonies. Linked to this, neonicotinoid exposure was significantly associated with a reduced propensity to swarm during the next spring. Both short-term and long-term effects of neonicotinoids on colony performance were significantly influenced by the honeybees’ genetic background. Conclusions/Significance Sublethal neonicotinoid exposure did not provoke increased winter losses. Yet

  10. Impact of chronic neonicotinoid exposure on honeybee colony performance and queen supersedure.

    PubMed

    Sandrock, Christoph; Tanadini, Matteo; Tanadini, Lorenzo G; Fauser-Misslin, Aline; Potts, Simon G; Neumann, Peter

    2014-01-01

    Honeybees provide economically and ecologically vital pollination services to crops and wild plants. During the last decade elevated colony losses have been documented in Europe and North America. Despite growing consensus on the involvement of multiple causal factors, the underlying interactions impacting on honeybee health and colony failure are not fully resolved. Parasites and pathogens are among the main candidates, but sublethal exposure to widespread agricultural pesticides may also affect bees. To investigate effects of sublethal dietary neonicotinoid exposure on honeybee colony performance, a fully crossed experimental design was implemented using 24 colonies, including sister-queens from two different strains, and experimental in-hive pollen feeding with or without environmentally relevant concentrations of thiamethoxam and clothianidin. Honeybee colonies chronically exposed to both neonicotinoids over two brood cycles exhibited decreased performance in the short-term resulting in declining numbers of adult bees (-28%) and brood (-13%), as well as a reduction in honey production (-29%) and pollen collections (-19%), but colonies recovered in the medium-term and overwintered successfully. However, significantly decelerated growth of neonicotinoid-exposed colonies during the following spring was associated with queen failure, revealing previously undocumented long-term impacts of neonicotinoids: queen supersedure was observed for 60% of the neonicotinoid-exposed colonies within a one year period, but not for control colonies. Linked to this, neonicotinoid exposure was significantly associated with a reduced propensity to swarm during the next spring. Both short-term and long-term effects of neonicotinoids on colony performance were significantly influenced by the honeybees' genetic background. Sublethal neonicotinoid exposure did not provoke increased winter losses. Yet, significant detrimental short and long-term impacts on colony performance and queen

  11. Value of Neonicotinoid Insecticide Seed Treatments in Mid-South Soybean (Glycine max) Production Systems.

    PubMed

    North, J H; Gore, J; Catchot, A L; Stewart, S D; Lorenz, G M; Musser, F R; Cook, D R; Kerns, D L; Dodds, D M

    2016-04-18

    Early-season insect management is complex in the Mid-South region of the United States. A complex of multiple pest species generally occurs simultaneously at subthreshold levels in most fields. Neonicotinoids are the only insecticide seed treatment widely used in soybean,Glycine maxL., production. An analysis was performed on 170 trials conducted in Arkansas, Louisiana, Mississippi, and Tennessee from 2005 to 2014 to determine the impact of neonicotinoid seed treatments in soybean. The analysis compared soybean seed treated with a neonicotinoid insecticide and a fungicide with soybean seed only treated with the same fungicide. When analyzed by state, soybean yields were significantly greater in all states when neonicotinoid seed treatments were used compared with fungicide-only treatments. Soybean treated with neonicotinoid treatments yielded 112.0 kg ha(-1), 203.0 kg ha(-1), 165.0 kg ha(-1), and 70.0 kg ha(-1), higher than fungicide-only treatments for Arkansas, Louisiana, Mississippi, and Tennessee, respectively. Across all states, neonicotinoid seed treatments yielded 132.0 kg ha(-1)more than with fungicide-only treated seed. Net returns from neonicotinoid seed treatment usage were US$1,203 per ha(-1)compared with US$1,172 per ha(-1)for fungicide-only treated seed across the Mid-South. However, economic returns for neonicotinoid seed treatments were significantly greater than fungicide-only treated seed in 4 out of the 10 yr. When analyzed by state economic returns the neonicotinoid seed treatments were significantly greater than fungicide-only treated seed in Louisiana and Mississippi. These data show that in some areas and years, neonicotinoid seed treatments provide significant economic benefits in Mid-South soybean. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.

  12. Synthesis, insecticidal activity, and QSAR of novel nitromethylene neonicotinoids with tetrahydropyridine fixed cis configuration and exo-ring ether modification.

    PubMed

    Tian, Zhongzhen; Shao, Xusheng; Li, Zhong; Qian, Xuhong; Huang, Qingchun

    2007-03-21

    To keep the nitro group in the cis position, a series of nitromethylene neonicotinoids containing a tetrahydropyridine ring with exo-ring ether modifications were designed and synthesized. All of the compounds were characterized and confirmed by 1H NMR, high-resolution mass spectroscopy, elemental analysis, and IR. The bioassay tests showed that some of them exhibited good insecticidal activities against pea aphids. On the basis of 10 nitromethylene derivatives, the quantitative structure-bioactivity relationship (QSAR) was analyzed and established. The results suggested that AlogP98 and Dipole_Mopac might be the important parameters related with biological activities.

  13. Neonicotinoid Insecticide Imidacloprid Causes Outbreaks of Spider Mites on Elm Trees in Urban Landscapes

    PubMed Central

    Szczepaniec, Adrianna; Creary, Scott F.; Laskowski, Kate L.; Nyrop, Jan P.; Raupp, Michael J.

    2011-01-01

    Background Attempts to eradicate alien arthropods often require pesticide applications. An effort to remove an alien beetle from Central Park in New York City, USA, resulted in widespread treatments of trees with the neonicotinoid insecticide imidacloprid. Imidacloprid's systemic activity and mode of entry via roots or trunk injections reduce risk of environmental contamination and limit exposure of non-target organisms to pesticide residues. However, unexpected outbreaks of a formerly innocuous herbivore, Tetranychus schoenei (Acari: Tetranychidae), followed imidacloprid applications to elms in Central Park. This undesirable outcome necessitated an assessment of imidacloprid's impact on communities of arthropods, its effects on predators, and enhancement of the performance of T. schoenei. Methodology/Principal Findings By sampling arthropods in elm canopies over three years in two locations, we document changes in the structure of communities following applications of imidacloprid. Differences in community structure were mostly attributable to increases in the abundance of T. schoenei on elms treated with imidacloprid. In laboratory experiments, predators of T. schoenei were poisoned through ingestion of prey exposed to imidacloprid. Imidacloprid's proclivity to elevate fecundity of T. schoenei also contributed to their elevated densities on treated elms. Conclusions/Significance This is the first study to report the effects of pesticide applications on the arthropod communities in urban landscapes and demonstrate that imidacloprid increases spider mite fecundity through a plant-mediated mechanism. Laboratory experiments provide evidence that imidacloprid debilitates insect predators of spider mites suggesting that relaxation of top-down regulation combined with enhanced reproduction promoted a non-target herbivore to pest status. With global commerce accelerating the incidence of arthropod invasions, prophylactic applications of pesticides play a major role in

  14. Neonicotinoid insecticide removal by prairie strips in row-cropped watersheds with historical seed coating use

    USGS Publications Warehouse

    Hladik, Michelle; Bradbury, Steven; Schulte, Lisa A.; Helmers, Matthew; Witte, Christopher; Kolpin, Dana W.; Garrett, Jessica D.; Harris, Mary

    2017-01-01

    Neonicotinoids are a widely used class of insecticides that are commonly applied as seed coatings for agricultural crops. Such neonicotinoid use may pose a risk to non-target insects, including pollinators and natural enemies of crop pests, and ecosystems. This study assessed neonicotinoid residues in groundwater, surface runoff water, soil, and native plants adjacent to corn and soybean crop fields with a history of being planted with neonicotinoid-treated seeds from 2008-2013. Data from six sites with the same crop management history, three with and three without in-field prairie strips, were collected in 2015-2016, 2-3 years after neonicotinoid (clothianidin and imidacloprid) seed treatments were last used. Three of the six neonicotinoids analyzed were detected in at least one environmental matrix: the two applied as seed coatings on the fields (clothianidin and imidacloprid) and another widely used neonicotinoid (thiamethoxam). Sites with prairie strips generally had lower concentrations of neonicotinoids: groundwater and footslope soil neonicotinoid concentrations were significantly lower in the sites with prairie strips than those without; mean concentrations for groundwater were 11 and 20 ng/L (p = 0.048) and <1 and 6 ng/g (p = 0.0004) for soil, respectively. Surface runoff water concentrations were not significantly (p = 0.38) different for control sites (44 ng/L) or sites with prairie strips (140 ng/L). Consistent with the decreased inputs of neonicotinoids, concentrations tended to decrease over the sampling timeframe. Two sites recorded concentration increases, however, potentially due to disturbance of previous applications or influence from nearby fields where use of seed treatments continued. There were no detections (limit of detection: 1 ng/g) of neonicotinoids in the foliage or roots of plants comprising prairie strips, indicating a low likelihood of exposure to pollinators and other insects visiting these plants following the cessation of seed

  15. A critical review of neonicotinoid insecticides for developmental neurotoxicity.

    PubMed

    Sheets, Larry P; Li, Abby A; Minnema, Daniel J; Collier, Richard H; Creek, Moire R; Peffer, Richard C

    2016-02-01

    A comprehensive review of published and previously unpublished studies was performed to evaluate the neonicotinoid insecticides for evidence of developmental neurotoxicity (DNT). These insecticides have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in insects, poor penetration of the mammalian blood-brain barrier, and low application rates. Nevertheless, examination of this issue is warranted, due to their insecticidal mode of action and potential exposure with agricultural and residential uses. This review identified in vitro, in vivo, and epidemiology studies in the literature and studies performed in rats in accordance with GLP standards and EPA guidelines with imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran, which are all the neonicotinoids currently registered in major markets. For the guideline-based studies, treatment was administered via the diet or gavage to primiparous female rats at three dose levels, plus a vehicle control (≥20/dose level), from gestation day 0 or 6 to lactation day 21. F1 males and females were evaluated using measures of motor activity, acoustic startle response, cognition, brain morphometry, and neuropathology. The principal effects in F1 animals were associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition. No common effects were identified among the neonicotinoids that were consistent with DNT or the neurodevelopmental effects associated with nicotine. Findings at high doses were associated with evidence of systemic toxicity, which indicates that these insecticides do not selectively affect the developing nervous system.

  16. A critical review of neonicotinoid insecticides for developmental neurotoxicity

    PubMed Central

    Sheets, Larry P.; Li, Abby A.; Minnema, Daniel J.; Collier, Richard H.; Creek, Moire R.; Peffer, Richard C.

    2016-01-01

    Abstract A comprehensive review of published and previously unpublished studies was performed to evaluate the neonicotinoid insecticides for evidence of developmental neurotoxicity (DNT). These insecticides have favorable safety profiles, due to their preferential affinity for nicotinic receptor (nAChR) subtypes in insects, poor penetration of the mammalian blood–brain barrier, and low application rates. Nevertheless, examination of this issue is warranted, due to their insecticidal mode of action and potential exposure with agricultural and residential uses. This review identified in vitro, in vivo, and epidemiology studies in the literature and studies performed in rats in accordance with GLP standards and EPA guidelines with imidacloprid, acetamiprid, thiacloprid, clothianidin, thiamethoxam, and dinotefuran, which are all the neonicotinoids currently registered in major markets. For the guideline-based studies, treatment was administered via the diet or gavage to primiparous female rats at three dose levels, plus a vehicle control (≥20/dose level), from gestation day 0 or 6 to lactation day 21. F1 males and females were evaluated using measures of motor activity, acoustic startle response, cognition, brain morphometry, and neuropathology. The principal effects in F1 animals were associated with decreased body weight (delayed sexual maturation, decreased brain weight, and morphometric measurements) and acute toxicity (decreased activity during exposure) at high doses, without neuropathology or impaired cognition. No common effects were identified among the neonicotinoids that were consistent with DNT or the neurodevelopmental effects associated with nicotine. Findings at high doses were associated with evidence of systemic toxicity, which indicates that these insecticides do not selectively affect the developing nervous system. PMID:26513508

  17. Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing

    PubMed Central

    Samson-Robert, Olivier; Labrie, Geneviève; Mercier, Pierre-Luc; Chagnon, Madeleine; Derome, Nicolas; Fournier, Valérie

    2015-01-01

    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication. PMID:26223214

  18. First national-scale reconnaissance of neonicotinoid insecticides in streams across the USA

    USGS Publications Warehouse

    Hladik, Michelle; Kolpin, Dana W.

    2015-01-01

     To better understand the fate and transport of neonicotinoid insecticides, water samples were collected from streams across the United States. In a nationwide study, at least one neonicotinoid was detected in 53 % of the samples collected, with imidacloprid detected most frequently (37 %), followed by clothianidin (24 %), thiamethoxam (21 %), dinotefuran (13 %), acetamiprid (3 %) and thiacloprid (0 %). Clothianidin and thiamethoxam concentrations were positively related to the percentage of the land use in cultivated crop production and imidacloprid concentrations were positively related to the percentage of urban area within the basin. Additional sampling was also conducted in targeted research areas to complement these national-scale results, including determining: (1) neonicotinoid concentrations during elevated flow conditions in an intensely agricultural region; (2) temporal patterns of neonicotinoids in heavily urbanised basins; (3) neonicotinoid concentrations in agricultural basins in a nationally important ecosystem; and (4) in-stream transport of neonicotinoids near a wastewater treatment plant. Across all study areas, at least one neonicotinoid was detected in 63 % of the 48 streams sampled.

  19. Ecological and Landscape Drivers of Neonicotinoid Insecticide Detections and Concentrations in Canada's Prairie Wetlands.

    PubMed

    Main, Anson R; Michel, Nicole L; Headley, John V; Peru, Kerry M; Morrissey, Christy A

    2015-07-21

    Neonicotinoids are commonly used seed treatments on Canada's major prairie crops. Transported via surface and subsurface runoff into wetlands, their ultimate aquatic fate remains largely unknown. Biotic and abiotic wetland characteristics likely affect neonicotinoid presence and environmental persistence, but concentrations vary widely between wetlands that appear ecologically (e.g., plant composition) and physically (e.g., depth) similar for reasons that remain unclear. We conducted intensive surveys of 238 wetlands, and documented 59 wetland (e.g., dominant plant species) and landscape (e.g., surrounding crop) characteristics as part of a novel rapid wetland assessment system. We used boosted regression tree (BRT) analysis to predict both probability of neonicotinoid analytical detection and concentration. BRT models effectively predicted the deviance in neonicotinoid detection (62.4%) and concentration (74.7%) from 21 and 23 variables, respectively. Detection was best explained by shallow marsh plant species identity (34.8%) and surrounding crop (13.9%). Neonicotinoid concentration was best explained by shallow marsh plant species identity (14.9%) and wetland depth (14.2%). Our research revealed that plant composition is a key indicator and/or driver of neonicotinoid presence and concentration in Prairie wetlands. We recommend wetland buffers consisting of diverse native vegetation be retained or restored to minimize neonicotinoid transport and retention in wetlands, thereby limiting their potential effects on wetland-dependent organisms.

  20. Resistance of insect pests to neonicotinoid insecticides: current status and future prospects.

    PubMed

    Nauen, Ralf; Denholm, Ian

    2005-04-01

    The first neonicotinoid insecticide introduced to the market was imidacloprid in 1991 followed by several others belonging to the same chemical class and with the same mode of action. The development of neonicotinoid insecticides has provided growers with invaluable new tools for managing some of the world's most destructive crop pests, primarily those of the order Hemiptera (aphids, whiteflies, and planthoppers) and Coleoptera (beetles), including species with a long history of resistance to earlier-used products. To date, neonicotinoids have proved relatively resilient to the development of resistance, especially when considering aphids such as Myzus persicae and Phorodon humuli. Although the susceptibility of M. persicae may vary up to 20-fold between populations, this does not appear to compromise the field performance of neonicotinoids. Stronger resistance has been confirmed in some populations of the whitefly, Bemisia tabaci, and the Colorado potato beetle, Leptinotarsa decemlineata. Resistance in B- and Q-type B. tabaci appears to be linked to enhanced oxidative detoxification of neonicotinoids due to overexpression of monooxygenases. No evidence for target-site resistance has been found in whiteflies, whereas the possibility of target-site resistance in L. decemlineata is being investigated further. Strategies to combat neonicotinoid resistance must take account of the cross-resistance characteristics of these mechanisms, the ecology of target pests on different host plants, and the implications of increasing diversification of the neonicotinoid market due to a continuing introduction of new molecules. Copyright 2005 Wiley-Liss, Inc.

  1. The environmental risks of neonicotinoid pesticides: a review of the evidence post 2013.

    PubMed

    Wood, Thomas James; Goulson, Dave

    2017-07-01

    Neonicotinoid pesticides were first introduced in the mid-1990s, and since then, their use has grown rapidly. They are now the most widely used class of insecticides in the world, with the majority of applications coming from seed dressings. Neonicotinoids are water-soluble, and so can be taken up by a developing plant and can be found inside vascular tissues and foliage, providing protection against herbivorous insects. However, only approximately 5% of the neonicotinoid active ingredient is taken up by crop plants and most instead disperses into the wider environment. Since the mid-2000s, several studies raised concerns that neonicotinoids may be having a negative effect on non-target organisms, in particular on honeybees and bumblebees. In response to these studies, the European Food Safety Authority (EFSA) was commissioned to produce risk assessments for the use of clothianidin, imidacloprid and thiamethoxam and their impact on bees. These risk assessments concluded that the use of these compounds on certain flowering crops poses a high risk to bees. On the basis of these findings, the European Union adopted a partial ban on these substances in May 2013. The purpose of the present paper is to collate and summarise scientific evidence published since 2013 that investigates the impact of neonicotinoids on non-target organisms. Whilst much of the recent work has focused on the impact of neonicotinoids on bees, a growing body of evidence demonstrates that persistent, low levels of neonicotinoids can have negative impacts on a wide range of free-living organisms.

  2. Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing.

    PubMed

    Samson-Robert, Olivier; Labrie, Geneviève; Mercier, Pierre-Luc; Chagnon, Madeleine; Derome, Nicolas; Fournier, Valérie

    2015-07-30

    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥ 3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.

  3. Does Waterborne Exposure Explain Effects Caused by Neonicotinoid-Contaminated Plant Material in Aquatic Systems?

    PubMed

    Englert, Dominic; Zubrod, Jochen P; Link, Moritz; Mertins, Saskia; Schulz, Ralf; Bundschuh, Mirco

    2017-05-16

    Neonicotinoids are increasingly applied on trees as protection measure against insect pests. Consequently, neonicotinoids are inevitably transferred into aquatic environments either via spray drift or surface runoff or (due to neonicotinoids' systemic nature) via senescent leaves. There particularly leaf-shredding invertebrates may be exposed to neonicotinoids through both the water phase and the consumption of contaminated leaves. In 7 day bioassays (n = 30), we examined ecotoxicological differences between these two exposure scenarios for an amphipod and an insect nymph with their feeding rate as the response variable. Organisms either experienced waterborne neonicotinoid (i.e., imidacloprid, thiacloprid, and acetamiprid) exposure only or a combined exposure (waterborne and dietary) through both the consumption of contaminated leaves and neonicotinoids leaching from leaves into water. The amphipod (7 day EC50s from 0.3 to 8.4 μg/L) was more sensitive than the insect nymph (7 day EC50s from 7.0 to 19.4 μg/L). Moreover, for both species, concentration-response models derived from water concentrations indicated higher effects under the combined exposure. Together with the observed inability of shredders to avoid neonicotinoid-contaminated leaves, our results emphasize the relevance of dietary exposure (e.g., via leaves) for systemic insecticides. Thus, it would be prudent to consider dietary exposure during the registration of systemic insecticides to safeguard ecosystem integrity.

  4. Widespread use and frequent detection of neonicotinoid insecticides in wetlands of Canada's Prairie Pothole Region.

    PubMed

    Main, Anson R; Headley, John V; Peru, Kerry M; Michel, Nicole L; Cessna, Allan J; Morrissey, Christy A

    2014-01-01

    Neonicotinoids currently dominate the insecticide market as seed treatments on Canada's major Prairie crops (e.g., canola). The potential impact to ecologically significant wetlands in this dominantly agro-environment has largely been overlooked while the distribution of use, incidence and level of contamination remains unreported. We modelled the spatial distribution of neonicotinoid use across the three Prairie Provinces in combination with temporal assessments of water and sediment concentrations in wetlands to measure four active ingredients (clothianidin, thiamethoxam, imidacloprid and acetamiprid). From 2009 to 2012, neonicotinoid use was increasing; by 2012, applications covered an estimated ∼11 million hectares (44% of Prairie cropland) with >216,000 kg of active ingredients. Thiamethoxam, followed by clothianidin, were the dominant seed treatments by mass and area. Areas of high neonicotinoid use were identified as high density canola or soybean production. Water sampled four times from 136 wetlands (spring, summer, fall 2012 and spring 2013) across four rural municipalities in Saskatchewan similarly revealed clothianidin and thiamethoxam in the majority of samples. In spring 2012 prior to seeding, 36% of wetlands contained at least one neonicotinoid. Detections increased to 62% in summer 2012, declined to 16% in fall, and increased to 91% the following spring 2013 after ice-off. Peak concentrations were recorded during summer 2012 for both thiamethoxam (range: neonicotinoid concentrations (which did not exceed 20 ng/L). Wetlands situated in barley, canola and oat fields consistently contained higher mean concentrations of neonicotinoids than in grasslands, but no individual crop singularly influenced overall detections or concentrations. Distribution maps indicate neonicotinoid use is increasing and becoming

  5. Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment.

    PubMed

    Blacquière, Tjeerd; Smagghe, Guy; van Gestel, Cornelis A M; Mommaerts, Veerle

    2012-05-01

    Neonicotinoid insecticides are successfully applied to control pests in a variety of agricultural crops; however, they may not only affect pest insects but also non-target organisms such as pollinators. This review summarizes, for the first time, 15 years of research on the hazards of neonicotinoids to bees including honey bees, bumble bees and solitary bees. The focus of the paper is on three different key aspects determining the risks of neonicotinoid field concentrations for bee populations: (1) the environmental neonicotinoid residue levels in plants, bees and bee products in relation to pesticide application, (2) the reported side-effects with special attention for sublethal effects, and (3) the usefulness for the evaluation of neonicotinoids of an already existing risk assessment scheme for systemic compounds. Although environmental residue levels of neonicotinoids were found to be lower than acute/chronic toxicity levels, there is still a lack of reliable data as most analyses were conducted near the detection limit and for only few crops. Many laboratory studies described lethal and sublethal effects of neonicotinoids on the foraging behavior, and learning and memory abilities of bees, while no effects were observed in field studies at field-realistic dosages. The proposed risk assessment scheme for systemic compounds was shown to be applicable to assess the risk for side-effects of neonicotinoids as it considers the effect on different life stages and different levels of biological organization (organism versus colony). Future research studies should be conducted with field-realistic concentrations, relevant exposure and evaluation durations. Molecular markers may be used to improve risk assessment by a better understanding of the mode of action (interaction with receptors) of neonicotinoids in bees leading to the identification of environmentally safer compounds.

  6. Cumulative toxicity of neonicotinoid insecticide mixtures to Chironomus dilutus under acute exposure scenarios.

    PubMed

    Maloney, Erin M; Morrissey, Christy A; Headley, John V; Peru, Kerry M; Liber, Karsten

    2017-06-21

    Extensive agricultural use of neonicotinoid insecticide products has resulted in the presence of neonicotinoid mixtures in surface waters worldwide. Although many aquatic insect species are known to be sensitive to neonicotinoids, the impact of neonicotinoid mixtures is poorly understood. In the present study, the cumulative toxicities of binary and ternary mixtures of select neonicotinoids (imidacloprid, clothianidin, and thiamethoxam) were characterized under acute (96-h) exposure scenarios using the larval midge Chironomus dilutus as a representative aquatic insect species. Using the MIXTOX approach, predictive parametric models were fitted and statistically compared with observed toxicity in subsequent mixture tests. Single-compound toxicity tests yielded median lethal concentration (LC50) values of 4.63, 5.93, and 55.34 μg/L for imidacloprid, clothianidin, and thiamethoxam, respectively. Because of the similar modes of action of neonicotinoids, concentration-additive cumulative mixture toxicity was the predicted model. However, we found that imidacloprid-clothianidin mixtures demonstrated response-additive dose-level-dependent synergism, clothianidin-thiamethoxam mixtures demonstrated concentration-additive synergism, and imidacloprid-thiamethoxam mixtures demonstrated response-additive dose-ratio-dependent synergism, with toxicity shifting from antagonism to synergism as the relative concentration of thiamethoxam increased. Imidacloprid-clothianidin-thiamethoxam ternary mixtures demonstrated response-additive synergism. These results indicate that, under acute exposure scenarios, the toxicity of neonicotinoid mixtures to C. dilutus cannot be predicted using the common assumption of additive joint activity. Indeed, the overarching trend of synergistic deviation emphasizes the need for further research into the ecotoxicological effects of neonicotinoid insecticide mixtures in field settings, the development of better toxicity models for neonicotinoid mixture

  7. Widespread Use and Frequent Detection of Neonicotinoid Insecticides in Wetlands of Canada's Prairie Pothole Region

    PubMed Central

    Main, Anson R.; Headley, John V.; Peru, Kerry M.; Michel, Nicole L.; Cessna, Allan J.; Morrissey, Christy A.

    2014-01-01

    Neonicotinoids currently dominate the insecticide market as seed treatments on Canada's major Prairie crops (e.g., canola). The potential impact to ecologically significant wetlands in this dominantly agro-environment has largely been overlooked while the distribution of use, incidence and level of contamination remains unreported. We modelled the spatial distribution of neonicotinoid use across the three Prairie Provinces in combination with temporal assessments of water and sediment concentrations in wetlands to measure four active ingredients (clothianidin, thiamethoxam, imidacloprid and acetamiprid). From 2009 to 2012, neonicotinoid use was increasing; by 2012, applications covered an estimated ∼11 million hectares (44% of Prairie cropland) with >216,000 kg of active ingredients. Thiamethoxam, followed by clothianidin, were the dominant seed treatments by mass and area. Areas of high neonicotinoid use were identified as high density canola or soybean production. Water sampled four times from 136 wetlands (spring, summer, fall 2012 and spring 2013) across four rural municipalities in Saskatchewan similarly revealed clothianidin and thiamethoxam in the majority of samples. In spring 2012 prior to seeding, 36% of wetlands contained at least one neonicotinoid. Detections increased to 62% in summer 2012, declined to 16% in fall, and increased to 91% the following spring 2013 after ice-off. Peak concentrations were recorded during summer 2012 for both thiamethoxam (range: neonicotinoid concentrations (which did not exceed 20 ng/L). Wetlands situated in barley, canola and oat fields consistently contained higher mean concentrations of neonicotinoids than in grasslands, but no individual crop singularly influenced overall detections or concentrations. Distribution maps indicate neonicotinoid use is increasing and

  8. Binding of imidacloprid, thiamethoxam and N-desmethylthiamethoxam to nicotinic receptors of Myzus persicae: pharmacological profiling using neonicotinoids, natural agonists and antagonists.

    PubMed

    Kayser, Hartmut; Lehmann, Katrin; Gomes, Marilyne; Schleicher, Wolfgang; Dotzauer, Karin; Moron, Margarethe; Maienfisch, Peter

    2016-11-01

    The increasing structural diversity of the neonicotinoid class of insecticides presently used in crop protection calls for a more detailed analysis of their mode of action at their cellular targets, the nicotinic acetylcholine receptors. Comparative radioligand binding studies using membranes of Myzus persicae (Sulzer) and representatives of the chloropyridyl subclass (imidacloprid), the chlorothiazolyl subclass (thiamethoxam), the tetrahydrofuranyl subclass (dinotefuran), as well as the novel sulfoximine type (sulfoxaflor), which is not a neonicotinoid, reveal significant differences in the number of binding sites, the displacing potencies and the mode of binding interference. Furthermore, the mode of interaction of [(3) H]thiamethoxam and the nicotinic antagonists methyllycaconitine and dihydro-β-erythroidine is unique, with Hill values of >1, clearly different to the values of around unity for [(3) H]imidacloprid and [(3) H]N-desmethylthiamethoxam. The interaction of [(3) H]N-desmethylthiamethoxam with the agonist (-)nicotine is also characterised by a Hill value of >1. There is no single conserved site or mode of binding of neonicotinoids and related nicotinic ligands to their target receptor, but a variety of binding pockets depending on the combination of receptor subunits, the receptor subtype, its functional state, as well as the structural flexibility of both the binding pockets and the ligands. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  9. Structural and functional insight into the universal stress protein family

    PubMed Central

    Tkaczuk, Karolina L; A Shumilin, Igor; Chruszcz, Maksymilian; Evdokimova, Elena; Savchenko, Alexei; Minor, Wladek

    2013-01-01

    We present the crystal structures of two universal stress proteins (USP) from Archaeoglobus fulgidus and Nitrosomonas europaea in both apo- and ligand-bound forms. This work is the first complete synthesis of the structural properties of 26 USP available in the Protein Data Bank, over 75% of which were determined by structure genomics centers with no additional information provided. The results of bioinformatic analyses of all available USP structures and their sequence homologs revealed that these two new USP structures share overall structural similarity with structures of USPs previously determined. Clustering and cladogram analyses, however, show how they diverge from other members of the USP superfamily and show greater similarity to USPs from organisms inhabiting extreme environments. We compared them with other archaeal and bacterial USPs and discuss their similarities and differences in context of structure, sequential motifs, and potential function. We also attempted to group all analyzed USPs into families, so that assignment of the potential function to those with no experimental data available would be possible by extrapolation. PMID:23745136

  10. Dinotefuran: a potential neonicotinoid insecticide against resistant mosquitoes.

    PubMed

    Corbel, Vincent; Duchon, Stephane; Zaim, Morteza; Hougard, Jean-Marc

    2004-07-01

    Because pyrethroid, organophosphate, and carbamate resistance is more and more developed in mosquitoes of medical importance, there is an urgent need for alternative insecticides for vector control. Dinotefuran, a new neonicotinoid insecticide commercialized by Mitsui Chemicals (Tokyo, Japan), could be a useful candidate in public health because it shows low mammalian toxicity and great insecticidal activity against a broad range of pests. In this study, the intrinsic toxicity of dinotefuran was evaluated by larval bioassay and topical application against different mosquito strains of Anopheles gambiae Giles, Culex quinquefasciatus Say, and Aedes aegypti L. having none, one, or several resistance mechanisms, respectively, to insecticides. The results showed that dinotefuran was less toxic than most of the commonly used insecticides (e.g., deltamethrin, carbosulfan, and temephos) against the susceptible mosquitoes tested (between 6- and 100-fold at the LD50 level). However, the toxicity of dinotefuran was not strongly affected by the presence of common resistance mechanism, i.e., kdr mutation and insensitive acetylcholinesterase (resistance ratio [RR] from 1.3 to 2.3). More interestingly, the carbamate-resistant strain of Cx. quinquefasciatus was significantly more affected by dinotefuran than the susceptible strain (RR = 0.70), probably because the insensitive acetylcholinesterase is less efficient to degrade nicotinic substrates than normal acetylcholinesterase. Despite the relatively low toxicity of dinotefuran against susceptible mosquitoes, the absence of cross-resistance with common insecticides (pyrethroids, carbamates, and organophosphates) makes neonicotinoids potential candidates for disease vector control, especially in area where mosquitoes are resistant to insecticides.

  11. Chemistry and biology of thiamethoxam: a second generation neonicotinoid.

    PubMed

    Maienfisch, P; Angst, M; Brandl, F; Fischer, W; Hofer, D; Kayser, H; Kobel, W; Rindlisbacher, A; Senn, R; Steinemann, A; Widmer, H

    2001-10-01

    Thiamethoxam is the first commercial neonicotinoid insecticide from the thianicotinyl subclass. It was discovered in the course of our optimisation program on neonicotinoids started in 1985. Novel variations of the nitroimino-heterocycle of imidacloprid led to 4-nitroimino-1,3,5-oxadiazinanes exhibiting high insecticidal activity. Among these, thiamethoxam (CGA 293433) was identified as the best compound and selected for worldwide development. The compound can be synthesised in only a few steps and high yield from easily accessible starting materials. Thiamethoxam acts by binding to nicotinic acetylcholine receptors. It exhibits exceptional systemic characteristics and provides excellent control of a broad range of commercially important pests, such as aphids, jassids, whiteflies, thrips, rice hoppers, Colorado potato beetle, flea beetles and wireworms, as well as some lepidopteran species. In addition, a strong preventative effect on some virus transmissions has been demonstrated. Thiamethoxam is developed both for foliar/soil applications and as a seed treatment for use in most agricultural crops all over the world. Low use rates, flexible application methods, excellent efficacy, long-lasting residual activity and favourable safety profile make this new insecticide well-suited for modern integrated pest management programmes in many cropping systems.

  12. Value of neonicotinoid seed treatments to US soybean farmers.

    PubMed

    Hurley, Terrance; Mitchell, Paul

    2017-01-01

    The benefits of neonicotinoid seed treatment to soybean farmers have received increased scrutiny. Rather than use data from small-plot experiments, this research uses survey data from 500 US farmers to estimate the benefit of neonicotinoid seed treatments to them. As seed treatment users, farmers are familiar with their benefits in the field and have economic incentives to only use them if they provide value. Of the surveyed farmers, 51% used insecticide seed treatments, averaging 87% of their soybean area. Farmers indicated that human and environmental safety is an important consideration affecting their pest management decisions and reported aphids as the most managed and important soybean pest. Asking farmers who used seed treatments to state how much value they provided gives an estimate of $US 28.04 ha(-1) treated in 2013, net of seed treatment costs. Farmer-reported average yields provided an estimated average yield gain of 128.0 kg ha(-1) treated in 2013, or about $US 42.20 ha(-1) treated, net of seed treatment costs. These estimates using different data and methods are consistent and suggest the value of insecticide seed treatments to the US soybean farmers who used them in 2013 was around $US 28-42 ha(-1) treated, net of seed treatment costs. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  13. Structural insights on biologically relevant cationic membranes by ESR spectroscopy.

    PubMed

    Rozenfeld, Julio H K; Duarte, Evandro L; Oliveira, Tiago R; Lamy, M Teresa

    2017-08-23

    Cationic bilayers have been used as models to study membrane fusion, templates for polymerization and deposition of materials, carriers of nucleic acids and hydrophobic drugs, microbicidal agents and vaccine adjuvants. The versatility of these membranes depends on their structure. Electron spin resonance (ESR) spectroscopy is a powerful technique that employs hydrophobic spin labels to probe membrane structure and packing. The focus of this review is the extensive structural characterization of cationic membranes prepared with dioctadecyldimethylammonium bromide or diC14-amidine to illustrate how ESR spectroscopy can provide important structural information on bilayer thermotropic behavior, gel and fluid phases, phase coexistence, presence of bilayer interdigitation, membrane fusion and interactions with other biologically relevant molecules.

  14. Recent Structural Insights into Cytochrome P450 Function

    PubMed Central

    Peter Guengerich, F.; Waterman, Michael R.; Egli, Martin

    2016-01-01

    Cytochrome P450 (P450) enzymes are important in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides, and many other types of chemicals. Their catalytic activities are important issues in areas such as drug-drug interactions and endocrine function. During the past 30 years, structures of P450s have been very helpful in understanding function, particularly the mammalian P450 structures available in the past 15 years. We review recent activity in this area, focusing on the past two years (2014–2015). Structural work with microbial P450s includes studies related to the biosynthesis of natural products and the use of parasitic and fungal P450 structures as targets for drug discovery. Studies on mammalian P450s include the utilization of information about ‘drug-metabolizing’ P450s to improve drug development and also to understand the molecular bases of endocrine dysfunction. PMID:27267697

  15. Insights from modeling the tertiary structure of human BACE2.

    PubMed

    Chou, Kuo-Chen

    2004-01-01

    BACE1, or beta-secretase, is a putative prime therapeutic target for the treatment of Alzheimer's disease. Mapping to the Down syndrome critical region (chromosome 21) and identified as a homologue of BACE1, BACE2 also cleaves amyloid precursor protein at the beta-site. Thus, BACE2, named also as Asp1 or Memapsin1, represents a second beta-secretase candidate. In this paper, the tertiary structure of the protease domain of BACE2 was developed. Although the overall structural topology between BACE1 and BACE2 protease domains is quite similar, the former contains 3 disulfide bonds but the latter only two. Particularly, a subtle structural difference around the DTG/DSG active site between the two structures has been observed that is useful for the in-depth selectivity study of BACE1 and BACE2 inhibitors, stimulating new therapeutic strategies for the treatment of Alzheimer's disease and Down syndrome as well.

  16. Animal NLRs provide structural insights into plant NLR function.

    PubMed

    Bentham, Adam; Burdett, Hayden; Anderson, Peter A; Williams, Simon J; Kobe, Bostjan

    2017-03-01

    The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat [LRR]/nucleotide-binding oligomerization domain [NOD]-like receptors) to detect effector proteins secreted into the plant cell by potential pathogens. Activated plant NLRs trigger a range of immune responses, collectively known as the hypersensitive response (HR), which culminates in death of the infected cell. Plant NLRs show structural and functional resemblance to animal NLRs involved in inflammatory and innate immune responses. Therefore, knowledge of the activation and regulation of animal NLRs can help us understand the mechanism of action of plant NLRs, and vice versa. This review provides an overview of the innate immune pathways in plants and animals, focusing on the available structural and biochemical information available for both plant and animal NLRs. We highlight the gap in knowledge between the animal and plant systems, in particular the lack of structural information for plant NLRs, with crystal structures only available for the N-terminal domains of plant NLRs and an integrated decoy domain, in contrast to the more complete structures available for animal NLRs. We assess the similarities and differences between plant and animal NLRs, and use the structural information on the animal NLR pair NAIP/NLRC4 to derive a plausible model for plant NLR activation. Signalling by cooperative assembly formation (SCAF) appears to operate in most innate immunity pathways, including plant and animal NLRs. Our proposed model of plant NLR activation includes three key steps: (1) initially, the NLR exists in an inactive auto-inhibited state; (2) a combination of binding by activating elicitor and ATP leads to a structural rearrangement of the NLR; and (3) signalling occurs through cooperative assembly of the resistosome. Further studies, structural and biochemical in particular, will be required to provide additional evidence for the different features of this model and

  17. Structural insights into antibody recognition of mycobacterial polysaccharides.

    PubMed

    Murase, Tomohiko; Zheng, Ruixiang Blake; Joe, Maju; Bai, Yu; Marcus, Sandra L; Lowary, Todd L; Ng, Kenneth K S

    2009-09-18

    Mycobacteria are major human pathogens responsible for such serious and widespread diseases as tuberculosis and leprosy. Among the evolutionary adaptations essential for pathogenicity in mycobacteria is a complex carbohydrate-rich cell-wall structure that contains as a major immunomodulatory molecule the polysaccharide lipoarabinomannan (LAM). We report here crystal structures of three fragments from the non-reducing termini of LAM in complex with a murine antibody Fab fragment (CS-35Fab). These structures reveal for the first time the three-dimensional structures of key components of LAM and the molecular basis of LAM recognition at between 1.8- and 2.0-A resolution. The antigen-binding site of CS-35Fab forms three binding pockets that show a high degree of complementarity to the reducing end, the branch point and one of the non-reducing ends of the Y-shaped hexasaccharide moiety found at most of the non-reducing termini of LAM. Structures of CS-35Fab bound to two additional tetrasaccharides confirm the general mode of binding seen in the hexasaccharide and indicate how different parts of LAM are recognized. Altogether, these structures provide a rational basis for understanding the overall architecture of LAM and identify the key elements of an epitope that may be exploited for the development of novel and more effective anti-mycobacterial vaccines. Moreover, this study represents the first high-resolution X-ray crystallographic investigation of oligofuranoside-protein recognition.

  18. Comparative analysis of neonicotinoid binding to insect membranes: II. An unusual high affinity site for [3H]thiamethoxam in Myzus persicae and Aphis craccivora.

    PubMed

    Wellmann, Henning; Gomes, Marilyne; Lee, Connie; Kayser, Hartmut

    2004-10-01

    Neonicotinoids represent a class of insect-selective ligands of nicotinic acetylcholine receptors. Imidacloprid, the first commercially used neonicotinoid insecticide, has been studied on neuronal preparations from many insects to date. Here we report first intrinsic binding data of thiamethoxam, using membranes from Myzus persicae Sulzer and Aphis craccivora Koch. In both aphids, specific binding of [3H]thiamethoxam was sensitive to temperature, while the absolute level of non-specific binding was not affected. In M persicae, binding capacity (Bmax) for [3H]thiamethoxam was ca 450 fmol mg(-1) of protein at 22 degrees C and ca 700 fmol mg(-1) of protein at 2 degrees C. The negative effect of increased temperature was reversible and hence not due to some destructive process. The affinity for [3H]thiamethoxam was less affected by temperature: Kd was ca 11 nM at 2 degrees C and ca 15 nM at 22 degrees C. The membranes also lost binding sites for [3H]thiamethoxam during prolonged storage at room temperature, and upon freezing and thawing. In A craccivora, [3H]thiamethoxam was bound with a capacity of ca 1000 fmol mg(-1) protein and an affinity of ca 90 nM, as measured at 2 degrees C. Overall, the in vitro temperature sensitivity of [3H]thiamethoxam binding was in obvious contrast to the behaviour of [3H]imidacloprid studied in parallel. Moreover, the binding of [3H]thiamethoxam was inhibited by imidacloprid in a non-competitive mode, as shown with M persicae. In our view, these differences demonstrate that thiamethoxam and imidacloprid, which represent different structural sub-classes of neonicotinoids, do not share the same binding site or mode. This holds also for other neonicotinoids, as we report in a companion article.

  19. Structural insight into the PTS sugar transporter EIIC

    PubMed Central

    McCoy, Jason G.; Levin, Elena J.; Zhou, Ming

    2014-01-01

    Background The enzyme IIC component (EIIC) of the phosphotransferase system (PTS) is responsible for selectively transporting sugar molecules across the inner bacterial membrane. This is accomplished in parallel with phosphorylation of the sugar, which prevents efflux of the sugar back across the membrane. This process is a key part of an extensive signaling network that allows bacteria to efficiently utilize preferred carbohydrate sources. Scope of review The goal of this review is to examine the current understanding of the structural features of EIIC and how it mediates concentrative, selective sugar transport. The crystal structure of an N,N’-diacetylchitobiose transporter is used as a structural template for the glucose superfamily of PTS transporters. Major conclusions Comparison of protein sequences in context with the known EIIC structure suggests members of the glucose superfamily of PTS transporters may exhibit variations in topology. Despite these differences, a conserved histidine and glutamate appear to have roles shared across the superfamily in sugar binding and phosphorylation. In the proposed transport model, a rigid body motion between two structural domains and movement of an intracellular loop provide the substrate binding site with alternating access, and reveal a surface required for interaction with the phosphotransfer protein required for catalysis. General significance The structural and functional data discussed here give a preliminary understanding of how transport in EIIC is achieved. However, given the great sequence diversity between varying glucose-superfamily PTS transporters and lack of data on conformational changes needed for transport, additional structures of other members and conformations are still required. PMID:24657490

  20. Structural insights into bacterial flagellar hooks similarities and specificities

    PubMed Central

    Yoon, Young-Ho; Barker, Clive S.; Bulieris, Paula V.; Matsunami, Hideyuki; Samatey, Fadel A.

    2016-01-01

    Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments. PMID:27759043

  1. Insights to primitive replication derived from structures of small oligonucleotides

    NASA Technical Reports Server (NTRS)

    Smith, G. K.; Fox, G. E.

    1995-01-01

    Available information on the structure of small oligonucleotides is surveyed. It is observed that even small oligomers typically exhibit defined structures over a wide range of pH and temperature. These structures rely on a plethora of non-standard base-base interactions in addition to the traditional Watson-Crick pairings. Stable duplexes, though typically antiparallel, can be parallel or staggered and perfect complementarity is not essential. These results imply that primitive template directed reactions do not require high fidelity. Hence, the extensive use of Watson-Crick complementarity in genes rather than being a direct consequence of the primitive condensation process, may instead reflect subsequent selection based on the advantage of accuracy in maintaining the primitive genetic machinery once it arose.

  2. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation.

    PubMed

    Mulder, David W; Shepard, Eric M; Meuser, Jonathan E; Joshi, Neelambari; King, Paul W; Posewitz, Matthew C; Broderick, Joan B; Peters, John W

    2011-08-10

    Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.

  3. Structural insights into bacterial modulation of the host cytoskeleton.

    PubMed

    Stebbins, C Erec

    2004-12-01

    Many bacterial pathogens manipulate the host cell cytoskeleton during infection. Such cytoskeletal modulation can occur at several points of contact between the pathogen and the host, and involves extracellular receptors, intracellular signal transduction and cytoskeletal proteins themselves. The field of bacterial pathogenesis has progressed dramatically over the past decade, such that structural knowledge is both timely and essential for a full appreciation of the biology at the pathogen-host interface. Several recent examples involving bacterial proteins that target actin, Rho family GTPases and extracellular receptors have contributed to a structural understanding of eukaryotic cytoskeletal modulation by pathogens.

  4. Structural Insight into OprD Substrate Specifity

    SciTech Connect

    Biswas,S.; Mohammad, M.; Patel, D.; Movileanu, L.; van den Berg, B.

    2007-01-01

    OprD proteins form a large family of substrate-specific outer-membrane channels in Gram-negative bacteria. We report here the X-ray crystal structure of OprD from Pseudomonas aeruginosa, which reveals a monomeric 18-stranded beta-barrel characterized by a very narrow pore constriction, with a positively charged basic ladder on one side and an electronegative pocket on the other side. The location of highly conserved residues in OprD suggests that the structure represents the general architecture of OprD channels.

  5. Bismuth modified carbon-based electrodes for the determination of selected neonicotinoid insecticides.

    PubMed

    Guzsvány, Valéria; Papp, Zsigmond; Zbiljić, Jasmina; Vajdle, Olga; Rodić, Marko

    2011-05-27

    Two types of bismuth modified electrodes, a bismuth-film modified glassy carbon (BiF-GCE) and a bismuth bulk modified carbon paste, were applied for the determination of selected nitroguanidine neonicotinoid insecticides. The method based on an ex situ prepared BiF-GCE operated in the differential pulse voltammetric (DPV) mode was applied to determine clothianidin in the concentration range from 2.5 to 23 μg cm⁻³ with a relative standard deviation (RSD) not exceeding 1.5%. The tricresyl phosphate-based carbon paste electrodes (TCP-CPEs), bulk modified with 5 and 20 w/w% of bismuth, showed a different analytical performance in the determination of imidacloprid, regarding the peak shape, potential window, and noise level. The TCP-CPE with 5% Bi was advantageous, and the developed DPV method based on it allowed the determination in the concentration range from 1.7 to 60 μg cm⁻³ with an RSD of 2.4%. To get a deeper insight into the morphology of the bismuth-based sensor surfaces, scanning electron microscopic measurements were performed of both the surface film and the bulk modified electrodes.

  6. Widespread occurrence of neonicotinoid insecticides in streams in a high corn and soybean producing region, USA

    USGS Publications Warehouse

    Hladik, Michelle; Kolpin, Dana W.; Kuivila, Kathryn

    2014-01-01

    Neonicotinoid insecticides are of environmental concern, but little is known about their occurrence in surface water. An area of intense corn and soybean production in the Midwestern United States was chosen to study this issue because of the high agricultural use of neonicotinoids via both seed treatments and other forms of application. Water samples were collected from nine stream sites during the 2013 growing season. The results for the 79 water samples documented similar patterns among sites for both frequency of detection and concentration (maximum:median) with clothianidin (75%, 257 ng/L:8.2 ng/L) > thiamethoxam (47%, 185 ng/L: imidacloprid (23%, 42.7 ng/L: <2 ng/L). Neonicotinoids were detected at all nine sites sampled even though the basin areas spanned four orders of magnitude. Temporal patterns in concentrations reveal pulses of neonicotinoids associated with rainfall events during crop planting, suggesting seed treatments as their likely source.

  7. The neonicotinoid clothianidin interferes with navigation of the solitary bee Osmia cornuta in a laboratory test.

    PubMed

    Jin, Nanxiang; Klein, Simon; Leimig, Fabian; Bischoff, Gabriela; Menzel, Randolf

    2015-09-01

    Pollinating insects provide a vital ecosystem service to crops and wild plants. Exposure to low doses of neonicotinoid insecticides has sub-lethal effects on social pollinators such as bumblebees and honeybees, disturbing their navigation and interfering with their development. Solitary Hymenoptera are also very important ecosystem service providers, but the sub-lethal effects of neonicotinoids have not yet been studied well in those animals. We analyzed the ability of walking Osmia to remember a feeding place in a small environment and found that Osmia remembers the feeding place well after 4 days of training. Uptake of field-realistic amounts of the neonicotinoid clothianidin (0.76 ng per bee) altered the animals' sensory responses to the visual environment and interfered with the retrieval of navigational memory. We conclude that the neonicotinoid clothianidin compromises visual guidance and the use of navigational memory in the solitary bee Osmia cornuta. © 2015. Published by The Company of Biologists Ltd.

  8. Insight into the antiferromagnetic structure manipulated by electronic reconstruction

    NASA Astrophysics Data System (ADS)

    Cui, B.; Li, F.; Song, C.; Peng, J. J.; Saleem, M. S.; Gu, Y. D.; Li, S. N.; Wang, K. L.; Pan, F.

    2016-10-01

    Antiferromagnetic (AFM) materials, with robust rigidity to magnetic field perturbations and ultrafast spin dynamics, show great advantages in information storage and have developed into a fast-emerging field of AFM spintronics. However, a direct characterization of spin alignments in AFM films has been challenging, and their manipulation by lattice distortion and magnetic proximity is inevitably accompanied by "ferromagnetic" features within the AFM matrix. Here we resolve the G -type AFM structure of SrCo O2.5 and find that the interfacial AFM structure could be modulated intrinsically from in plane to out of plane with a canted angle of 60∘ by the charge transfer and orbital reconstruction in SrCo O2.5/L a2 /3S r1 /3Mn O3 heterostructures both experimentally and theoretically. Such an interfacial AFM reconfiguration caused by electronic reconstruction does not cause the ferromagnetic feature and changes the magnetization switching process of L a2 /3S r1 /3Mn O3 from in plane to perpendicular to the plane, in turn. Our study not only reveals the coupling between charge, orbital, and AFM structure, but also provides a unique approach to manipulating AFM structure.

  9. Spatial structure of cyclosporin A and insight into its flexibility

    NASA Astrophysics Data System (ADS)

    Efimov, S. V.; Karataeva, F. Kh.; Aganov, A. V.; Berger, S.; Klochkov, V. V.

    2013-03-01

    The molecule of immunosuppressant drug cyclosporin A (CsA) exhibits different properties when dissolved in different media. In apolar solvents it is stabilized by intramolecular hydrogen bonds, but there also exist some less populated conformations. Existence of minor forms is clearly seen from 1H NMR spectra. Using nuclear Overhauser effect (NOE) spectroscopy and analysis of residual dipolar couplings, we obtained data on the molecular structure of the dominant conformers. Based on these data, the spatial structure of the main conformer of cyclosporin in chloroform was determined by molecular dynamics simulation. The kinetics of exchange between the major and minor forms was also studied. Energy barrier (ΔG‡) between the two states is 81 ± 2 kJ/mol. The conformation of CsA in complex with sodium dodecyl sulphate micelles was determined from NOE data. Use of independent structural data improves the reliability of the simulated results. The structure of the minor forms, which exist in organic solvents and also in micellar solution, cannot be assessed by means of nuclear magnetic resonance. Spectroscopic and thermodynamic parameters, however, point to their certain properties. In particular, the minor conformer of CsA in chloroform differs from the main one by a peptide bond (in cis- rather than trans-conformation) in the region of residues from 4 to 7.

  10. Structural insights into reptarenavirus cap-snatching machinery

    PubMed Central

    Rosenthal, Maria; Gogrefe, Nadja; Reguera, Juan; Rauschenberger, Bianka; Günther, Stephan

    2017-01-01

    Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed. PMID:28505175

  11. Structural insights into reptarenavirus cap-snatching machinery.

    PubMed

    Rosenthal, Maria; Gogrefe, Nadja; Vogel, Dominik; Reguera, Juan; Rauschenberger, Bianka; Cusack, Stephen; Günther, Stephan; Reindl, Sophia

    2017-05-01

    Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.

  12. Improved prediction of RNA tertiary structure with insights into native state dynamics.

    PubMed

    Bida, John Paul; Maher, L James

    2012-03-01

    The importance of RNA tertiary structure is evident from the growing number of published high resolution NMR and X-ray crystallographic structures of RNA molecules. These structures provide insights into function and create a knowledge base that is leveraged by programs such as Assemble, ModeRNA, RNABuilder, NAST, FARNA, Mc-Sym, RNA2D3D, and iFoldRNA for tertiary structure prediction and design. While these methods sample native-like RNA structures during simulations, all struggle to capture the native RNA conformation after scoring. We propose RSIM, an improved RNA fragment assembly method that preserves RNA global secondary structure while sampling conformations. This approach enhances the quality of predicted RNA tertiary structure, provides insights into the native state dynamics, and generates a powerful visualization of the RNA conformational space. RSIM is available for download from http://www.github.com/jpbida/rsim.

  13. Structural insights into the YAP and TEAD complex.

    PubMed

    Li, Ze; Zhao, Bin; Wang, Ping; Chen, Fei; Dong, Zhenghong; Yang, Huirong; Guan, Kun-Liang; Xu, Yanhui

    2010-02-01

    The Yes-associated protein (YAP) transcriptional coactivator is a key regulator of organ size and a candidate human oncogene inhibited by the Hippo tumor suppressor pathway. The TEAD family of transcription factors binds directly to and mediates YAP-induced gene expression. Here we report the three-dimensional structure of the YAP (residues 50-171)-TEAD1 (residues 194-411) complex, in which YAP wraps around the globular structure of TEAD1 and forms extensive interactions via three highly conserved interfaces. Interface 3, including YAP residues 86-100, is most critical for complex formation. Our study reveals the biochemical nature of the YAP-TEAD interaction, and provides a basis for pharmacological intervention of YAP-TEAD hyperactivation in human diseases.

  14. Structural insights into the YAP and TEAD complex

    PubMed Central

    Li, Ze; Zhao, Bin; Wang, Ping; Chen, Fei; Dong, Zhenghong; Yang, Huirong; Guan, Kun-Liang; Xu, Yanhui

    2010-01-01

    The Yes-associated protein (YAP) transcriptional coactivator is a key regulator of organ size and a candidate human oncogene inhibited by the Hippo tumor suppressor pathway. The TEAD family of transcription factors binds directly to and mediates YAP-induced gene expression. Here we report the three-dimensional structure of the YAP (residues 50–171)–TEAD1 (residues 194–411) complex, in which YAP wraps around the globular structure of TEAD1 and forms extensive interactions via three highly conserved interfaces. Interface 3, including YAP residues 86–100, is most critical for complex formation. Our study reveals the biochemical nature of the YAP–TEAD interaction, and provides a basis for pharmacological intervention of YAP–TEAD hyperactivation in human diseases. PMID:20123905

  15. Structural Insights into Ail-Mediated Adhesion in Yersinia pestis

    SciTech Connect

    Yamashita, Satoshi; Lukacik, Petra; Barnard, Travis J.; Noinaj, Nicholas; Felek, Suleyman; Tsang, Tiffany M.; Krukonis, Eric S.; Hinnebusch, B. Joseph; Buchanan, Susan K.

    2012-01-30

    Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.

  16. New insight on the structural trends of polyphosphate coacervation processes.

    PubMed

    Silva, Maurício A P; Franco, Douglas F; de Oliveira, Luiz Fernando C

    2008-06-19

    In this study new compositions of polyphosphate coacervates were obtained with the ions Ni (2+) and Co (2+). Samples of the glassy systems were prepared with proportions P/M (+2) varying between 0.5 and 10. The qualitative and quantitative description of the first coordination shells of the transition metal were obtained through extended X-ray absorption fine structure spectroscopy (EXAFS) analysis, performed at the Ni (2+) and Co (2+) K-edges. An analysis of the symmetric stretching vibrations of terminal P-O t and bridging P-O b groups performed through Raman spectroscopy revealed the different phases of the coacervation process in terms of bond strengths and corroborates the EXAFS results. The results obtained permitted a detailed structural description of these materials as well as the role played by the metallic ions on the coacervation process.

  17. The nuclear pore complex: understanding its function through structural insight.

    PubMed

    Beck, Martin; Hurt, Ed

    2017-02-01

    Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. They are large macromolecular assemblies with a complex composition and diverse functions. Apart from facilitating nucleocytoplasmic transport, NPCs are involved in chromatin organization, the regulation of gene expression and DNA repair. Understanding the molecular mechanisms underlying these functions has been hampered by a lack of structural knowledge about the NPC. The recent convergence of crystallographic and biochemical in vitro analysis of nucleoporins (NUPs), the components of the NPC, with cryo-electron microscopic imaging of the entire NPC in situ has provided first pseudo-atomic view of its central core and revealed that an unexpected network of short linear motifs is an important spatial organization principle. These breakthroughs have transformed the way we understand NPC structure, and they provide an important base for functional investigations, including the elucidation of the molecular mechanisms underlying clinically manifested mutations of the nucleocytoplasmic transport system.

  18. Structural Insights into the Role of Mutations in Amyloidogenesis

    SciTech Connect

    Baden, Elizabeth M.; Randles, Edward G.; Aboagye, Awo K.; Thompson, James R.; Ramirez-Alvarado, Marina

    2009-08-14

    Mechanisms of amyloidogenesis are not well understood, including potential structural contributions of mutations in the process. Our previous research indicated that the dimer interface of amyloidogenic immunoglobulin light chain protein AL-09 is twisted 90{sup o} relative to the protein from its germline sequence, {kappa}I O18/O8. Here we report a systematic restoration of AL-09 to its germline sequence by mutating the non-conservative somatic mutations located in the light chain dimer interface. Among these mutants, we find a correlation between increased thermodynamic stability and an increase in the lag time for fibril formation. The restorative mutant AL-09 H87{gamma} completes the trifecta and restores the dimer interface observed in ?I O18/O8, emphasizing the potential importance of the structural integrity of these proteins to protect against amyloidogenicity. We also find that adding amyloidogenic mutations into the germline protein illustrates mutational cooperativity in promoting amyloidogenesis.

  19. Structural Insights into Fibronectin Type III Domain Mediated Signaling

    PubMed Central

    Bencharit, Sompop; Cui, Cai Bin; Siddiqui, Adnan; Howard-Williams, Escher L.; Sondek, John; Zuobi-Hasona, Kheir; Aukhil, Ikramuddin

    2007-01-01

    The alternatively spliced type-III extradomain B (EIIIB) of Fibronectin (FN) is only expressed during embryogenesis, wound healing and tumorigenesis. The biological function of this domain remains unclear. We describe here the first crystal structure of the interface between alternatively-spliced domain EIIIB and its adjacent FN type-III domain 8 (FN B-8). The opened CC′ loop of EIIIB and the rotation and tilt of EIIIB domain allows good access to the FG loop of FN-8 which is normally hindered by the CC′ loop of FN-7. In addition, the AGEGIP sequence of the CC′ loop of EIIIB replaces the NGQQGN sequence of the CC′ loop of FN-7. Finally, the CC” loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilagenous development, wound healing, and tumorigenesis, respectively. PMID:17261313

  20. Some insights into stellar structure from nonlinear pulsations

    NASA Astrophysics Data System (ADS)

    Goupil, M. J.

    1993-12-01

    Efficient tools of investigation of stellar pulsation are the integral relations which link oscillation frequencies to the static structure of stellar models, as provided by the linear theory of pulsation. Similarly, oscillation amplitudes and phases, which arise from nonlinear processes, can be related to the stellar structure by means of amplitude equation formalisms. For the simple case of a monoperiodic oscillation, involving only one unstable marginal mode, such a formalism shows that the (limit cycle) radius variations, at time t and mass level m, can be approximated, up to second order of approximation. The nonlinear, nonadiabatic coefficients, are integrals over mass of kernels which depend on eigenfrequencies, eigenfunctions, on second and third order Taylor quantities from the equations modelling the star. They can either be computed from static models (Klapp et al., 1985) or obtained by numerical fits of hydrodynamical results (Kovacs and Buchler, 1989).

  1. Structural insight for chain selection and stagger control in collagen

    PubMed Central

    Boudko, Sergei P.; Bächinger, Hans Peter

    2016-01-01

    Collagen plays a fundamental role in all known metazoans. In collagens three polypeptides form a unique triple-helical structure with a one-residue stagger to fit every third glycine residue in the inner core without disturbing the poly-proline type II helical conformation of each chain. There are homo- and hetero-trimeric types of collagen consisting of one, two or three distinct chains. Thus there must be mechanisms that control composition and stagger during collagen folding. Here, we uncover the structural basis for both chain selection and stagger formation of a collagen molecule. Three distinct chains (α1, α2 and α3) of the non-collagenous domain 2 (NC2) of type IX collagen are assembled to guide triple-helical sequences in the leading, middle and trailing positions. This unique domain opens the door for generating any fragment of collagen in its native composition and stagger. PMID:27897211

  2. Crustal structure of central Lake Baikal: Insights into intracontinental rifting

    USGS Publications Warehouse

    ten Brink, U.S.; Taylor, M.H.

    2002-01-01

    The Cenozoic rift system of Baikal, located in the interior of the largest continental mass on Earth, is thought to represent a potential analog of the early stage of breakup of supercontinents. We present a detailed P wave velocity structure of the crust and sediments beneath the Central Basin, the deepest basin in the Baikal rift system. The structure is characterized by a Moho depth of 39-42.5 km; an 8-km-thick, laterally continuous high-velocity (7.05-7.4 km/s) lower crust, normal upper mantle velocity (8 km/s), a sedimentary section reaching maximum depths of 9 km, and a gradual increase of sediment velocity with depth. We interpret the high-velocity lower crust to be part of the Siberian Platform that was not thinned or altered significantly during rifting. In comparison to published results from the Siberian Platform, Moho under the basin is elevated by <3 km. On the basis of these results we propose that the basin was formed by upper crustal extension, possibly reactivating structures in an ancient fold-and-thrust belt. The extent and location of upper mantle extension are not revealed by our data, and it may be offset from the rift. We believe that the Baikal rift structure is similar in many respects to the Mesozoic Atlantic rift system, the precursor to the formation of the North Atlantic Ocean. We also propose that the Central Baikal rift evolved by episodic fault propagation and basin enlargement, rather than by two-stage rift evolution as is commonly assumed.

  3. Structural insights into the evolution of an antibody combining site

    SciTech Connect

    Wedemayer, G.J.; Patten, P.A.; Wang, L.H.; Schultz, P.G.; Stevens, R.C. |

    1997-06-13

    The crystal structures of a germline antibody Fab fragment and its complex with hapten have been solved at 2.1 {angstrom} resolution. These structures are compared with the corresponding crystal structures of the affinity-matured antibody, 48G7, which has a 30,000 times higher affinity for hapten as a result of nine replacement somatic mutations. Significant changes in the configuration of the combining site occur upon binding of hapten to the germline antibody, whereas hapten binds to the mature antibody by a lock-and-key fit mechanism. The reorganization of the combining site that was nucleated by hapten binding is further optimized by somatic mutations that occur up to 15 {angstrom} from bound hapten. These results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarily.

  4. Insights into molecular structure and digestion rate of oat starch.

    PubMed

    Xu, Jinchuan; Kuang, Qirong; Wang, Kai; Zhou, Sumei; Wang, Shuo; Liu, Xingxun; Wang, Shujun

    2017-04-01

    The in vitro digestibility of oat starch and its relationship with starch molecular structure was investigated. The in vitro digestion results showed that the first-order kinetic constant (k) of oat starches (OS-1 and OS-2) was lower than that of rice starch. The size of amylose chains, amylose content and degree of branching (DB) of amylopectin in oat starch were significantly higher than the corresponding parameters in rice starch. The larger molecular size of oat starch may account for its lower digestion rate. The fine structure of amylopectin showed that oat starch had less chains of DP 6-12 and DP>36, which may explain the small difference in digestion rate between oat and rice starch. The biosynthesis model from oat amylopectin fine structure data suggested a lower starch branching enzyme (SBE) activity and/or a higher starch synthase (SS) activity, which may decrease the DB of oat starch and increase its digestion rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Structural insights into NusG regulating transcription elongation

    PubMed Central

    Liu, Bin; Steitz, Thomas A.

    2017-01-01

    NusG is an essential transcription factor that plays multiple key regulatory roles in transcription elongation, termination and coupling translation and transcription. The core role of NusG is to enhance transcription elongation and RNA polymerase processivity. Here, we present the structure of Escherichia coli RNA polymerase complexed with NusG. The structure shows that the NusG N-terminal domain (NGN) binds at the central cleft of RNA polymerase surrounded by the β' clamp helices, the β protrusion, and the β lobe domains to close the promoter DNA binding channel and constrain the β' clamp domain, but with an orientation that is different from the one observed in the archaeal β' clamp–Spt4/5 complex. The structure also allows us to construct a reliable model of the complete NusG-associated transcription elongation complex, suggesting that the NGN domain binds at the upstream fork junction of the transcription elongation complex, similar to σ2 in the transcription initiation complex, to stabilize the junction, and therefore enhances transcription processivity. PMID:27899640

  6. Structural Insights Into the Evolutionary Paths of Oxylipin Biosynthetic Enzymes

    SciTech Connect

    Lee, D.-S.; Nioche, P.; Hamberg, M.; Raman, C.S.

    2009-05-20

    The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic {pi}-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.

  7. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    SciTech Connect

    Goldsbury, C.; Wall, J.; Baxa, U.; Simon, M. N.; Steven, A. C.; Engel, A.; Aebi, U.; Muller, S. A.

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

  8. Structure of CD84 Provides Insight into SLAM Family Function

    SciTech Connect

    Yan,Q.; Malashkevich, V.; Fedorov, A.; Fedorov, E.; Cao, E.; Lary, J.; Cole, J.; Nathenson, S.; Almo, S.

    2007-01-01

    The signaling lymphocyte activation molecule (SLAM) family includes homophilic and heterophilic receptors that modulate both adaptive and innate immune responses. These receptors share a common ectodomain organization: a membrane-proximal immunoglobulin constant domain and a membrane-distal immunoglobulin variable domain that is responsible for ligand recognition. CD84 is a homophilic family member that enhances IFN-{gamma} secretion in activated T cells. Our solution studies revealed that CD84 strongly self-associates with a K{sub d} in the submicromolar range. These data, in combination with previous reports, demonstrate that the SLAM family homophilic affinities span at least three orders of magnitude and suggest that differences in the affinities may contribute to the distinct signaling behavior exhibited by the individual family members. The 2.0 {angstrom} crystal structure of the human CD84 immunoglobulin variable domain revealed an orthogonal homophilic dimer with high similarity to the recently reported homophilic dimer of the SLAM family member NTB-A. Structural and chemical differences in the homophilic interfaces provide a mechanism to prevent the formation of undesired heterodimers among the SLAM family homophilic receptors. These structural data also suggest that, like NTB-A, all SLAM family homophilic dimers adopt a highly kinked organization spanning an end-to-end distance of {approx}140 {angstrom}. This common molecular dimension provides an opportunity for all two-domain SLAM family receptors to colocalize within the immunological synapse and bridge the T cell and antigen-presenting cell.

  9. Electronic structure of hydrogenated diamond: Microscopical insight into surface conductivity

    NASA Astrophysics Data System (ADS)

    Iacobucci, S.; Alippi, Paola; Calvani, P.; Girolami, M.; Offi, F.; Petaccia, L.; Trucchi, D. M.

    2016-07-01

    We have correlated the surface conductivity of hydrogen-terminated diamond to the electronic structure in the Fermi region. Significant density of electronic states (DOS) in proximity of the Fermi edge has been measured by photoelectron spectroscopy (PES) on surfaces exposed to air, corresponding to a p -type electric conductive regime, while upon annealing a depletion of the DOS has been achieved, resembling the diamond insulating state. The surface and subsurface electronic structure has been determined, exploiting the different probing depths of PES applied in a photon energy range between 7 and 31 eV. Ab initio density functional calculations including surface charge depletion and band-bending effects favorably compare with electronic states measured by angular-resolved photoelectron spectroscopy. Such states are organized in the energy-momentum space in a twofold structure: one, bulk-derived, band disperses in the Γ -X direction with an average hole effective mass of (0.43 ±0.02 ) m0 , where m0 is the bare electron mass; a second flatter band, with an effective mass of (2.2 ±0.9 ) m0 , proves that a hole gas confined in the topmost layers is responsible for the conductivity of the (2 ×1 ) hydrogen-terminated diamond (100 ) surface.

  10. Molecular and structural insight into plasmodium falciparum RIO2 kinase.

    PubMed

    Chouhan, Devendra K; Sharon, Ashoke; Bal, Chandralata

    2013-02-01

    Among approximately 65 kinases of the malarial genome, RIO2 (right open reading frame) kinase belonging to the atypical class of kinase is unique because along with a kinase domain, it has a highly conserved N-terminal winged helix (wHTH) domain. The wHTH domain resembles the wing like domain found in DNA binding proteins and is situated near to the kinase domain. Ligand binding to this domain may reposition the kinase domain leading to inhibition of enzyme function and could be utilized as a novel allosteric site to design inhibitor. In the present study, we have generated a model of RIO2 kinase from Plasmodium falciparum utilizing multiple modeling, simulation approach. A novel putative DNA-binding site is identified for the first time in PfRIO2 kinase to understand the DNA binding events involving wHTH domain and flexible loop. Induced fit DNA docking followed by minimization, molecular dynamics simulation, energetic scoring and binding mode studies are used to reveal the structural basis of PfRIO2-ATP-DNA complex. Ser105 as a potential site of phosphorylation is revealed through the structural studies of ATP binding in PfRIO2. Overall the present study discloses the structural facets of unknown PfRIO2 complex and opens an avenue toward exploration of novel drug target.

  11. Structural insights into competitive antagonism in NMDA receptors

    PubMed Central

    Jespersen, Annie; Tajima, Nami; Fernandez-Cuervo, Gabriela; Garnier-Amblard, Ethel C.; Furukawa, Hiro

    2014-01-01

    Summary There has been a great level of enthusiasm to down-regulate overactive N-methyl-d-aspartate (NMDA) receptors to protect neurons from excitotoxicity. NMDA receptors play pivotal roles in basic brain development and functions as well as in neurological disorders and diseases. However, mechanistic understanding of antagonism in NMDA receptors is limited due to complete lack of antagonist-bound structures for the l-glutamate-binding GluN2 subunits. Here we report the crystal structures of GluN1/GluN2A NMDA receptor ligand-binding domain (LBD) heterodimers in complex with GluN1- and GluN2-targeting antagonists. The crystal structures reveal that the antagonists, D-(−)-2-Amino-5-phosphonopentanoic acid (d-AP5) and 1-(Phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), have discrete binding modes and mechanisms for opening of the bilobed architecture of GluN2A LBD compared to the agonist-bound form. The current study shows distinct ways by which the conformations of NMDA receptor LBDs may be controlled and coupled to receptor inhibition and provides possible strategies to develop therapeutic compounds with higher subtype-specificity. PMID:24462099

  12. Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes.

    PubMed

    Lee, Dong-Sun; Nioche, Pierre; Hamberg, Mats; Raman, C S

    2008-09-18

    The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic pi-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.

  13. Structural insights into µ-opioid receptor activation.

    PubMed

    Huang, Weijiao; Manglik, Aashish; Venkatakrishnan, A J; Laeremans, Toon; Feinberg, Evan N; Sanborn, Adrian L; Kato, Hideaki E; Livingston, Kathryn E; Thorsen, Thor S; Kling, Ralf C; Granier, Sébastien; Gmeiner, Peter; Husbands, Stephen M; Traynor, John R; Weis, William I; Steyaert, Jan; Dror, Ron O; Kobilka, Brian K

    2015-08-20

    Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

  14. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    PubMed Central

    Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N.; Steven, Alasdair C.; Engel, Andreas; Wall, Joseph S.; Aebi, Ueli; Müller, Shirley A.

    2010-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases like Alzheimer’s disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies like Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). PMID:20868754

  15. Structural insights into μ-opioid receptor activation

    PubMed Central

    Huang, Weijiao; Manglik, Aashish; Venkatakrishnan, A. J.; Laeremans, Toon; Feinberg, Evan N.; Sanborn, Adrian L.; Kato, Hideaki E.; Livingston, Kathryn E.; Thorsen, Thor S.; Kling, Ralf; Granier, Sébastien; Gmeiner, Peter; Husbands, Stephen M.; Traynor, John R.; Weis, William I.; Steyaert, Jan; Dror, Ron O.; Kobilka, Brian K.

    2015-01-01

    Summary Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To understand the structural basis for μOR activation, we obtained a 2.1 Å X-ray crystal structure of the μOR bound to the morphinan agonist BU72 and stabilized by a G protein-mimetic camelid-antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2 adrenergic receptor (β2AR) and the M2 muscarinic receptor (M2R). Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three GPCRs. PMID:26245379

  16. Amyloid structure and assembly: insights from scanning transmission electron microscopy.

    PubMed

    Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N; Steven, Alasdair C; Engel, Andreas; Wall, Joseph S; Aebi, Ueli; Müller, Shirley A

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Structural Insight into KCNQ (Kv7) Channel Assembly and Channelopathy

    PubMed Central

    Howard, Rebecca J.; Clark, Kimberly A.; Holton, James M.; Minor, Daniel L.

    2010-01-01

    Summary Kv7.x (KCNQ) voltage-gated potassium channels form the cardiac and auditory IKs current and the neuronal M-current. The five Kv7 subtypes have distinct assembly preferences encoded by a C-terminal cytoplasmic assembly domain, the A-domain Tail. Here, we present the high-resolution structure of the Kv7.4 A-domain Tail together with biochemical experiments that show that the domain is a self-assembling, parallel, four-stranded coiled coil. Structural analysis and biochemical studies indicate conservation of the coiled coil in all Kv7 subtypes and that a limited set of interactions encode assembly specificity determinants. Kv7 mutations have prominent roles in arrhythmias, deafness, and epilepsy. The structure together with biochemical data indicate that A-domain Tail arrhythmia mutations cluster on the solvent-accessible surface of the subunit interface at a likely site of action for modulatory proteins. Together, the data provide a framework for understanding Kv7 assembly specificity and the molecular basis of a distinct set of Kv7 channelopathies. PMID:17329207

  18. Potential application of immunoassays for simple, rapid and quantitative detections of phytoavailable neonicotinoid insecticides in cropland soils.

    PubMed

    Watanabe, Eiki; Seike, Nobuyasu; Motoki, Yutaka; Inao, Keiya; Otani, Takashi

    2016-10-01

    This study evaluated the applicability of commercially available kit-based enzyme-linked immunosorbent assay (ELISA) to simple, quick, and quantitative detection for three water-extractable (phytoavailable) neonicotinoid insecticides: dinotefuran, clothianidin, and imidacloprid in soils. ELISA showed excellent analytical sensitivity for determination, but with cross-reaction to structurally related neonicotinoid analogues, which might produce false positives. To analyze insecticides in soil samples of diverse physicochemical properties, they were extracted with water. The aqueous soil extracts were assayed directly with ELISA. No matrix interference was observed without additional dilution with water. Recovery experiments for the insecticides from aqueous soil extracts spiked at 2-10 ng/mL showed good accuracy (72-126%) and precision (<16%). Kit-based ELISAs were used to estimate soil-water distribution coefficients (Kd). Values estimated using this method showed positive correlation between organic carbon contents in soil and those for evaluated insecticides. Results indicate that the evaluated kit-based ELISA has applicability for simple, quick, and reliable detection of phytoavailable insecticides in soils and for estimating Kd values in soil. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Evidence for pollinator cost and farming benefits of neonicotinoid seed coatings on oilseed rape

    NASA Astrophysics Data System (ADS)

    Budge, G. E.; Garthwaite, D.; Crowe, A.; Boatman, N. D.; Delaplane, K. S.; Brown, M. A.; Thygesen, H. H.; Pietravalle, S.

    2015-08-01

    Chronic exposure to neonicotinoid insecticides has been linked to reduced survival of pollinating insects at both the individual and colony level, but so far only experimentally. Analyses of large-scale datasets to investigate the real-world links between the use of neonicotinoids and pollinator mortality are lacking. Moreover, the impacts of neonicotinoid seed coatings in reducing subsequent applications of foliar insecticide sprays and increasing crop yield are not known, despite the supposed benefits of this practice driving widespread use. Here, we combine large-scale pesticide usage and yield observations from oilseed rape with those detailing honey bee colony losses over an 11 year period, and reveal a correlation between honey bee colony losses and national-scale imidacloprid (a neonicotinoid) usage patterns across England and Wales. We also provide the first evidence that farmers who use neonicotinoid seed coatings reduce the number of subsequent applications of foliar insecticide sprays and may derive an economic return. Our results inform the societal discussion on the pollinator costs and farming benefits of prophylactic neonicotinoid usage on a mass flowering crop.

  20. Evidence for pollinator cost and farming benefits of neonicotinoid seed coatings on oilseed rape

    PubMed Central

    Budge, G. E.; Garthwaite, D.; Crowe, A.; Boatman, N. D.; Delaplane, K. S.; Brown, M. A.; Thygesen, H. H.; Pietravalle, S.

    2015-01-01

    Chronic exposure to neonicotinoid insecticides has been linked to reduced survival of pollinating insects at both the individual and colony level, but so far only experimentally. Analyses of large-scale datasets to investigate the real-world links between the use of neonicotinoids and pollinator mortality are lacking. Moreover, the impacts of neonicotinoid seed coatings in reducing subsequent applications of foliar insecticide sprays and increasing crop yield are not known, despite the supposed benefits of this practice driving widespread use. Here, we combine large-scale pesticide usage and yield observations from oilseed rape with those detailing honey bee colony losses over an 11 year period, and reveal a correlation between honey bee colony losses and national-scale imidacloprid (a neonicotinoid) usage patterns across England and Wales. We also provide the first evidence that farmers who use neonicotinoid seed coatings reduce the number of subsequent applications of foliar insecticide sprays and may derive an economic return. Our results inform the societal discussion on the pollinator costs and farming benefits of prophylactic neonicotinoid usage on a mass flowering crop. PMID:26270806

  1. Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments.

    PubMed

    Boily, Monique; Sarrasin, Benoit; Deblois, Christian; Aras, Philippe; Chagnon, Madeleine

    2013-08-01

    In Québec, as observed globally, abnormally high honey bee mortality rates have been reported recently. Several potential contributing factors have been identified, and exposure to pesticides is of increasing concern. In maize fields, foraging bees are exposed to residual concentrations of insecticides such as neonicotinoids used for seed coating. Highly toxic to bees, neonicotinoids are also reported to increase AChE activity in other invertebrates exposed to sub-lethal doses. The purpose of this study was therefore to test if the honey bee's AChE activity could be altered by neonicotinoid compounds and to explore possible effects of other common products used in maize fields: atrazine and glyphosate. One week prior to pollen shedding, beehives were placed near three different field types: certified organically grown maize, conventionally grown maize or non-cultivated. At the same time, caged bees were exposed to increasing sub-lethal doses of neonicotinoid insecticides (imidacloprid and clothianidin) and herbicides (atrazine and glyphosate) under controlled conditions. While increased AChE activity was found in all fields after 2 weeks of exposure, bees close to conventional maize crops showed values higher than those in both organic maize fields and non-cultivated areas. In caged bees, AChE activity increased in response to neonicotinoids, and a slight decrease was observed by glyphosate. These results are discussed with regard to AChE activity as a potential biomarker of exposure for neonicotinoids.

  2. Neonicotinoid insecticides differently modulate acetycholine-induced currents on mammalian α7 nicotinic acetylcholine receptor.

    PubMed

    Cartereau, Alison; Martin, Carine; Thany, Steeve H

    2017-08-29

    Neonicotinoid insecticides are described as poor agonists of mammalian nicotinic acetylcholine receptors. In this paper, we provide evidence that they diffenrently act on mammalian nicotinic receptors. Two-electrode voltage-clamp electrophysiology was used to characterized the pharmacology of neonicotinoid insecticides on α7 receptors expressed in Xenopus oocytes. Single and combined application of clothianidin, acetamiprid and thiamethoxam were tested. The neonicotinoid insecticides, clothianidin and acetamiprid were partial agonists of mammalian neuronal α7 nicotinic receptors and thiamethoxam, a neonicotinoid insecticide, which is converted to clothianidin in insect and plant tissues had no effect. Pretreatment of 10 μM clothianidin and acetamiprid with 100 μM acetylcholine, significantly enhanced the subsequent acetylcholine-evoked currents whereas, 10 μM thiamethoxam reduced acetylcholine-induced current amplitudes. Moreover, the combinations of the three neonicotinoids decreased the ACh evoked currents. The present findings suggest that neonicotinoid insecticides differently affect α7 nicotinic acetylcholine receptors and can modulate acetylcholine-induced current. In final, the data indicate a previous unknown modulation of mammalian α7 receptors by combined application of clothianidin, acetamiprid and thiamethoxam. This article is protected by copyright. All rights reserved.

  3. Exposure to neonicotinoids influences the motor function of adult worker honeybees.

    PubMed

    Williamson, Sally M; Willis, Sarah J; Wright, Geraldine A

    2014-10-01

    Systemic pesticides such as neonicotinoids are commonly used on flowering crops visited by pollinators, and their use has been implicated in the decline of insect pollinator populations in Europe and North America. Several studies show that neonicotinoids affect navigation and learning in bees but few studies have examined whether these substances influence their basic motor function. Here, we investigated how prolonged exposure to sublethal doses of four neonicotinoid pesticides (imidacloprid, thiamethoxam, clothianidin, dinotefuran) and the plant toxin, nicotine, affect basic motor function and postural control in foraging-age worker honeybees. We used doses of 10 nM for each neonicotinoid: field-relevant doses that we determined to be sublethal and willingly consumed by bees. The neonicotinoids were placed in food solutions given to bees for 24 h. After the exposure period, bees were more likely to lose postural control during the motor function assay and fail to right themselves if exposed to imidacloprid, thiamethoxam, clothianidin. Bees exposed to thiamethoxam and nicotine also spent more time grooming. Other behaviours (walking, sitting and flying) were not significantly affected. Expression of changes in motor function after exposure to imidacloprid was dose-dependent and affected all measured behaviours. Our data illustrate that 24 h exposure to sublethal doses of neonicotinoid pesticides has a subtle influence on bee behaviour that is likely to affect normal function in a field setting.

  4. Evidence for pollinator cost and farming benefits of neonicotinoid seed coatings on oilseed rape.

    PubMed

    Budge, G E; Garthwaite, D; Crowe, A; Boatman, N D; Delaplane, K S; Brown, M A; Thygesen, H H; Pietravalle, S

    2015-08-13

    Chronic exposure to neonicotinoid insecticides has been linked to reduced survival of pollinating insects at both the individual and colony level, but so far only experimentally. Analyses of large-scale datasets to investigate the real-world links between the use of neonicotinoids and pollinator mortality are lacking. Moreover, the impacts of neonicotinoid seed coatings in reducing subsequent applications of foliar insecticide sprays and increasing crop yield are not known, despite the supposed benefits of this practice driving widespread use. Here, we combine large-scale pesticide usage and yield observations from oilseed rape with those detailing honey bee colony losses over an 11 year period, and reveal a correlation between honey bee colony losses and national-scale imidacloprid (a neonicotinoid) usage patterns across England and Wales. We also provide the first evidence that farmers who use neonicotinoid seed coatings reduce the number of subsequent applications of foliar insecticide sprays and may derive an economic return. Our results inform the societal discussion on the pollinator costs and farming benefits of prophylactic neonicotinoid usage on a mass flowering crop.

  5. Factors influencing the occurrence and distribution of neonicotinoid insecticides in surface waters of southern Ontario, Canada.

    PubMed

    Struger, John; Grabuski, Josey; Cagampan, Steve; Sverko, Ed; McGoldrick, Daryl; Marvin, Christopher H

    2017-02-01

    The widespread use of neonicotinoid insecticides and recent increased regulatory scrutiny requires the generation of monitoring data with sufficient scope and resolution to provide decision makers with a better understanding of occurrence and distribution in the environment. This study presents a wide-scale investigation of neonicotinoid insecticides used across the range of agricultural activities from fifteen surface water sites in southern Ontario. Using statistical analysis, the correlation of individual compounds with land use was investigated, and the relationship between neonicotinoid occurrence and hydrologic parameters in calibrated water courses was also assessed. Of the five neonicotinoids studied, imidacloprid, clothianidin and thiamethoxam exhibited detection rates above 90% at over half the sites sampled over a three year period (2012-2014). At two sites in southwestern Ontario, the Canadian Federal freshwater guideline value for imidacloprid (230 ng/L) was exceeded in roughly 75% of the samples collected. For some watersheds, there were correlations between the occurrence of neonicotinoids and precipitation and/or stream discharge. Some watersheds exhibited seasonal maxima in concentrations of neonicotinoids in spring and fall, particularly for those areas where row crop agriculture is predominant; these seasonal patterns were absent in some areas characterized by a broad range of agricultural activities.

  6. Structural insight into the Clostridium difficile ethanolamine utilisation microcompartment.

    PubMed

    Pitts, Alison C; Tuck, Laura R; Faulds-Pain, Alexandra; Lewis, Richard J; Marles-Wright, Jon

    2012-01-01

    Bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. The genome of the virulent, multidrug-resistant Clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. The C. difficile eut operon displays regulatory genetic elements and protein encoding regions in common with homologous loci found in the genomes of other bacteria, including the enteric pathogens Salmonella enterica and Enterococcus faecalis. The crystal structures of two microcompartment shell proteins, CD1908 and CD1918, and an uncharacterised protein with potential enzymatic activity, CD1925, were determined by X-ray crystallography. CD1908 and CD1918 display the same protein fold, though the order of secondary structure elements is permuted in CD1908 and this protein displays an N-terminal β-strand extension. These proteins form hexamers with molecules related by crystallographic and non-crystallographic symmetry. The structure of CD1925 has a cupin β-barrel fold and a putative active site that is distinct from the metal-ion dependent catalytic cupins. Thin-section transmission electron microscopy of Escherichia coli over-expressing eut proteins indicates that CD1918 is capable of self-association into arrays, suggesting an organisational role for CD1918 in the formation of this microcompartment. The work presented provides the basis for further study of the architecture and function of the C. difficile eut microcompartment, its role in metabolism and the wider consequences of intestinal colonisation and virulence in this pathogen.

  7. Structural insights into DNA replication without hydrogen bonds.

    PubMed

    Betz, Karin; Malyshev, Denis A; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Romesberg, Floyd E; Marx, Andreas

    2013-12-11

    The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.

  8. Structural insight into Ca2+ specificity in tetrameric cation channels

    PubMed Central

    Alam, Amer; Shi, Ning; Jiang, Youxing

    2007-01-01

    Apparent blockage of monovalent cation currents by the permeating blocker Ca2+ is a physiologically essential phenomenon relevant to cyclic nucleotide-gated (CNG) channels. The recently determined crystal structure of a bacterial homolog of CNG channel pores, the NaK channel, revealed a Ca2+ binding site at the extracellular entrance to the selectivity filter. This site is not formed by the side-chain carboxylate groups from the conserved acidic residue, Asp-66 in NaK, conventionally thought to directly chelate Ca2+ in CNG channels, but rather by the backbone carbonyl groups of residue Gly-67. Here we present a detailed structural analysis of the NaK channel with a focus on Ca2+ permeability and blockage. Our results confirm that the Asp-66 residue, although not involved in direct chelation of Ca2+, plays an essential role in external Ca2+ binding. Furthermore, we give evidence for the presence of a second Ca2+ binding site within the NaK selectivity filter where monovalent cations also bind, providing a structural basis for Ca2+ permeation through the NaK pore. Compared with other Ca2+-binding proteins, both sites in NaK present a novel mode of Ca2+ chelation, using only backbone carbonyl oxygen atoms from residues in the selectivity filter. The external site is under indirect control by an acidic residue (Asp-66), making it Ca2+-specific. These findings give us a glimpse of the possible underlying mechanisms allowing Ca2+ to act both as a permeating ion and blocker of CNG channels and raise the possibility of a similar chemistry governing Ca2+ chelation in Ca2+ channels. PMID:17878296

  9. Structural Insights into the Mechanism of Protein O-Fucosylation

    PubMed Central

    Lira-Navarrete, Erandi; Valero-González, Jessika; Villanueva, Raquel; Martínez-Júlvez, Marta; Tejero, Tomás; Merino, Pedro; Panjikar, Santosh; Hurtado-Guerrero, Ramon

    2011-01-01

    Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future. PMID:21966509

  10. Structural Insights into DNA Replication Without Hydrogen-Bonds

    PubMed Central

    Betz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Romesberg, Floyd E.; Marx, Andreas

    2014-01-01

    The genetic alphabet is comprised of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the pre-chemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple post-chemistry complexes in which the already formed unnatural base pair is positioned at the post-insertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the post-insertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by de-intercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation. PMID:24283923

  11. Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

    PubMed

    Moonens, Kristof; Gideonsson, Pär; Subedi, Suresh; Bugaytsova, Jeanna; Romaõ, Ema; Mendez, Melissa; Nordén, Jenny; Fallah, Mahsa; Rakhimova, Lena; Shevtsova, Anna; Lahmann, Martina; Castaldo, Gaetano; Brännström, Kristoffer; Coppens, Fanny; Lo, Alvin W; Ny, Tor; Solnick, Jay V; Vandenbussche, Guy; Oscarson, Stefan; Hammarström, Lennart; Arnqvist, Anna; Berg, Douglas E; Muyldermans, Serge; Borén, Thomas; Remaut, Han

    2016-01-13

    The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

  12. Structural insights into the bacterial carbon-phosphorus lyase machinery

    PubMed Central

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J.; Passmore, Lori A.; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E.

    2015-01-01

    Summary Phosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown. PMID:26280334

  13. Structural insights into the bacterial carbon-phosphorus lyase machinery.

    PubMed

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J; Passmore, Lori A; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E

    2015-09-03

    Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

  14. Structural and mechanistic insights into Mps1 kinase activation

    SciTech Connect

    Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

    2010-11-05

    Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

  15. Structural insights into transcription initiation by yeast RNA polymerase I.

    PubMed

    Sadian, Yashar; Tafur, Lucas; Kosinski, Jan; Jakobi, Arjen J; Wetzel, Rene; Buczak, Katarzyna; Hagen, Wim Jh; Beck, Martin; Sachse, Carsten; Müller, Christoph W

    2017-09-15

    In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  16. The Ccr4-Not Complex: Architecture and Structural Insights.

    PubMed

    Collart, Martine A; Panasenko, Olesya O

    2017-01-01

    The Ccr4-Not complex is an essential multi-subunit protein complex that plays a fundamental role in eukaryotic mRNA metabolism and has a multitude of different roles that impact eukaryotic gene expression . It has a conserved core of three Not proteins, the Ccr4 protein, and two Ccr4 associated factors, Caf1 and Caf40. A fourth Not protein, Not4, is conserved, but is only a stable subunit of the complex in yeast. Certain subunits have been duplicated during evolution, with functional divergence, such as Not3 in yeast, and Ccr4 or Caf1 in human. However the complex includes only one homolog for each protein. In addition, species-specific subunits are part of the complex, such as Caf130 in yeast or Not10 and Not11 in human. Two conserved catalytic functions are associated with the complex, deadenylation and ubiquitination . The complex adopts an L-shaped structure, in which different modules are bound to a large Not1 scaffold protein. In this chapter we will summarize our current knowledge of the architecture of the complex and of the structure of its constituents.

  17. Structural Insight into Polymorphic ABO Glycan Binding by Helicobacter pylori

    PubMed Central

    Moonens, Kristof; Gideonsson, Pär; Subedi, Suresh; Bugaytsova, Jeanna; Romaõ, Ema; Mendez, Melissa; Nordén, Jenny; Fallah, Mahsa; Rakhimova, Lena; Shevtsova, Anna; Lahmann, Martina; Castaldo, Gaetano; Brännström, Kristoffer; Coppens, Fanny; Lo, Alvin W.; Ny, Tor; Solnick, Jay V.; Vandenbussche, Guy; Oscarson, Stefan; Hammarström, Lennart; Arnqvist, Anna; Berg, Douglas E.; Muyldermans, Serge; Borén, Thomas; Remaut, Han

    2016-01-01

    Summary The Helicobacter pylori adhesin BabA binds mucosal ABO/Leb blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Leb binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Leb-expressing mice, providing perspectives on possible H. pylori eradication therapies. PMID:26764597

  18. Structural insights into Ail-mediated adhesion in Yersinia pestis.

    PubMed

    Yamashita, Satoshi; Lukacik, Petra; Barnard, Travis J; Noinaj, Nicholas; Felek, Suleyman; Tsang, Tiffany M; Krukonis, Eric S; Hinnebusch, B Joseph; Buchanan, Susan K

    2011-11-09

    Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Artemin Crystal Structure Reveals Insights into Heparan Sulfate Binding

    SciTech Connect

    Silvian,L.; Jin, P.; Carmillo, P.; Boriack-Sjodin, P.; Pelletier, C.; Rushe, M.; Gong, B.; Sah, D.; Pepinsky, B.; Rossomando, A.

    2006-01-01

    Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFR{alpha}3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.

  20. Structural insights into enzymatic degradation of oxidized polyvinyl alcohol.

    PubMed

    Yang, Yu; Ko, Tzu-Ping; Liu, Long; Li, Jianghua; Huang, Chun-Hsiang; Chan, Hsiu-Chien; Ren, Feifei; Jia, Dongxu; Wang, Andrew H-J; Guo, Rey-Ting; Chen, Jian; Du, Guocheng

    2014-09-05

    The ever-increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel α/β-hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA-degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid-like β-ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the CC bond of β-diketone, although it has a catalytic triad similar to that of most α/β-hydrolases. Analysis of the crystal structures suggests a double-oxyanion-hole mechanism, previously only found in thiolase cleaving β-ketoacyl-CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Structural insights into the catalytic mechanism of human squalene synthase.

    PubMed

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Shih, Min-Fang; Ko, Tzu-Ping; Wang, Andrew H-J

    2014-02-01

    Squalene synthase (SQS) is a divalent metal-ion-dependent enzyme that catalyzes the two-step reductive `head-to-head' condensation of two molecules of farnesyl pyrophosphate to form squalene using presqualene diphosphate (PSPP) as an intermediate. In this paper, the structures of human SQS and its mutants in complex with several substrate analogues and intermediates coordinated with Mg2+ or Mn2+ are presented, which stepwise delineate the biosynthetic pathway. Extensive study of the SQS active site has identified several critical residues that are involved in binding reduced nicotinamide dinucleotide phosphate (NADPH). Based on mutagenesis data and a locally closed (JK loop-in) structure observed in the hSQS-(F288L)-PSPP complex, an NADPH-binding model is proposed for SQS. The results identified four major steps (substrate binding, condensation, intermediate formation and translocation) of the ordered sequential mechanisms involved in the `1'-1' isoprenoid biosynthetic pathway. These new findings clarify previous hypotheses based on site-directed mutagenesis and biochemical analysis.

  2. Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

    PubMed Central

    Roret, Thomas; Tsan, Pascale; Couturier, Jérémy; Zhang, Bo; Johnson, Michael K.; Rouhier, Nicolas; Didierjean, Claude

    2014-01-01

    BolA proteins are defined as stress-responsive transcriptional regulators, but they also participate in iron metabolism. Although they can form [2Fe-2S]-containing complexes with monothiol glutaredoxins (Grx), structural details are lacking. Three Arabidopsis thaliana BolA structures were solved. They differ primarily by the size of a loop referred to as the variable [H/C] loop, which contains an important cysteine (BolA_C group) or histidine (BolA_H group) residue. From three-dimensional modeling and spectroscopic analyses of A. thaliana GrxS14-BolA1 holo-heterodimer (BolA_H), we provide evidence for the coordination of a Rieske-type [2Fe-2S] cluster. For BolA_C members, the cysteine could replace the histidine as a ligand. NMR interaction experiments using apoproteins indicate that a completely different heterodimer was formed involving the nucleic acid binding site of BolA and the C-terminal tail of Grx. The possible biological importance of these complexes is discussed considering the physiological functions previously assigned to BolA and to Grx-BolA or Grx-Grx complexes. PMID:25012657

  3. Structural and spectroscopic insights into BolA-glutaredoxin complexes.

    PubMed

    Roret, Thomas; Tsan, Pascale; Couturier, Jérémy; Zhang, Bo; Johnson, Michael K; Rouhier, Nicolas; Didierjean, Claude

    2014-08-29

    BolA proteins are defined as stress-responsive transcriptional regulators, but they also participate in iron metabolism. Although they can form [2Fe-2S]-containing complexes with monothiol glutaredoxins (Grx), structural details are lacking. Three Arabidopsis thaliana BolA structures were solved. They differ primarily by the size of a loop referred to as the variable [H/C] loop, which contains an important cysteine (BolA_C group) or histidine (BolA_H group) residue. From three-dimensional modeling and spectroscopic analyses of A. thaliana GrxS14-BolA1 holo-heterodimer (BolA_H), we provide evidence for the coordination of a Rieske-type [2Fe-2S] cluster. For BolA_C members, the cysteine could replace the histidine as a ligand. NMR interaction experiments using apoproteins indicate that a completely different heterodimer was formed involving the nucleic acid binding site of BolA and the C-terminal tail of Grx. The possible biological importance of these complexes is discussed considering the physiological functions previously assigned to BolA and to Grx-BolA or Grx-Grx complexes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Structural Insights into Substrate Recognition by Clostridium difficile Sortase

    PubMed Central

    Yin, Jui-Chieh; Fei, Chun-Hsien; Lo, Yen-Chen; Hsiao, Yu-Yuan; Chang, Jyun-Cyuan; Nix, Jay C.; Chang, Yuan-Yu; Yang, Lee-Wei; Huang, I-Hsiu; Wang, Shuying

    2016-01-01

    Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26–PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB. PMID:27921010

  5. Structural insights into RNA recognition by RIG-I

    PubMed Central

    Luo, Dahai; Ding, Steve C.; Vela, Adriana; Kohlway, Andrew; Lindenbach, Brett D.; Pyle, Anna Marie

    2011-01-01

    Summary Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP-2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA. The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of super family 2 of helicases and this structure reveals complex interplay between motor domains, accessory mechanical domains and RNA that has implications for understanding the nanomechanical function this protein family and other ATPases more broadly. PMID:22000018

  6. FRESH INSIGHTS ON THE STRUCTURE OF THE SOLAR CORE

    SciTech Connect

    Basu, Sarbani; Chaplin, William J.; Elsworth, Yvonne; New, Roger; Serenelli, Aldo M. E-mail: w.j.chaplin@bham.ac.uk E-mail: r.new@shu.ac.uk

    2009-07-10

    We present new results on the structure of the solar core, obtained with new sets of frequencies of solar low-degree p modes obtained from the BiSON network. We find that different methods used in extracting the different sets of frequencies cause shifts in frequencies, but the shifts are not large enough to affect solar structure results. We find that the BiSON frequencies show that the solar sound speed in the core is slightly larger than that inferred from data from Michelson Doppler Imager low-degree modes, and the uncertainties on the inversion results are smaller. Density results also change by a larger amount, and we find that solar models now tend to show smaller differences in density compared to the Sun. The result is seen at all radii, a result of the fact that conservation of mass implies that density differences in one region have to cancel out density differences in others, since our models are constructed to have the same mass as the Sun. The uncertainties on the density results are much smaller too. We attribute the change in results to having more, and lower frequency, low-degree mode frequencies available. These modes provide greater sensitivity to conditions in the core.

  7. Structural and functional insights into nuclear receptor signaling

    PubMed Central

    Jin, Lihua; Li, Yong

    2010-01-01

    Nuclear receptors are important transcriptional factors that share high sequence identity and conserved domains, including a DNA-binding domain (DBD) and a ligand-binding domain (LBD). The LBD plays a crucial role in ligand-mediated nuclear receptor activity. Hundreds of different crystal structures of nuclear receptors have revealed a general mechanism for the molecular basis of ligand binding and ligand-mediated regulation of nuclear receptors. Despite the conserved fold of nuclear receptor LBDs, the ligand-binding pocket is the least conserved region among different nuclear receptor LBDs. Structural comparison and analysis show that several features of the pocket, like the size and also the shape, have contributed to the ligand binding affinity and specificity. In addition, the plastic nature of the ligand-binding pockets in many nuclear receptors provides greater flexibility to further accommodate specific ligands with a variety of conformations. Nuclear receptor coactivators usually contain multiple LXXLL motifs that are used to interact with nuclear receptors. The nuclear receptors respond differently to distinct ligands and readily exchange their ligands in different environments. The conformational flexibility of the AF-2 helix allows the nuclear receptor to sense the presence of the bound ligands, either an agonist or an antagonist, and to recruit the coactivators or corepressors that ultimately determine the transcriptional activation or repression of nuclear receptors. PMID:20723571

  8. Structural insights into the evolution of the adaptive immune system.

    PubMed

    Deng, Lu; Luo, Ming; Velikovsky, Alejandro; Mariuzza, Roy A

    2013-01-01

    The adaptive immune system, which is based on highly diverse antigen receptors that are generated by somatic recombination, arose approximately 500 Mya at the dawn of vertebrate evolution. In jawed vertebrates, adaptive immunity is mediated by antibodies and T cell receptors (TCRs), which are composed of immunoglobulin (Ig) domains containing hypervariable loops that bind antigen. In striking contrast, the adaptive immune receptors of jawless vertebrates, termed variable lymphocyte receptors (VLRs), are constructed from leucine-rich repeat (LRR) modules. Structural studies of VLRs have shown that these LRR-based receptors bind antigens though their concave surface, in addition to a unique hypervariable loop in the C-terminal LRR capping module. These studies have revealed a remarkable example of convergent evolution in which jawless vertebrates adopted the LRR scaffold to recognize as broad a spectrum of antigens as the Ig-based antibodies and TCRs of jawed vertebrates, with altogether comparable affinity and specificity.

  9. Structural insight into the sequence dependence of nucleosome positioning.

    PubMed

    Wu, Bin; Mohideen, Kareem; Vasudevan, Dileep; Davey, Curt A

    2010-03-14

    Nucleosome positioning displays sequence dependency and contributes to genomic regulation in a site-specific manner. We solved the structures of nucleosome core particle composed of strong positioning TTTAA elements flanking the nucleosome center. The positioning strength of the super flexible TA dinucleotide is consistent with its observed central location within minor groove inward regions, where it can contribute maximally to energetically challenging minor groove bending, kinking and compression. The marked preference for TTTAA and positioning power of the site 1.5 double helix turns from the nucleosome center relates to a unique histone protein motif at this location, which enforces a sustained, extremely narrow minor groove via a hydrophobic "sugar clamp." Our analysis sheds light on the basis of nucleosome positioning and indicates that the histone octamer has evolved not to fully minimize sequence discrimination in DNA binding.

  10. Au-Based Catalysts: Electrochemical Characterization for Structural Insights.

    PubMed

    Pifferi, Valentina; Chan-Thaw, Carine E; Campisi, Sebastiano; Testolin, Anna; Villa, Alberto; Falciola, Luigi; Prati, Laura

    2016-02-25

    Au-based catalysts are widely used in important processes because of their peculiar characteristics. The catalyst performance depends strongly on the nature and structure of the metal nanoparticles, especially in the case of bimetallic catalysts where synergistic effects between the two metals can be occasionally seen. In this paper, it is shown that electrochemical characterisation (cyclovoltammetry CV and electrochemical impedance spectroscopy EIS) of AuPd systems can be used to determine the presence of an electronic interaction between the two metals, thus providing a strong support in the determination of the nature of the synergy between Au and Pd in the liquid phase oxidation of alcohols. However, it seems likely that the strong difference in the catalytic behavior between the single metals and the bimetallic system is connected not only to the redox behaviour, but also to the energetic balance between the different elementary steps of the reaction.

  11. Structural Insights into Rab27 Recruitment by its Effectors

    NASA Astrophysics Data System (ADS)

    M. G. Chavas, Leonard; Ihara, Kentaro; Kawasaki, Masato; Wakatsuki, Soichi

    An increasing number of Rab GTPases associated with partial dysfunction has been linked to several human diseases characterized by a diminution in vesicle transport. Due to its direct implication in human disorders, the Rab27 subfamily is considered as a standard for vesicle docking studies. By which mechanism Rab27 effectors distinguish among the pool of Rab GTPases? What is the underneath machinery rendering the interaction of eleven distinct effectors specific of Rab27 when compared to other Rabs of the secretory pathway? By solving the X-ray structures of Rab27, both in its inactive form and active form bound to the effector protein Slp2-a, attempts have been given to unravel the molecular basis of regulation of the delivering process of vesicles to fusion by the Rab27 subfamily.

  12. Microscopic insight into the structure of gallium isotopes

    NASA Astrophysics Data System (ADS)

    Verma, Preeti; Sharma, Chetan; Singh, Suram; Bharti, Arun; Khosa, S. K.

    2012-07-01

    Projected Shell Model technique has been applied to odd-A71-81Ga nuclei with the deformed single-particle states generated by the standard Nilsson potential. Various nuclear structure quantities have been calculated with this technique and compared with the available experimental data in the present work. The known experimental data of the yrast bands in these nuclei are persuasively described and the band diagrams obtained for these nuclei show that the yrast bands in these odd-A Ga isotopes don't belong to the single intrinsic state only but also have multi-particle states. The back-bending in moment of inertia and the electric quadrupole transitions are also calculated.

  13. Structural insights into the regulation of aromatic amino acid hydroxylation.

    PubMed

    Fitzpatrick, Paul F

    2015-12-01

    The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase are homotetramers, with each subunit containing a homologous catalytic domain and a divergent regulatory domain. The solution structure of the regulatory domain of tyrosine hydroxylase establishes that it contains a core ACT domain similar to that in phenylalanine hydroxylase. The isolated regulatory domain of tyrosine hydroxylase forms a stable dimer, while that of phenylalanine hydroxylase undergoes a monomer-dimer equilibrium, with phenylalanine stabilizing the dimer. These solution properties are consistent with the regulatory mechanisms of the two enzymes, in that phenylalanine hydroxylase is activated by phenylalanine binding to an allosteric site, while tyrosine hydroxylase is regulated by binding of catecholamines in the active site.

  14. Dynamic insight into protein structure utilizing red edge excitation shift.

    PubMed

    Chattopadhyay, Amitabha; Haldar, Sourav

    2014-01-21

    Proteins are considered the workhorses in the cellular machinery. They are often organized in a highly ordered conformation in the crowded cellular environment. These conformations display characteristic dynamics over a range of time scales. An emerging consensus is that protein function is critically dependent on its dynamics. The subtle interplay between structure and dynamics is a hallmark of protein organization and is essential for its function. Depending on the environmental context, proteins can adopt a range of conformations such as native, molten globule, unfolded (denatured), and misfolded states. Although protein crystallography is a well established technique, it is not always possible to characterize various protein conformations by X-ray crystallography due to transient nature of these states. Even in cases where structural characterization is possible, the information obtained lacks dynamic component, which is needed to understand protein function. In this overall scenario, approaches that reveal information on protein dynamics are much appreciated. Dynamics of confined water has interesting implications in protein folding. Interfacial hydration combines the motion of water molecules with the slow moving protein molecules. The red edge excitation shift (REES) approach becomes relevant in this context. REES is defined as the shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption spectrum. REES arises due to slow rates (relative to fluorescence lifetime) of solvent relaxation (reorientation) around an excited state fluorophore in organized assemblies such as proteins. Consequently, REES depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. In the case of a protein, the confined water in the protein creates a dipolar field that acts as the solvent for a fluorophore

  15. Ribonuclease revisited: structural insights into ribonuclease III family enzymes.

    PubMed

    MacRae, Ian J; Doudna, Jennifer A

    2007-02-01

    Ribonuclease III (RNase III) enzymes occur ubiquitously in biology and are responsible for processing RNA precursors into functional RNAs that participate in protein synthesis, RNA interference and a range of other cellular activities. Members of the RNase III enzyme family, including Escherichia coli RNase III, Rnt1, Dicer and Drosha, share the ability to recognize and cleave double-stranded RNA (dsRNA), typically at specific positions or sequences. Recent biochemical and structural data have shed new light on how RNase III enzymes catalyze dsRNA hydrolysis and how substrate specificity is achieved. A major theme emerging from these studies is that accessory domains present in different RNase III enzymes are the key determinants of substrate selectivity, which in turn dictates the specialized biological function of each type of RNase III protein.

  16. The water-benzene interaction: insight from electronic structure theories.

    PubMed

    Ma, Jie; Alfè, Dario; Michaelides, Angelos; Wang, Enge

    2009-04-21

    Weak noncovalent interactions such as van der Waals and hydrogen bonding are ubiquitous in nature, yet their accurate description with electronic structure theories is challenging. Here we assess the ability of a variety of theories to describe a water-benzene binding energy curve. Specifically, we test Hartree-Fock, second-order Møller-Plesset perturbation theory, coupled cluster, density functional theory with several exchange-correlation functionals with and without empirical vdW corrections, and quantum Monte Carlo (QMC). Given the relative paucity of QMC reports for noncovalent interactions, it is interesting to see that QMC and coupled cluster with single, double, and perturbative triple excitations [CCSD(T)] are in very good agreement for most of the binding energy curve, although at short distances there are small deviations on the order of 20 meV.

  17. Structural insight into histone recognition by the ING PHD fingers.

    PubMed

    Champagne, Karen S; Kutateladze, Tatiana G

    2009-05-01

    The Inhibitor of Growth (ING) tumor suppressors are implicated in oncogenesis, control of DNA damage repair, cellular senescence and apoptosis. All members of the ING family contain unique amino-terminal regions and a carboxy-terminal plant homeodomain (PHD) finger. While the amino-terminal domains associate with a number of protein effectors including distinct components of histone deacetylase (HDAC) and histone acetyltransferase (HAT) complexes, the PHD finger binds strongly and specifically to histone H3 trimethylated at lysine 4 (H3K4me3). In this review we describe the molecular mechanism of H3K4me3 recognition by the ING1-5 PHD fingers, analyze the determinants of the histone specificity and compare the biological activities and structures within subsets of PHD fingers. The atomic-resolution structures of the ING PHD fingers in complex with a H3K4me3 peptide reveal that the histone tail is bound in a large and deep binding site encompassing nearly one-third of the protein surface. An extensive network of intermolecular hydrogen bonds, hydrophobic and cation-pi contacts, and complementary surface interactions coordinate the first six residues of the H3K4me3 peptide. The trimethylated Lys4 occupies an elongated groove, formed by the highly conserved aromatic and hydrophobic residues of the PHD finger, whereas the adjacent groove accommodates Arg2. The two grooves are connected by a narrow channel, the small size of which defines the PHD finger's specificity, excluding interactions with other modified histone peptides. Binding of the ING PHD fingers to H3K4me3 plays a critical role in regulating chromatin acetylation. The ING proteins function as tethering molecules that physically link the HDAC and HAT enzymatic complexes to chromatin. In this review we also highlight progress recently made in understanding the molecular basis underlying biological and tumorigenic activities of the ING tumor suppressors.

  18. Toxicity of a neonicotinoid insecticide, guadipyr, in earthworm (Eisenia fetida).

    PubMed

    Wang, Kai; Mu, Xiyan; Qi, Suzhen; Chai, Tingting; Pang, Sen; Yang, Yang; Wang, Chengju; Jiang, Jiazhen

    2015-04-01

    Neonicotinoid insecticides are new class of pesticides and it is very meaningful to evaluate the toxicity of guadipyr to earthworm (Eisenia fetida). In the present study, effects of guadipyr on reproduction, growth, catalase(CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE) and DNA damage in earthworm were assessed using an artificial soil medium. Guadipyr showed low toxicity to earthworms and did not elicit an effect on earthworm reproduction or growth in artificial soils at concentrations <100mg/kg. However, after exposure to guadipyr, the activity of SOD and CAT in earthworm increased and then decreased to control level. AChE activity decreased at day 3 at 50 and 100mg/kg and then increased to control level. Our data indicate that guadipyr did not induce DNA damage in earthworms at concentration of <100mg/kg.

  19. Oxetanes in drug discovery: structural and synthetic insights.

    PubMed

    Wuitschik, Georg; Carreira, Erick M; Wagner, Björn; Fischer, Holger; Parrilla, Isabelle; Schuler, Franz; Rogers-Evans, Mark; Müller, Klaus

    2010-04-22

    An oxetane can trigger profound changes in aqueous solubility, lipophilicity, metabolic stability, and conformational preference when replacing commonly employed functionalities such as gem-dimethyl or carbonyl groups. The magnitude of these changes depends on the structural context. Thus, by substitution of a gem-dimethyl group with an oxetane, aqueous solubility may increase by a factor of 4 to more than 4000 while reducing the rate of metabolic degradation in most cases. The incorporation of an oxetane into an aliphatic chain can cause conformational changes favoring synclinal rather than antiplanar arrangements of the chain. Additionally spirocyclic oxetanes (e.g., 2-oxa-6-aza-spiro[3.3]heptane) bear remarkable analogies to commonly used fragments in drug discovery, such as morpholine, and are even able to supplant the latter in its solubilizing ability. A rich chemistry of oxetan-3-one and derived Michael acceptors provide venues for the preparation of a broad variety of novel oxetanes not previously documented, thus providing the foundation for their broad use in chemistry and drug discovery.

  20. Thermal structure of the ITM: old challenges and new insights

    NASA Astrophysics Data System (ADS)

    Waldrop, L.; Qin, J.

    2016-12-01

    Upper atmospheric temperature is well known to be a fundamental state parameter of the ITM system, driven by solar and geomagnetic energy inputs and itself a driver of numerous chemical reactions as well as the hydrostatic equilibrium distribution of thermospheric constituents. Although it is widely recognized that the ITM region spans a transition from efficient collisional thermalization of neutral species at low altitudes to a nearly collisionless, non-Maxwellian regime, empirical quantification of the thermal structure across this transition is notoriously sparse. Doppler broadened airglow emission lines have long been used to derive the mean kinetic energy of the emitters integrated along the viewing column, and these data are the foundation of contemporary climatologies of thermospheric temperature. Recently, comprehensive observations of thermospheric and exospheric density distributions, derived from TIMED/GUVI measurements of UV airglow emissions along the limb, have revealed unexpectedly large atomic hydrogen scale heights during low solar activity. We will discuss the implications of this finding in the context of historical temperature estimation, challenge the long held assumption that departures from Maxwellian distributions in the thermosphere are small, and motivate a careful re-examination of the concept of neutral temperature in the presence of incomplete thermalization and non-thermal energization processes.

  1. Insights into G protein structure, function, and regulation.

    PubMed

    Cabrera-Vera, Theresa M; Vanhauwe, Jurgen; Thomas, Tarita O; Medkova, Martina; Preininger, Anita; Mazzoni, Maria R; Hamm, Heidi E

    2003-12-01

    In multicellular organisms from Caenorhabditis elegans to Homo sapiens, the maintenance of homeostasis is dependent on the continual flow and processing of information through a complex network of cells. Moreover, in order for the organism to respond to an ever-changing environment, intercellular signals must be transduced, amplified, and ultimately converted to the appropriate physiological response. The resolution of the molecular events underlying signal response and integration forms the basis of the signal transduction field of research. An evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide-binding proteins (G proteins) are key determinants of the specificity and temporal characteristics of many signaling processes and are the topic of this review. Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to heptahelical membrane receptors coupled to heterotrimeric G proteins. These highly specialized transducers can modulate the activity of multiple signaling pathways leading to diverse biological responses. In vivo, specific combinations of G alpha- and G beta gamma-subunits are likely required for connecting individual receptors to signaling pathways. The structural determinants of receptor-G protein-effector specificity are not completely understood and, in addition to involving interaction domains of these primary acting proteins, also require the participation of scaffolding and regulatory proteins.

  2. Structural Insights into Central Hypertension Regulation by Human Aminopeptidase A*

    PubMed Central

    Yang, Yang; Liu, Chang; Lin, Yi-Lun; Li, Fang

    2013-01-01

    Hypertension is regulated through both the central and systemic renin-angiotensin systems. In the central renin-angiotensin system, zinc-dependent aminopeptidase A (APA) up-regulates blood pressure by specifically cleaving the N-terminal aspartate, but not the adjacent arginine, from angiotensin II, a process facilitated by calcium. Here, we determined the crystal structures of human APA and its complexes with different ligands and identified a calcium-binding site in the S1 pocket of APA. Without calcium, the S1 pocket can bind both acidic and basic residues through formation of salt bridges with the charged side chains. In the presence of calcium, the binding of acidic residues is enhanced as they ligate the cation, whereas the binding of basic residues is no longer favorable due to charge repulsion. Of the peptidomimetic inhibitors of APA, amastatin has higher potency than bestatin by fitting better in the S1 pocket and interacting additionally with the S3′ subsite. These results explain the calcium-modulated substrate specificity of APA in central hypertension regulation and can guide the design and development of brain-targeting antihypertensive APA inhibitors. PMID:23888046

  3. Insight into SUCNR1 (GPR91) structure and function.

    PubMed

    Gilissen, Julie; Jouret, François; Pirotte, Bernard; Hanson, Julien

    2016-03-01

    SUCNR1 (or GPR91) belongs to the family of G protein-coupled receptors (GPCR), which represents the largest group of membrane proteins in human genome. The majority of marketed drugs targets GPCRs, directly or indirectly. SUCNR1 has been classified as an orphan receptor until a landmark study paired it with succinate, a citric acid cycle intermediate. According to the current paradigm, succinate triggers SUCNR1 signaling pathways to indicate local stress that may affect cellular metabolism. SUCNR1 implication has been well documented in renin-induced hypertension, ischemia/reperfusion injury, inflammation and immune response, platelet aggregation and retinal angiogenesis. In addition, the SUCNR1-induced increase of blood pressure may contribute to diabetic nephropathy or cardiac hypertrophy. The understanding of SUCNR1 activation, signaling pathways and functions remains largely elusive, which calls for deeper investigations. SUCNR1 shows a high potential as an innovative drug target and is probably an important regulator of basic physiology. In order to achieve the full characterization of this receptor, more specific pharmacological tools such as small-molecules modulators will represent an important asset. In this review, we describe the structural features of SUCNR1, its current ligands and putative binding pocket. We give an exhaustive overview of the known and hypothetical signaling partners of the receptor in different in vitro and in vivo systems. The link between SUCNR1 intracellular pathways and its pathophysiological roles are also extensively discussed.

  4. Structure of the Tongariro Volcanic system: Insights from magnetotelluric imaging

    NASA Astrophysics Data System (ADS)

    Hill, Graham J.; Bibby, Hugh M.; Ogawa, Yasuo; Wallin, Erin L.; Bennie, Stewart L.; Caldwell, T. Grant; Keys, Harry; Bertrand, Edward A.; Heise, Wiebke

    2015-12-01

    The dynamics of magma reservoirs (the main repositories for eruptible magma) play a fundamental role in the style and behaviour of volcanic systems. A key first step in understanding these systems is to identify their location and size accurately. We present results from a broadband magnetotelluric study of the Tongariro Volcanic system and discuss how the results fit within current petrological models. The Tongariro Volcanic system is a composite andesitic cone complex, located at the southern end of the Taupo Volcanic Zone in the central North Island of New Zealand. We use data from 136 broadband magnetotelluric soundings within a 25 × 35 km area covering the volcanic system to construct a 3D image of the magmatic system of the Tongariro Volcanic Complex including Mount Ngauruhoe. The structure of the Tongariro magmatic system has been determined from 3D forward and inverse modelling of the magnetotelluric data and allowed for an estimation of the melt fraction present within the system. 3D inverse modelling of the magnetotelluric data shows: a well-developed shallow low resistivity zone outlining the geothermal system; a zone of even lower resistivity representing a shallow crustal magma accumulation zone located at a depth of ∼4-12 km offset to the east of the Tongariro vent system; and a zone with a slightly higher resistivity connecting these two components of the magmatic system providing the path for magmatic fluids from the deeper source region to reach the surface during eruptive events.

  5. Structures and functions in the crowded nucleus: new biophysical insights

    NASA Astrophysics Data System (ADS)

    Hancock, Ronald

    2014-09-01

    Concepts and methods from the physical sciences have catalysed remarkable progress in understanding the cell nucleus in recent years. To share this excitement with physicists and encourage their interest in this field, this review offers an overview of how the physics which underlies structures and functions in the nucleus is becoming more clear thanks to methods which have been developed to simulate and study macromolecules, polymers, and colloids. The environment in the nucleus is very crowded with macromolecules, making entropic (depletion) forces major determinants of interactions. Simulation and experiments are consistent with their key role in forming membraneless compartments such as nucleoli, PML and Cajal bodies, and discrete "territories" for chromosomes. The chromosomes, giant linear polyelectrolyte polymers, exist in vivo in a state like a polymer melt. Looped conformations are predicted in crowded conditions, and have been confirmed experimentally and are central to the regulation of gene expression. Polymer theory has revealed how the chromosomes are so highly compacted in the nucleus, forming a "crumpled globule" with fractal properties which avoids knots and entanglements in DNA while allowing facile accessibility for its replication and transcription. Entropic repulsion between looped polymers can explain the confinement of each chromosome to a discrete region of the nucleus. Crowding and looping are predicted to facilitate finding the specific targets of factors which modulate activities of DNA. Simulation shows that entropic effects contribute to finding and repairing potentially lethal double-strand breaks in DNA by increasing the mobility of the broken ends, favouring their juxtaposition for repair. Signaling pathways are strongly influenced by crowding, which favours a processive mode of response (consecutive reactions without releasing substrates). This new information contributes to understanding the sometimes counter-intuitive consequences.

  6. INSIGHTS ON SCRAPIE PRION PROTEIN (PrPSc) STRUCTURE OBTAINED BY LIMITED PROTEOLYSIS AND MASS SPECTROMETRY

    USDA-ARS?s Scientific Manuscript database

    Elucidation of the structure of PrPSc, essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research, and is hampered by the insolubility and polymeric character of PrPSc. Limited proteolysis is a useful tool to obtain insight on...

  7. Cognitive Structures of the Gifted: Theoretical Perspectives, Factor Analysis, Triarchic Theories of Intelligence, and Insight Issues.

    ERIC Educational Resources Information Center

    Shaughnessy, Michael F.

    The paper reviews research on the cognitive structures of gifted students. Theories of R. Sternberg and his triarchic model of intelligence are described. Sternberg asserts that three processes appear to account for insight: selective encoding, selective combination, and selective comparison. H. Gardner's perspective citing six types of…

  8. The Manifest Association Structure of the Single-Factor Model: Insights from Partial Correlations

    ERIC Educational Resources Information Center

    Salgueiro, Maria de Fatima; Smith, Peter W. F.; McDonald, John W.

    2008-01-01

    The association structure between manifest variables arising from the single-factor model is investigated using partial correlations. The additional insights to the practitioner provided by partial correlations for detecting a single-factor model are discussed. The parameter space for the partial correlations is presented, as are the patterns of…

  9. The Manifest Association Structure of the Single-Factor Model: Insights from Partial Correlations

    ERIC Educational Resources Information Center

    Salgueiro, Maria de Fatima; Smith, Peter W. F.; McDonald, John W.

    2008-01-01

    The association structure between manifest variables arising from the single-factor model is investigated using partial correlations. The additional insights to the practitioner provided by partial correlations for detecting a single-factor model are discussed. The parameter space for the partial correlations is presented, as are the patterns of…

  10. Validation of a QuECheRS method for analysis of neonicotinoids in small volumes of blood and assessment of exposure in Eurasian eagle owl (Bubo bubo) nestlings.

    PubMed

    Taliansky-Chamudis, A; Gómez-Ramírez, P; León-Ortega, M; García-Fernández, A J

    2017-10-01

    Neonicotinoid pesticides have gained great interest in the last years both for agricultural and domestic use. Since the information on their environmental distribution or the effects derived from exposure to ecosystems and biota is scarce, new analytical techniques are being developed for monitoring studies. In this sense, two extraction techniques based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology to detect the neonicotinoids authorised in Spain (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram and thiamethoxam) were adapted and compared: a) using acetate buffer (AB); and b) using citrate buffer (CB). For detection and quantification, high performance liquid chromatography coupled with time of flight mass spectrometry (HPLC/TOF-MS) was used. The CB method provided a wide range of recoveries (68-134%) and accuracy (4-9%). The AB method provided good recoveries (59-76%, 59% corresponded to clothianidin) precision (4-11%) linearity (0.987-0.998%) and limit of quantification (2-10ng/mL) for all the compounds. To test the effectiveness of the technique, we analysed 30 blood samples of free-ranging nestlings of Eurasian eagle owl (Bubo bubo). The only compound detected, in one nestling from a dry land farming area, was imidacloprid, with a concentration of 3.28ng/mL. To our knowledge, this is the first study of neonicotinoid pesticides in free-ranging birds of prey using non-destructive samples, providing the first insight for biomonitoring studies. Further studies, including toxicokinetics and toxicodynamics, are recommended to assess the risk for these species. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    PubMed Central

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2015-01-01

    SUMMARY Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases, but with notable differences such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional co-crystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently-bound transition state mimic, provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily. PMID:22035792

  12. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    SciTech Connect

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2011-12-22

    Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

  13. Quantifying Neonicotinoid Insecticide Residues Escaping during Maize Planting with Vacuum Planters.

    PubMed

    Xue, Yingen; Limay-Rios, Victor; Smith, Jocelyn; Baute, Tracey; Forero, Luis Gabriel; Schaafsma, Arthur

    2015-11-03

    Neonicotinoid residues escaping in vacuum-planter exhaust during maize planting were measured in 25 fields in southwestern Ontario in 2013-2014 using filter bags to collect planter exhaust dust and horizontal and vertical sticky traps to collect planter operation-generated dust. Atrazine residues were used to differentiate between neonicotinoid residues originating from seed or from disturbed soil. Recovery rates of seed-applied neonicotinoids in exhaust were 0.014 and 0.365% in 2013 and 2014, respectively, calculated on the basis of neonicotinoid concentrations in preplant soil and seed application rates. Neonicotinoid exhaust emission rates were 0.0036 and 0.1104 g/ha for 2013 and 2014, respectively, with 99.9472 and 99.7820% originating from treated seed in 2013 and 2014, respectively, calculated on the basis of the atrazine marker. Rates of recovery of seed-applied neonicotinoid residues by exhaust filter bags were 0.015 and 0.437% for 2013 and 2014, respectively. Neonicotinoid residues captured on horizontal and vertical traps were 1.10 ng/cm2 (0.1104 g/ha) and 1.45 ng/cm2 (0.0029 g/ha), respectively, with 92.31 and 93.03% originating from treated seed, respectively, representing 0.3896% of the original active ingredient applied to the seed planted. Exposure to pollinators can be best reduced by strategies to keep active ingredient on the seed, below the soil surface, and in the field where applied.

  14. Planned Products of the Mars Structure Service for the InSight Mission to Mars

    NASA Astrophysics Data System (ADS)

    Panning, Mark P.; Lognonné, Philippe; Bruce Banerdt, W.; Garcia, Raphaël; Golombek, Matthew; Kedar, Sharon; Knapmeyer-Endrun, Brigitte; Mocquet, Antoine; Teanby, Nick A.; Tromp, Jeroen; Weber, Renee; Beucler, Eric; Blanchette-Guertin, Jean-Francois; Bozdağ, Ebru; Drilleau, Mélanie; Gudkova, Tamara; Hempel, Stefanie; Khan, Amir; Lekić, Vedran; Murdoch, Naomi; Plesa, Ana-Catalina; Rivoldini, Atillio; Schmerr, Nicholas; Ruan, Youyi; Verhoeven, Olivier; Gao, Chao; Christensen, Ulrich; Clinton, John; Dehant, Veronique; Giardini, Domenico; Mimoun, David; Thomas Pike, W.; Smrekar, Sue; Wieczorek, Mark; Knapmeyer, Martin; Wookey, James

    2016-11-01

    The InSight lander will deliver geophysical instruments to Mars in 2018, including seismometers installed directly on the surface (Seismic Experiment for Interior Structure, SEIS). Routine operations will be split into two services, the Mars Structure Service (MSS) and Marsquake Service (MQS), which will be responsible, respectively, for defining the structure models and seismicity catalogs from the mission. The MSS will deliver a series of products before the landing, during the operations, and finally to the Planetary Data System (PDS) archive. Prior to the mission, we assembled a suite of a priori models of Mars, based on estimates of bulk composition and thermal profiles. Initial models during the mission will rely on modeling surface waves and impact-generated body waves independent of prior knowledge of structure. Later modeling will include simultaneous inversion of seismic observations for source and structural parameters. We use Bayesian inversion techniques to obtain robust probability distribution functions of interior structure parameters. Shallow structure will be characterized using the hammering of the heatflow probe mole, as well as measurements of surface wave ellipticity. Crustal scale structure will be constrained by measurements of receiver function and broadband Rayleigh wave ellipticity measurements. Core interacting body wave phases should be observable above modeled martian noise levels, allowing us to constrain deep structure. Normal modes of Mars should also be observable and can be used to estimate the globally averaged 1D structure, while combination with results from the InSight radio science mission and orbital observations will allow for constraint of deeper structure.

  15. Cross-cultural adaptation, validation and factor structure of the Insight Scale for Affective Disorders.

    PubMed

    de Assis da Silva, Rafael; Mograbi, Daniel C; Camelo, Evelyn V M; Morton, Gregory Duff; Landeira-Fernandez, J; Cheniaux, Elie

    2015-06-01

    In the last few decades, several tools for studying insight in bipolar disorders have been used. Olaya and colleagues developed the Insight Scale for Affective Disorders (ISAD), which consists of a scale measuring insight through hetero evaluation for patients with mood disorders. The objective of this work is to translate and adapt the original English version of the ISAD to Brazilian Portuguese (ISAD-BR) and to conduct an evaluation of its psychometric properties. Adaptation procedures included translation/back-translation and consultation with a panel of experts. 95 patients with the diagnosis of Type 1 bipolar disorder were evaluated with the final version of the ISAD-BR, which was applied, simultaneously, but independently, by two examiners. Internal consistency and inter-rater reliability were explored and the latent structure of the scale was investigated with principal axis factoring and promax rotation. A second-order factor analysis was conducted to test if the scale had a hierarchical factor structure. The ISAD-BR showed good internal consistency and good inter-rater reliability. The analysis pointed to a four-factor solution of the ISAD-BR: awareness of symptoms associated with activity/energy; awareness of having a disorder; awareness of self-esteem and feelings of pleasure; and awareness of social functioning and relationships. The second order factor analysis indicated a hierarchical factor structure for the ISAD-BR, with the four lower-order factors loading on a single higher-order factor. Insight into bipolar disorder is a multidimensional construct, covering different aspects of the condition and its symptomatology. Nevertheless, insight about activity/energy changes may be a crucial aspect of insight into bipolar disorder. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Insights into RNA structure and function from genome-wide studies.

    PubMed

    Mortimer, Stefanie A; Kidwell, Mary Anne; Doudna, Jennifer A

    2014-07-01

    A comprehensive understanding of RNA structure will provide fundamental insights into the cellular function of both coding and non-coding RNAs. Although many RNA structures have been analysed by traditional biophysical and biochemical methods, the low-throughput nature of these approaches has prevented investigation of the vast majority of cellular transcripts. Triggered by advances in sequencing technology, genome-wide approaches for probing the transcriptome are beginning to reveal how RNA structure affects each step of protein expression and RNA stability. In this Review, we discuss the emerging relationships between RNA structure and the regulation of gene expression.

  17. Evaluation of river pollution of neonicotinoids in Osaka City (Japan) by LC/MS with dopant-assisted photoionisation.

    PubMed

    Yamamoto, Atsushi; Terao, Tomoko; Hisatomi, Hirotaka; Kawasaki, Hideya; Arakawa, Ryuichi

    2012-08-01

    An atmospheric pressure photoionisation (APPI) source for liquid chromatography/mass spectrometry (LC/MS) was applied to determine neonicotinoid pesticides in the aquatic environment. Dopant-assisted APPI was very effective in the ionisation of neonicotinoids. Neonicotinoids generated protonated molecules in APPI with high sensitivity, while adduct ions, such as sodiated molecules, were predominantly generated in conventional electrospray ionisation. The ionisation of neonicotinoids was confirmed by ultra-high-resolution MS. An analytical method coupled with solid phase extraction was developed for acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, and thiamethoxam. Method detection limits were 0.47 to 2.1 ng L(-1) for six neonicotinoids. Dinotefuran was the most frequent and highest among the neonicotinoids examined in the aquatic environment in Osaka, Japan. The maximum concentration of dinotefuran was 220 ng L(-1). Given the toxicity of neonicotinoids for aquatic creatures, the concentrations observed here were substantially low. The change in concentrations was temporally coincident with the period of the neonicotinoid application. Although rapid photodegradation and some degradation products have been elucidated, the degradation products in the aquatic environment were not identified in the present study.

  18. Low expression of nicotinic acetylcholine receptor subunit Mdα2 in neonicotinoid-resistant strains of Musca domestica L.

    PubMed

    Markussen, Mette D K; Kristensen, Michael

    2010-11-01

    Neonicotinoid action as well as resistance involves interaction with nicotinic acetylcholine receptors (nAChRs). In the housefly, neonicotinoid resistance also involves cytochrome P450, as indicated by bioassay with synergist as well as altered expression. In bioassay, synergism was only partial and indicated possible target-site resistance. The nAChR α2 subunit is important in neonicotinoid toxicity to insects, and gene expression of the Mdα2 subunit was investigated in field populations and laboratory strains of neonicotinoid-resistant and insecticide-susceptible houseflies, Musca domestica L. The genomic sequence covering exon III-VII of Mdα2 was analysed for mutations. Gene expression profiling of Mdα2 revealed notable differences between neonicotinoid-resistant and insecticide-susceptible houseflies. On average, the neonicotinoid-resistant field population 766b and the imidacloprid selected strain 791imi had 60% lower copy numbers of Mdα2 compared with the susceptible reference strain. Sequencing of exon III-VII of the Mdα2, encoding acetylcholine binding-site regions and three out of four transmembrane domains, did not reveal any mutations explaining the increased neonicotinoid tolerance in the strains examined. Previous discoveries and the results of this study suggest that the neonicotinoid resistance mechanism in Danish houseflies involves both cytochrome P450 monooxygenase-mediated detoxification and reduced expression of the nAChR subunit α2. Copyright © 2010 Society of Chemical Industry.

  19. Functional, structural, and emotional correlates of impaired insight in cocaine addiction

    PubMed Central

    Moeller, Scott J.; Konova, Anna B.; Parvaz, Muhammad A.; Tomasi, Dardo; Lane, Richard D.; Fort, Carolyn; Goldstein, Rita Z.

    2014-01-01

    Context Individuals with cocaine use disorder (CUD) have difficulty monitoring ongoing behavior, possibly stemming from dysfunction of brain regions subserving insight and self-awareness [e.g., anterior cingulate cortex (ACC)]. Objective To test the hypothesis that CUD with impaired insight (iCUD) would show abnormal (A) ACC activity during error processing, assessed with functional magnetic resonance imaging during a classic inhibitory control task; (B) ACC gray matter integrity assessed with voxel-based morphometry; and (C) awareness of one’s own emotional experiences, assessed with the Levels of Emotional Awareness Scale (LEAS). Using a previously validated probabilistic choice task, we grouped 33 CUD according to insight [iCUD: N=15; unimpaired insight CUD: N=18]; we also studied 20 healthy controls, all with unimpaired insight. Design Multimodal imaging design. Setting Clinical Research Center at Brookhaven National Laboratory. Participants Thirty-three CUD and 20 healthy controls. Main Outcome Measure Functional magnetic resonance imaging, voxel-based morphometry, LEAS, and drug use variables. Results Compared with the other two study groups, iCUD showed lower (A) error-induced rostral ACC (rACC) activity as associated with more frequent cocaine use; (B) gray matter within the rACC; and (C) LEAS scores. Conclusions These results point to rACC functional and structural abnormalities, and diminished emotional awareness, in a subpopulation of CUD characterized by impaired insight. Because the rACC has been implicated in appraising the affective/motivational significance of errors and other types of self-referential processing, functional and structural abnormalities in this region could result in lessened concern (frequently ascribed to minimization and denial) about behavioral outcomes that could potentially culminate in increased drug use. Treatments targeting this CUD subgroup could focus on enhancing the salience of errors (e.g., lapses). PMID:24258223

  20. Neonicotinoid-Coated Zea mays Seeds Indirectly Affect Honeybee Performance and Pathogen Susceptibility in Field Trials.

    PubMed

    Alburaki, Mohamed; Boutin, Sébastien; Mercier, Pierre-Luc; Loublier, Yves; Chagnon, Madeleine; Derome, Nicolas

    2015-01-01

    Thirty-two honeybee (Apis mellifera) colonies were studied in order to detect and measure potential in vivo effects of neonicotinoid pesticides used in cornfields (Zea mays spp) on honeybee health. Honeybee colonies were randomly split on four different agricultural cornfield areas located near Quebec City, Canada. Two locations contained cornfields treated with a seed-coated systemic neonicotinoid insecticide while the two others were organic cornfields used as control treatments. Hives were extensively monitored for their performance and health traits over a period of two years. Honeybee viruses (brood queen cell virus BQCV, deformed wing virus DWV, and Israeli acute paralysis virus IAPV) and the brain specific expression of a biomarker of host physiological stress, the Acetylcholinesterase gene AChE, were investigated using RT-qPCR. Liquid chromatography-mass spectrometry (LC-MS) was performed to detect pesticide residues in adult bees, honey, pollen, and corn flowers collected from the studied hives in each location. In addition, general hive conditions were assessed by monitoring colony weight and brood development. Neonicotinoids were only identified in corn flowers at low concentrations. However, honeybee colonies located in neonicotinoid treated cornfields expressed significantly higher pathogen infection than those located in untreated cornfields. AChE levels showed elevated levels among honeybees that collected corn pollen from treated fields. Positive correlations were recorded between pathogens and the treated locations. Our data suggests that neonicotinoids indirectly weaken honeybee health by inducing physiological stress and increasing pathogen loads.

  1. Neonicotinoid-Coated Zea mays Seeds Indirectly Affect Honeybee Performance and Pathogen Susceptibility in Field Trials

    PubMed Central

    Alburaki, Mohamed; Boutin, Sébastien; Mercier, Pierre-Luc; Loublier, Yves; Chagnon, Madeleine; Derome, Nicolas

    2015-01-01

    Thirty-two honeybee (Apis mellifera) colonies were studied in order to detect and measure potential in vivo effects of neonicotinoid pesticides used in cornfields (Zea mays spp) on honeybee health. Honeybee colonies were randomly split on four different agricultural cornfield areas located near Quebec City, Canada. Two locations contained cornfields treated with a seed-coated systemic neonicotinoid insecticide while the two others were organic cornfields used as control treatments. Hives were extensively monitored for their performance and health traits over a period of two years. Honeybee viruses (brood queen cell virus BQCV, deformed wing virus DWV, and Israeli acute paralysis virus IAPV) and the brain specific expression of a biomarker of host physiological stress, the Acetylcholinesterase gene AChE, were investigated using RT-qPCR. Liquid chromatography-mass spectrometry (LC-MS) was performed to detect pesticide residues in adult bees, honey, pollen, and corn flowers collected from the studied hives in each location. In addition, general hive conditions were assessed by monitoring colony weight and brood development. Neonicotinoids were only identified in corn flowers at low concentrations. However, honeybee colonies located in neonicotinoid treated cornfields expressed significantly higher pathogen infection than those located in untreated cornfields. AChE levels showed elevated levels among honeybees that collected corn pollen from treated fields. Positive correlations were recorded between pathogens and the treated locations. Our data suggests that neonicotinoids indirectly weaken honeybee health by inducing physiological stress and increasing pathogen loads. PMID:25993642

  2. Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors.

    PubMed

    Tomizawa, Motohiro; Casida, John E

    2003-01-01

    Neonicotinoids, the most important new class of synthetic insecticides of the past three decades, are used to control sucking insects both on plants and on companion animals. Imidacloprid (the principal example), nitenpyram, acetamiprid, thiacloprid, thiamethoxam, and others act as agonists at the insect nicotinic acetylcholine receptor (nAChR). The botanical insecticide nicotine acts at the same target without the neonicotinoid level of effectiveness or safety. Fundamental differences between the nAChRs of insects and mammals confer remarkable selectivity for the neonicotinoids. Whereas ionized nicotine binds at an anionic subsite in the mammalian nAChR, the negatively tipped ("magic" nitro or cyano) neonicotinoids interact with a proposed unique subsite consisting of cationic amino acid residue(s) in the insect nAChR. Knowledge reviewed here of the functional architecture and molecular aspects of the insect and mammalian nAChRs and their neonicotinoid-binding site lays the foundation for continued development and use of this new class of safe and effective insecticides.

  3. Sex allocation theory reveals a hidden cost of neonicotinoid exposure in a parasitoid wasp

    PubMed Central

    Whitehorn, Penelope R.; Cook, Nicola; Blackburn, Charlotte V.; Gill, Sophie M.; Green, Jade; Shuker, David M.

    2015-01-01

    Sex allocation theory has proved to be one the most successful theories in evolutionary ecology. However, its role in more applied aspects of ecology has been limited. Here we show how sex allocation theory helps uncover an otherwise hidden cost of neonicotinoid exposure in the parasitoid wasp Nasonia vitripennis. Female N. vitripennis allocate the sex of their offspring in line with Local Mate Competition (LMC) theory. Neonicotinoids are an economically important class of insecticides, but their deployment remains controversial, with evidence linking them to the decline of beneficial species. We demonstrate for the first time to our knowledge, that neonicotinoids disrupt the crucial reproductive behaviour of facultative sex allocation at sub-lethal, field-relevant doses in N. vitripennis. The quantitative predictions we can make from LMC theory show that females exposed to neonicotinoids are less able to allocate sex optimally and that this failure imposes a significant fitness cost. Our work highlights that understanding the ecological consequences of neonicotinoid deployment requires not just measures of mortality or even fecundity reduction among non-target species, but also measures that capture broader fitness costs, in this case offspring sex allocation. Our work also highlights new avenues for exploring how females obtain information when allocating sex under LMC. PMID:25925105

  4. The Neonicotinoid Insecticide Imidacloprid Repels Pollinating Flies and Beetles at Field-Realistic Concentrations

    PubMed Central

    Easton, Amy H.; Goulson, Dave

    2013-01-01

    Neonicotinoids are widely used systemic insecticides which, when applied to flowering crops, are translocated to the nectar and pollen where they may impact upon pollinators. Given global concerns over pollinator declines, this potential impact has recently received much attention. Field exposure of pollinators to neonicotinoids depends on the concentrations present in flowering crops and the degree to which pollinators choose to feed upon them. Here we describe a simple experiment using paired yellow pan traps with or without insecticide to assess whether the commonly used neonicotinoid imidacloprid repels or attracts flying insects. Both Diptera and Coleoptera exhibited marked avoidance of traps containing imidacloprid at a field-realistic dose of 1 µg L−1, with Diptera avoiding concentrations as low as 0.01 µg L−1. This is to our knowledge the first evidence for any biological activity at such low concentrations, which are below the limits of laboratory detection using most commonly available techniques. Catch of spiders in pan traps was also slightly reduced by the highest concentrations of imidacloprid used (1 µg L−1), but catch was increased by lower concentrations. It remains to be seen if the repellent effect on insects occurs when neonicotinoids are present in real flowers, but if so then this could have implications for exposure of pollinators to neonicotinoids and for crop pollination. PMID:23382980

  5. Sex allocation theory reveals a hidden cost of neonicotinoid exposure in a parasitoid wasp.

    PubMed

    Whitehorn, Penelope R; Cook, Nicola; Blackburn, Charlotte V; Gill, Sophie M; Green, Jade; Shuker, David M

    2015-05-22

    Sex allocation theory has proved to be one the most successful theories in evolutionary ecology. However, its role in more applied aspects of ecology has been limited. Here we show how sex allocation theory helps uncover an otherwise hidden cost of neonicotinoid exposure in the parasitoid wasp Nasonia vitripennis. Female N. vitripennis allocate the sex of their offspring in line with Local Mate Competition (LMC) theory. Neonicotinoids are an economically important class of insecticides, but their deployment remains controversial, with evidence linking them to the decline of beneficial species. We demonstrate for the first time to our knowledge, that neonicotinoids disrupt the crucial reproductive behaviour of facultative sex allocation at sub-lethal, field-relevant doses in N. vitripennis. The quantitative predictions we can make from LMC theory show that females exposed to neonicotinoids are less able to allocate sex optimally and that this failure imposes a significant fitness cost. Our work highlights that understanding the ecological consequences of neonicotinoid deployment requires not just measures of mortality or even fecundity reduction among non-target species, but also measures that capture broader fitness costs, in this case offspring sex allocation. Our work also highlights new avenues for exploring how females obtain information when allocating sex under LMC.

  6. Sublethal effects on wood frogs chronically exposed to environmentally relevant concentrations of two neonicotinoid insecticides.

    PubMed

    Robinson, Stacey A; Richardson, Sarah D; Dalton, Rebecca L; Maisonneuve, France; Trudeau, Vance L; Pauli, Bruce D; Lee-Jenkins, Stacey S Y

    2017-04-01

    Neonicotinoids are prophylactically used globally on a variety of crops, and there is concern for the potential impacts of neonicotinoids on aquatic ecosystems. The intensive use of pesticides on crops has been identified as a contributor to population declines of amphibians, but currently little is known regarding the sublethal effects of chronic neonicotinoid exposure on amphibians. The objective of the present study was to characterize the sublethal effect(s) of exposure to 3 environmentally relevant concentrations (1 μg/L, 10 μg/L, and 100 μg/L) of 2 neonicotinoids on larval wood frogs (Lithobates sylvaticus) using outdoor mesocosms. We exposed tadpoles to solutions of 2 commercial formulations containing imidacloprid and thiamethoxam, and assessed survival, growth, and development. Exposure to imidacloprid at 10 μg/L and 100 μg/L increased survival and delayed completion of metamorphosis compared with controls. Exposure to thiamethoxam did not influence amphibian responses. There was no significant effect of any treatment on body mass or size of the metamorphs. The results suggest that current usage of imidacloprid and thiamethoxam does not pose a threat to wood frogs. However, further assessment of both direct and indirect effects on subtle sublethal endpoints, and the influence of multiple interacting stressors at various life stages, is needed to fully understand the effects of neonicotinoids on amphibians. Environ Toxicol Chem 2017;36:1101-1109. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

  7. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    PubMed Central

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-01-01

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of long-term (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain. PMID:27782041

  8. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum.

    PubMed

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-10-04

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  9. High Levels of Resistance in the Common Bed Bug, Cimex lectularius (Hemiptera: Cimicidae), to Neonicotinoid Insecticides.

    PubMed

    Romero, Alvaro; Anderson, Troy D

    2016-05-01

    The rapid increase of bed bug populations resistant to pyrethroids demands the development of novel control tactics. Products combining pyrethroids and neonicotinoids have become very popular for bed bug control in the United States, but there are concerns about evolution of resistance to these compounds. Laboratory assays were used to measure the toxicity of topical applications of four neonicotinoids to a susceptible population and three pyrethroid-resistant populations. Activity of esterases, glutathione S-transferases, and cytochrome P450s of all strains was also evaluated. High levels of resistance to four neonicotinoids, acetamiprid, imidacloprid, dinotefuran, and thiamethoxam, relative to the susceptible Fort Dix population, were detected in populations collected from human dwellings in Cincinnati and Michigan. Because activity of detoxifying enzymes was increased in these two populations, our results suggest that these enzymes have some involvement in neonicotinoid resistance, but other resistance mechanisms might be involved as well. Detection of high levels of resistance to neonicotinoids further limits the options for chemical control of bed bugs. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

  10. Behavioural avoidance and enhanced dispersal in neonicotinoid-resistant Myzus persicae (Sulzer).

    PubMed

    Fray, Lucy M; Leather, Simon R; Powell, Glen; Slater, Russell; McIndoe, Eddie; Lind, Robert J

    2014-01-01

    The peach potato aphid Myzus persicae is a major agricultural pest capable of transmitting over 100 plant viruses to a wide range of crops. Control relies largely upon treatment with neonicotinoid insecticides such as thiamethoxam (TMX). In 2009, a strain denoted FRC, which exhibits between 255- and 1679-fold resistance to current neonicotinoids previously linked to metabolic and target site resistance, was discovered in France. Dispersal behaviour may potentially further enhance the resistance of this strain. This study investigated this possibility and is the first to compare the dispersal behaviour of aphid clones of the same species with differing levels of neonicotinoid resistance. Comparing the dispersal behaviour of the FRC strain with that of a clone of lower neonicotinoid resistance (5191A), and a susceptible clone (US1L) highlighted several differences. Most importantly, the FRC strain exhibited an increased ability to locate untreated areas when presented with an environment consisting of both TMX-treated and untreated plant tissue. The altered dispersal behaviour of the FRC may partially account for the high level of neonicotinoid resistance exhibited by this strain in the field. Since the dispersal of aphid vectors is key to the transmission of viruses across crop fields this has implications for current crop protection practice. © 2013 Society of Chemical Industry.

  11. Incidence and characterisation of resistance to neonicotinoid insecticides and pymetrozine in the greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae).

    PubMed

    Karatolos, Nikos; Denholm, Ian; Williamson, Martin; Nauen, Ralf; Gorman, Kevin

    2010-12-01

    Trialeurodes vaporariorum (Westwood), also known as the greenhouse whitefly, is a serious pest of protected vegetable and ornamental crops in most temperate regions of the world. Neonicotinoid insecticides are used widely to control this species, although resistance has been reported and may be becoming widespread. Mortality rates of UK and European strains of T. vaporariorum to a range of neonicotinoids and pymetrozine, a compound with a different mode of action, were calculated, and significant resistance was found in some of those strains. A strong association was found between neonicotinoids and pymetrozine, and reciprocal selection experiments confirmed this finding. Expression of resistance to the neonicotinoid imidacloprid and pymetrozine was age specific, and resistance in nymphs did not compromise recommended application rates. This study indicates strong parallels in the phenotypic characteristics of neonicotinoid resistance in T. vaporariorum and the tobacco whitefly Bemisia tabaci Gennadius, suggesting possible parallels in the underlying mechanisms. Copyright © 2010 Society of Chemical Industry.

  12. New Insights about Enzyme Evolution from Large Scale Studies of Sequence and Structure Relationships*

    PubMed Central

    Brown, Shoshana D.; Babbitt, Patricia C.

    2014-01-01

    Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes. PMID:25210038

  13. New insights about enzyme evolution from large scale studies of sequence and structure relationships.

    PubMed

    Brown, Shoshana D; Babbitt, Patricia C

    2014-10-31

    Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes.

  14. Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides.

    PubMed

    Crespin, Lucille; Legros, Christian; List, Olivier; Tricoire-Leignel, Hélène; Mattei, César

    2016-01-01

    Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the

  15. Insights from molecular structure predictions of the infectious bronchitis virus S1 spike glycoprotein.

    PubMed

    Leyson, Christina Lora M; Jordan, Brian J; Jackwood, Mark W

    2016-12-01

    Infectious bronchitis virus is an important respiratory pathogen in chickens. The IBV S1 spike is a viral structural protein that is responsible for attachment to host receptors and is a major target for neutralizing antibodies. To date, there is no experimentally determined structure for the IBV S1 spike. In this study, we sought to find a predicted tertiary structure for IBV S1 using I-TASSER, which is an automated homology modeling platform. We found that the predicted structures obtained were robust and consistent with experimental data. For instance, we observed that all four residues (38, 43, 63, and 68) that have been shown to be critical for binding to host tissues, were found at the surface of the predicted structure of Massachusetts (Mass) S1 spike. Together with antigenicity index analysis, we were also able to show that Ma5 vaccine has higher antigenicity indices at residues close to the receptor-binding region than M41 vaccine, thereby providing a possible mechanism on how Ma5 achieves better protection against challenge. Examination of the predicted structure of the Arkansas IBV S1 spike also gave insights on the effect of polymorphisms at position 43 on the surface availability of receptor binding residues. This study showcases advancements in protein structure prediction and contributes useful, inexpensive tools to provide insights into the biology of IBV. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Increasing neonicotinoid use and the declining butterfly fauna of lowland California

    PubMed Central

    Cousens, Bruce; Harrison, Joshua G.; Anderson, Kayce; Thorne, James H.; Waetjen, Dave; Nice, Chris C.; De Parsia, Matthew; Meese, Robert; van Vliet, Heidi; Shapiro, Arthur M.

    2016-01-01

    The butterfly fauna of lowland Northern California has exhibited a marked decline in recent years that previous studies have attributed in part to altered climatic conditions and changes in land use. Here, we ask if a shift in insecticide use towards neonicotinoids is associated with butterfly declines at four sites in the region that have been monitored for four decades. A negative association between butterfly populations and increasing neonicotinoid application is detectable while controlling for land use and other factors, and appears to be more severe for smaller-bodied species. These results suggest that neonicotinoids could influence non-target insect populations occurring in proximity to application locations, and highlights the need for mechanistic work to complement long-term observational data. PMID:27531159

  17. Increasing neonicotinoid use and the declining butterfly fauna of lowland California

    USGS Publications Warehouse

    Forister, Matthew L.; Cousens, Bruce; Harrison, Joshua G.; Anderson, Kayce; Thorne, James H.; Waetjen, Dave; Nice, Chris C.; De Parsia, Matthew; Hladik, Michelle; Meese, Robert; van Vliet, Heidi; Shapiro, Arthur M.

    2016-01-01

    The butterfly fauna of lowland Northern California has exhibited a marked decline in recent years that previous studies have attributed in part to altered climatic conditions and changes in land use. Here, we ask if a shift in insecticide use towards neonicotinoids is associated with butterfly declines at four sites in the region that have been monitored for four decades. A negative association between butterfly populations and increasing neonicotinoid application is detectable while controlling for land use and other factors, and appears to be more severe for smaller-bodied species. These results suggest that neonicotinoids could influence non-target insect populations occurring in proximity to application locations, and highlights the need for mechanistic work to complement long-term observational data.

  18. Increasing neonicotinoid use and the declining butterfly fauna of lowland California.

    PubMed

    Forister, Matthew L; Cousens, Bruce; Harrison, Joshua G; Anderson, Kayce; Thorne, James H; Waetjen, Dave; Nice, Chris C; De Parsia, Matthew; Hladik, Michelle L; Meese, Robert; van Vliet, Heidi; Shapiro, Arthur M

    2016-08-01

    The butterfly fauna of lowland Northern California has exhibited a marked decline in recent years that previous studies have attributed in part to altered climatic conditions and changes in land use. Here, we ask if a shift in insecticide use towards neonicotinoids is associated with butterfly declines at four sites in the region that have been monitored for four decades. A negative association between butterfly populations and increasing neonicotinoid application is detectable while controlling for land use and other factors, and appears to be more severe for smaller-bodied species. These results suggest that neonicotinoids could influence non-target insect populations occurring in proximity to application locations, and highlights the need for mechanistic work to complement long-term observational data.

  19. Biodegradation of neonicotinoid insecticide, imidacloprid by restriction enzyme mediated integration (REMI) generated Trichoderma mutants.

    PubMed

    He, Xiangfeng; Wubie, Abebe Jenberie; Diao, Qingyun; Li, Wei; Xue, Fei; Guo, Zhanbo; Zhou, Ting; Xu, Shufa

    2014-10-01

    REMI (restriction enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS-hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p<0.01). More specifically, seven single-copied T. atroviride T23 transformants have confirmed a 30% higher degradation rate than their parent isolate. Among all transformed mutants, a 95% imidacloprid degradation rate was identified as the highest. This study, thus, provided an effective approach for improving neonicotinoid insecticide-degrading capability using REMI transformed T. atroviride mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Insights into Surface Structure and Performance of Fluorinated Silicates from Cohesive Energy Studies

    DTIC Science & Technology

    2016-03-17

    fluorinated silicates from cohesive energy studies 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Andrew J...Insights into surface structure and performance of fluorinated silicates from cohesive energy studies 17 March 2016 Andrew J. Guenthner,1 Timothy...distribution is unlimited. PA Clearance Number 16153 Comparison of Surface Energy Parameters for Fluorosilicates 5 • Typical Surface Energies : -CF3 = 6

  1. Atomic Force Microscopy in Microbiology: New Structural and Functional Insights into the Microbial Cell Surface

    PubMed Central

    2014-01-01

    ABSTRACT Microbial cells sense and respond to their environment using their surface constituents. Therefore, understanding the assembly and biophysical properties of cell surface molecules is an important research topic. With its ability to observe living microbial cells at nanometer resolution and to manipulate single-cell surface molecules, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Here, we survey major breakthroughs made in cell surface microbiology using AFM techniques, emphasizing the most recent structural and functional insights. PMID:25053785

  2. Insight into DEG/ENaC channel gating from genetics and structure.

    PubMed

    Eastwood, Amy L; Goodman, Miriam B

    2012-10-01

    The founding members of the superfamily of DEG/ENaC ion channel proteins are C. elegans proteins that form mechanosensitive channels in touch and pain receptors. For more than a decade, the research community has used mutagenesis to identify motifs that regulate gating. This review integrates insight derived from unbiased in vivo mutagenesis screens with recent crystal structures to develop new models for activation of mechanically gated DEGs.

  3. Neonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees

    PubMed Central

    Stanley, Dara A.; Garratt, Michael P.D.; Wickens, Jennifer B.; Wickens, Victoria J.; Potts, Simon G.; Raine, Nigel E.

    2015-01-01

    Recent concern over global pollinator declines has led to considerable research on the effects of pesticides on bees1-5. Although pesticides are typically not encountered at lethal levels in the field, there is growing evidence indicating that exposure to field-realistic levels can have sub-lethal effects on bees affecting their foraging behaviour1,6,7, homing ability8,9 and reproductive success2,5. Bees are essential for the pollination of a wide variety of crops and the majority of wild flowering plants10-12, but until now research on pesticide impacts has been limited to direct effects on bees themselves and not on the pollination services they provide. Here we show the first evidence that pesticide exposure can reduce the pollination services bumblebees deliver to apples, a crop of global economic importance. Colonies exposed to a neonicotinoid pesticide provided lower visitation rates to apple trees and collected pollen less often. Most importantly these pesticide exposed colonies produced apples containing fewer seeds demonstrating a reduced delivery of pollination services. Our results also suggest reduced pollination service delivery is not due to pesticide-induced changes in individual bee behaviour but most likely due to impacts at the colony level. These findings show that pesticide exposure can impair the ability of bees to provide pollination services, with important implications for both the sustained delivery of stable crop yields and the function of natural ecosystems. PMID:26580009

  4. Neonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees.

    PubMed

    Stanley, Dara A; Garratt, Michael P D; Wickens, Jennifer B; Wickens, Victoria J; Potts, Simon G; Raine, Nigel E

    2015-12-24

    Recent concern over global pollinator declines has led to considerable research on the effects of pesticides on bees. Although pesticides are typically not encountered at lethal levels in the field, there is growing evidence indicating that exposure to field-realistic levels can have sublethal effects on bees, affecting their foraging behaviour, homing ability and reproductive success. Bees are essential for the pollination of a wide variety of crops and the majority of wild flowering plants, but until now research on pesticide effects has been limited to direct effects on bees themselves and not on the pollination services they provide. Here we show the first evidence to our knowledge that pesticide exposure can reduce the pollination services bumblebees deliver to apples, a crop of global economic importance. Bumblebee colonies exposed to a neonicotinoid pesticide provided lower visitation rates to apple trees and collected pollen less often. Most importantly, these pesticide-exposed colonies produced apples containing fewer seeds, demonstrating a reduced delivery of pollination services. Our results also indicate that reduced pollination service delivery is not due to pesticide-induced changes in individual bee behaviour, but most likely due to effects at the colony level. These findings show that pesticide exposure can impair the ability of bees to provide pollination services, with important implications for both the sustained delivery of stable crop yields and the functioning of natural ecosystems.

  5. Photodegradation of neonicotinoid insecticides in water by semiconductor oxides.

    PubMed

    Fenoll, José; Garrido, Isabel; Hellín, Pilar; Flores, Pilar; Navarro, Simón

    2015-10-01

    The photocatalytic degradation of three neonicotinoid insecticides (NIs), thiamethoxam (TH), imidacloprid (IM) and acetamiprid (AC), in pure water has been studied using zinc oxide (ZnO) and titanium dioxide (TiO2) as photocatalysts under natural sunlight and artificial light irradiation. Photocatalytic experiments showed that the addition of these chalcogenide oxides in tandem with the electron acceptor (Na2S2O8) strongly enhances the degradation rate of these compounds in comparison with those carried out with ZnO and TiO2 alone and photolytic tests. Comparison of catalysts showed that ZnO is the most efficient for the removal of such insecticides in optimal conditions and at constant volumetric rate of photon absorption. Thus, the complete disappearance of all the studied compounds was achieved after 10 and 30 min of artificial light irradiation, in the ZnO/Na2S2O8 and TiO2/Na2S2O8 systems, respectively. The highest degradation rate was noticed for IM, while the lowest rate constant was obtained for AC under artificial light irradiation. In addition, solar irradiation was more efficient compared to artificial light for the removal of these insecticides from water. The main photocatalytic intermediates detected during the degradation of NIs were identified.

  6. Atmospheric Chemistry of Neonicotinoids Used in Urban Areas

    NASA Astrophysics Data System (ADS)

    Finlayson-Pitts, B. J.; Aregahegn, K.; Shemesh, D.; Gerber, R. B.

    2016-12-01

    Neonicotinoid (NN) pesticides are used extensively in both urban and agricultural settings to control sucking pests such as aphids, as well as for flea control for domestic pets. As a result, they are commonly found on surfaces that are exposed to the atmosphere. Imidacloprid (IMD) is one of the major NNs in pest control formulations. While there have been a number of studies of IMD reactions in solution, there are relatively few surface reactions that are relevant to atmospheric exposures. We report here laboratory studies of the photochemistry of IMD on surfaces in which quantum yields are measured and combined with absorption cross sections to estimate tropospheric lifetimes with respect to photolysis. Products identified using a combination of ATR-FTIR, DART-MS and ESI-MS include the desnitro and urea derivatives in the solid, and N2O in the gas phase. Quantum chemical calculations suggest a mechanism for the photolysis and production of these products. The implications for altering toxicity through atmospheric reactions will be discussed.

  7. Neonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees

    NASA Astrophysics Data System (ADS)

    Stanley, Dara A.; Garratt, Michael P. D.; Wickens, Jennifer B.; Wickens, Victoria J.; Potts, Simon G.; Raine, Nigel E.

    2015-12-01

    Recent concern over global pollinator declines has led to considerable research on the effects of pesticides on bees. Although pesticides are typically not encountered at lethal levels in the field, there is growing evidence indicating that exposure to field-realistic levels can have sublethal effects on bees, affecting their foraging behaviour, homing ability and reproductive success. Bees are essential for the pollination of a wide variety of crops and the majority of wild flowering plants, but until now research on pesticide effects has been limited to direct effects on bees themselves and not on the pollination services they provide. Here we show the first evidence to our knowledge that pesticide exposure can reduce the pollination services bumblebees deliver to apples, a crop of global economic importance. Bumblebee colonies exposed to a neonicotinoid pesticide provided lower visitation rates to apple trees and collected pollen less often. Most importantly, these pesticide-exposed colonies produced apples containing fewer seeds, demonstrating a reduced delivery of pollination services. Our results also indicate that reduced pollination service delivery is not due to pesticide-induced changes in individual bee behaviour, but most likely due to effects at the colony level. These findings show that pesticide exposure can impair the ability of bees to provide pollination services, with important implications for both the sustained delivery of stable crop yields and the functioning of natural ecosystems.

  8. Reduction of neonicotinoid insecticide residues in Prairie wetlands by common wetland plants.

    PubMed

    Main, Anson R; Fehr, Jessica; Liber, Karsten; Headley, John V; Peru, Kerry M; Morrissey, Christy A

    2017-02-01

    Neonicotinoid insecticides are frequently detected in wetlands during the early to mid-growing period of the Canadian Prairie cropping season. These detections also overlap with the growth of macrophytes that commonly surround agricultural wetlands which we hypothesized may reduce neonicotinoid transport and retention in wetlands. We sampled 20 agricultural wetlands and 11 macrophyte species in central Saskatchewan, Canada, over eight weeks to investigate whether macrophytes were capable of reducing movement of neonicotinoids from cultivated fields and/or reducing concentrations in surface water by accumulating insecticide residues into their tissues. Study wetlands were surrounded by clothianidin-treated canola and selected based on the presence (n=10) or absence (n=10) of a zonal plant community. Neonicotinoids were positively detected in 43% of wetland plants, and quantified in 8% of all plant tissues sampled. Three plant species showed high rates of detection: 78% Equisetum arvense (clothianidin, range: neonicotinoid concentrations in Prairie wetlands over time using linear mixed-effects models. Clothianidin concentrations were significantly lower in wetlands surrounded by taller plants (β±S.E.: -0.57±0.12, P≤0.001). The results of this study suggest that macrophytes can play an important role in mitigating water contamination by accumulating neonicotinoids and possibly slowing transport to wetlands during the growing season.

  9. Neonicotinoid insecticides inhibit cholinergic neurotransmission in a molluscan (Lymnaea stagnalis) nervous system.

    PubMed

    Vehovszky, Á; Farkas, A; Ács, A; Stoliar, O; Székács, A; Mörtl, M; Győri, J

    2015-10-01

    Neonicotinoids are highly potent and selective systemic insecticides, but their widespread use also has a growing impact on non-target animals and contaminates the environment, including surface waters. We tested the neonicotinoid insecticides commercially available in Hungary (acetamiprid, Mospilan; imidacloprid, Kohinor; thiamethoxam, Actara; clothianidin, Apacs; thiacloprid, Calypso) on cholinergic synapses that exist between the VD4 and RPeD1 neurons in the central nervous system of the pond snail Lymnaea stagnalis. In the concentration range used (0.01-1 mg/ml), neither chemical acted as an acetylcholine (ACh) agonist; instead, both displayed antagonist activity, inhibiting the cholinergic excitatory components of the VD4-RPeD1 connection. Thiacloprid (0.01 mg/ml) blocked almost 90% of excitatory postsynaptic potentials (EPSPs), while the less effective thiamethoxam (0.1 mg/ml) reduced the synaptic responses by about 15%. The ACh-evoked membrane responses of the RPeD1 neuron were similarly inhibited by the neonicotinoids, confirming that the same ACh receptor (AChR) target was involved. We conclude that neonicotinoids act on nicotinergic acetylcholine receptors (nAChRs) in the snail CNS. This has been established previously in the insect CNS; however, our data indicate differences in the background mechanism or the nAChR binding site in the snail. Here, we provide the first results concerning neonicotinoid-related toxic effects on the neuronal connections in the molluscan nervous system. Aquatic animals, including molluscs, are at direct risk while facing contaminated surface waters, and snails may provide a suitable model for further studies of the behavioral/neuronal consequences of intoxication by neonicotinoids. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Assessment of cross-resistance potential to neonicotinoid insecticides in Bemisia tabaci (Hemiptera: Aleyrodidae).

    PubMed

    Prabhaker, N; Castle, S; Henneberry, T J; Toscano, N C

    2005-12-01

    Laboratory bioassays were carried out with four neonicotinoid insecticides on multiple strains of Bemisia tabaci (Gennadius) to evaluate resistance and cross-resistance patterns. Three imidacloprid-resistant strains and field populations from three different locations in the southwestern USA were compared in systemic uptake bioassays with acetamiprid, dinotefuran, imidacloprid and thiamethoxam. An imidacloprid-resistant strain (IM-R) with 120-fold resistance originally collected from Imperial Valley, California, did not show cross-resistance to acetamiprid, dinotefuran or thiamethoxam. The Guatemala-resistant strain (GU-R) that was also highly resistant to imidacloprid (RR=109-fold) showed low levels of cross-resistance when bioassayed with acetamiprid and thiamethoxam. However, dinotefuran was more toxic than either imidacloprid or thiamethoxam to both IM-R and GU-R strains as indicated by low LC50s. By contrast, a Q-biotype Spanish-resistant strain (SQ-R) of B. tabaci highly resistant to imidacloprid demonstrated high cross-resistance to the two related neonicotinoids. Field populations from Imperial Valley (California), Maricopa and Yuma (Arizona), showed variable susceptibility to imidacloprid (LC50s ranging from 3.39 to 115 microg ml(-1)) but did not exhibit cross-resistance to the three neonicotinoids suggesting that all three compounds would be effective in managing whiteflies. Yuma populations were the most susceptible to imidacloprid. Dinotefuran was the most toxic of the four neonicotinoids against field populations. Although differences in binding at the target site and metabolic pathways may influence the variability in cross-resistance patterns among whitefly populations, comparison of whitefly responses from various geographic regions to the four neonicotinoids indicates the importance of ecological and operational factors on development of cross-resistance to the neonicotinoids.

  11. Impact of neonicotinoid insecticides on natural enemies in greenhouse and interiorscape environments.

    PubMed

    Cloyd, Raymond A; Bethke, James A

    2011-01-01

    The neonicotinoid insecticides imidacloprid, acetamiprid, dinotefuran, thiamethoxam and clothianidin are commonly used in greenhouses and/or interiorscapes (plant interiorscapes and conservatories) to manage a wide range of plant-feeding insects such as aphids, mealybugs and whiteflies. However, these systemic insecticides may also be harmful to natural enemies, including predators and parasitoids. Predatory insects and mites may be adversely affected by neonicotinoid systemic insecticides when they: (1) feed on pollen, nectar or plant tissue contaminated with the active ingredient; (2) consume the active ingredient of neonicotinoid insecticides while ingesting plant fluids; (3) feed on hosts (prey) that have consumed leaves contaminated with the active ingredient. Parasitoids may be affected negatively by neonicotinoid insecticides because foliar, drench or granular applications may decrease host population levels so that there are not enough hosts to attack and thus sustain parasitoid populations. Furthermore, host quality may be unacceptable for egg laying by parasitoid females. In addition, female parasitoids that host feed may inadvertently ingest a lethal concentration of the active ingredient or a sublethal dose that inhibits foraging or egg laying. There are, however, issues that require further consideration, such as: the types of plant and flower that accumulate active ingredients, and the concentrations in which they are accumulated; the influence of flower age on the level of exposure of natural enemies to the active ingredient; the effect of neonicotinoid metabolites produced within the plant. As such, the application of neonicotinoid insecticides in conjunction with natural enemies in protected culture and interiorscape environments needs further investigation. Copyright © 2010 Society of Chemical Industry.

  12. Structural Insights into Intermediate Steps in the Sir2 Deacetylation Reaction

    SciTech Connect

    Hawse, William F.; Hoff, Kevin G.; Fatkins, David G.; Daines, Alison; Zubkova, Olga V.; Schramm, Vern L.; Zheng, Weiping; Wolberger, Cynthia

    2010-07-22

    Sirtuin enzymes comprise a unique class of NAD{sup +}-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD{sup +} analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.

  13. New insights into RNA secondary structure in the alternative splicing of pre-mRNAs.

    PubMed

    Jin, Yongfeng; Yang, Yun; Zhang, Peng

    2011-01-01

    Alternative splicing is an important mechanism in generating proteomic diversity, and RNA secondary structure is an important element in splicing regulation. The use of high-throughput sequencing and other approaches has increased the number of known pre-mRNA secondary structures by several orders of magnitude, and we now have new insights into the role of RNA secondary structure in alternative splicing and the mechanisms involved (e.g., physical competition, long-range RNA pairing, the structural splicing code, and co-transcriptional splicing). Furthermore, an RNA pairing-based mechanism ensures the selection of only one of several available exons (e.g., Dscam splicing). Here we review several recent discoveries related to the role of RNA secondary structure in alternative splicing and the underlying mechanisms.

  14. Natural Products at Work: Structural Insights into Inhibition of the Bacterial Membrane Protein MraY.

    PubMed

    Koppermann, Stefan; Ducho, Christian

    2016-09-19

    Natural(ly) fit: The X-ray crystal structure of the bacterial membrane protein MraY in complex with its natural product inhibitor muraymycin D2 is discussed. MraY catalyzes one of the membrane-associated steps in peptidoglycan biosynthesis and, therefore, represents a promising target for novel antibiotics. Structural insights derived from the protein-inhibitor complex might now pave the way for the development of new antimicrobial drugs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Extracellular regulation of type IIa receptor protein tyrosine phosphatases: mechanistic insights from structural analyses

    PubMed Central

    Coles, Charlotte H.; Jones, E. Yvonne; Aricescu, A. Radu

    2016-01-01

    The receptor protein tyrosine phosphatases (RPTPs) exhibit a wide repertoire of cellular signalling functions. In particular, type IIa RPTP family members have recently been highlighted as hubs for extracellular interactions in neurons, regulating neuronal extension and guidance, as well as synaptic organisation. In this review, we will discuss the recent progress of structural biology investigations into the architecture of type IIa RPTP ectodomains and their interactions with extracellular ligands. Structural insights, in combination with biophysical and cellular studies, allow us to begin to piece together molecular mechanisms for the transduction and integration of type IIa RPTP signals and to propose hypotheses for future experimental validation. PMID:25234613

  16. Atom by atom: HRTEM insights into inorganic nanotubes and fullerene-like structures.

    PubMed

    Bar Sadan, Maya; Houben, Lothar; Enyashin, Andrey N; Seifert, Gotthard; Tenne, Reshef

    2008-10-14

    The characterization of nanostructures down to the atomic scale is essential to understand some physical properties. Such a characterization is possible today using direct imaging methods such as aberration-corrected high-resolution transmission electron microscopy (HRTEM), when iteratively backed by advanced modeling produced by theoretical structure calculations and image calculations. Aberration-corrected HRTEM is therefore extremely useful for investigating low-dimensional structures, such as inorganic fullerene-like particles and inorganic nanotubes. The atomic arrangement in these nanostructures can lead to new insights into the growth mechanism or physical properties, where imminent commercial applications are unfolding. This article will focus on two structures that are symmetric and reproducible. The first structure that will be dealt with is the smallest stable symmetric closed-cage structure in the inorganic system, a MoS(2) nanooctahedron. It is investigated by means of aberration-corrected microscopy which allowed validating the suggested DFTB-MD model. It will be shown that structures diverging from the energetically most stable structures are present in the laser ablated soot and that the alignment of the different shells is parallel, unlike the bulk material where the alignment is antiparallel. These findings correspond well with the high-energy synthetic route and they provide more insight into the growth mechanism. The second structure studied is WS(2) nanotubes, which have already been shown to have a unique structure with very desirable mechanical properties. The joint HRTEM study combined with modeling reveals new information regarding the chirality of the different shells and provides a better understanding of their growth mechanism.

  17. Atom by atom: HRTEM insights into inorganic nanotubes and fullerene-like structures

    PubMed Central

    Sadan, Maya Bar; Houben, Lothar; Enyashin, Andrey N.; Seifert, Gotthard; Tenne, Reshef

    2008-01-01

    The characterization of nanostructures down to the atomic scale is essential to understand some physical properties. Such a characterization is possible today using direct imaging methods such as aberration-corrected high-resolution transmission electron microscopy (HRTEM), when iteratively backed by advanced modeling produced by theoretical structure calculations and image calculations. Aberration-corrected HRTEM is therefore extremely useful for investigating low-dimensional structures, such as inorganic fullerene-like particles and inorganic nanotubes. The atomic arrangement in these nanostructures can lead to new insights into the growth mechanism or physical properties, where imminent commercial applications are unfolding. This article will focus on two structures that are symmetric and reproducible. The first structure that will be dealt with is the smallest stable symmetric closed-cage structure in the inorganic system, a MoS2 nanooctahedron. It is investigated by means of aberration-corrected microscopy which allowed validating the suggested DFTB-MD model. It will be shown that structures diverging from the energetically most stable structures are present in the laser ablated soot and that the alignment of the different shells is parallel, unlike the bulk material where the alignment is antiparallel. These findings correspond well with the high-energy synthetic route and they provide more insight into the growth mechanism. The second structure studied is WS2 nanotubes, which have already been shown to have a unique structure with very desirable mechanical properties. The joint HRTEM study combined with modeling reveals new information regarding the chirality of the different shells and provides a better understanding of their growth mechanism. PMID:18838681

  18. Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations

    PubMed Central

    Zhai, Liting; Feng, Lingling; Xia, Lin; Yin, Huiyong; Xiang, Song

    2016-01-01

    Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170 kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII. PMID:27088557

  19. Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation.

    PubMed

    Olal, Daniel; Daumke, Oliver

    2016-03-08

    Hantaviruses are etiological agents of life-threatening hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. The nucleoprotein (N) of hantavirus is essential for viral transcription and replication, thus representing an attractive target for therapeutic intervention. We have determined the crystal structure of hantavirus N to 3.2 Å resolution. The structure reveals a two-lobed, mostly α-helical structure that is distantly related to that of orthobunyavirus Ns. A basic RNA binding pocket is located at the intersection between the two lobes. We provide evidence that oligomerization is mediated by amino- and C-terminal arms that bind to the adjacent monomers. Based on these findings, we suggest a model for the oligomeric ribonucleoprotein (RNP) complex. Our structure provides mechanistic insights into RNA encapsidation in the genus Hantavirus and constitutes a template for drug discovery efforts aimed at combating hantavirus infections.

  20. Structural insights into functional overlapping and differentiation among myosin V motors.

    PubMed

    Nascimento, Andrey F Z; Trindade, Daniel M; Tonoli, Celisa C C; de Giuseppe, Priscila O; Assis, Leandro H P; Honorato, Rodrigo V; de Oliveira, Paulo S L; Mahajan, Pravin; Burgess-Brown, Nicola A; von Delft, Frank; Larson, Roy E; Murakami, Mario T

    2013-11-22

    Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.

  1. Structural Insights into Functional Overlapping and Differentiation among Myosin V Motors*

    PubMed Central

    Nascimento, Andrey F. Z.; Trindade, Daniel M.; Tonoli, Celisa C. C.; de Giuseppe, Priscila O.; Assis, Leandro H. P.; Honorato, Rodrigo V.; de Oliveira, Paulo S. L.; Mahajan, Pravin; Burgess-Brown, Nicola A.; von Delft, Frank; Larson, Roy E.; Murakami, Mario T.

    2013-01-01

    Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors. PMID:24097982

  2. Structural insights into the assembly and regulation of distinct viral capsid complexes

    PubMed Central

    Sarker, Subir; Terrón, María C.; Khandokar, Yogesh; Aragão, David; Hardy, Joshua M.; Radjainia, Mazdak; Jiménez-Zaragoza, Manuel; de Pablo, Pedro J.; Coulibaly, Fasséli; Luque, Daniel; Raidal, Shane R.; Forwood, Jade K.

    2016-01-01

    The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from the smallest and simplest known viruses capable of autonomously replicating in animal cells, circoviruses, to establish structural and mechanistic insights into capsid morphogenesis and regulation. The beak and feather disease virus, like many circoviruses, encode only two genes: a capsid protein and a replication initiation protein. The capsid protein forms distinct macromolecular assemblies during replication and here we elucidate these structures at high resolution, showing that these complexes reverse the exposure of the N-terminal arginine rich domain responsible for DNA binding and nuclear localization. We show that assembly of these complexes is regulated by single-stranded DNA (ssDNA), and provide a structural basis of capsid assembly around single-stranded DNA, highlighting novel binding interfaces distinct from the highly positively charged N-terminal ARM domain. PMID:27698405

  3. Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation

    PubMed Central

    Lawrence, Sara L.; Feil, Susanne C.; Morton, Craig J.; Farrand, Allison J.; Mulhern, Terrence D.; Gorman, Michael A.; Wade, Kristin R.; Tweten, Rodney K.; Parker, Michael W.

    2015-01-01

    Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface. PMID:26403197

  4. Structure of the cathelicidin motif of protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein.

    PubMed

    Sanchez, Jean-Frédéric; Hoh, François; Strub, Marie-Paule; Aumelas, André; Dumas, Christian

    2002-10-01

    Cathelicidins are a family of antimicrobial proteins isolated from leucocytes and epithelia cells that contribute to the innate host defense mechanisms in mammalians. Located in the C-terminal part of the holoprotein, the cathelicidin-derived antimicrobial peptide is liberated by a specific protease cleavage. Here, we report the X-ray structure of the cathelicidin motif of protegrin-3 solved by MAD phasing using the selenocysteine-labeled protein. Its overall structure represents a fold homologous to the cystatin family and adopts two native states, a monomer, and a domain-swapped dimer. This crystal structure is the first example of a structural characterization of the highly conserved cathelicidin motif and thus provides insights into the possible mechanism of activation of the antimicrobial protegrin peptide.

  5. Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity.

    PubMed

    Bong, Seoung Min; Son, Kka-bi; Yang, Seung-Won; Park, Jae-Won; Cho, Jea-Won; Kim, Kyung-Tae; Kim, Hackyoung; Kim, Seung Jun; Kim, Young Jun; Lee, Byung Il

    2016-01-01

    Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)5R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P2 over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.

  6. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    SciTech Connect

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  7. Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2011-08-01

    The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues. The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  8. Structural insight into dephosphorylation by trehalose 6-phosphate phosphatase (OtsB2) from Mycobacterium tuberculosis.

    PubMed

    Shan, Shan; Min, Haowei; Liu, Ting; Jiang, Dunquan; Rao, Zihe

    2016-12-01

    Trehalose serves as a key structural component in the cell wall of Mycobacterium tuberculosis. M. tuberculosis trehalose-6-phosphate phosphatase (MtbTPP), an essential enzyme in the trehalose biosynthesis OtsAB pathway, catalyzes the dephosphorylation of trehalose-6-phosphate (trehalose-6-P) to generate trehalose, and plays a critical role in M. tuberculosis survival-associated cell wall formation and permeability. Therefore, MtbTPP (OtsB2) is considered a promising potential target for discovery of antimicrobial drugs. However, the absence of structural information of MtbTPP restrains our understanding of its underlying catalytic mechanism. Here, we report the high-resolution crystal structures of apo active MtbTPP and its trehalose-6-P bound complex. The apo structure presents a canonical haloacid dehalogenase superfamily structural fold plus an extra N-terminal domain. The catalytic center is located in a positively charged cleft between the hydrolase domain and the cap domain, demonstrating a highly conserved substrate binding pocket. The role of residues interacting with the substrate in catalysis were probed by site-directed mutagenesis. Asp147, Asp149, Asp330, and Asp331 were found to be pivotal for the enzymatic activity of MtbTPP. The MtbTPP structures reported here provide insight into a key step in the biosynthesis of trehalose, which would facilitate future development of anti-TB therapeutics.-Shan, S., Min, H., Liu, T., Jiang, D., Rao, Z. Structural insight into dephosphorylation by trehalose 6-phosphate phosphatase (OtsB2) from Mycobacterium tuberculosis.

  9. Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris)

    PubMed Central

    Moffat, Christopher; Pacheco, Joao Goncalves; Sharp, Sheila; Samson, Andrew J.; Bollan, Karen A.; Huang, Jeffrey; Buckland, Stephen T.; Connolly, Christopher N.

    2015-01-01

    The global decline in the abundance and diversity of insect pollinators could result from habitat loss, disease, and pesticide exposure. The contribution of the neonicotinoid insecticides (e.g., clothianidin and imidacloprid) to this decline is controversial, and key to understanding their risk is whether the astonishingly low levels found in the nectar and pollen of plants is sufficient to deliver neuroactive levels to their site of action: the bee brain. Here we show that bumblebees (Bombus terrestris audax) fed field levels [10 nM, 2.1 ppb (w/w)] of neonicotinoid accumulate between 4 and 10 nM in their brains within 3 days. Acute (minutes) exposure of cultured neurons to 10 nM clothianidin, but not imidacloprid, causes a nicotinic acetylcholine receptor-dependent rapid mitochondrial depolarization. However, a chronic (2 days) exposure to 1 nM imidacloprid leads to a receptor-dependent increased sensitivity to a normally innocuous level of acetylcholine, which now also causes rapid mitochondrial depolarization in neurons. Finally, colonies exposed to this level of imidacloprid show deficits in colony growth and nest condition compared with untreated colonies. These findings provide a mechanistic explanation for the poor navigation and foraging observed in neonicotinoid treated bumblebee colonies.—Moffat, C., Pacheco, J. G., Sharp, S., Samson, A. J., Bollan, K. A., Huang, J., Buckland, S. T., Connolly, C. N. Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris). PMID:25634958

  10. Neonicotinoid insecticide interact with honeybee odorant-binding protein: Implication for olfactory dysfunction.

    PubMed

    Li, Hongliang; Wu, Fan; Zhao, Lei; Tan, Jing; Jiang, Hongtao; Hu, Fuliang

    2015-11-01

    The decline of bee population has caused great concern in recent years. A noticeable factor points to the neonicotinoid insecticides, which remain in the nectar and pollen of plants and impair the olfactory cognition of foraging bees. However, it remains elusive that if and how neonicotinoid insecticides interact with the olfactory system of bees. Herein, we studied the binding interaction between neonicotinoid imidacloprid and ASP2, one odorant-binding protein in eastern bees, Apis cerana, by multispectroscopic methods. The results indicate that imidacloprid significantly quenched the intrinsic fluorescence of ASP2 as the static quenching mode, and expanded the conformation of ASP2 measured by the circular dichroism (CD) spectra. The acting force is mainly driven by hydrophobic force based on thermodynamic analysis. Docking analysis predicts a formation of a hydrogen bond, while the corresponding site-directed mutagenesis indicated that the hydrogen bond is not main force here. Moreover, imidacloprid with a sublethal dose (0.8ng/bee) clearly decreased the binding affinity of ASP2 to a floral volatile, β-ionone, which had been identified to strongly bind with the wild ASP2 before. This study may benefit to evaluate the effect of neonicotinoid insecticides on the olfactory cognitive behavior of bees involved in the crops pollination. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.).

    PubMed

    Brandt, Annely; Gorenflo, Anna; Siede, Reinhold; Meixner, Marina; Büchler, Ralph

    2016-03-01

    A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24h oral exposure, 200 μg/l or 2000 μg/l) and imidacloprid (1 μg/l or 10 μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50-200 μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity.

  12. Neonicotinoid-induced resurgence of rice leaffolder, Cnaphalocrocis medinalis (Guénee).

    PubMed

    Chintalapati, Padmavathi; Katti, Gururaj; Puskur, Raghuveer Rao; Nagella Venkata, Krishnaiah

    2016-01-01

    Among the neonicotinoids, imidacloprid and thiamethoxam have been frequently used in planthopper endemic areas. Wherever leaffolder incidence occurs along with planthoppers in rice fields, use of neonicotinoids has resulted in increase in leaffolder population. The present study was carried out to verify and confirm the resurgence, as well as to identify factors contributing to resurgence. In imidacloprid- and thiamethoxam-applied plots, a 17.5-217.5% increase in leaffolder population over the untreated control was observed. Neonicotinoids showed moderate toxicity to eggs with <50% hatching, and less toxicity to first instars with >60% survival, while 37-60% of larvae reached adult stage. The larval duration was also reduced. Fecundity was stimulated, with a 6.2-37.21% increase over the untreated control. A significant positive correlation was observed between larval population and total soluble sugars in thiamethoxam treatment (r = 0.9984, P ≤ 0.01). Stimulated fecundity on neonicotinoid-sprayed plants, coupled with reduced larval duration and low egg toxicity, could be the major factors contributing to the upsurge of leaffolder. This study aids in cautioning farmers to be more vigilant while using imidacloprid and thiamethoxam, particularly in rice fields where leaffolder exists alongside planthoppers. © 2015 Society of Chemical Industry.

  13. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

    PubMed Central

    Moffat, Christopher; Buckland, Stephen T.; Samson, Andrew J.; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A.; Huang, Jeffrey T.-J.; Connolly, Christopher N.

    2016-01-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species. PMID:27124107

  14. Rapid analysis of neonicotinoid insecticides in guttation drops of corn seedlings obtained from coated seeds.

    PubMed

    Tapparo, Andrea; Giorio, Chiara; Marzaro, Matteo; Marton, Daniele; Soldà, Lidia; Girolami, Vincenzo

    2011-06-01

    Regarding the hypothesis that neonicotinoid insecticides used for seed coating of agricultural crops - mainly corn, sunflower and seed rape - are related to the extensive death of honey bees, the phenomenon of corn seedling guttation has been recently considered as a possible route of exposure of bees to these systemic insecticides. In the present study, guttation drops of corn plants obtained from commercial seeds coated with thiamethoxam, clothianidin, imidacloprid and fipronil have been analyzed by an optimized fast UHPLC-DAD procedure showing excellent detection limits and accuracy, both adequate for the purpose. The young plants grown both in pots - in greenhouse - and in open field from coated seeds, produced guttation solutions containing high levels of the neonicotinoid insecticides (up to 346 mg L(-1) for imidacloprid, 102 mg L(-1) for clothianidin and 146 mg L(-1) for thiamethoxam). These concentration levels may represent lethal doses for bees that use guttation drops as a source of water. The neonicotinoid concentrations in guttation drops progressively decrease during the first 10-15 days after the emergence of the plant from the soil. Otherwise fipronil, which is a non-systemic phenylpyrazole insecticide, was never detected into guttation drops. Current results confirm that the physiological fluids of the corn plant can effectively transfer neonicotinoid insecticides from the seed onto the surface of the leaves, where guttation drops may expose bees and other insects to elevated doses of neurotoxic insecticides.

  15. Neonicotinoid-contaminated pollinator strips adjacent to cropland reduce honey bee nutritional status

    USDA-ARS?s Scientific Manuscript database

    Worldwide pollinator declines are attributed to a number of factors, including pesticide exposures. Neonicotinoid insecticides specifically have been detected in surface waters, non-target vegetation, and bee products, but the risks posed by environmental exposures are still not well understood. Pol...

  16. Activity of selected neonicotinoids and dicrotophos on nontarget arthropods in cotton: implications in insect management.

    PubMed

    Kilpatrick, A L; Hagerty, A M; Turnipseed, S G; Sullivan, M J; Bridges, W C

    2005-06-01

    Certain neonicotinoids are used in cotton, Gossypium hirsutum (L.), to control various piercing-sucking pests. We conducted field studies using three neonicotinoids (acetamiprid, thiamethoxam, and imidacloprid) and an organophosphate (dicrotophos) to assess the activity of these insecticides against nontarget arthropods, particularly predators, and to determine the potential economic consequences of such activity. Mortality among populations of the big-eyed bug, Geocoris punctipes (Say), and the red imported fire ant, Solenopsis invicta Buren, was highest after thiamethoxam and dicrotophos treatments. Numbers of arachnids were consistently lower after dicrotophos treatments, whereas none of the neonicotinoids caused appreciable mortality. Total predators in pooled data from five separate studies revealed that numbers, compared with untreated plots, were reduced by -75% in dicrotophos, 55-60% in thiamethoxam, and only 30% in both acetamiprid and imidacloprid plots. Acetamiprid and thiamethoxam exhibited significant mortality against field-deposited eggs of bollworm, Helicoverpa zea (Boddie). Both thiamethoxam and dicrotophos plots exhibited bollworm numbers that were approximately three times higher than treatment thresholds (three per 100 plants), whereas numbers in untreated plots were below threshold levels. In one study on Bt cotton, a significant negative correlation was observed between numbers of predators and bollworm larvae. Results demonstrated that neonicotinoids differ in activity against predaceous arthropods and bollworm eggs and that high predator mortality can result in resurgence of bollworm larvae and additional insecticide costs.

  17. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees.

    PubMed

    Moffat, Christopher; Buckland, Stephen T; Samson, Andrew J; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A; Huang, Jeffrey T-J; Connolly, Christopher N

    2016-04-28

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.

  18. Field-Evolved Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Carbodiimide and Neonicotinoids in Pakistan.

    PubMed

    Ahmad, Mushtaq; Khan, Rashid A

    2017-03-03

    The evolution of resistance to carbodiimide (a toxic metabolite of diafenthiuron) and four neonicotinoids imidacloprid, acetamiprid, thiamethoxam, and thiacloprid in the Pakistani populations of sweetpotato whitefly (Bemisia tabaci Gennadius) was monitored from 1996 to 2015 using a leaf-dip bioassay. Diafenthiuron, imidacloprid, and acetamiprid were introduced into Pakistani agriculture in mid-1990s and heavily used since then, because B. tabaci resistance and consequently control failures to conventional insecticides such as organophosphates, carbamates, and pyrethroids were widespread during the 1990s. According to the current studies, resistance to carbodiimide, imidacloprid, and acetamiprid during 1996-2010 and to thiamethoxam during 1999-2007 remained very low, but then it rose sharply, and by the year 2015, the B. tabaci resistance increased to very high levels. Among neonicotinoids, thiacloprid was the latest introduction in Pakistan in 2002. There was no thiacloprid resistance in 2002 and 2003, a low to moderate resistance during 2004-2006, and a very high resistance during 2007-2010 that even exceeded resistance to previous neonicotinoids. We may conclude that diafenthiuron and neonicotinoids remained effective against B. tabaci for 15 yr following their intensive use under field conditions, before a significant resistance, leading to their field failures, occurred in Pakistan.

  19. Alternatives to neonicotinoid insecticides for pest control: case studies in agriculture and forestry.

    PubMed

    Furlan, Lorenzo; Kreutzweiser, David

    2015-01-01

    Neonicotinoid insecticides are widely used for control of insect pests around the world and are especially pervasive in agricultural pest management. There is a growing body of evidence indicating that the broad-scale and prophylactic uses of neonicotinoids pose serious risks of harm to beneficial organisms and their ecological function. This provides the impetus for exploring alternatives to neonicotinoid insecticides for controlling insect pests. We draw from examples of alternative pest control options in Italian maize production and Canadian forestry to illustrate the principles of applying alternatives to neonicotinoids under an integrated pest management (IPM) strategy. An IPM approach considers all relevant and available information to make informed management decisions, providing pest control options based on actual need. We explore the benefits and challenges of several options for management of three insect pests in maize crops and an invasive insect pest in forests, including diversifying crop rotations, altering the timing of planting, tillage and irrigation, using less sensitive crops in infested areas, applying biological control agents, and turning to alternative reduced risk insecticides. Continued research into alternatives is warranted, but equally pressing is the need for information transfer and training for farmers and pest managers and the need for policies and regulations to encourage the adoption of IPM strategies and their alternative pest control options.

  20. Compatibility of Two Systematic Neonicotinoids, Imidacloprid and Thiamethoxam with various Natural Enemies of Agricultural Pests.

    USDA-ARS?s Scientific Manuscript database

    Two systemic neonicotinoids, imidacloprid and thiamethoxam, are widely used for residual control of a number of insect pests in cotton, vegetables, and citrus. We evaluated their impact on six species of beneficial arthropods including four parasitoid species, Aphytis melinus Gonatocerus ashmeadi, ...

  1. Responses of neonicotinoid resistant and susceptible Frankliniella fusca life stages to multiple insecticide groups in cotton.

    PubMed

    Huseth, Anders S; D'Ambrosio, Damon A; Kennedy, George G

    2017-10-01

    Detection of neonicotinoid resistance in populations of tobacco thrips, Frankliniella fusca Hinds, throughout the southeastern USA has motivated an examination of alternative insecticides to control problematic infestations on seedling cotton. The objective of this study was to refine understanding of stage-specific mortality and reduced oviposition of several common insecticides (acephate, abamectin, cyantraniliprole, spinetoram, imidacloprid, imidacloprid+thiodicarb, thiamethoxam) on neonicotinoid resistant and susceptible F. fusca populations under laboratory and field conditions. Laboratory studies revealed that the average number of eggs per female and larval or adult survivorship responses differed by insecticide and were dependent on the resistance status of the population. In the presence of neonicotinoids, the resistant F. fusca populations exhibited lower mortality and higher egg counts than the susceptible population. In the field study, similar patterns of oviposition suppression were observed, indicating that some insecticides may impact reproductive rate. This study shows that insecticides have different effects on F. fusca oviposition events, larval and adult mortality that are dependent on neonicotinoid resistance status. Because insecticides tested in this study have varied activity on specific F. fusca life stages (e.g. oviposition suppression, larvicidal activity, adulticidal activity), knowledge of stage-specific activity can be used to improve control and enhance long-term product stewardship. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Neonicotinoids act like endocrine disrupting chemicals in newly-emerged bees and winter bees.

    PubMed

    Baines, Danica; Wilton, Emily; Pawluk, Abbe; de Gorter, Michael; Chomistek, Nora

    2017-09-08

    Accumulating evidence suggests that neonicotinoids may have long-term adverse effects on bee health, yet our understanding of how this could occur is incomplete. Pesticides can act as endocrine disrupting chemicals (EDCs) in animals providing characteristic multiphasic dose-response curves and non-lethal endpoints in toxicity studies. However, it is not known if neonicotinoids act as EDCs in bees. To address this issue, we performed oral acute and chronic toxicity studies including concentrations recorded in nectar and pollen, applying acetamiprid, clothianidin, imidacloprid, and thiamethoxam to bumble bees, honey bees and leafcutter bees, the three most common bee species managed for pollination. In acute toxicity studies, late-onset symptoms, such as ataxia, were recorded as non-lethal endpoints for all three bee species. Clothianidin and thiamethoxam produced biphasic dose-response curves for all three bee species. Clothianidin and thiamethoxam were extremely toxic to winter worker honey bees prior to brood production in spring, making this the most sensitive bee stage identified to date. Chronic exposure to field-realistic levels of neonicotinoids reduced bee survival and caused significant late-onset symptoms for all three bee species. Given these findings, neonicotinoid risk should be reevaluated to address the EDC-like behavior and the sensitivity of winter worker honey bees.

  3. Neonicotinoid Residues in Wildflowers, a Potential Route of Chronic Exposure for Bees.

    PubMed

    Botías, Cristina; David, Arthur; Horwood, Julia; Abdul-Sada, Alaa; Nicholls, Elizabeth; Hill, Elizabeth; Goulson, Dave

    2015-11-03

    In recent years, an intense debate about the environmental risks posed by neonicotinoids, a group of widely used, neurotoxic insecticides, has been joined. When these systemic compounds are applied to seeds, low concentrations are subsequently found in the nectar and pollen of the crop, which are then collected and consumed by bees. Here we demonstrate that the current focus on exposure to pesticides via the crop overlooks an important factor: throughout spring and summer, mixtures of neonicotinoids are also found in the pollen and nectar of wildflowers growing in arable field margins, at concentrations that are sometimes even higher than those found in the crop. Indeed, the large majority (97%) of neonicotinoids brought back in pollen to honey bee hives in arable landscapes was from wildflowers, not crops. Both previous and ongoing field studies have been based on the premise that exposure to neonicotinoids would occur only during the blooming period of flowering crops and that it may be diluted by bees also foraging on untreated wildflowers. Here, we show that exposure is likely to be higher and more prolonged than currently recognized because of widespread contamination of wild plants growing near treated crops.

  4. Effects of Neonicotinoids and Crop Rotation for Managing Wireworms in Wheat Crops.

    PubMed

    Esser, Aaron D; Milosavljević, Ivan; Crowder, David W

    2015-08-01

    Soil-dwelling insects are severe pests in many agroecosystems. These pests have cryptic life cycles, making sampling difficult and damage hard to anticipate. The management of soil insects is therefore often based on preventative insecticides applied at planting or cultural practices. Wireworms, the subterranean larvae of click beetles (Coleoptera: Elateridae), have re-emerged as problematic pests in cereal crops in the Pacific Northwestern United States. Here, we evaluated two management strategies for wireworms in long-term field experiments: 1) treating spring wheat seed with the neonicotinoid thiamethoxam and 2) replacing continuous spring wheat with a summer fallow and winter wheat rotation. Separate experiments were conducted for two wireworm species--Limonius californicus (Mannerheim) and Limonius infuscatus (Motschulsky). In the experiment with L. californicus, spring wheat yields and economic returns increased by 24-30% with neonicotinoid treatments. In contrast, in the experiment with L. infuscatus, spring wheat yields and economic returns did not increase with neonicotinoids despite an 80% reduction in wireworms. Thus, the usefulness of seed-applied neonicotinoids differed based on the wireworm species present. In experiments with both species, we detected significantly fewer wireworms with a no-till summer fallow and winter wheat rotation compared with continuous spring wheat. This suggests that switching from continuous spring wheat to a winter wheat and summer fallow rotation may aid in wireworm management. More generally, our results show that integrated management of soil-dwelling pests such as wireworms may require both preventative insecticide treatments and cultural practices.

  5. Enantioselective absorption and transformation of a novel chiral neonicotinoid [(14)C]-cycloxaprid in rats.

    PubMed

    Wu, Chengchen; Huang, Lei; Tang, Shenghua; Li, Zhong; Ye, Qingfu

    2016-06-01

    Neonicotinoid pesticides caused hazardous effects on pollinators and aquatic ecosystem. The new developed chiral cis-neonicotinoid cycloxaprid(CYC) is a highly potent substitute for low toxicity to bees and high efficiency on target-insects, but little is known about the metabolic dynamics of racemic CYC and its 2 enantiomers(SR and RS) in animal models. In this study, chiral separation of (14)C-labeled racemic CYC was performed in high-performance liquid chromatography under optimal conditions. For the first time that the stereoselectivity of the chiral neonicotinoid insecticide CYC was exhibited in rats after single dose oral administration using (14)C-labeled isotope trace technique. Enantioselective behaviors of racemic CYC, SR and RS were observed in blood metabolism, tissue distribution and excretion. The major deposition of (14)C were found in liver, lung, kidney and heart. After 24 h, skin and fat showed a strong bioaccumulation effect, and total excreted urine and feces of CYC, SR and RS were 50.4%, 59.7% and 74.5%, respectively. Enantiomer RS had the fastest absorption and elimination rates, and it was least bioaccumulated in rats. The results provide scientific basis and practical techniques for environmental risk assessment of chiral pesticides, especially neonicotinoids. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

    NASA Astrophysics Data System (ADS)

    Moffat, Christopher; Buckland, Stephen T.; Samson, Andrew J.; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A.; Huang, Jeffrey T.-J.; Connolly, Christopher N.

    2016-04-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.

  7. Design, multicomponent synthesis, and bioactivities of novel neonicotinoid analogues with 1,4-dihydropyridine scaffold.

    PubMed

    Zhang, Wenwen; Yang, Xiaobao; Chen, Weidong; Xu, Xiaoyong; Li, Lu; Zhai, Hongbin; Li, Zhong

    2010-03-10

    Novel neonicotinoid analogues bearing a 1,4-dihydropridine scaffold were designed and synthesized by multicomponent reactions (MCRs) to enhance pi-pi stacking. The synthesized compounds were identified by (1)H NMR, (13)C NMR, high-resolution mass spectroscopy, and elemental analysis. Bioassay tests showed that some of them exhibited high insecticidal activities against pea aphid ( Aphis craccivora ).

  8. Widespread occurrence of neonicotinoid insecticides in streams in a high corn and soybean producing region, USA.

    PubMed

    Hladik, Michelle L; Kolpin, Dana W; Kuivila, Kathryn M

    2014-10-01

    Neonicotinoid insecticides are of environmental concern, but little is known about their occurrence in surface water. An area of intense corn and soybean production in the Midwestern United States was chosen to study this issue because of the high agricultural use of neonicotinoids via both seed treatments and other forms of application. Water samples were collected from nine stream sites during the 2013 growing season. The results for the 79 water samples documented similar patterns among sites for both frequency of detection and concentration (maximum:median) with clothianidin (75%, 257 ng/L:8.2 ng/L) > thiamethoxam (47%, 185 ng/L:<2 ng/L) > imidacloprid (23%, 42.7 ng/L: <2 ng/L). Neonicotinoids were detected at all nine sites sampled even though the basin areas spanned four orders of magnitude. Temporal patterns in concentrations reveal pulses of neonicotinoids associated with rainfall events during crop planting, suggesting seed treatments as their likely source.

  9. Crystal structure of microsomal prostaglandin E2 synthase provides insight into diversity in the MAPEG superfamily

    PubMed Central

    Sjögren, Tove; Nord, Johan; Ek, Margareta; Johansson, Patrik; Liu, Gang; Geschwindner, Stefan

    2013-01-01

    Prostaglandin E2 (PGE2) is a key mediator in inflammatory response. The main source of inducible PGE2, microsomal PGE2 synthase-1 (mPGES-1), has emerged as an interesting drug target for treatment of pain. To support inhibitor design, we have determined the crystal structure of human mPGES-1 to 1.2 Å resolution. The structure reveals three well-defined active site cavities within the membrane-spanning region in each monomer interface of the trimeric structure. An important determinant of the active site cavity is a small cytosolic domain inserted between transmembrane helices I and II. This extra domain is not observed in other structures of proteins within the MAPEG (Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily but is likely to be present also in microsomal GST-1 based on sequence similarity. An unexpected feature of the structure is a 16-Å-deep cone-shaped cavity extending from the cytosolic side into the membrane-spanning region. We suggest a potential role for this cavity in substrate access. Based on the structure of the active site, we propose a catalytic mechanism in which serine 127 plays a key role. We have also determined the structure of mPGES-1 in complex with a glutathione-based analog, providing insight into mPGES-1 flexibility and potential for structure-based drug design. PMID:23431194

  10. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    SciTech Connect

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-04-01

    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  11. Structure of Protein Related to DAN and Cerberus (PRDC): Insights into the Mechanism of BMP Antagonism

    PubMed Central

    Nolan, Kristof; Kattamuri, Chandramohan; Luedeke, David M.; Deng, Andy; Jagpal, Amrita; Zhang, Fuming; Linhardt, Robert; Kenny, Alan P.; Zorn, Aaron M.; Thompson, Thomas B.

    2013-01-01

    Summary The Bone Morphogenetic Proteins (BMP) are secreted ligands largely known for their functional roles in embryogenesis and tissue development. A number of structurally diverse extracellular antagonists inhibit BMP ligands to regulate signaling. The DAN family of antagonists represents the largest group of BMP inhibitors, however, little is known for how they mechanistically inhibit BMP ligands. Here, we present the structure of the DAN family member Protein Related to Dan and Cerberus (PRDC) solved by X-ray crystallography. The structure reveals an unexpected growth factor-like appearance with a novel dimerization mechanism that is formed through extensive β-strand contacts. Using site-directed mutagenesis coupled with in vitro and in vivo activity assays, we identified a BMP binding epitope on PRDC. We also determined that PRDC binds heparin with high affinity and that heparin binding to PRDC interferes with BMP antagonism. These results offer insight for how DAN family antagonists functionally inhibit BMP ligands. PMID:23850456

  12. Crystal structure of a claudin provides insight into the architecture of tight junctions.

    PubMed

    Suzuki, Hiroshi; Nishizawa, Tomohiro; Tani, Kazutoshi; Yamazaki, Yuji; Tamura, Atsushi; Ishitani, Ryuichiro; Dohmae, Naoshi; Tsukita, Sachiko; Nureki, Osamu; Fujiyoshi, Yoshinori

    2014-04-18

    Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

  13. New insights into the evolution and structure of Colletotrichum plant-like subtilisins (CPLSs)

    PubMed Central

    Armijos Jaramillo, Vinicio D; Vargas, Walter A; Sukno, Serenella A; Thon, Michael R

    2013-01-01

    The Colletotrichum plant-like subtilisins (CPLSs) are a family of proteins found only in species of the phytopathogenic fungus Colletotrichum. CPLSs have high similarity to plant subtilisins and our previous work has shown that they were acquired by an ancient horizontal gene transfer event from plants. The rapid growth of sequence data in public databases enabled us to reexamine the structure and evolution of the CPLSs. A new plant subtilisin structural model aided us in refining the tertiary structure of CPLSs. Also, new information about protein interactions of plant subtilisin has provided new insights into the putative function of CPLSs. The availability of new genome sequences of members of the genus Colletotrichum gave us the opportunity to further validate our hypothesis that the CPLSs are unique to the Colletotrichum lineage. Together, this information furthers our knowledge of the potential role of the CPLSs in pathogenicity and the role of HGT in the genome evolution of plant pathogenic fungi. PMID:24563701

  14. The structure of phosphoinositide phosphatases: Insights into substrate specificity and catalysis

    PubMed Central

    Hsu, FoSheng; Mao, Yuxin

    2014-01-01

    Phosphoinositides (PIs) are a group of key signaling and structural lipid molecules involved in a myriad of cellular processes. PI phosphatases, together with PI kinases, are responsible for the conversion of PIs between distinctive phosphorylation states. PI phosphatases are a large collection of enzymes that are evolved from at least two disparate ancestors. One group is distantly related to endonucleases, which applies divalent metal ions for phosphoryl transfer. The other group is related to protein tyrosine phosphatases, which contains a highly conserved active site motif Cys-X5-Arg (CX5R). In this review, we focus on structural insights to illustrate current understandings of the molecular mechanisms of each PI phosphatase family, with emphasis on their structural basis for substrate specificity determinants and catalytic mechanisms. PMID:25264170

  15. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

    NASA Astrophysics Data System (ADS)

    Roccatano, Danilo

    2015-07-01

    The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions.

  16. Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2011-07-19

    The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  17. The structure of phosphoinositide phosphatases: Insights into substrate specificity and catalysis.

    PubMed

    Hsu, FoSheng; Mao, Yuxin

    2015-06-01

    Phosphoinositides (PIs) are a group of key signaling and structural lipid molecules involved in a myriad of cellular processes. PI phosphatases, together with PI kinases, are responsible for the conversion of PIs between distinctive phosphorylation states. PI phosphatases are a large collection of enzymes that are evolved from at least two disparate ancestors. One group is distantly related to endonucleases, which apply divalent metal ions for phosphoryl transfer. The other group is related to protein tyrosine phosphatases, which contain a highly conserved active site motif Cys-X5-Arg (CX5R). In this review, we focus on structural insights to illustrate current understandings of the molecular mechanisms of each PI phosphatase family, with emphasis on their structural basis for substrate specificity determinants and catalytic mechanisms. This article is part of a Special Issue entitled Phosphoinositides.

  18. Structural insights into the role of domain flexibility in human DNA ligase IV.

    PubMed

    Ochi, Takashi; Wu, Qian; Chirgadze, Dimitri Y; Grossmann, J Günter; Bolanos-Garcia, Victor M; Blundell, Tom L

    2012-07-03

    Knowledge of the architecture of DNA ligase IV (LigIV) and interactions with XRCC4 and XLF-Cernunnos is necessary for understanding its role in the ligation of double-strand breaks during nonhomologous end joining. Here we report the structure of a subdomain of the nucleotidyltrasferase domain of human LigIV and provide insights into the residues associated with LIG4 syndrome. We use this structural information together with the known structures of the BRCT/XRCC4 complex and those of LigIV orthologs to interpret small-angle X-ray scattering of LigIV in complex with XRCC4 and size exclusion chromatography of LigIV, XRCC4, and XLF-Cernunnos. Our results suggest that the flexibility of the catalytic region is limited in a manner that affects the formation of the LigIV/XRCC4/XLF-Cernunnos complex.

  19. Structural Insights into Substrate Binding of Brown Spider Venom Class II Phospholipases D.

    PubMed

    Coronado, M A; Ullah, A; da Silva, L S; Chaves-Moreira, D; Vuitika, L; Chaim, O M; Veiga, S S; Chahine, J; Murakami, M T; Arni, R K

    2015-01-01

    Phospholipases D (PLDs), the major dermonecrotic factors from brown spider venoms, trigger a range of biological reactions both in vitro and in vivo. Despite their clinical relevance in loxoscelism, structural data is restricted to the apo-form of these enzymes, which has been instrumental in understanding the functional differences between the class I and II spider PLDs. The crystal structures of the native class II PLD from Loxosceles intermedia complexed with myo-inositol 1-phosphate and the inactive mutant H12A complexed with fatty acids indicate the existence of a strong ligand-dependent conformation change of the highly conserved aromatic residues, Tyr 223 and Trp225 indicating their roles in substrate binding. These results provided insights into the structural determinants for substrate recognition and binding by class II PLDs.

  20. M2 protein from influenza A: from multiple structures to biophysical and functional insights.

    PubMed

    Cross, Timothy A; Dong, Hao; Sharma, Mukesh; Busath, David D; Zhou, Huan-Xiang

    2012-04-01

    The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.

  1. Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases.

    PubMed

    Bloudoff, Kristjan; Alonzo, Diego A; Schmeing, T Martin

    2016-03-17

    Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Three dimensional structural insight of laser drilled orifices in osmotic pump tablets.

    PubMed

    Wu, Li; Wang, Lebing; Wang, Shuxia; Xiao, Tiqiao; Chen, Min; Shao, Qun; York, Peter; Singh, Vikaramjeet; Yin, Xianzhen; Gu, Jingkai; Zhang, Jiwen

    2016-10-10

    The orifice drilled in the membrane as a channel for drug delivery is the key functional part of the osmotic pumps for a controlled drug release system. Reported conventional microscopic evaluations of these orifices have been limited to measurement of two-dimensional cross-section diameters. This study was aimed at establishing a novel method to measure quantitatively the three-dimensional architectures of orifices based on synchrotron radiation X-ray microcomputed tomography (SR-μCT). Quantitative analysis of architectures extracted from captopril osmotic pumps drilled by a range of operating parameters indicated that laser power correlated with the cross section area, volume, surface area and depth of the orifices, while scanning speed of laser beam showed inverse relationships with the above structure characters. The synchrotron radiation based Fourier transform infrared microspectroscopy mapping showed that there was no apparent chemical change in the surrounding area of the orifice compared with the normal membrane region. Thus SR-μCT was successfully applied to marketed felodipine osmotic pumps for architectural evaluation of the orifices. In conclusion, the first three-dimensional structural insight of orifices in osmotic pump tablets by SR-μCT and structural reconstruction for the architectures has provided deeper insight into improving the design of advanced osmotic pumps for controlled drug release. Copyright © 2016. Published by Elsevier B.V.

  3. The core structure of Mars as expected to be seen by InSight's VBB seismometer

    NASA Astrophysics Data System (ADS)

    Hempel, Stefanie; Garcia, Raphael; Wieczorek, Mark; Murdoch, Naomi

    2016-04-01

    The question of the Martian core concerns our basic understanding of the planet's thermal evolution, dynamo models for the past and present, the composition of the Martian mantle, especially in regards to its iron content and prevalent phase transitions, which in turn constrain possible regimes of mantle convection. So far the (outer) core radius of Mars is uncertain to about 250 kilometers (Sohl et al., 2005), and evidence neither supports nor falsifies the existence of an inner core (Defraigne et al., 2003). We apply our extensions of the ray tracing toolbox TauP (Crotwell et al., 1999) to compute amplitude loss, ellipticity, crustal and topography corrections in combination with existing models of seismic activity on Mars (Golombek, 1992, Knapmeyer et al., 2006), crustal thickness models (Wieczorek, 2007) and structure models (e.g. Okal and Anderson, 1978, Zharkov and Gudkova, 2000, Rivoldini et al., 2011). In preparation for NASA's discovery mission InSight, we simulate the detected relative travel-time curves at a single seismic station in Elysium Planitia for several models of Martian structure, seismicity, environmental and instrumental noise. We discuss possibilities and difficulties of considering the effects of Martian ellipticity and topography up to degree 8 and 30, respectively. Furthermore, we demonstrate the effect of low velocity layers, as well as the relevance of modeling the effects of ellipticity and crustal thickness during the interpretation of seismic data acquired by InSight's SEIS instrument on Mars, especially concerning seismic phases which provide direct evidence on the core structure of Mars.

  4. Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: a review.

    PubMed

    Morrissey, Christy A; Mineau, Pierre; Devries, James H; Sanchez-Bayo, Francisco; Liess, Matthias; Cavallaro, Michael C; Liber, Karsten

    2015-01-01

    Neonicotinoids, broad-spectrum systemic insecticides, are the fastest growing class of insecticides worldwide and are now registered for use on hundreds of field crops in over 120 different countries. The environmental profile of this class of pesticides indicate that they are persistent, have high leaching and runoff potential, and are highly toxic to a wide range of invertebrates. Therefore, neonicotinoids represent a significant risk to surface waters and the diverse aquatic and terrestrial fauna that these ecosystems support. This review synthesizes the current state of knowledge on the reported concentrations of neonicotinoids in surface waters from 29 studies in 9 countries world-wide in tandem with published data on their acute and chronic toxicity to 49 species of aquatic insects and crustaceans spanning 12 invertebrate orders. Strong evidence exists that water-borne neonicotinoid exposures are frequent, long-term and at levels (geometric means=0.13μg/L (averages) and 0.63μg/L (maxima)) which commonly exceed several existing water quality guidelines. Imidacloprid is by far the most widely studied neonicotinoid (66% of the 214 toxicity tests reviewed) with differences in sensitivity among aquatic invertebrate species ranging several orders of magnitude; other neonicotinoids display analogous modes of action and similar toxicities, although comparative data are limited. Of the species evaluated, insects belonging to the orders Ephemeroptera, Trichoptera and Diptera appear to be the most sensitive, while those of Crustacea (although not universally so) are less sensitive. In particular, the standard test species Daphnia magna appears to be very tolerant, with 24-96hour LC50 values exceeding 100,000μg/L (geometric mean>44,000μg/L), which is at least 2-3 orders of magnitude higher than the geometric mean of all other invertebrate species tested. Overall, neonicotinoids can exert adverse effects on survival, growth, emergence, mobility, and behavior of many

  5. Splitsville: structural and functional insights into the dynamic bacterial Z ring

    PubMed Central

    Haeusser, Daniel P.; Margolin, William

    2017-01-01

    Preface Bacteria must divide in order to increase in number and colonize their niche. Binary fission is the most widespread means of bacterial cell division, but even this relatively simple mechanism displays many variations on a theme. In most bacteria, the tubulin homolog FtsZ assembles into a ring structure (Z ring) at the site of cytokinesis and recruits additional proteins to form a large protein machine (divisome) that spans the membrane. Here we discuss current insights into the regulation of Z ring assembly and how the divisome drives membrane invagination and septal cell wall growth while still flexibly responding to various cellular inputs. PMID:27040757

  6. Structural and compositional heterogeneities in liquid aluminosilicate: insight from a grain structure model

    NASA Astrophysics Data System (ADS)

    Van Nguyen, Hong; Tran, Duong Thuy; Pham, Hung Khac

    2017-02-01

    Network structure as well as structural and compositional heterogeneities in aluminosilicate (Al2O3-2SiO2) under compression is investigated by analysis and visualization of simulation data. Structural and compositional heterogeneities are clarified through analysis of topology structure and size distribution of TO x -clusters ( T = Si, Al; x = 3, 4, 5, 6) as well as O T y -clusters ( y = 2, 3, 4). The TO x -cluster can be considered as TO x -grains. It appears that the structure of aluminosilicate is the mixture of TO x -grains with a different short-range order structure and this is the origin of structural heterogeneity. Regarding their composition, the OSi y - and OAl y -clusters can be considered as silica- and alumina-grains respectively, and the structure of aluminosilicate can thus be considered to be formed from silica- and alumina-grains. This results in compositional heterogeneity. Moreover, the degree of polymerization and polyamorphism as well as dynamic heterogeneity is also discussed in detail.

  7. Structural insights into the nucleotide base specificity of P2X receptors

    PubMed Central

    Kasuya, Go; Fujiwara, Yuichiro; Tsukamoto, Hisao; Morinaga, Satoshi; Ryu, Satoshi; Touhara, Kazushige; Ishitani, Ryuichiro; Furutani, Yuji; Hattori, Motoyuki; Nureki, Osamu

    2017-01-01

    P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors. PMID:28332633

  8. Structural characterisation of Tpx from Yersinia pseudotuberculosis reveals insights into the binding of salicylidene acylhydrazide compounds.

    PubMed

    Gabrielsen, Mads; Beckham, Katherine S H; Feher, Victoria A; Zetterström, Caroline E; Wang, Dai; Müller, Sylke; Elofsson, Mikael; Amaro, Rommie E; Byron, Olwyn; Roe, Andrew J

    2012-01-01

    Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for "forced" reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds.

  9. Structural Characterisation of Tpx from Yersinia pseudotuberculosis Reveals Insights into the Binding of Salicylidene Acylhydrazide Compounds

    PubMed Central

    Feher, Victoria A.; Zetterström, Caroline E.; Wang, Dai; Müller, Sylke; Elofsson, Mikael; Amaro, Rommie E.; Byron, Olwyn; Roe, Andrew J.

    2012-01-01

    Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for “forced” reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds. PMID:22384182

  10. Neonicotinoid-contaminated puddles of water represent a risk of intoxication for honey bees.

    PubMed

    Samson-Robert, Olivier; Labrie, Geneviève; Chagnon, Madeleine; Fournier, Valérie

    2014-01-01

    In recent years, populations of honey bees and other pollinators have been reported to be in decline worldwide. A number of stressors have been identified as potential contributing factors, including the extensive prophylactic use of neonicotinoid insecticides, which are highly toxic to bees, in agriculture. While multiple routes of exposure to these systemic insecticides have been documented for honey bees, contamination from puddle water has not been investigated. In this study, we used a multi-residue method based on LC-MS/MS to analyze samples of puddle water taken in the field during the planting of treated corn and one month later. If honey bees were to collect and drink water from these puddles, our results showed that they would be exposed to various agricultural pesticides. All water samples collected from corn fields were contaminated with at least one neonicotinoid compound, although most contained more than one systemic insecticide. Concentrations of neonicotinoids were higher in early spring, indicating that emission and drifting of contaminated dust during sowing raises contamination levels of puddles. Although the overall average acute risk of drinking water from puddles was relatively low, concentrations of neonicotinoids ranged from 0.01 to 63 µg/L and were sufficient to potentially elicit a wide array of sublethal effects in individuals and colony alike. Our results also suggest that risk assessment of honey bee water resources underestimates the foragers' exposure and consequently miscalculates the risk. In fact, our data shows that honey bees and native pollinators are facing unprecedented cumulative exposure to these insecticides from combined residues in pollen, nectar and water. These findings not only document the impact of this route of exposure for honey bees, they also have implications for the cultivation of a wide variety of crops for which the extensive use of neonicotinoids is currently promoted.

  11. Neonicotinoid-Contaminated Puddles of Water Represent a Risk of Intoxication for Honey Bees

    PubMed Central

    Samson-Robert, Olivier; Labrie, Geneviève; Chagnon, Madeleine; Fournier, Valérie

    2014-01-01

    In recent years, populations of honey bees and other pollinators have been reported to be in decline worldwide. A number of stressors have been identified as potential contributing factors, including the extensive prophylactic use of neonicotinoid insecticides, which are highly toxic to bees, in agriculture. While multiple routes of exposure to these systemic insecticides have been documented for honey bees, contamination from puddle water has not been investigated. In this study, we used a multi-residue method based on LC-MS/MS to analyze samples of puddle water taken in the field during the planting of treated corn and one month later. If honey bees were to collect and drink water from these puddles, our results showed that they would be exposed to various agricultural pesticides. All water samples collected from corn fields were contaminated with at least one neonicotinoid compound, although most contained more than one systemic insecticide. Concentrations of neonicotinoids were higher in early spring, indicating that emission and drifting of contaminated dust during sowing raises contamination levels of puddles. Although the overall average acute risk of drinking water from puddles was relatively low, concentrations of neonicotinoids ranged from 0.01 to 63 µg/L and were sufficient to potentially elicit a wide array of sublethal effects in individuals and colony alike. Our results also suggest that risk assessment of honey bee water resources underestimates the foragers' exposure and consequently miscalculates the risk. In fact, our data shows that honey bees and native pollinators are facing unprecedented cumulative exposure to these insecticides from combined residues in pollen, nectar and water. These findings not only document the impact of this route of exposure for honey bees, they also have implications for the cultivation of a wide variety of crops for which the extensive use of neonicotinoids is currently promoted. PMID:25438051

  12. Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist

    PubMed Central

    Shao, Xusheng; Xia, Shanshan; Durkin, Kathleen A.; Casida, John E.

    2013-01-01

    The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [3H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [3H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site. PMID:24108354

  13. Monitoring changes in Bemisia tabaci (Hemiptera: Aleyrodidae) susceptibility to neonicotinoid insecticides in Arizona and California.

    PubMed

    Castle, S J; Prabhaker, N

    2013-06-01

    Bemisia tabaci (Gennadius) biotype B is a highly prolific and polyphagous whitefly that established in much of North America during the 1980s. Neonicotinoid insecticides have been fundamental in regaining control over outbreak populations of B. tabaci, but resistance threatens their sustainability. Susceptibility of B. tabaci in the southwestern United States to four neonicotinoid insecticides varied considerably across populations within each year over a 3 yr period. Using a variability ratio of highest LC50 to lowest LC50 in field-collected whitefly adults from Arizona and California, the ranges of LC50(s) across all tests within compounds were highest to imidacloprid and lowest to thiamethoxam. Patterns of susceptibility were similar among all four neonicotinoid insecticides, but the greater variability in responses to imidacloprid and significantly higher LC50(s) attained indicated higher resistance levels to imidacloprid in all field populations. Further evidence of differential toxicities of neonicotinoids was observed in multiple tests of dinotefuran against imidacloprid-resistant lab strains that yielded significant differences in the LC50(s) of dinotefuran and imidacloprid in simultaneous bioassays. To test the possibility that resistance expression in field-collected insects was sometimes masked by stressful conditions, field strains cultured in a greenhouse without insecticide exposure produced significantly higher LC50(s) to all neonicotinoids compared with LC50(s) attained directly from the field. In harsh climates such as the American southwest, resistance expression in field-collected test insects may be strongly influenced by environmental stresses such as high temperatures, overcrowding, and declining host plant quality.

  14. Sensitivity of the early-life stages of freshwater mollusks to neonicotinoid and butenolide insecticides.

    PubMed

    Prosser, R S; de Solla, S R; Holman, E A M; Osborne, R; Robinson, S A; Bartlett, A J; Maisonneuve, F J; Gillis, P L

    2016-11-01

    Neonicotinoid insecticides can be transported from agricultural fields, where they are used as foliar sprays or seed treatments, to surface waters by surface or sub-surface runoff. Few studies have investigated the toxicity of neonicotinoid or the related butenolide insecticides to freshwater mollusk species. The current study examined the effect of neonicotinoid and butenolide exposures to the early-life stages of the ramshorn snail, Planorbella pilsbryi, and the wavy-rayed lampmussel, Lampsilis fasciola. Juvenile P. pilsbryi were exposed to imidacloprid, clothianidin, or thiamethoxam for 7 or 28 d and mortality, growth, and biomass production were measured. The viability of larval (glochidia) L. fasciola was monitored during a 48 h exposure to six neonicotinoids (imidacloprid, thiamethoxam, clothianidin, acetamiprid, thiacloprid, or dinotefuran), or a butenolide (flupyradifurone). The 7-d LC50s of P. pilsbryi for imidacloprid, clothianidin, and thiamethoxam were ≥4000 μg/L and the 28-d LC50s were ≥182 μg/L. Growth and biomass production were considerably more sensitive endpoints than mortality with EC50s ranging from 33.2 to 122.0 μg/L. The 48-h LC50s for the viability of glochidia were ≥456 μg/L for all seven insecticides tested. Our data indicate that neonicotinoid and butenolide insecticides pose less of a hazard with respect to mortality of the two species of mollusk compared to the potential hazard to other non-target aquatic insects. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  15. Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA

    SciTech Connect

    Sidhu, Navdeep S.; Schreiber, Kathrin; Pröpper, Kevin; Becker, Stefan; Usón, Isabel; Sheldrick, George M.; Gärtner, Jutta; Krätzner, Ralph Steinfeld, Robert

    2014-05-01

    Mucopolysaccharidosis IIIA is a fatal neurodegenerative disease that typically manifests itself in childhood and is caused by mutations in the gene for the lysosomal enzyme sulfamidase. The first structure of this enzyme is presented, which provides insight into the molecular basis of disease-causing mutations, and the enzymatic mechanism is proposed. Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

  16. Effects of sub-clinical psychosis and cognitive insight on psychological well-being: a structural equation model.

    PubMed

    Weintraub, Marc J; Weisman de Mamani, Amy

    2015-03-30

    Psychological well-being has been widely researched along the psychosis spectrum, and increased psychotic symptoms are generally associated with worse well-being. Additionally, the construct of insight has been extensively studied in schizophrenia. While having greater insight has many benefits for those with schizophrenia, a paradox exists in which greater insight is also associated with poorer psychological well-being. However, it is unclear whether the link between insight and poor well-being occurs only once serious psychopathology has been established, or whether this is a more universal process seen even at lower levels on the psychosis spectrum. We used a structural modeling approach in an ethnically diverse, non-clinical sample of 420 undergraduates to evaluate the association between sub-clinical psychosis, cognitive insight and psychological well-being. As hypothesized, results indicated that sub-clinical psychotic symptoms were negatively associated with psychological well-being. The insight paradox was also substantiated, as greater cognitive insight was associated with worse psychological well-being. However, cognitive insight did not moderate the association between symptoms and well-being. The link between sub-clinical psychotic symptoms and psychological well-being as well as the insight paradox appears to emerge even before reaching threshold for a psychotic disorder. Research and clinical implications are discussed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Insights into Medium-chain Acyl-CoA Dehydrogenase Structure by Molecular Dynamics Simulations.

    PubMed

    Bonito, Cátia A; Leandro, Paula; Ventura, Fátima V; Guedes, Rita C

    2016-08-01

    The medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that catalyzes the first step of mitochondrial fatty acid β-oxidation (mFAO) pathway. Its deficiency is the most common genetic disorder of mFAO. Many of the MCAD disease-causing variants, including the most common p.K304E variant, show loss of function due to protein misfolding. Herein, we used molecular dynamics simulations to provide insights into the structural stability and dynamic behavior of MCAD wild-type (MCADwt) and validate a structure that would allow reliable new studies on its variants. Our results revealed that in both proteins the flavin adenine dinucleotide (FAD) has an important structural role on the tetramer stability and also in maintaining the volume of the enzyme catalytic pockets. We confirmed that the presence of substrate changes the dynamics of the catalytic pockets and increases FAD affinity. A comparison between the porcine MCADwt (pMCADwt) and human MCADwt (hMCADwt) structures revealed that both proteins are essentially similar and that the reversion of the double mutant E376G/T255E of hMCAD enzyme does not affect the structure of the protein neither its behavior in simulation. Our validated hMCADwt structure is crucial for complementing and accelerating the experimental studies aiming for the discovery and development of potential stabilizers of MCAD variants as candidates for the treatment of MCAD deficiency (MCADD).

  18. Mesophile versus thermophile: insights into the structural mechanisms of kinetic stability.

    PubMed

    Kelch, Brian A; Agard, David A

    2007-07-20

    Obtaining detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. Comparisons between proteins adapted to survive extreme temperatures with their mesophilic homologs are likely to provide valuable information on the interactions relevant to the unfolding transition. For kinetically stable proteins such as alpha-lytic protease (alphaLP) and its family members, their large free energy barrier to unfolding is central to their biological function. To gain new insights into the mechanisms that underlie kinetic stability, we have determined the structure and high temperature unfolding kinetics of a thermophilic homolog, Thermobifida fusca protease A (TFPA). These studies led to the identification of a specific structural element bridging the N and C-terminal domains of the protease (the "domain bridge") proposed to be associated with the enhanced high temperature kinetic stability in TFPA. Mutagenesis experiments exchanging the TFPA domain bridge into alphaLP validate this hypothesis and illustrate key structural details that contribute to TFPA's increased kinetic thermostability. These results lead to an updated model for the unfolding transition state structure for this important class of proteases in which domain bridge undocking and unfolding occurs at or before the TS. The domain bridge appears to be a structural element that can modulate the degree of kinetic stability of the different members of this class of proteases.

  19. Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli.

    PubMed

    Berry, Andrea A; Yang, Yi; Pakharukova, Natalia; Garnett, James A; Lee, Wei-chao; Cota, Ernesto; Marchant, Jan; Roy, Saumendra; Tuittila, Minna; Liu, Bing; Inman, Keith G; Ruiz-Perez, Fernando; Mandomando, Inacio; Nataro, James P; Zavialov, Anton V; Matthews, Steve

    2014-09-01

    Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.

  20. Insights into the role of protein molecule size and structure on interfacial properties using designed sequences

    PubMed Central

    Dwyer, Mirjana Dimitrijev; He, Lizhong; James, Michael; Nelson, Andrew; Middelberg, Anton P. J.

    2013-01-01

    Mixtures of a large, structured protein with a smaller, unstructured component are inherently complex and hard to characterize at interfaces, leading to difficulties in understanding their interfacial behaviours and, therefore, formulation optimization. Here, we investigated interfacial properties of such a mixed system. Simplicity was achieved using designed sequences in which chemical differences had been eliminated to isolate the effect of molecular size and structure, namely a short unstructured peptide (DAMP1) and its longer structured protein concatamer (DAMP4). Interfacial tension measurements suggested that the size and bulk structuring of the larger molecule led to much slower adsorption kinetics. Neutron reflectometry at equilibrium revealed that both molecules adsorbed as a monolayer to the air–water interface (indicating unfolding of DAMP4 to give a chain of four connected DAMP1 molecules), with a concentration ratio equal to that in the bulk. This suggests the overall free energy of adsorption is equal despite differences in size and bulk structure. At small interfacial extensional strains, only molecule packing influenced the stress response. At larger strains, the effect of size became apparent, with DAMP4 registering a higher stress response and interfacial elasticity. When both components were present at the interface, most stress-dissipating movement was achieved by DAMP1. This work thus provides insights into the role of proteins' molecular size and structure on their interfacial properties, and the designed sequences introduced here can serve as effective tools for interfacial studies of proteins and polymers. PMID:23303222

  1. Structural and Functional Insights into Small, Glutamine-Rich, Tetratricopeptide Repeat Protein Alpha

    PubMed Central

    Roberts, Joanna D.; Thapaliya, Arjun; Martínez-Lumbreras, Santiago; Krysztofinska, Ewelina M.; Isaacson, Rivka L.

    2015-01-01

    The small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA) is an emerging player in the quality control of secretory and membrane proteins mislocalized to the cytosol, with established roles in tail-anchored (TA) membrane protein biogenesis. SGTA consists of three structural domains with individual functions, an N-terminal dimerization domain that assists protein sorting pathways, a central tetratricopeptide repeat (TPR) domain that mediates interactions with heat-shock proteins, proteasomal, and hormonal receptors, and viral proteins, and a C-terminal glutamine rich region that binds hydrophobic substrates. SGTA has been linked to viral lifecycles and hormone receptor signaling, with implications in the pathogenesis of various disease states. Thus far, a range of biophysical techniques have been employed to characterize SGTA structure in some detail, and to investigate its interactions with binding partners in different biological contexts. A complete description of SGTA structure, together with further investigation into its function as a co-chaperone involved quality control, could provide us with useful insights into its role in maintaining cellular proteostasis, and broaden our understanding of mechanisms underlying associated pathologies. This review describes how some structural features of SGTA have been elucidated, and what this has uncovered about its cellular functions. A brief background on the structure and function of SGTA is given, highlighting its importance to biomedicine and related fields. The current level of knowledge and what remains to be understood about the structure and function of SGTA is summarized, discussing the potential direction of future research. PMID:26734616

  2. Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

    DOE PAGES

    Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.; ...

    2017-07-13

    The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the a CTD may play a role in Mtb transcription regulation. Here, our results reveal the structure of an Actinobacteria-unique insert ofmore » the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.« less

  3. Structural Insights into Dissimilatory Sulfite Reductases: Structure of Desulforubidin from Desulfomicrobium Norvegicum

    PubMed Central

    Oliveira, Tânia F.; Franklin, Edward; Afonso, José P.; Khan, Amir R.; Oldham, Neil J.; Pereira, Inês A. C.; Archer, Margarida

    2011-01-01

    Dissimilatory sulfite reductases (dSiRs) are crucial enzymes in bacterial sulfur-based energy metabolism, which are likely to have been present in some of the earliest life forms on Earth. Several classes of dSiRs have been proposed on the basis of different biochemical and spectroscopic properties, but it is not clear whether this corresponds to actual physiological or structural differences. Here, we describe the first structure of a dSiR from the desulforubidin class isolated from Desulfomicrobium norvegicum. The desulforubidin (Drub) structure is assembled as α2β2γ2, in which two DsrC proteins are bound to the core [DsrA]2[DsrB]2 unit, as reported for the desulfoviridin (Dvir) structure from Desulfovibrio vulgaris. Unlike Dvir, four sirohemes and eight [4Fe–4S] clusters are present in Drub. However, the structure indicates that only two of the Drub coupled siroheme-[4Fe–4S] cofactors are catalytically active. Mass spectrometry studies of purified Drub and Dvir show that both proteins present different oligomeric complex forms that bind two, one, or no DsrC proteins, providing an explanation for conflicting spectroscopic and biochemical results in the literature, and further indicating that DsrC is not a subunit of dSiR, but rather a protein with which it interacts. PMID:21833321

  4. Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction.

    PubMed

    Casino, Patricia; Rubio, Vicente; Marina, Alberto

    2009-10-16

    The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.

  5. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    SciTech Connect

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  6. Structural Insights into the Anti-methicillin-resistant Staphylococcus aureus (MRSA) Activity of Ceftobiprole*

    PubMed Central

    Lovering, Andrew L.; Gretes, Michael C.; Safadi, Susan S.; Danel, Franck; de Castro, Liza; Page, Malcolm G. P.; Strynadka, Natalie C. J.

    2012-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant strain of S. aureus afflicting hospitals and communities worldwide. Of greatest concern is its development of resistance to current last-line-of-defense antibiotics; new therapeutics are urgently needed to combat this pathogen. Ceftobiprole is a recently developed, latest generation cephalosporin and has been the first to show activity against MRSA by inhibiting essential peptidoglycan transpeptidases, including the β-lactam resistance determinant PBP2a, from MRSA. Here we present the structure of the complex of ceftobiprole bound to PBP2a. This structure provides the first look at the molecular details of an effective β-lactam-resistant PBP interaction, leading to new insights into the mechanism of ceftobiprole efficacy against MRSA. PMID:22815485

  7. Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes.

    PubMed

    Miliara, Xeni; Garnett, James A; Tatsuta, Takashi; Abid Ali, Ferdos; Baldie, Heather; Pérez-Dorado, Inmaculada; Simpson, Peter; Yague, Ernesto; Langer, Thomas; Matthews, Stephen

    2015-07-01

    The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1-SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.

  8. Protein Fibrillar Nanopolymers: Molecular-Level Insights into Their Structural, Physical and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.

    2015-09-01

    Amyloid fibrils represent a generic class of mechanically strong and stable biomaterials with extremely advantageous properties. Although amyloids were initially associated only with severe neurological disorders, the role of these structures nowadays is shifting from health debilitating to highly beneficial both in biomedical and technological aspects. Intensive involvement of fibrillar assemblies into the wide range of pathogenic and functional processes strongly necessitate the molecular level characterization of the structural, physical and elastic features of protein nanofibrils. In the present contribution, we made an attempt to highlight the up-to-date progress in the understanding of amyloid properties from the polymer physics standpoint. The fundamental insights into protein fibril behavior are essential not only for development of therapeutic strategies to combat the protein misfolding disorders but also for rational and precise design of novel biodegradable protein-based nanopolymers.

  9. Experimental Insights into the Origin of Defect-Structured Hibonites Found in Meteorites

    NASA Technical Reports Server (NTRS)

    Han. J.; Keller, L. P.; Danielson, L. R.

    2016-01-01

    Hibonite (CaAl12O19) is a primary, highly refractory phase occurring in many Ca-Al-rich inclusions (CAIs). Previous microstructural studies of hibonite in CAIs and their Wark-Lovering (WL) rims showed the presence of numerous stacking defects in hibonites. These defects are interpreted as the modification of the stacking sequences of spinel and Ca-containing blocks within the ideal hexagonal hibonite structure due to the presence of wider spinel blocks [3], as shown by experimental studies of reaction-sintered compounds in the CaO-Al2O3 system. We performed a series of experiments in the CaO-Al2O3-MgO system in order to provide additional in-sights into the formation processes and conditions of defect-structured hibonites found in meteorites.

  10. New insights into structural determinants of prion protein folding and stability.

    PubMed

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  11. Structural insights on two hypothetical secretion chaperones from Xanthomonas axonopodis pv. citri.

    PubMed

    Fattori, Juliana; Prando, Alessandra; Assis, Leandro H P; Aparicio, Ricardo; Tasic, Ljubica

    2011-06-01

    Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.

  12. Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes

    PubMed Central

    Miliara, Xeni; Garnett, James A; Tatsuta, Takashi; Abid Ali, Ferdos; Baldie, Heather; Pérez-Dorado, Inmaculada; Simpson, Peter; Yague, Ernesto; Langer, Thomas; Matthews, Stephen

    2015-01-01

    The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1–SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops. PMID:26071602

  13. Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis.

    PubMed

    Lovering, Andrew L; de Castro, Liza H; Lim, Daniel; Strynadka, Natalie C J

    2007-03-09

    Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the "holy grail" of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition.

  14. Large Scale Overturned Structures and Frontal Thrusts Structures in Taiwan: Insight from Sandbox Models

    NASA Astrophysics Data System (ADS)

    Lu, C.; Malavieille, J.; Chen, L.; Huang, C.

    2013-12-01

    In Taiwan today, the subduction of the Chinese continental margin under the Philippine Sea plate results in the progressive growth of an active orogenic wedge. It is one of the best places to study the complex relationships that occur between the tectono-metamorphic processes controlling deformation (plate rheology and kinematics) and surface processes (erosion and sedimentation). In the Central Range of Taiwan, foliation and lineation traces outline the geometry and kinematics of deformation in both, the foreland and hinterland of the orogenic wedge. The foliation dip and the strain ellipsoids distribution show the fan shape of a large pop-up structure characterizing the effects of oblique plate convergence. On the eastern flank, regionally developed penetrative cleavage dips, isotope data and sedimentary structures demonstrating regional overturned structures. Two mélange units, the Kenting and Lichi mélange are exposed at the south and east of the Central Range respectively. Experiments allow the study of interactions between tectonics and surface processes. Accounting for various boundary conditions and parameters such as sedimentation, erosion, basal friction, and décollement level. We present the results of 2D sandbox models designed to investigate the complex deformation characterizing the active Taiwan orogenic wedge and to demonstrate the development of those mélanges, overturned structures and mountain frontal thrusts. Models are analyzed using pictures, movies and PIV (Particle Image Velocimetry software). We then characterize the exhumation patterns, the mode of fault propagation and displacement patterns by strain partitioning of those mélanges and overturned structures. The preliminary conclusions are: 1.the Lichi mélange may result from mega-back thrust/ back fold, with the ophiolite source from the western oceanic lithosphere. 2. Double subduction are necessary to produce large E-vergent and overturned sequence. 3. The frontal fault

  15. Large Scale Overturned Structures Structures in Taiwan: Insight from Sandbox Models

    NASA Astrophysics Data System (ADS)

    Lu, Chia-Yu; Malavieille, Jacques; Chen, Liwen; Huang, Chinhuang

    2014-05-01

    In Taiwan today, the subduction of the Chinese continental margin under the Philippine Sea plate results in the progressive growth of an active orogenic wedge. It is one of the best places to study the complex relationships that occur between the tectono-metamorphic processes controlling deformation (plate rheology and kinematics) and surface processes (erosion and sedimentation). In the Central Range of Taiwan, foliation and lineation traces outline the geometry and kinematics of deformation in both, the foreland and hinterland of the orogenic wedge. The foliation dip and the strain ellipsoids distribution show the fan shape of a large pop-up structure characterizing the effects of oblique plate convergence. On the eastern flank, regionally developed penetrative cleavage dips, isotope data and sedimentary structures demonstrating regional overturned structures. Two mélange units, the Kenting and Lichi mélange are exposed at the south and east of the Central Range respectively. Experiments allow the study of interactions between tectonics and surface processes. Accounting for various boundary conditions and parameters such as sedimentation, erosion, basal friction, and décollement level. We present the results of 2D sandbox models designed to investigate the complex deformation characterizing the active Taiwan orogenic wedge and to demonstrate the development of those mélanges, overturned structures and mountain frontal thrusts. Models are analyzed using pictures, movies and PIV (Particle Image Velocimetry software). We then characterize the exhumation patterns, the mode of fault propagation and displacement patterns by strain partitioning of those mélanges and overturned structures. The preliminary conclusions are: 1.the Lichi mélange may result from mega-back thrust/ back fold, with the ophiolite source from the western oceanic lithosphere. 2. Double subduction favors to produce large E-vergent and overturned sequence. 3. The structures in Suao area and

  16. Neonicotinoid Insecticides and Their Impacts on Bees: A Systematic Review of Research Approaches and Identification of Knowledge Gaps.

    PubMed

    Lundin, Ola; Rundlöf, Maj; Smith, Henrik G; Fries, Ingemar; Bommarco, Riccardo

    2015-01-01

    It has been suggested that the widespread use of neonicotinoid insecticides threatens bees, but research on this topic has been surrounded by controversy. In order to synthesize which research approaches have been used to examine the effect of neonicotinoids on bees and to identify knowledge gaps, we systematically reviewed research on this subject that was available on the Web of Science and PubMed in June 2015. Most of the 216 primary research studies were conducted in Europe or North America (82%), involved the neonicotinoid imidacloprid (78%), and concerned the western honey bee Apis mellifera (75%). Thus, little seems to be known about neonicotinoids and bees in areas outside Europe and North America. Furthermore, because there is considerable variation in ecological traits among bee taxa, studies on honey bees are not likely to fully predict impacts of neonicotinoids on other species. Studies on crops were dominated by seed-treated maize, oilseed rape (canola) and sunflower, whereas less is known about potential side effects on bees from the use of other application methods on insect pollinated fruit and vegetable crops, or on lawns and ornamental plants. Laboratory approaches were most common, and we suggest that their capability to infer real-world consequences are improved when combined with information from field studies about realistic exposures to neonicotinoids. Studies using field approaches often examined only bee exposure to neonicotinoids and more field studies are needed that measure impacts of exposure. Most studies measured effects on individual bees. We suggest that effects on the individual bee should be linked to both mechanisms at the sub-individual level and also to the consequences for the colony and wider bee populations. As bees are increasingly facing multiple interacting pressures future research needs to clarify the role of neonicotinoids in relative to other drivers of bee declines.

  17. Neonicotinoid Insecticides and Their Impacts on Bees: A Systematic Review of Research Approaches and Identification of Knowledge Gaps

    PubMed Central

    Lundin, Ola; Rundlöf, Maj; Smith, Henrik G.; Fries, Ingemar; Bommarco, Riccardo

    2015-01-01

    It has been suggested that the widespread use of neonicotinoid insecticides threatens bees, but research on this topic has been surrounded by controversy. In order to synthesize which research approaches have been used to examine the effect of neonicotinoids on bees and to identify knowledge gaps, we systematically reviewed research on this subject that was available on the Web of Science and PubMed in June 2015. Most of the 216 primary research studies were conducted in Europe or North America (82%), involved the neonicotinoid imidacloprid (78%), and concerned the western honey bee Apis mellifera (75%). Thus, little seems to be known about neonicotinoids and bees in areas outside Europe and North America. Furthermore, because there is considerable variation in ecological traits among bee taxa, studies on honey bees are not likely to fully predict impacts of neonicotinoids on other species. Studies on crops were dominated by seed-treated maize, oilseed rape (canola) and sunflower, whereas less is known about potential side effects on bees from the use of other application methods on insect pollinated fruit and vegetable crops, or on lawns and ornamental plants. Laboratory approaches were most common, and we suggest that their capability to infer real-world consequences are improved when combined with information from field studies about realistic exposures to neonicotinoids. Studies using field approaches often examined only bee exposure to neonicotinoids and more field studies are needed that measure impacts of exposure. Most studies measured effects on individual bees. We suggest that effects on the individual bee should be linked to both mechanisms at the sub-individual level and also to the consequences for the colony and wider bee populations. As bees are increasingly facing multiple interacting pressures future research needs to clarify the role of neonicotinoids in relative to other drivers of bee declines. PMID:26313444

  18. Structural insights into the dual-targeting mechanism of Nutlin-3

    SciTech Connect

    Shin, Jae-Sun; Ha, Ji-Hyang; He, Fahu; Muto, Yutaka; Ryu, Kyoung-Seok; Yoon, Ho Sup; Kang, Sunghyun; Park, Sung Goo; Park, Byoung Chul; Choi, Sang-Un; Chi, Seung-Wook

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Universal binding of Nutlin-3 with diverse anti-apoptotic Bcl-2 family proteins. Black-Right-Pointing-Pointer Nutlin-3 binds to the BH3 peptide-binding grooves of Bcl-2 family proteins. Black-Right-Pointing-Pointer A conserved Bcl-X{sub L} binding mechanism of the Nutlin-3 and BH3-mimetic compounds. Black-Right-Pointing-Pointer A molecular basis for the transcription-independent apoptosis by Nutlin-3. Black-Right-Pointing-Pointer Structural insights into the dual-targeting mechanism of Nutlin-3. -- Abstract: Multi-targeting therapy is an emerging strategy of drug discovery to improve therapeutic efficacy, safety and resistance profiles. In this study, we monitored the binding of a potent MDM2 inhibitor Nutlin-3 with anti-apoptotic Bcl-2 family proteins using NMR spectroscopy. Our results showed the universal binding of Nutlin-3 with diverse anti-apoptotic Bcl-2 family proteins. Taken together with the binding data for Nutlin-3 analogs, the structural model of the Bcl-X{sub L}/Nutlin-3 complex showed that the binding mode of Nutlin-3 resembles that of the Bcl-X{sub L}/Bcl-2 inhibitors, suggesting the molecular mechanism of transcription-independent mitochondrial apoptosis by Nutlin-3. Finally, our structural comparison provides structural insights into the dual-targeting mechanism of how Nutlin-3 can bind to two different target proteins, MDM2 and anti-apoptotic Bcl-2 family proteins in a similar manner.

  19. Structural Insight into the Dioxygenation of Nitroarene Compounds: the Crystal Structure of Nitrobenzene Dioxygenase

    SciTech Connect

    Friemann, Rosmarie; Ivkovic-Jensen, Maja M.; Lessner, Daniel J.; Yu, Chi-Li; Gibson, David T.; Parales, Rebecca E.; Eklund, Hans; Ramaswamy, S.

    2010-07-19

    Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.

  20. Further insights on the French WISC-IV factor structure through Bayesian structural equation modeling.

    PubMed

    Golay, Philippe; Reverte, Isabelle; Rossier, Jérôme; Favez, Nicolas; Lecerf, Thierry

    2013-06-01

    The interpretation of the Wechsler Intelligence Scale for Children--Fourth Edition (WISC-IV) is based on a 4-factor model, which is only partially compatible with the mainstream Cattell-Horn-Carroll (CHC) model of intelligence measurement. The structure of cognitive batteries is frequently analyzed via exploratory factor analysis and/or confirmatory factor analysis. With classical confirmatory factor analysis, almost all cross-loadings between latent variables and measures are fixed to zero in order to allow the model to be identified. However, inappropriate zero cross-loadings can contribute to poor model fit, distorted factors, and biased factor correlations; most important, they do not necessarily faithfully reflect theory. To deal with these methodological and theoretical limitations, we used a new statistical approach, Bayesian structural equation modeling (BSEM), among a sample of 249 French-speaking Swiss children (8-12 years). With BSEM, zero-fixed cross-loadings between latent variables and measures are replaced by approximate zeros, based on informative, small-variance priors. Results indicated that a direct hierarchical CHC-based model with 5 factors plus a general intelligence factor better represented the structure of the WISC-IV than did the 4-factor structure and the higher order models. Because a direct hierarchical CHC model was more adequate, it was concluded that the general factor should be considered as a breadth rather than a superordinate factor. Because it was possible for us to estimate the influence of each of the latent variables on the 15 subtest scores, BSEM allowed improvement of the understanding of the structure of intelligence tests and the clinical interpretation of the subtest scores. PsycINFO Database Record (c) 2013 APA, all rights reserved.

  1. Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

    PubMed

    Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Yongchao; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

    2016-09-16

    Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums.

  2. An Insight into the Pharmacophores of Phosphodiesterase-5 Inhibitors from Synthetic and Crystal Structural Studies

    SciTech Connect

    Chen,G.; Wang, H.; Robinson, H.; Cai, J.; Wan, Y.; Ke, H.

    2008-01-01

    Selective inhibitors of cyclic nucleotide phosphodiesterase-5 (PDE5) have been used as drugs for treatment of male erectile dysfunction and pulmonary hypertension. An insight into the pharmacophores of PDE5 inhibitors is essential for development of second generation of PDE5 inhibitors, but has not been completely illustrated. Here we report the synthesis of a new class of the sildenafil derivatives and a crystal structure of the PDE5 catalytic domain in complex with 5-(2-ethoxy-5-(sulfamoyl)-3-thienyl)-1-methyl-3-propyl-1, 6-dihydro-7H-pyrazolo[4, 3-d]pyrimidin-7-one (12). Inhibitor 12 induces conformational change of the H-loop (residues 660-683), which is different from any of the known PDE5 structures. The pyrazolopyrimidinone groups of 12 and sildenafil are well superimposed, but their sulfonamide groups show a positional difference of as much as 1.5 Angstroms . The structure-activity analysis suggests that a small hydrophobic pocket and the H-loop of PDE5 are important for the inhibitor affinity, in addition to two common elements for binding of almost all the PDE inhibitors: the stack against the phenylalanine and the hydrogen bond with the invariant glutamine. However, the PDE5-12 structure does not provide a full explanation to affinity changes of the inhibitors. Thus alternatives such as conformational change of the M-loop are open and further structural study is required.

  3. Insights into the Mechanism of Type I Dehydroquinate Dehydratases from Structures of Reaction Intermediates

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Duban, Mark-Eugene; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-02-27

    The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre- and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.

  4. Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

    NASA Astrophysics Data System (ADS)

    Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

    2014-12-01

    Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

  5. Structure of the Taz2 domain of p300: insights into ligand binding

    SciTech Connect

    Miller, Maria; Dauter, Zbigniew; Cherry, Scott; Tropea, Joseph E.; Wlodawer, Alexander

    2009-12-01

    The crystal structure of the Taz2 zinc-finger domain of the human p300 transcriptional coactivator was determined using the anomalous diffraction signal of the bound Zn ions. Crystal contacts suggested a possible novel mode of Taz2–peptide ligand interactions. CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723–1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813–1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed.

  6. Structural Insights into the Anti-HIV Activity of the Oscillatoria agardhii Agglutinin Homolog Lectin Family*

    PubMed Central

    Koharudin, Leonardus M. I.; Kollipara, Sireesha; Aiken, Christopher; Gronenborn, Angela M.

    2012-01-01

    Oscillatoria agardhii agglutinin homolog (OAAH) proteins belong to a recently discovered lectin family. All members contain a sequence repeat of ∼66 amino acids, with the number of repeats varying among different family members. Apart from data for the founding member OAA, neither three-dimensional structures, information about carbohydrate binding specificities, nor antiviral activity data have been available up to now for any other members of the OAAH family. To elucidate the structural basis for the antiviral mechanism of OAAHs, we determined the crystal structures of Pseudomonas fluorescens and Myxococcus xanthus lectins. Both proteins exhibit the same fold, resembling the founding family member, OAA, with minor differences in loop conformations. Carbohydrate binding studies by NMR and x-ray structures of glycan-lectin complexes reveal that the number of sugar binding sites corresponds to the number of sequence repeats in each protein. As for OAA, tight and specific binding to α3,α6-mannopentaose was observed. All the OAAH proteins described here exhibit potent anti-HIV activity at comparable levels. Altogether, our results provide structural details of the protein-carbohydrate interaction for this novel lectin family and insights into the molecular basis of their HIV inactivation properties. PMID:22865886

  7. Structural insights into 5' flap DNA unwinding and incision by the human FAN1 dimer.

    PubMed

    Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

    2014-12-11

    Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5' flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5' flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

  8. Structure-based Insights into the Catalytic Power and Conformational Dexterity of Peroxiredoxins

    PubMed Central

    Hall, Andrea; Nelson, Kimberly; Poole, Leslie B.

    2011-01-01

    Abstract Peroxiredoxins (Prxs), some of nature's dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic SN2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly. Antioxid. Redox Signal. 15, 795–815. PMID:20969484

  9. Structural insights into the reaction mechanism of S-adenosyl-L-homocysteine hydrolase

    PubMed Central

    Kusakabe, Yoshio; Ishihara, Masaaki; Umeda, Tomonobu; Kuroda, Daisuke; Nakanishi, Masayuki; Kitade, Yukio; Gouda, Hiroaki; Nakamura, Kazuo T.; Tanaka, Nobutada

    2015-01-01

    S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues—3’-keto-aristeromycin (3KA) and noraristeromycin (NRN)—at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3’-keto-4’,5’-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer & Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme. PMID:26573329

  10. Neonicotinoids impact bumblebee colony fitness in the field; a reanalysis of the UK's Food & Environment Research Agency 2012 experiment.

    PubMed

    Goulson, Dave

    2015-01-01

    The causes of bee declines remain hotly debated, particularly the contribution of neonicotinoid insecticides. In 2013 the UK's Food & Environment Research Agency made public a study of the impacts of exposure of bumblebee colonies to neonicotinoids. The study concluded that there was no clear relationship between colony performance and pesticide exposure, and the study was subsequently cited by the UK government in a policy paper in support of their vote against a proposed moratorium on some uses of neonicotinoids. Here I present a simple re-analysis of this data set. It demonstrates that these data in fact do show a negative relationship between both colony growth and queen production and the levels of neonicotinoids in the food stores collected by the bees. Indeed, this is the first study describing substantial negative impacts of neonicotinoids on colony performance of any bee species with free-flying bees in a field realistic situation where pesticide exposure is provided only as part of normal farming practices. It strongly suggests that wild bumblebee colonies in farmland can be expected to be adversely affected by exposure to neonicotinoids.

  11. Neonicotinoid concentrations in urine from chronic kidney disease patients in the North Central Region of Sri Lanka.

    PubMed

    Kabata, Risako; Nanayakkara, Shanika; Senevirathna, Stmld; Harada, Kouji H; Chandrajith, Rohana; Hitomi, Toshiaki; Abeysekera, Tilak; Takasuga, Takumi; Koizumi, Akio

    2016-01-01

    Neonicotinoid insecticides have been widely used around the world since the 1990s. Reports have been made since the 1990s of rice paddy farmers in the North Central Region (NCR) of Sri Lanka suffering from chronic kidney disease with unknown etiology (CKDu). A preliminary evaluation of the exposure of local farmers in the NCR of Sri Lanka to neonicotinoids was performed. We analyzed neonicotinoid and neonicotinoid metabolite concentrations in spot urine samples. We selected 40 samples, 10 from farmers with CKDu and 10 from controls from each of two areas, Medawachchiya and Girandurukotte. Imidacloprid and desmethyl-acetamiprid were found at significantly higher concentrations in the control samples (with medians of 51 ng/l and 340 ng/l, respectively) than in the CKDu samples (medians of 15 ng/l and 150 ng/l, respectively) when the results were not adjusted for the creatinine contents. None of the six compounds that were measured in the urine samples were found at significantly higher concentrations in the CKDu samples than in the control samples. None of the neonicotinoid concentrations in the samples analyzed in this study exceeded the concentrations that have been found in samples from the general population of Japan. Farmers (both with and without CKDu) living in CKDu-endemic areas in the NCR of Sri Lanka are exposed to lower neonicotinoid concentrations than non-occupationally exposed residents of Japan.

  12. Understanding the structural setting in the Southern Apennines (Italy): insight from Gravity Gradient Tensor

    NASA Astrophysics Data System (ADS)

    Fedi, Maurizio; Ferranti, Luigi; Florio, Giovanni; Giori, Italiano; Italiano, Francesco

    2005-03-01

    The Lucania Apennines form the frontal part of the Apennines in Southern Italy. This orogenic belt was formed in response to late Miocene-Early Pleistocene shortening and allochthon emplacement toward the northeast, and was subsequently affected by extensional faulting which migrated to northeast ahead of the thrust system. As a result, contraction structures are found associated to Pliocene-Early Pleistocene thrust-top basins in the NE part of the area, whereas extensional faults shape Middle Pleistocene-Holocene basin on the SW sector. The main structural grain of the belt is NW-SE, but E-W structures are widespread in the area as a result of phases of non-coaxial thrusting and strike-slip faulting. The analysis, one by one, of the components of the Gravity Gradient Tensor (GGT) has proved to yield a fine image of the structural setting of the investigated area. GGT is a second rank tensor containing the second spatial derivatives of the gravity potential and in this paper is used instead than the more traditional gravity data. The Tzz component allows an accurate description of the location and of the shape of basins and other structures, but different components of the GGT provide even more detailed insights for such structures. In particular, we found that the Tzy and Tyy locate well those structures trending closer to the E-W direction for both thrust-top and extensional basins, and their termination against the main NW-SE structures. The combined use of components of GGT provides a finer definition of the anomaly sources particularly if a good knowledge of their strike and depth is independently estimated with other geological and geophysical investigations.

  13. Structural Insight into Anaphase Promoting Complex 3 Structure and Docking with a Natural Inhibitory Compound

    PubMed Central

    Rahimi, Hamzeh; Shokrgozar, Mohammad Ali; Madadkar-Sobhani, Armin; Mahdian, Reza; Foroumadi, Alireza; Karimipoor, Morteza

    2017-01-01

    Background: Anaphase promoting complex (APC) is the biggest Cullin-RING E3 ligase and is very important in cell cycle control; many anti-cancer agents target this. APC controls the onset of chromosome separation and mitotic exit through securin and cyclin B degradation, respectively. Its APC3 subunit identifies the APC activators-Cdh1 and Cdc20. Materials and Methods: The structural model of the APC3 subunit of APC was developed by means of computational techniques; the binding of a natural inhibitory compound to APC3 was also investigated. Results: It was found that APC3 structure consists of numerous helices organized in anti-parallel and the overall model is superhelical of tetratrico-peptide repeat (TPR) domains. Furthermore, binding pocket of the natural inhibitory compound as APC3 inhibitor was shown. Conclusion: The findings are beneficial to understand the mechanism of the APC activation and design inhibitory compounds. PMID:28401073

  14. The X-Ray Crystal Structure of Escherichia coli Succinic Semialdehyde Dehydrogenase; Structural Insights into NADP+/Enzyme Interactions

    PubMed Central

    Langendorf, Christopher G.; Key, Trevor L. G.; Fenalti, Gustavo; Kan, Wan-Ting; Buckle, Ashley M.; Caradoc-Davies, Tom; Tuck, Kellie L.; Law, Ruby H. P.; Whisstock, James C.

    2010-01-01

    Background In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells. Methodology/Principal Findings Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site. Conclusions/Significance Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease. PMID:20174634

  15. Crystal structure of Ruminococcus albus cellobiose 2-epimerase: structural insights into epimerization of unmodified sugar.

    PubMed

    Fujiwara, Takaaki; Saburi, Wataru; Inoue, Sota; Mori, Haruhide; Matsui, Hirokazu; Tanaka, Isao; Yao, Min

    2013-04-02

    Enzymatic epimerization is an important modification for carbohydrates to acquire diverse functions attributable to their stereoisomers. Cellobiose 2-epimerase (CE) catalyzes interconversion between d-glucose and d-mannose residues at the reducing end of β-1,4-linked oligosaccharides. Here, we solved the structure of Ruminococcus albus CE (RaCE). The structure of RaCE showed strong similarity to those of N-acetyl-D-glucosamine 2-epimerase and aldose-ketose isomerase YihS with a high degree of conservation of residues around the catalytic center, although sequence identity between them is low. Based on structural comparison, we found that His184 is required for RaCE activity as the third histidine added to two essential histidines in other sugar epimerases/isomerases. This finding was confirmed by mutagenesis, suggesting a new catalytic mechanism for CE involving three histidines. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

    PubMed

    Gutiérrez-Fernández, Javier; Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús; Hermoso, Juan A

    2014-09-01

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.

  17. Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations.

    PubMed

    Balasco, Nicole; Barone, Daniela; Vitagliano, Luigi

    2015-01-01

    Recent structural investigations have shown that the C-terminal domain (CTD) of the transcription factor RfaH undergoes unique structural modifications that have a profound impact into its functional properties. These modifications cause a complete change in RfaH(CTD) topology that converts from an α-hairpin to a β-barrel fold. To gain insights into the determinants of this major structural conversion, we here performed computational studies (protein structure prediction and molecular dynamics simulations) on RfaH(CTD). Although these analyses, in line with literature data, suggest that the isolated RfaH(CTD) has a strong preference for the β-barrel fold, they also highlight that a specific region of the protein is endowed with a chameleon conformational behavior. In particular, the Leu-rich region (residues 141-145) has a good propensity to adopt both α-helical and β-structured states. Intriguingly, in the RfaH homolog NusG, whose CTD uniquely adopts the β-barrel fold, the corresponding region is rich in residues as Val or Ile that present a strong preference for the β-structure. On this basis, we suggest that the presence of this Leu-rich element in RfaH(CTD) may be responsible for the peculiar structural behavior of the domain. The analysis of the sequences of RfaH family (PfamA code PF02357) unraveled that other members potentially share the structural properties of RfaH(CTD). These observations suggest that the unusual conformational behavior of RfaH(CTD) may be rare but not unique.

  18. Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

    PubMed Central

    Schellenberger, Pascale; Sauter, Claude; Lorber, Bernard; Bron, Patrick; Trapani, Stefano; Bergdoll, Marc; Marmonier, Aurélie; Schmitt-Keichinger, Corinne; Lemaire, Olivier; Demangeat, Gérard; Ritzenthaler, Christophe

    2011-01-01

    Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector. PMID:21625570

  19. Structural insights into the interaction of IL-33 with its receptors.

    PubMed

    Liu, Xi; Hammel, Michal; He, Yanfeng; Tainer, John A; Jeng, U-Ser; Zhang, Linqi; Wang, Shuying; Wang, Xinquan

    2013-09-10

    Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand-receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray-scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33-ST2-IL-1RAcP complex from small-angle X-ray-scattering analysis resembles IL-1β-IL-1RII-IL-1RAcP and IL-1β-IL-1RI-IL-1RAcP crystal structures. The collective results confer IL-33 structure-function relationships, supporting and extending a general model for ligand-receptor assembly and activation in the IL-1 family.

  20. Crystal structure of a TSH receptor monoclonal antibody: insight into Graves' disease pathogenesis.

    PubMed

    Chen, Chun-Rong; Hubbard, Paul A; Salazar, Larry M; McLachlan, Sandra M; Murali, Ramachandran; Rapoport, Basil

    2015-01-01

    The TSH receptor (TSHR) A-subunit is more effective than the holoreceptor in inducing thyroid-stimulating antibodies (TSAb) that cause Graves' disease. A puzzling phenomenon is that 2 recombinant, eukaryotic forms of A-subunits (residues 22-289), termed active and inactive, are recognized mutually exclusively by pathogenic TSAb and mouse monoclonal antibody 3BD10, respectively. Understanding the structural difference between these TSHR A-subunit forms could provide insight into Graves' disease pathogenesis. The 3-dimensional structure of the active A-subunit (in complex with a human TSAb Fab, M22) is known, but the structural difference with inactive A-subunits is unknown. We solved the 3BD10 Fab 3-dimensional crystal structure. Guided by prior knowledge of a portion of its epitope, 3BD10 docked in silico with the known active TSHR-289 monomeric structure. Because both TSAb and 3BD10 recognize the active TSHR A-subunit monomer, this form of the molecule can be excluded as the basis for the active-inactive dichotomy, suggesting, instead a role for A-subunit quaternary structure. Indeed, in silico analysis revealed that M22, but not 3BD10, bound to a TSHR-289 trimer. In contrast, 3BD10, but not M22, bound to a TSHR-289 dimer. The validity of these models is supported experimentally by the temperature-dependent balance between active and inactive TSHR-289. In summary, we provide evidence for a structural basis to explain the conformational heterogeneity of TSHR A-subunits (TSHR-289). The pathophysiologic importance of these findings is that affinity maturation of pathogenic TSAb in Graves' disease is likely to involve a trimer of the shed TSHR A-subunit.

  1. Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

    SciTech Connect

    Li, Yongchao; Wang, Yang; Jiang, Haobo; Deng, Junpeng

    2010-02-22

    Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 {angstrom}, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.

  2. Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

    PubMed

    Sidhu, Navdeep S; Schreiber, Kathrin; Pröpper, Kevin; Becker, Stefan; Usón, Isabel; Sheldrick, George M; Gärtner, Jutta; Krätzner, Ralph; Steinfeld, Robert

    2014-05-01

    Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

  3. New Insights into the Structure of Multimetallic Nanoparticles and their Advanced Characterization

    NASA Astrophysics Data System (ADS)

    Khanal, Subarna; Bhattarai, Nabraj; Velázquez-Salazar, Jesus; Guisbiers, Gregory; Jose-Yacaman, Miguel

    2015-03-01

    Noble multimetallic nanoparticles have led to exciting progress in a versatile array of applications. For the purpose of better tailoring of nanoparticles activities and understanding the correlation between their structures and properties, control over the composition, shape, size and architecture of bimetallic and multimetallic nanomaterials plays an important role on revealing their new or enhanced functions for potentials application. Advance electron microscopy techniques were used to provide atomic scale insights into the structure-properties of different materials: Pt-Pd, Au-Au3Cu, Cu-Pt, AgPd-Pt and AuCu/Pt nanoparticles. These multimetallic nanoparticles have raised interest for their various applications in fuel cells, ethanol and methanol oxidation reactions, hydrogen storage, and so on. The nanostructures were analyzed by transmission electron microscopy (TEM) and by aberration-corrected scanning transmission electron microscopy (Cs-corrected STEM), in combination with high angle annular dark field (HAADF), bright field (BF), energy dispersive X-ray spectroscopy (EDS), and electron energy loss spectroscopy (EELS) detectors. These techniques allowed us to probe the structure at the atomic level of the nanoparticles revealing new structural information and elemental composition of the nanoparticles.

  4. Structural insight into nucleotide recognition by human death-associated protein kinase

    SciTech Connect

    McNamara, Laurie K.; Watterson, D.M.; Brunzelle, Joseph S.

    2009-06-01

    Death-associated protein kinase (DAPK) is a member of the Ca{sup 2+}/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg{sup 2+} at 1.85 {angstrom} resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK-ADP-Mg{sup 2+} was compared with a newly determined DAPK-AMP-PNP-Mg{sup 2+} structure and the previously determined apo DAPK structure (PDB code 1 jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

  5. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

    PubMed Central

    Fernandes, Carlos A. H.; Pazin, Wallance M.; Dreyer, Thiago R.; Bicev, Renata N.; Cavalcante, Walter L. G.; Fortes-Dias, Consuelo L.; Ito, Amando S.; Oliveira, Cristiano L. P.; Fernandez, Roberto Morato; Fontes, Marcos R. M.

    2017-01-01

    Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation. PMID:28256632

  6. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism.

    PubMed

    Fernandes, Carlos A H; Pazin, Wallance M; Dreyer, Thiago R; Bicev, Renata N; Cavalcante, Walter L G; Fortes-Dias, Consuelo L; Ito, Amando S; Oliveira, Cristiano L P; Fernandez, Roberto Morato; Fontes, Marcos R M

    2017-03-03

    Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

  7. The formation, function and regulation of amyloids: insights from structural biology.

    PubMed

    Landreh, M; Sawaya, M R; Hipp, M S; Eisenberg, D S; Wüthrich, K; Hartl, F U

    2016-08-01

    Amyloid diseases are characterized by the accumulation of insoluble, β-strand-rich aggregates. The underlying structural conversions are closely associated with cellular toxicity, but can also drive the formation of functional protein assemblies. In recent years, studies in the field of structural studies have revealed astonishing insights into the origins, mechanisms and implications of amyloid formation. Notably, high-resolution crystal structures of peptides in amyloid-like fibrils and prefibrillar oligomers have become available despite their challenging chemical nature. Nuclear magnetic resonance spectroscopy has revealed that dynamic local polymorphisms in the benign form of the prion protein affect the transformation into amyloid fibrils and the transmissibility of prion diseases. Studies of the structures and interactions of chaperone proteins help us to understand how the cellular proteostasis network is able to recognize different stages of aberrant protein folding and prevent aggregation. In this review, we will focus on recent developments that connect the different aspects of amyloid biology and discuss how understanding the process of amyloid formation and the associated defence mechanisms can reveal targets for pharmacological intervention that may become the first steps towards clinically viable treatment strategies. © 2016 The Association for the Publication of the Journal of Internal Medicine.

  8. Insights into disease-associated mutations in the human proteome through protein structural analysis

    PubMed Central

    Gao, Mu; Zhou, Hongyi; Skolnick, Jeffrey

    2015-01-01

    Summary Most known disease-associated mutations are missense mutations involving changes of amino acids of proteins encoded by their genes. Given the plethora of genetic studies, sequenced exomes, and new protein structures determined each year, it is appropriate to revisit the role that structure plays in providing insights into the molecular basis of disease associated mutations. In that regard, a large-scale structural analysis on 6,025 disease-associated mutations as well as 4,536 neutral variations for comparison was performed. While buried amino acids are common among the disease-associated mutations as reported previously, more are statistically significantly enriched at observed or predicted functional sites. Interesting findings are that ligand-binding sites adjacent to protein-protein interfaces and residues involved in enzymatic function are especially vulnerable to disease-associated mutations. Finally, a compositional analysis of disease-associated mutations in comparison to variants identified in the 1000 Genomes Project provides a structural rationalization of the most disease-associated residue types. PMID:26027735

  9. Structural insights into binding of small molecule inhibitors to Enhancer of Zeste Homolog 2

    NASA Astrophysics Data System (ADS)

    Kalinić, Marko; Zloh, Mire; Erić, Slavica

    2014-11-01

    Enhancer of Zeste Homolog 2 (EZH2) is a SET domain protein lysine methyltransferase (PKMT) which has recently emerged as a chemically tractable and therapeutically promising epigenetic target, evidenced by the discovery and characterization of potent and highly selective EZH2 inhibitors. However, no experimental structures of the inhibitors co-crystallized to EZH2 have been resolved, and the structural basis for their activity and selectivity remains unknown. Considering the need to minimize cross-reactivity between prospective PKMT inhibitors, much can be learned from understanding the molecular basis for selective inhibition of EZH2. Thus, to elucidate the binding of small-molecule inhibitors to EZH2, we have developed a model of its fully-formed cofactor binding site and used it to carry out molecular dynamics simulations of protein-ligand complexes, followed by molecular mechanics/generalized born surface area calculations. The obtained results are in good agreement with biochemical inhibition data and reflect the structure-activity relationships of known ligands. Our findings suggest that the variable and flexible post-SET domain plays an important role in inhibitor binding, allowing possibly distinct binding modes of inhibitors with only small variations in their structure. Insights from this study present a good basis for design of novel and optimization of existing compounds targeting the cofactor binding site of EZH2.

  10. Structural insight into the mechanisms of enveloped virus tethering by tetherin

    SciTech Connect

    Yang, Haitao; Wang, Jimin; Jia, Xiaofei; McNatt, Matthew W.; Zang, Trinity; Pan, Baocheng; Meng, Wuyi; Wang, Hong-Wei; Bieniasz, Paul D.; Xiong, Yong

    2010-11-10

    Tetherin/BST2 is a type-II membrane protein that inhibits the release of a range of enveloped viruses, including HIV-1. Here we report three crystal structures of human tetherin, including the full-length ectodomain, a triple cysteine mutant and an ectodomain truncation. These structures show that tetherin forms a continuous alpha helix encompassing almost the entire ectodomain. Tetherin helices dimerize into parallel coiled coils via interactions throughout the C-terminal portion of the ectodomain. A comparison of the multiple structures of the tetherin dimer reveals inherent constrained flexibility at two hinges positioned at residues A88 and G109. In the crystals, two tetherin ectodomain dimers associate into a tetramer by forming an antiparallel four-helix bundle at their N termini. However, mutagenesis studies suggest that the tetrametric form of tetherin, although potentially contributing to, is not essential for its antiviral activity. Nonetheless, the structural and chemical properties of the N terminus of the ectodomain are important for optimal tethering function. This study provides detailed insight into the mechanisms by which this broad-spectrum antiviral restriction factor can function.

  11. Structural Insight into the Mechanisms of Enveloped Virus Tethering by Tetherin

    SciTech Connect

    H Yang; J Wang; X Jia; M McNatt; T Zang; B Pan; W Meng; H Wang; P Bieniasz; Y Xiong

    2011-12-31

    Tetherin/BST2 is a type-II membrane protein that inhibits the release of a range of enveloped viruses, including HIV-1. Here we report three crystal structures of human tetherin, including the full-length ectodomain, a triple cysteine mutant and an ectodomain truncation. These structures show that tetherin forms a continuous alpha helix encompassing almost the entire ectodomain. Tetherin helices dimerize into parallel coiled coils via interactions throughout the C-terminal portion of the ectodomain. A comparison of the multiple structures of the tetherin dimer reveals inherent constrained flexibility at two hinges positioned at residues A88 and G109. In the crystals, two tetherin ectodomain dimers associate into a tetramer by forming an antiparallel four-helix bundle at their N termini. However, mutagenesis studies suggest that the tetrametric form of tetherin, although potentially contributing to, is not essential for its antiviral activity. Nonetheless, the structural and chemical properties of the N terminus of the ectodomain are important for optimal tethering function. This study provides detailed insight into the mechanisms by which this broad-spectrum antiviral restriction factor can function.

  12. Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: INSIGHTS ON LEUCINE AND ALIPHATIC GLUCOSINOLATE BIOSYNTHESIS.

    PubMed

    Lee, Soon Goo; Nwumeh, Ronald; Jez, Joseph M

    2016-06-24

    Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different β-hydroxyacids in the leucine- and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here, we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg(2+) or NAD(+) These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys-232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys-232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the β-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.

  13. Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

    PubMed

    Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

    2015-10-01

    Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

  14. Crystal Structure of Human Senescence Marker Protein 30: Insights Linking Structural, Enzymatic, and Physiological Functions

    SciTech Connect

    Chakraborti, Subhendu; Bahnson, Brian J.

    2010-05-25

    Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 {angstrom}. The protein has a 6-bladed {beta}-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca{sup 2+} or a Zn{sup 2+} atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k{sub cat} preference of divalent cations in the order Zn{sup 2+} > Mn{sup 2+} > Ca{sup 2+} > Mg{sup 2+}. Notably, the Ca{sup 2+} had a significantly higher value of K{sub d} compared to those of the other metal ions tested (566, 82, 7, and 0.6 {micro}m for Ca{sup 2+}, Mg{sup 2+}, Zn{sup 2+}, and Mn{sup 2+}, respectively), suggesting that the Ca{sup 2+}-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.

  15. Lipid-free Apolipoprotein A-I Structure: Insights into HDL Formation and Atherosclerosis Development

    PubMed Central

    Mei, Xiaohu; Atkinson, David

    2015-01-01

    Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important role during the process of reverse cholesterol transport (RCT). Knowledge of the high-resolution structure of full-length apoA-I is vital for a molecular understanding of the function of HDL at the various steps of the RCT pathway. Due to the flexible nature of apoA-I and aggregation properties, the structure of full-length lipid-free apoA-I has evaded description for over three decades. Sequence analysis of apoA-I suggested that the amphipathic α-helix is the structural motif of exchangeable apolipoprotein, and NMR, X-ray and MD simulation studies have confirmed this. Different laboratories have used different methods to probe the secondary structure distribution and organization of both the lipid-free and lipid-bound apoA-I structure. Mutation analysis, synthetic peptide models, surface chemistry and crystal structures have converged on the lipid-free apoA-I domain structure and function: the N-terminal domain [1–184] forms a helix bundle while the C-terminal domain [185–243] mostly lacks defined structure and is responsible for initiating lipid-binding, aggregation and is also involved in cholesterol efflux. The first 43 residues of apoA-I are essential to stabilize the lipid-free structure. In addition, the crystal structure of C-terminally truncated apoA-I suggests a monomer-dimer conversation mechanism mediated through helix 5 reorganization and dimerization during the formation of HDL. Based on previous research, we have proposed a structural model for full-length monomeric apoA-I in solution and updated the HDL formation mechanism through three intermediate states. Mapping the known natural mutations on the full-length monomeric apoA-I model provides insight into atherosclerosis development through disruption of the N-terminal helix bundle or deletion of the C-terminal lipid-binding domain. PMID:26048453

  16. Recent Insights into the Structure, Regulation and Function of the V-ATPases

    PubMed Central

    Cotter, Kristina; Stransky, Laura; McGuire, Christina; Forgac, Michael

    2015-01-01

    The V-ATPases are ATP-dependent proton pumps that acidify intracellular compartments and are also present at the plasma membrane. They function in such processes as membrane traffic, protein degradation, viral and toxin entry, bone resorption, pH homeostasis and tumor cell invasion. V-ATPases are large, multi-subunit complexes composed of an ATP-hydrolytic domain (V1) and proton translocation domain (V0) and operate by a rotary mechanism. This review focuses on recent insights into their structure and mechanism, the mechanisms that regulate V-ATPase activity (particularly regulated assembly and trafficking) and the role of V-ATPases in such processes as cell signaling and cancer. These developments have highlighted the potential of V-ATPases as a therapeutic target for a variety of human diseases. PMID:26410601

  17. Group-theoretic insights on the vibration of symmetric structures in engineering

    PubMed Central

    Zingoni, Alphose

    2014-01-01

    Group theory has been used to study various problems in physics and chemistry for many years. Relatively recently, applications have emerged in engineering, where problems of the vibration, bifurcation and stability of systems exhibiting symmetry have been studied. From an engineering perspective, the main attraction of group-theoretic methods has been their potential to reduce computational effort in the analysis of large-scale problems. In this paper, we focus on vibration problems in structural mechanics and reveal some of the insights and qualitative benefits that group theory affords. These include an appreciation of all the possible symmetries of modes of vibration, the prediction of the number of modes of a given symmetry type, the identification of modes associated with the same frequencies, the prediction of nodal lines and stationary points of a vibrating system, and the untangling of clustered frequencies. PMID:24379427

  18. Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

    PubMed

    Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A; Muench, Stephen P

    2015-03-03

    Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.

  19. New insight into the structure of dispersed titania by combining normal-mode analysis with experiment

    NASA Astrophysics Data System (ADS)

    Nitsche, David; Hess, Christian

    2014-11-01

    Normal-mode analysis has been combined with experiment to gain new insight into the vibrational structure of dispersed titania. For the calculations, double- and tri-grafted hydroxylated titania species have been adapted to a model silica support based on polyhedral oligomeric silsesquioxane (POSS). The choice of hydroxylated models was validated by IR detection of the Osbnd H stretching band of dispersed titania (0.7 Ti/nm2). UV resonance Raman experiments have identified three titania-related vibrational features within the spectral region 900-1100 cm-1 due to Tisbnd Osbnd Si interphase, Tisbnd Osbnd Si in-phase and out-of-phase stretching vibrations. This behaviour is fully consistent with the results obtained by the normal-mode analysis.

  20. Structural insight into the role of the Ton complex in energy transduction

    PubMed Central

    Celia, Hervé; Noinaj, Nicholas; Zakharov, Stanislav D.; Bordignon, Enrica; Botos, Istvan; Santamaria, Monica; Barnard, Travis J.; Cramer, William A.; Lloubes, Roland; Buchanan, Susan K.

    2016-01-01

    Summary In Gram-negative bacteria, outer membrane (OM) transporters import nutrients by coupling to an inner membrane (IM) protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force (pmf) at the IM to transduce energy to the OM via TonB. Here, we structurally characterize the Ton complex from E. coli using X-ray crystallography, electron microscopy, DEER spectroscopy, and crosslinking, revealing a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels, providing insight to the mechanism by which it may harness the pmf for energy production. PMID:27654919

  1. Insights from the DNA databases: approaches to the phylogenetic structure of Acanthamoeba.

    PubMed

    Fuerst, Paul A

    2014-11-01

    Species of Acanthamoeba have been traditionally described using morphology (primarily cyst structure), or cytology of nuclear division (used by Pussard and Pons, 1977). Twenty-plus putative species were proposed based on such criteria. Morphology, however, is often plastic, dependent upon culture conditions. DNA sequences of the nuclear small subunit (18S) rRNA that can be used for the study of the phylogeny of Acanthamoeba have increased from a single sequence in 1986 to more than 1800 in 2013. Some of the patterns of the sequence data for Acanthamoeba are reviewed, and some of the insights that this data illuminates are illustrated. In particular, the data suggest the existence of 20 or more genotypic types, a number not dissimilar to the number of named species of Acanthamoeba. However, molecular studies make clear that the relationship between phylogenetic relatedness and species names as we know them for Acanthamoeba is tenuous at best.

  2. Splitting up the powerhouse: structural insights into the mechanism of mitochondrial fission.

    PubMed

    Richter, Viviane; Singh, Abeer P; Kvansakul, Marc; Ryan, Michael T; Osellame, Laura D

    2015-10-01

    Mitochondria are dynamic organelles whose shape is regulated by the opposing processes of fission and fusion, operating in conjunction with organelle distribution along the cytoskeleton. The importance of fission and fusion homeostasis has been highlighted by a number of disease states linked to mutations in proteins involved in regulating mitochondrial morphology, in addition to changes in mitochondrial dynamics in Alzheimer's, Huntington's and Parkinson's diseases. While a number of mitochondrial morphology proteins have been identified, how they co-ordinate to assemble the fission apparatus is not clear. In addition, while the master mediator of mitochondrial fission, dynamin-related protein 1, is conserved throughout evolution, the adaptor proteins involved in its mitochondrial recruitment are not. This review focuses on our current understanding of mitochondrial fission and the proteins that regulate this process in cell homeostasis, with a particular focus on the recent mechanistic insights based on protein structures.

  3. Genetic, structural, and molecular insights into the function of ras of complex proteins domains.

    PubMed

    Civiero, Laura; Dihanich, Sybille; Lewis, Patrick A; Greggio, Elisa

    2014-07-17

    Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson's disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural information available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

  4. Structural Insights into High Density Lipoprotein: Old Models and New Facts

    PubMed Central

    Gogonea, Valentin

    2016-01-01

    The physiological link between circulating high density lipoprotein (HDL) levels and cardiovascular disease is well-documented, albeit its intricacies are not well-understood. An improved appreciation of HDL function and overall role in vascular health and disease requires at its foundation a better understanding of the lipoprotein's molecular structure, its formation, and its process of maturation through interactions with various plasma enzymes and cell receptors that intervene along the pathway of reverse cholesterol transport. This review focuses on summarizing recent developments in the field of lipid free apoA-I and HDL structure, with emphasis on new insights revealed by newly published nascent and spherical HDL models constructed by combining low resolution structures obtained from small angle neutron scattering (SANS) with contrast variation and geometrical constraints derived from hydrogen–deuterium exchange (HDX), crosslinking mass spectrometry, electron microscopy, Förster resonance energy transfer, and electron spin resonance. Recently published low resolution structures of nascent and spherical HDL obtained from SANS with contrast variation and isotopic labeling of apolipoprotein A-I (apoA-I) will be critically reviewed and discussed in terms of how they accommodate existing biophysical structural data from alternative approaches. The new low resolution structures revealed and also provided some answers to long standing questions concerning lipid organization and particle maturation of lipoproteins. The review will discuss the merits of newly proposed SANS based all atom models for nascent and spherical HDL, and compare them with accepted models. Finally, naturally occurring and bioengineered mutations in apoA-I, and their impact on HDL phenotype, are reviewed and discuss together with new therapeutics employed for restoring HDL function. PMID:26793109

  5. Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure.

    PubMed

    Stockand, James D; Staruschenko, Alexander; Pochynyuk, Oleh; Booth, Rachell E; Silverthorn, Dee U

    2008-09-01

    The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.

  6. Comparative riftology: insights from crustal structure into the evolution of continental rifts and passive continental margins

    NASA Astrophysics Data System (ADS)

    Kley, Jonas; Stein, Carol; Stein, Seth; Keller, Randy; Wysession, Michael; Frederiksen, Andrew

    2017-04-01

    Continental rifts evolve to seafloor spreading and are preserved in passive margins, or fail and remain as fossil features in continents. Rifts at different stages give insight into these different evolutionary paths. Of particular interest is how volcanic passive margins evolve. These features are characterized by sequences of volcanic rocks yielding magnetic anomalies landward of and sometimes larger than the oldest spreading anomalies. Seaward-dipping reflectors (SDR) occur in stretched continental crust landward of the oldest oceanic crust and are underplated by high-velocity lower crustal bodies. How and when these features form remains unclear. Insights are given by the Midcontinent Rift (MCR), formed by 1.1 Ga rifting of Amazonia from Laurentia, that failed once seafloor spreading was established elsewhere. MCR volcanics are much thicker than other continental flood basalts, due to deposition in a narrow rift rather than a broad region, giving a rift's geometry but a LIP's magma volume. The MCR provides a snapshot of the deposition of a thick highly magnetized volcanic section during rifting. Surface exposures and seismic-reflection data in and near Lake Superior show a rift basin filled by inward-dipping flood basalt layers. Had the rift evolved to seafloor spreading, the basin would have split into two sets of volcanics with opposite-facing SDRs, each with a strong magnetic anomaly. Because the rift formed as a series of alternating half-grabens, structural asymmetries between conjugate margins can naturally occur. Hence the MCR shows that many features form prior to breakup. Because the MCR was massively inverted by regional compression long after it failed and was uplifted, its structure is better known than failed rifts that incurred lesser degrees of inversion. It provides an end member for the evolution of actively extending rifts, characterized by upwelling mantle and negative gravity anomalies, in contrast to failed and inverted rifts without

  7. Computation Sheds Insight into Iron Porphyrin Carbenes' Electronic Structure, Formation, and N-H Insertion Reactivity.

    PubMed

    Sharon, Dina A; Mallick, Dibyendu; Wang, Binju; Shaik, Sason

    2016-08-03

    Iron porphyrin carbenes constitute a new frontier of species with considerable synthetic potential. Exquisitely engineered myoglobin and cytochrome P450 enzymes can generate these complexes and facilitate the transformations they mediate. The current work harnesses density functional theoretical methods to provide insight into the electronic structure, formation, and N-H insertion reactivity of an iron porphyrin carbene, [Fe(Por)(SCH3)(CHCO2Et)](-), a model of a complex believed to exist in an experimentally studied artificial metalloenzyme. The ground state electronic structure of the terminal form of this complex is an open-shell singlet, with two antiferromagnetically coupled electrons residing on the iron center and carbene ligand. As we shall reveal, the bonding properties of [Fe(Por)(SCH3)(CHCO2Et)](-) are remarkably analogous to those of ferric heme superoxide complexes. The carbene forms by dinitrogen loss from ethyl diazoacetate. This reaction occurs preferentially through an open-shell singlet transition state: iron donates electron density to weaken the C-N bond undergoing cleavage. Once formed, the iron porphyrin carbene accomplishes N-H insertion via nucleophilic attack. The resulting ylide then rearranges, using an internal carbonyl base, to form an enol that leads to the product. The findings rationalize experimentally observed reactivity trends reported in artificial metalloenzymes employing iron porphyrin carbenes. Furthermore, these results suggest a possible expansion of enzymatic substrate scope, to include aliphatic amines. Thus, this work, among the first several computational explorations of these species, contributes insights and predictions to the surging interest in iron porphyrin carbenes and their synthetic potential.

  8. Unique Insights into the Origin and Expansion of Coronal Structures from Total Solar Eclipse Observations

    NASA Astrophysics Data System (ADS)

    Habbal, S. R.; Morgan, H.; Druckmuller, M.; Ding, A.

    2015-12-01

    The enigma surrounding the solar corona continues to be the focus of observations from space and the ground, as well as model studies. The astounding spatial and temporal resolution of recent space-based imaging experiments, in particular in the ultraviolet and extreme ultraviolet, have added significantly to the complexity and dynamics of coronal structures. Their main limitation, however, is the coverage of the inner corona over a distance range limited to half a solar radius at best from the solar surface. They thus fall short in placing the observed structures within the context of the expanding corona from the solar surface out into interplanetary space. This presentation will review the unique advantages of total solar eclipse observations in yielding novel insights into the bewildering variety of seemingly disparate coronal structures. This is achieved through the outstanding imaging and diagnostic potentials of the continuum and spectral line emission over a distance range range covering a few solar radii starting from the solar surface, coupled with state of the art image processing tools.

  9. Revealing New Structural Insights from Surfactant Micelles through DLS, Microrheology and Raman Spectroscopy

    PubMed Central

    Amin, Samiul; Blake, Steven; Kennel, Rachel C.; Lewis, E. Neil

    2015-01-01

    The correlation between molecular changes and microstructural evolution of rheological properties has been demonstrated for the first time in a mixed anionic/zwitterionic surfactant-based wormlike micellar system. Utilizing a novel combination of DLS-microrheology and Raman Spectroscopy, the effect of electrostatic screening on these properties of anionic (SLES) and zwitterionic (CapB) surfactant mixtures was studied by modulating the NaCl concentration. As Raman Spectroscopy delivers information about the molecular structure and DLS-microrheology characterizes viscoelastic properties, the combination of data delivered allows for a deeper understanding of the molecular changes underlying the viscoelastic ones. The high frequency viscoelastic response obtained through DLS-microrheology has shown the persistence of the Maxwell fluid response for low viscosity solutions at high NaCl concentrations. The intensity of the Raman band at 170 cm−1 exhibits very strong correlation with the viscosity variation. As this Raman band is assigned to hydrogen bonding, its variation with NaCl concentration additionally indicates differences in water structuring due to potential microstructural differences at low and high NaCl concentrations. The microstructural differences at low and high NaCl concentrations are further corroborated by persistence of a slow mode at the higher NaCl concentrations as seen through DLS measurements. The study illustrates the utility of the combined DLS, DLS-optical microrheology and Raman Spectroscopy in providing new molecular structural insights into the self-assembly process in complex fluids.

  10. Insights into Caco-2 cell culture structure using coherent anti-Stokes Raman scattering (CARS) microscopy.

    PubMed

    Saarinen, Jukka; Sözeri, Erkan; Fraser-Miller, Sara J; Peltonen, Leena; Santos, Hélder A; Isomäki, Antti; Strachan, Clare J

    2017-05-15

    We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey

    PubMed Central

    Zhou, Huiqing; Hintze, Bradley J.; Kimsey, Isaac J.; Sathyamoorthy, Bharathwaj; Yang, Shan; Richardson, Jane S.; Al-Hashimi, Hashim M.

    2015-01-01

    Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson–Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1′–C1′ distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1′–C1′ distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5′-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA. PMID:25813047

  12. Functional and structural insight into properdin control of complement alternative pathway amplification.

    PubMed

    Pedersen, Dennis V; Roumenina, Lubka; Jensen, Rasmus K; Gadeberg, Trine Af; Marinozzi, Chiara; Picard, Capucine; Rybkine, Tania; Thiel, Steffen; Sørensen, Uffe Bs; Stover, Cordula; Fremeaux-Bacchi, Veronique; Andersen, Gregers R

    2017-03-06

    Properdin (FP) is an essential positive regulator of the complement alternative pathway (AP) providing stabilization of the C3 and C5 convertases, but its oligomeric nature challenges structural analysis. We describe here a novel FP deficiency (E244K) caused by a single point mutation which results in a very low level of AP activity. Recombinant FP E244K is monomeric, fails to support bacteriolysis, and binds weakly to C3 products. We compare this to a monomeric unit excised from oligomeric FP, which is also dysfunctional in bacteriolysis but binds the AP proconvertase, C3 convertase, C3 products and partially stabilizes the convertase. The crystal structure of such a FP-convertase complex suggests that the major contact between FP and the AP convertase is mediated by a single FP thrombospondin repeat and a small region in C3b. Small angle X-ray scattering indicates that FP E244K is trapped in a compact conformation preventing its oligomerization. Our studies demonstrate an essential role of FP oligomerization in vivo while our monomers enable detailed structural insight paving the way for novel modulators of complement.

  13. Structural insights into human Kif7, a kinesin involved in Hedgehog signalling

    SciTech Connect

    Klejnot, Marta Kozielski, Frank

    2012-02-01

    The human Kif7 motor domain structure provides insights into a kinesin of medical significance. Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melano@@gaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance.

  14. Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase

    NASA Astrophysics Data System (ADS)

    Klein, Tobias; Vajpai, Navratna; Phillips, Jonathan J.; Davies, Gareth; Holdgate, Geoffrey A.; Phillips, Chris; Tucker, Julie A.; Norman, Richard A.; Scott, Andrew D.; Higazi, Daniel R.; Lowe, David; Thompson, Gary S.; Breeze, Alexander L.

    2015-07-01

    Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called `DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a `DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and `molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

  15. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.

    PubMed

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A; Rasmussen, Søren G F; Thian, Foon Sun; Kobilka, Tong Sun; Choi, Hee-Jung; Yao, Xiao-Jie; Weis, William I; Stevens, Raymond C; Kobilka, Brian K

    2007-11-23

    The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

  16. Structural Insight into Caenorhabditis elegans Sex-determining Protein FEM-2*♦

    PubMed Central

    Zhang, Yi; Zhao, Haifeng; Wang, Jia; Ge, Jingpeng; Li, Yang; Gu, Jinke; Li, Peng; Feng, Yue; Yang, Maojun

    2013-01-01

    In the nematode Caenorhabditis elegans, fem-1, fem-2, and fem-3 play crucial roles in male sexual development. Among these three genes, fem-2 encodes a PP2C (serine/threonine phosphatase type 2C)-like protein, whose activity promotes the development of masculinity. Different from the canonical PP2Cs, FEM-2 consists of an additional N-terminal domain (NTD) apart from its C-terminal catalytic domain. Interestingly, genetic studies have indicated indispensable roles for both of these two domains of FEM-2 in promoting male development, but the underlying mechanism remains unknown. In the present study, we solved the crystal structure of full-length FEM-2, which revealed a novel structural fold formed by its NTD. Structural and functional analyses demonstrated that the NTD did not directly regulate the in vitro dephosphorylation activity of FEM-2, but instead functioned as a scaffold domain in the assembly of the FEM-1/2/3 complex, the executioner in the final step of the sex determination pathway. Biochemical studies further identified the regions in the NTD involved in FEM-1 and FEM-3 interactions. Our results not only identified a novel fold formed by the extra domain of a noncanonical PP2C enzyme, but also provided important insights into the molecular mechanism of how the NTD works in mediating the sex-determining role of FEM-1/2/3 complex. PMID:23760267

  17. Structured Ionomer Thin Films at Water Interface: Molecular Dynamics Simulation Insight

    DOE PAGES

    Aryal, Dipak; Agrawal, Anupriya; Perahia, Dvora; ...

    2017-08-23

    Controlling the structure and dynamics of thin films of ionizable polymers at water interfaces is critical to their many applications. As the chemical diversity within one polymer is increased, controlling the structure and dynamics of the polymer, which is a key to their use, becomes a challenge. Here molecular dynamics simulations (MD) are used to obtain molecular insight into the structure and dynamics of thin films of one such macromolecule at the interface with water. The polymer consists of an ABCBA topology with randomly sulfonated polystyrene (C), tethered symmetrically to flexible poly(ethylene-r-propylene) blocks (B), and end-capped by a poly(t-butylstyrene) blockmore » (A). The compositions of the interfacial and bulk regions of thin films of the ABCBA polymers are followed as a function of exposure time to water. We find that interfacial rearrangements take place where buried ionic segments migrate toward the water interface. The hydrophobic blocks collapse and rearrange to minimize their exposure to water. In conclusion, the water that initially drives interfacial reengagements breaks the ionic clusters within the film, forming a dynamic hydrophilic internal network within the hydrophobic segments.« less

  18. Molecular and evolutionary insights into the structural organization of cation chloride cotransporters

    PubMed Central

    Hartmann, Anna-Maria; Nothwang, Hans Gerd

    2015-01-01

    Cation chloride cotransporters (CCC) play an essential role for neuronal chloride homeostasis. K+-Cl− cotransporter (KCC2), is the principal Cl−-extruder, whereas Na+-K+-Cl− cotransporter (NKCC1), is the major Cl−-uptake mechanism in many neurons. As a consequence, the action of the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine strongly depend on the activity of these two transporters. Knowledge of the mechanisms involved in ion transport and regulation is thus of great importance to better understand normal and disturbed brain function. Although no overall 3-dimensional crystal str