Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7-biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels.

Formation of mononuclear and chloro-bridged binuclear copper(II) complexes of patellamide D, a naturally occurring cyclic peptide: influence of anion and solvent

Patellamide D (patH(4)) is a cyclic octapeptide isolated from the ascidian Lissoclinum patella. The peptide possesses a 24-azacrown-8 macrocyclic structure containing two oxazoline and two thiazole rings, each separated by an amino acid. The present spectrophotometric, electron paramagnetic resonance (EPR) and mass spectral studies show that patellamide D reacts with CuCl, and triethylamine in acetonitrile to form mononuclear and binuclear copper(II) complexes containing chloride. Molecular modelling and EPR studies suggest that the chloride anion bridges the copper(II) ions in the binuclear complex [Cu-2(patH(2))(mu-Cl)](+). These results contrast with a previous study employing both base and methanol, the latter substituting for chloride in the copper(II) complexes en route to the stable mu-carbonato binuclear copper(II) complex [Cu-2 (patH(2))(mu-CO3)]. Solvent clearly plays an important role in both stabilising these metal ion complexes and influencing their chemical reactivities. (C) 2004 Elsevier Inc. All rights reserved.

Synthetic molecular mimics of naturally occurring cyclopentenones exhibit antifungal activity towards pathogenic fungi

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100-200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent. © 2011 SGM.

Memory for emotional faces in naturally occurring dysphoria and induced sadness

The aim was to establish if the memory bias for sad faces, reported in clinically depressed patients (Gilboa-Schechtman, Erhard Weiss, & Jeczemien, 2002; Ridout, Astell, Reid, Glen, & O'Carroll, 2003) generalises to sub-clinical depression (dysphoria) and experimentally induced sadness. Study 1: dysphoric (n = 24) and non-dysphoric (n = 20) participants were presented with facial stimuli, asked to identify the emotion portrayed and then given a recognition memory test for these faces. At encoding, dysphoric participants (DP) exhibited impaired identification of sadness and neutral affect relative to the non-dysphoric group (ND). At memory testing, DP exhibited superior memory for sad faces relative to happy and neutral. They also exhibited enhanced memory for sad faces and impaired memory for happy relative to the ND. Study 2: non-depressed participants underwent a positive (n = 24) or negative (n = 24) mood induction (MI) and were assessed on the same tests as Study 1. At encoding, negative MI participants showed superior identification of sadness, relative to neutral affect and compared to the positive MI group. At memory testing, the negative MI group exhibited enhanced memory for the sad faces relative to happy or neutral and compared to the positive MI group. Conclusion: MCM bias for sad faces generalises from clinical depression to these sub-clinical affective states.

Examining the impact of thought substitution on intentional forgetting in induced and naturally occurring dysphoria

Two experiments were conducted to determine if natural and induced dysphoria is associated with impaired forgetting and, whether a thought-substitution strategy would ameliorate any observed deficits. Study 1: 36 dysphoric & 36 non-dysphoric participants learnt a series of emotional word pairs. Participants were subsequently presented with some of the cues and were asked to recall the targets or prevent the targets from coming to mind. Half of the participants were provided with substitute words to recall instead of the original targets (aided suppression). At final memory testing, participants were asked to recall the targets to all cues. Dysphoric participants exhibited impaired forgetting, even when using a thought substitution strategy. Non-dysphoric participants, however, were able to use substitutes to suppress words. Study 2: 50 healthy participants initially completed the aided condition of the forgetting task. Participants were then given a positive or negative mood-induction, followed by another version of the forgetting task. Although all participants showed a forgetting effect prior to the mood-induction, only the positive group was successful at forgetting after the mood induction. Taken together, these findings do not support the utility of thought-substitution as an aid to forgetting in individuals in a naturally or induced dysphoric mood.

Naturally occurring data as research instrument:analyzing examination responses to study the novice programmer

In New Zealand and Australia, the BRACElet project has been investigating students' acquisition of programming skills in introductory programming courses. The project has explored students' skills in basic syntax, tracing code, understanding code, and writing code, seeking to establish the relationships between these skills. This ITiCSE working group report presents the most recent step in the BRACElet project, which includes replication of earlier analysis using a far broader pool of naturally occurring data, refinement of the SOLO taxonomy in code-explaining questions, extension of the taxonomy to code-writing questions, extension of some earlier studies on students' 'doodling' while answering exam questions, and exploration of a further theoretical basis for work that until now has been primarily empirical.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

International audience

Domain Bundle Boundaries in Single Crystal BaTiO3 Lamellae: Searching for Naturally Forming Dipole Flux-Closure / Quadrupole Chains

Naturally occurring boundaries between bundles of 90o stripe domains, which form in BaTiO3 lamellae on cooling through the Curie Temperature, have been characterised using both piezoresponse force microscopy (PFM) and scanning transmission electron microscopy (STEM). Detailed interpretation of the dipole configurations present at these boundaries (using data taken from PFM) shows that, in the vast majority of cases, they are composed of simple zigzag 180° domain walls. Topological information from STEM shows that, occasionally, domain bundle boundaries can support chains of dipole flux closure and quadrupole nanostructures, but these kinds of boundaries are comparatively rare; when such chains do exist, it is notable that singularities at the cores of the dipole structures are avoided. The symmetry of the boundary shows that diads and centres of inversion exist at positions where core singularities should have been expected.

Domain Bundle Boundaries in Single Crystal BaTiO3 Lamellae: Searching for Naturally Forming Dipole Flux-Closure/Quadrupole Chains

Naturally occurring boundaries between bundles of 90° stripe domains, which form in BaTiO3 lamellae on cooling through the Curie Temperature, have been characterized using both piezoresponse force microscopy (PFM) and scanning transmission electron microscopy (STEM). Detailed interpretation of the dipole configurations present at these boundaries (using data taken from PFM) shows that in the vast majority of cases they are composed of simple zigzag 180° domain walls. Topological information from STEM shows that occasionally domain bundle boundaries can support chains of dipole flux closure and quadrupole nanostructures, but these kinds of boundaries are comparatively rare; when such chains do exist, it is notable that singularities at the cores of the dipole
structures are avoided. The symmetry of the boundary shows that diads and centers of inversion exist at positions where core singularities should have been expected.