Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9)

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wildtype and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.

A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier Ltd. All rights reserved.

The complexity of drinking: Interactions between the cognitive and behavioural determinants of alcohol consumption

This study expanded the earlier work conducted by this laboratory ( Hasking, P.A. and Oei, T.P.S. (2002a) . The differential role of alcohol expectancies, drinking refusal self-efficacy and coping resources in predicting alcohol consumption in community and clinical samples. Addiction Research and Theory , 10 , 465-494), by examining the independent and interactive effects of avoidant coping strategies, positive and negative expectancies and self-efficacy, in predicting volume and frequency of alcohol consumption in a sample of community drinkers. Differential relationships were found between the variables when predicting the two consumption measures. Specifically, while self-efficacy, seeking social support for emotional reasons and using drugs or alcohol to cope were independently related to both volume and frequency of drinking, complex interactions with positive and negative alcohol expectancies were also found. These interactions are discussed in terms of the cognitive and behavioural mechanisms thought to underlie drinking behaviour.

Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

The determinants of export performance:evidence for manufacturing plants in Ireland and Northern Ireland

The dramatic GDP and export growth of Ireland over the last decade forms a marked contrast with that of its nearest neighbour Northern Ireland. In Ireland, export volume growth averaged 15.5% p.a. from 1991 to 1999 compared with 6.3% from Northern Ireland. Using data on individual manufacturing plants this paper considers the determinants of export performance in the two areas. Larger, externally owned plants with higher skill levels are found to have the highest export propensities in both areas. Other influences (plant age, R&D, etc.) prove more strongly conditional on location, plant size, and ownership. Structural factors (e.g. ownership, industry) explain almost all of the difference in export propensity between larger plants in Northern Ireland and Ireland but only around one-third of that between smaller plants. Significant differences are also evident between plants in terms of their sources of new technology. For indigenously owned plants, inhouse R&D is important. For externally owned plants, R&D conducted elsewhere in the group - typically outside Ireland and Northern Ireland - proves more significant. This external dependency and lower than expected export propensity on the part of small plants in Northern Ireland represent significant policy challenges for the future.© 2006 Scottish Economic Society. Published by Blackwell Publishing Ltd.

Corporate social responsibility in China:impact of regulations, market orientation and ownership structure

Purpose – This paper aims to report on a study that contributes to the understanding of the determinants of corporate social responsibility (CSR) in the largest emerging market, namely China. Design/methodology/approach – The approach is a survey of 600 hotels that resulted in 143 returned responses from top managers. Findings – Market orientation is the most significant predicator of CSR followed by government regulations. In contrast, ownership structure is found to have little effect. Originality/value – Previous research on CSR focuses on its nature and impact on business performance, and is carried out mainly in developed countries. This research contributes to one's understanding of the determinants of CSR in emerging markets like China. © 2007, Emerald Group Publishing Limited

An exploration of the determinants of innovation:the top management team, organisational climate and organisational learning

Some researchers argue that the top team, rather than the CEO, is a better predictor of an organisation’s fate (Finkelstein & Hambrick, 1996; Knight et al., 1999). However, others suggest that the importance of the top management team (TMT) composition literature is exaggerated (West & Schwenk, 1996). This has stimulated a need for further research on TMTs. While the importance of TMT is well documented in the innovation literature, the organisational environment also plays a key role in determining organisational outcomes. Therefore, the inclusion of both TMT characteristics and organisational variables (climate and organisational learning) in this study provides a more holistic picture of innovation. The research methodologies employed includes (i) interviews with TMT members in 35 Irish software companies (ii) a survey completed by managerial respondents and core workers in these companies (iii) in-depth interviews with TMT members from five companies. Data were gathered in two phases, time 1 (1998-2000) and time 2 (2003). The TMT played an important part in fostering innovation. However, it was a group process, rather than team demography, that was most strongly associated with innovation. Task reflexivity was an important predictor of innovation time 1, time 2). Only one measure of TMT diversity was associated with innovation - tenure diversity -in time 2 only. Organisational context played an important role in determining innovation. This was positively associated with innovation - but with one dimension of organisational learning only. The ability to share information (access to information) was not associated with innovation but the motivation to share information was (perceiving the sharing of information to be valuable). Innovative climate was also associated with innovation. This study suggests that this will lead to innovative outcomes if employees perceive the organisation to support risk, experimentation and other innovative behaviours.

Reliability of vegetation community information derived using decorana ordination and fuzzy c-means clustering

Descriptions of vegetation communities are often based on vague semantic terms describing species presence and dominance. For this reason, some researchers advocate the use of fuzzy sets in the statistical classification of plant species data into communities. In this study, spatially referenced vegetation abundance values collected from Greek phrygana were analysed by ordination (DECORANA), and classified on the resulting axes using fuzzy c-means to yield a point data-set representing local memberships in characteristic plant communities. The fuzzy clusters matched vegetation communities noted in the field, which tended to grade into one another, rather than occupying discrete patches. The fuzzy set representation of the community exploited the strengths of detrended correspondence analysis while retaining richer information than a TWINSPAN classification of the same data. Thus, in the absence of phytosociological benchmarks, meaningful and manageable habitat information could be derived from complex, multivariate species data. We also analysed the influence of the reliability of different surveyors' field observations by multiple sampling at a selected sample location. We show that the impact of surveyor error was more severe in the Boolean than the fuzzy classification. © 2007 Springer.