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    ABSTRACT: The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 isassociated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise the accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both withinand around the cell nucleus,was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathionelevels by treatment with BSOFurthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents β-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes. In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 proteinaccumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress.
    Biochemical and Biophysical Research Communications 01/2014;
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    ABSTRACT: Currently, there are relatively few tasks suitable for testing planned problem solving in children. We presented 4- to 10-year-old children (N = 172) with two planning tasks (sequential planning and advance planning) using the paddle-box apparatus, which was originally designed to investigate the planning skills of nonhuman apes. First, we were interested in the development of children’s performance in the two tasks and whether the strategies children used to succeed differed among age groups. Performance improved significantly across age groups in both tasks. Strategies for success in the advance planning task differed among age groups, with 4- and 5-year-olds performing more excess actions, and a greater proportion of irrelevant excess actions, than older children. Findings are discussed in relation to the development of performance in tower tasks, which are a commonly used test of planning ability in humans. Second, based on previous findings with apes, we predicted that introducing measures to reduce the inhibitory demands of the advance planning task would improve children’s performance. Therefore, in this study we introduced two methodological alterations that have been shown to improve children’s performance in other tasks with inhibitory demands: (a) imposing a short delay before a child is allowed to act and (b) replacing reward items with symbolic tokens. Surprisingly, neither of these measures improved the performance of children in any of the age groups, suggesting that, contrary to our prediction, inhibitory control might not be a key performance-limiting factor in the advance planning paddle-box task.
    Journal of Experimental Child Psychology 01/2014;
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    ABSTRACT: Flavonoids are group of plant-derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio-accumulate within humans and are believed to have beneficial health effects including cancer chemo-protection. One mechanism that has been proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca(2+) ATPase. An investigation into the mechanism of inhibition of 3 flavonoids, quercetin, galangin and 3,6 dihydroxyflavone (3,6-DHF) was undertaken. Each inhibited the Ca(2+) ATPase with Ki values of 8.7 μM, 10.3 μM and 5.4 μM, respectively, showing cooperative inhibition with n~2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E1 conformation and reduced [(32) P]-ATP binding. However, both galangin and 3,6-DHF increased Ca(2+) affinity to the ATPase by decreasing the Ca(2+) -dissociation rate constant, while quercetin had little effect. Ca(2+) -induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6-DHF and galangin (but not with quercetin), indicating that Ca(2+) -associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP-dependent phosphorylation and dephosphorylation, while 3,6-DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites, one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesise that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids. This article is protected by copyright. All rights reserved.
    FEBS Journal 11/2013;
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    ABSTRACT: The production of recombinant proteins is crucial for both the development of new protein drugs and the structural determination of drug targets. As such, recombinant protein production has a major role in drug development. Bacterial hosts are commonly used for the production of recombinant proteins, accounting for approximately 30% of current biopharmaceuticals on the market. In this review, I introduce fundamental concepts in recombinant protein production in bacteria, from drug development to production scales. Recombinant protein production processes can often fail, but how can this failure be minimised to deliver rapidly maximum yields of high-quality protein and so accelerate drug discovery?
    Drug discovery today 11/2013;
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    ABSTRACT: Progress in microbiology has always been driven by technological advances, ever since Antonie van Leeuwenhoek discovered bacteria by making an improved compound microscope. Yet until very recently, we have not been able to identify microbes and record their mostly invisible activities such as nutrient consumption or toxin production on the level of the single cell, not even in the laboratory. This is now changing with the rapid rise of exciting new technologies for single-cell microbiology (1, 2), which enables microbiologists to do what plant and animal ecologists have been doing for a long time: observe who does what, when, where and next to whom. Single cells taken from the environment can be identified and even their genome sequenced. Ex situ, their size, elemental and biochemical composition, and other characteristics, can be measured with high-throughput and cells sorted accordingly. Even better, individual microbes can be observed in situ with a range of novel microscopic and spectroscopic methods, enabling localization, identification or functional characterization of cells in a natural sample, combined with detecting uptake of labeled compounds. Alternatively, they can be placed into fabricated microfluidic environments, where they can be positioned, exposed to stimuli, monitored, and their interactions controlled "in microfluido". By introducing genetically engineered reporter cells into a fabricated landscape or a microcosm taken from nature, their reproductive success or activity can be followed, or their sensing of their local environment recorded.
    Proceedings of the National Academy of Sciences 11/2013; 110(45):18027-8.
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    ABSTRACT: Hexabromocyclododecane (HBCD) is a widely utilised brominated flame retardant (BFR). It has been shown to bio-accumulate within organisms, including man, and possibly cause neurological disorders. The acute neurotoxicity of HBCD, and 6 other unrelated BFRs, were assessed in SH-SY5Y human neuroblastoma cells by 24 hour viability assays and HBCD proved to be the most lethal (LC50, 3μM). In addition, the effects of these BFRs were also assessed for their potency at inhibiting the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA) derived from the SH-SY5Y cells and again HBCD was the most potent (IC50, 2.7μM). The data for the other BFRs tested showed a direct correlation (coefficient 0.94) between the potencies of inducing cell death and inhibiting the Ca(2+) ATPase, indicating that SERCA is likely to be the molecular target for acute toxicity. Mechanistic studies of HBCD on the Ca(2+) ATPase suggest that it affects ATP binding, phosphorylation as well as the E2 to E1 transition step.
    Chemico-biological interactions 11/2013;
  • Nature 10/2013; 502(7472):448.
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    ABSTRACT: The ability to identify an appropriate sequence of actions or to consider alternative possible action sequences might be particularly useful during problem solving in the physical domain. We developed a new 'paddle-box' task to test the ability of different ape species to plan an appropriate sequence of physical actions (rotating paddles) to retrieve a reward from a goal location. The task had an adjustable difficulty level and was not dependent on species-specific behaviours (e.g. complex tool use). We investigated the planning abilities of captive orangutans (Pongo pygmaeus) and bonobos (Pan paniscus) using the paddle-box. In experiment 1, subjects had to rotate one or two paddles before rotating the paddle with the reward on. Subjects of both species performed poorly, though orangutans rotated more non-food paddles, which may be related to their greater exploratory tendencies and bolder temperament compared with bonobos. In experiment 2 subjects could always rotate the paddle with the reward on first and still succeed, and most subjects of both species performed appropriate sequences of up to three paddle rotations to retrieve the reward. Poor performance in experiment 1 may have been related to subjects' difficulty in inhibiting the prepotent response to act on the reward immediately.
    Behavioural processes 10/2013;
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    ABSTRACT: Both genetic and epigenetic responses of organisms to environmental factors, including chemical exposures, influence adaptation, susceptibility to toxicity and biodiversity. In model organisms, it is established that epigenetic alterations, including changes to the methylome, can create a memory of the received signal. This is partly evidenced through the analysis of epigenetic differences that develop between identical twins throughout their lifetime. The epigenetic marks induce alterations to the gene expression profile, which, in addition to mediating homeostatic responses, have the potential to promote an abnormal physiology either immediately or at a later stage of development, for example leading to an adult onset of disease. Although this has been well established, epigenetic mechanisms are not considered in chemical risk assessment or utilised in the monitoring of the exposure and effects of chemicals and environmental change. In this review, epigenetic factors, specifically DNA methylation, are highlighted as mechanisms of adaptation and response to environmental factors and which, if persistent, have the potential, retrospectively, to reflect previous stress exposures. Thus, it is proposed that epigenetic "foot-printing" of organisms could identify classes of chemical contaminants to which they have been exposed throughout their lifetime. In some cases, the potential for persistent transgenerational modification of the epigenome may also inform on parental germ cell exposures. It is recommended that epigenetic mechanisms, alongside genetic mechanisms, should eventually be considered in environmental toxicity safety assessments and in biomonitoring studies. This will assist in determining the mode of action of toxicants, no observed adverse effect level and identification of biomarkers of toxicity for early detection and risk assessment in toxicology but there are critical areas that remain to be explored before this can be achieved.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2013;
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    ABSTRACT: In eukaryotes the 40S and 60S ribosomal subunits are assembled in the nucleolus, but there appear to be mechanisms preventing mRNA binding, 80S formation, and initiation of translation in the nucleus. To visualize association between ribosomal subunits, we tagged pairs of Drosophila ribosomal proteins (RPs) located in different subunits with mutually complementing halves of fluorescent proteins. Pairs of tagged RPs expected to interact, or be adjacent in the 80S structure, showed strong fluorescence, while pairs that were not in close proximity did not. Moreover, the complementation signal is found in ribosomal fractions and it was enhanced by translation elongation inhibitors and reduced by initiation inhibitors. Our technique achieved 80S visualization both in cultured cells and in fly tissues in vivo. Notably, while the main 80S signal was in the cytoplasm, clear signals were also seen in the nucleolus and at other nuclear sites. Furthermore, we detected rapid puromycin incorporation in the nucleolus and at transcription sites, providing an independent indication of functional 80S in the nucleolus and 80S association with nascent transcripts.
    RNA 10/2013;
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