Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye (TM) and analysed by two-dimensional difference gel. electrophoresis. Gel images analysed off-line, using the DeCyder (TM) image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Flavonoids and brain health: multiple effects underpinned by common mechanisms

The neuroprotective actions of dietary flavonoids involve a number of effects within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. This multiplicity of effects appears to be underpinned by two processes. Firstly, they interact with important neuronal signalling cascades leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and differentiation. These interactions include selective actions on a number of protein kinase and lipid kinase signalling cascades, most notably the PI3K/Akt and MAP kinase pathways which regulate pro-survival transcription factors and gene expression. Secondly, they induce peripheral and cerebral vascular blood flow in a manner which may lead to the induction of angiogenesis, and new nerve cell growth in the hippocampus. Therefore, the consumption of flavonoid-rich foods, such as berries and cocoa, throughout life holds a potential to limit the neurodegeneration associated with a variety of neurological disorders and to prevent or reverse normal or abnormal deteriorations in cognitive performance.

Effect of apoE genotype and vitamin E on biomarkers of oxidative stress in cultured neuronal cells and the brain of targeted replacement mice

The aetiology of apoE4 genotype-Alzheimer's disease (AD) association are complex. The current study emphasizes the impact of apoE genotype and potential beneficial effects of vitamin E (VE) in relation to oxidative stress. Agonist induced neuronal cell death was examined 1) in the presence of conditioned media containing equal amounts of apoE3 or apoE4 obtained from stably transfected macrophages, and 2) after pretreatment with alpha- and gamma-tocopherol, and -tocotrienol. ApoE3 and apoE4 transgenic mice were fed a diet poor or rich in VE to study the interplay of both apoE genotype and VE status, on membrane lipid peroxidation, antioxidative enzyme activity and glutathione levels in the brain. Cytotoxicity of hydrogen peroxide and glutamate was higher in neuronal cells cultured with apoE4 than apoE3 conditioned media. VE pre-treatment of neurons counteracted the cytotoxicity of a peroxide challenge but not of nitric oxide. No significant effects of apoE genotype or VE supplementation were observed on lipid peroxidation or antioxidative status in the brain of apoE3 and apoE4 mice. VE protects against oxidative insults in vitro, however, no differences in brain oxidative status were observed in mice. Unlike in cultured cells, apoE4 may not contribute to higher neuronal oxidative stress in the brain of young targeted replacement mice.

Genistein reverses changes of the proteome induced by oxidized-LDL in EA center dot hy 926 human endothelial cells

Endothelial cells are primary targets for pro-atherosclerotic stressors such as oxidized LDL (ox-LDL). The isoflavone genistein, on the other hand, is suggested to prevent a variety of processes underlying atherosclerosis and cardiovascular diseases. By analyzing the proteome of EA(.)hy 926 endothelial cells, here we show, that genistein reverses the ox-LDL-induced changes of the steady-state levels of several proteins involved in atherosclerosis. These alterations caused by genistein are functionally linked to the inhibition of ox-LDL induced apoptosis.

Genistein blocks homocysteine-induced alterations in the proteome of human endothelial cells

Dietary isoflavones from soy are suggested to protect endothelial cells from damaging effects of endothelial stressors and thereby to prevent atherosclerosis. In search of the molecular targets of isoflavone action, we analyzed the effects of the major soy isoflavone, genistein, on changes in protein expression levels induced by the endothelial stressor homocysteine (Hcy) in EA.hy 926 endothelial cells. Proteins from cells exposed for 24 h to 25 mu M Hcy alone or in combination with 2.5 mu M genistein were separated by two-dimensional gel electrophoresis and those with altered spot intensities were identified by peptide mass fingerprinting, Genistein reversed Hcy-induced changes of proteins involved in metabolism, detoxification, and gene regulation: and some of those effects can be linked functionally to the antiatherosclerotic properties of the soy isoflavone. Alterations of steady-state levels of cytoskeletal proteins by genistein suggested an effect oil apoptosis. As a matter of fact genistein caused inhibition of Hcy-mediated apoptotic cell death as indicated by inhibition of DNA fragmentation and chromatin condensation. In conclusion, proteome analysis allows the rapid identification of cellular target proteins of genistein action in endothelial cells exposed to the endothelial stressor Hcy and therefore enables the identification of molecular pathways of its antiatherosclerotic action

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine

Background Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. Aim of the study To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells ( HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). Methods HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 muM) or by pharmacological intervention ( 25 muM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. Results Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. Conclusions Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Age-associated changes in protein oxidation and proteasome activities in rat brain: modulation by antioxidants

The free radical theory of ageing postulates that age-associated neurodegeneration is caused by an imbalance between pro-oxidants and antioxidants resulting in oxidative stress. The current study showed regional variation in brain susceptibility to age-associated oxidative stress as shown by increased lipofuscin deposition and protein carbonyl levels in male rats of age 15-16 months compared to control ones (3-5 months). The hippocampus is the area most vulnerable to change compared to the cortex and cerebellum. However, proteasomal enzyme activity was not affected by age in any of the brain regions studied. Treatment with melatonin or coenzyme Q10 for 4 weeks reduced the lipofuscin content of the hippocampus and carbonyl level. However, both melatonin and coenzyme Q10 treatments inhibited beta-glutamyl peptide hydrolase activity. This suggests that these molecules can alter proteasome function independently of their antioxidant actions. (c) 2005 Elsevier Inc. All rights reserved.

Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels

Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway. (c) 2008 Elsevier Inc. All Fights reserved.

Theory of mind in children with traumatic brain injury

Objective: This study was designed to examine the existence of deficits in mentalizing or theory of mind (ToM) in children with traumatic brain injury (TBI). Research design: ToM functioning was assessed in 12 children aged 6-12 years with TBI and documented frontal lobe damage and compared to 12 controls matched for age, sex and verbal ability. Brief measures of attention and memory were also included. Main outcome and results: The TBI group was significantly impaired relative to controls on the advanced ToM measure and a measure of basic emotion recognition. No difference was found in a basic measure of ToM. Conclusion: Traumatic brain damage in childhood may disrupt the developmental acquisition of emotion recognition and advanced ToM skills. The clinical and theoretical importance of these findings is discussed and the implications for the assessment and treatment of children who have experienced TBI are outlined.

fMRI delineation of working memory for emotional prosody in the brain: commonalities with the lexico-semantic emotion network

Decoding emotional prosody is crucial for successful social interactions, and continuous monitoring of emotional intent via prosody requires working memory. It has been proposed by Ross and others that emotional prosody cognitions in the right hemisphere are organized in an analogous fashion to propositional language functions in the left hemisphere. This study aimed to test the applicability of this model in the context of prefrontal cortex working memory functions. BOLD response data were therefore collected during performance of two emotional working memory tasks by participants undergoing fMRI. In the prosody task, participants identified the emotion conveyed in pre-recorded sentences, and working memory load was manipulated in the style of an N-back task. In the matched lexico-semantic task, participants identified the emotion conveyed by sentence content. Block-design neuroimaging data were analyzed parametrically with SPM5. At first, working memory for emotional prosody appeared to be right-lateralized in the PFC, however, further analyses revealed that it shared much bilateral prefrontal functional neuroanatomy with working memory for lexico-semantic emotion. Supplementary separate analyses of males and females suggested that these language functions were less bilateral in females, but their inclusion did not alter the direction of laterality. It is concluded that Ross et al.'s model is not applicable to prefrontal cortex working memory functions, that evidence that working memory cannot be subdivided in prefrontal cortex according to material type is increased, and that incidental working memory demands may explain the frontal lobe involvement in emotional prosody comprehension as revealed by neuroimaging studies. (c) 2007 Elsevier Inc. All rights reserved.

How does the brain mediate interpretation of incongruent auditory emotions? The neural response to prosody in the presence of conflicting lexico-semantic cues

We frequently encounter conflicting emotion cues. This study examined how the neural response to emotional prosody differed in the presence of congruent and incongruent lexico-semantic cues. Two hypotheses were assessed: (i) decoding emotional prosody with conflicting lexico-semantic cues would activate brain regions associated with cognitive conflict (anterior cingulate and dorsolateral prefrontal cortex) or (ii) the increased attentional load of incongruent cues would modulate the activity of regions that decode emotional prosody (right lateral temporal cortex). While the participants indicated the emotion conveyed by prosody, functional magnetic resonance imaging data were acquired on a 3T scanner using blood oxygenation level-dependent contrast. Using SPM5, the response to congruent cues was contrasted with that to emotional prosody alone, as was the response to incongruent lexico-semantic cues (for the 'cognitive conflict' hypothesis). The right lateral temporal lobe region of interest analyses examined modulation of activity in this brain region between these two contrasts (for the 'prosody cortex' hypothesis). Dorsolateral prefrontal and anterior cingulate cortex activity was not observed, and neither was attentional modulation of activity in right lateral temporal cortex activity. However, decoding emotional prosody with incongruent lexico-semantic cues was strongly associated with left inferior frontal gyrus activity. This specialist form of conflict is therefore not processed by the brain using the same neural resources as non-affective cognitive conflict and neither can it be handled by associated sensory cortex alone. The recruitment of inferior frontal cortex may indicate increased semantic processing demands but other contributory functions of this region should be explored.

How the brain blinks: towards a neurocognitive model of the attentional blink

When people monitor a visual stream of rapidly presented stimuli for two targets (T1 and T2), they often miss T2 if it falls into a time window of about half a second after T1 onset-the attentional blink (AB). We provide an overview of recent neuroscientific studies devoted to analyze the neural processes underlying the AB and their temporal dynamics. The available evidence points to an attentional network involving temporal, right-parietal and frontal cortex, and suggests that the components of this neural network interact by means of synchronization and stimulus-induced desynchronization in the beta frequency range. We set up a neurocognitive scenario describing how the AB might emerge and why it depends on the presence of masks and the other event(s) the targets are embedded in. The scenario supports the idea that the AB arises from "biased competition", with the top-down bias being generated by parietal-frontal interactions and the competition taking place between stimulus codes in temporal cortex.