Flavonoids: antioxidants or signalling molecules?

Many studies are accumulating that report the neuroprotective, cardioprotective, and chemopreventive actions of dietary flavonoids. While there has been a major focus on the antioxidant properties, there is an emerging view that flavonoids, and their in vivo metabolites, do not act as conventional hydrogen-donating antioxidants but may exert modulatory actions in cells through actions at protein kinase and lipid kinase signalling pathways. Flavonoids, and more recently their metabolites, have been reported to act at phosphoinositide 3-kinase (PI 3-kinase), Akt/protein kinase B (Akt/PKB), tyrosine kinases, protein kinase C (PKC), and mitogen activated protein kinase (MAP kinase) signalling cascades. Inhibitory or stimulatory actions at these pathways are likely to affect cellular function profoundly by altering the phosphorylation state of target molecules and by modulating gene expression. A clear understanding of the mechanisms of action of flavonoids, either as antioxidants or modulators of cell signalling, and the influence of their metabolism on these properties are key to the evaluation of these potent biomolecules as anticancer agents, cardioprotectants, and inhibitors of neurodegeneration (C) 2004 Elsevier Inc. All rights reserved.

Reduced heart rate responding to trauma reliving in trauma survivors with PTSD: correlates and consequences

The authors investigated whether heart rate (HR) responses to voluntary recall of trauma memories (a) are related to posttraumatic stress disorder (PTSD), and (b) predict recovery 6 months later. Sixty-two assault survivors completed a recall task modeled on imaginal reliving in the initial weeks postassault. Possible cognitive modulators of HR responsivity were assessed; dissociation, rumination, trauma memory disorganization. Individuals with PTSD showed a reduced HR response to reliving compared to those without PTSD, but reported greater distress. Notably, higher HR response but not self-reported distress during reliving predicted greater symptom reduction at follow-up in participants with PTSD. Engagement in rumination was the only cognitive factor that predicted lower HR response. The data are in contrast to studies using trauma reminders to trigger memories, which have found greater physiological reactivity in PTSD. The authors' observations are consistent with models of PTSD that highlight differences between cued or stimulus-driven retrieval and intentional trauma recall, and with E B. Foa and M.J. Kozak (1986) hypothesis that full activation of trauma memories facilitates emotional processing.

Postprandial lipemia and cardiovascular disease risk: interrelationships between dietary, physiological and genetic determinants

Although the independence of the association and causality has not been fully established, non-fasting (postprandial) triglyceride (TG) concentrations have emerged as a clinically significant cardiovascular disease (CVD) risk factor. In the current review, findings from three insightful prospective studies in the area, namely the Women's Health Study, the Copenhagen City Heart Study and the Norwegian Counties Study, are discussed. An overview is provided as to the likely etiological basis for the association between postprandial TG and CVD, with a focus on both lipid and non-lipid (inflammation, hemostasis and vascular function) risk factors. The impact of various lifestyle and physiological determinants are considered, in particular genetic variation and meal fat composition. Furthermore, although data is limited some information is provided as to the relative and interactive impact of a number of modulators of lipemia. It is evident that relative to age, gender and body mass index (known modulators of postprandial lipemia), the contribution of identified gene variants to the heterogeneity observed in the postprandial response is likely to be relatively small. Finally, we highlight the need for the development of a standardised ‘fat tolerance test’ for use in clinical trials, to allow the integration and comparison of data from individual studies

Gap junction-mediated spontaneous Ca(2+) waves in differentiated cholinergic SN56 cells

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca(2+) waves. These waves were unaffected by tetrodotoxin (1microM), but blocked by removal of extracellular Ca(2+), or addition of non-specific Ca(2+) channel inhibitors (Cd(2+) (0.1mM) or Ni(2+) (1mM)). Combined application of antagonists of NMDA receptors (AP5; 100microM), AMPA/kainate receptors (NBQX; 20microM), nicotinic AChR receptors (hexamethonium; 100microM) or inotropic purinoceptors (brilliant blue; 100nM) was also without effect. However, Ca(2+) waves were fully prevented by carbenoxolone (200microM), halothane (3mM) or niflumic acid (100microM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd(2+), Ni(2+) or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca(2+) waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.

Targeting Staphylococcus aureus quorum sensing with non-peptidic small molecule inhibitors

A series of 3-oxo-C12-HSL, tetramic acid and tetronic acid analogues was synthesized to gain insights into the structural requirements for quorum sensing inhibition in Staphylococcus aureus. Compounds active against agr were non-competitive inhibitors of the auto-inducing peptide (AIP)-activated AgrC receptor, by altering the activation efficacy of the cognate AIP-1. They appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17 which reduced nasal cell colonization and arthritis in a murine infection model.

Modulation of potassium channels inhibits bunyavirus infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Effects of short chain fatty acids on effector mechanisms of neutrophils

Short chain fatty acids (SCFAs) are metabolic by products of anerobic bacteria fermentation. These fatty acids, despite being an important fuel for colonocytes, are also modulators of leukocyte function. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate, and butyrate) on function of neutrophils, and the possible mechanisms involved. Neutrophils obtained from rats by intraperitoneal lavage 4 h after injection of oyster glycogen solution (1%) were treated with non toxic concentrations of the fatty acids. After that, the following measurements were performed: phagocytosis and destruction of Candida albicans, production of ROS (O(2)(center dot-), H(2)O(2), and HOCl) and degranulation. Gene expression (p47(phox) and p22(phox)) and protein phosphorylation (p47(phox)) were analyzed by real time reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. Butyrate inhibited phagocytosis and killing of C. albicans. This SCFA also had an inhibitory effect on production of O(2)(center dot-), H(2)O(2), and HOCI by neutrophils stimulated with PMA or fMLP. This effect of butyrate was not caused by modulation of expression of NADPH oxidase subunits (p47(phox) and p22(phox)) but it was in part due to reduced levels of p47(phox) phosphorylation and an increase in the concentration of cyclic AMP. Acetate increased the production of O(2)(center dot-) and H(2)O(2), in the absence of stimuli but had no effect on phagocytosis and killing of C. albicans. Propionate had no effect on the parameters studied. These results suggest that butyrate can modulate neutrophil function, and thus could be important in inflammatory neutrophil-associated diseases. Copyright (C) 2008 John Wiley & Sons, Ltd.

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-2 (CINC-2 alpha beta)] by rat neutrophils. The involvement of nuclear factor kappa B (NF-kappa B) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-alpha, CINC-2 alpha beta and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-kappa B activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

CHANGES OF GENE EXPRESSION IN ELECTRICALLY STIMULATED AND CONTRALATERAL RAT SOLEUS MUSCLES

In this study we investigate the effect of a single session of high-intensity contractions on expression of pleiotropic genes and, in particular, those genes associated with metabolism in soleus muscle from electrically stimulated (ES) and contralateral (CL) limbs. The right limbs of male Wistar rats were submitted to contractions by 200-ms trains of electrical stimulation at 100-Hz frequency with pulses of 0.1 ms (voltage 24 3 V) delivered each second for 1 hour. Soleus muscles were isolated 1 hour after contraction, and gene expression was analyzed by a macroarray technique (Atlas Toxicology 1.2 Array; Clontech Laboratories). Electrical stimulation increased expression in 92 genes (16% of the genes present in the membrane). Sixty-six genes were upregulated in both ES and CL soleus muscles, and expression of 26 genes was upregulated in the ES muscle only. The most altered genes were those related to stress response and metabolism. Electrical stimulation also raised expression of transcription factors, translation and posttranslational modification of proteins, ribosomal proteins, and intracellular transducers/effectors/modulators. The results indicate that a single session of electrical stimulation upregulated expression of genes related to metabolism and oxidative stress in soleus muscle from both ES and CL limbs. These findings may indicate an association with tissue hypertrophy and metabolic adaptations induced by physical exercise training not only in the ES but also in the CL non-stimulated muscle, suggesting a cross-education phenomenon. Muscle Nerve 40: 838-846, 2009

Phosphoproteomics Profiling Suggests a Role for Nuclear beta IPKC in Transcription Processes of Undifferentiated Murine Embryonic Stem Cells

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal and differentiation However, the function of specific PKC Isoenzymes have yet to be determined Of the PKCs expressed in undifferentiated ESCs, beta IPKC was the only isoenzyme abundantly expressed in the nuclei To investigate the role of beta IPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one beta IPKC-specific inhibitor peptide We identified 13 nuclear proteins that are direct or indirect beta IPKC substrates in undifferentiated ESCs These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation Inhibiting beta IPKC had no effect on DNA synthesis in undifferentiated ESCs However, upon differentiation many cells seized to express beta IPKC and beta IPKC was frequently found in the cytoplasm Taken together, our results suggest that beta IPKC takes part in the processes that maintain ESCs in their undifferentiated state

Modularity and robustness of bone networks

Cortical bones, essential for mechanical support and structure in many animals, involve a large number of canals organized in intricate fashion. By using state-of-the art image analysis and computer graphics, the 3D reconstruction of a whole bone (phalange) of a young chicken was obtained and represented in terms of a complex network where each canal was associated to an edge and every confluence of three or more canals yielded a respective node. The representation of the bone canal structure as a complex network has allowed several methods to be applied in order to characterize and analyze the canal system organization and the robustness. First, the distribution of the node degrees (i.e. the number of canals connected to each node) confirmed previous indications that bone canal networks follow a power law, and therefore present some highly connected nodes (hubs). The bone network was also found to be partitioned into communities or modules, i.e. groups of nodes which are more intensely connected to one another than with the rest of the network. We verified that each community exhibited distinct topological properties that are possibly linked with their specific function. In order to better understand the organization of the bone network, its resilience to two types of failures (random attack and cascaded failures) was also quantified comparatively to randomized and regular counterparts. The results indicate that the modular structure improves the robustness of the bone network when compared to a regular network with the same average degree and number of nodes. The effects of disease processes (e. g., osteoporosis) and mutations in genes (e.g., BMP4) that occur at the molecular level can now be investigated at the mesoscopic level by using network based approaches.

Structure-Based Approach for the Study of Estrogen Receptor Binding Affinity and Subtype Selectivity

Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpa (hER alpha) and beta (hER beta). Because the levels and relative proportion of hER alpha and hER beta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hER alpha and hER beta. Significant statistical coefficients were obtained (hER alpha, q(2) = 0.76; hER beta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hER alpha and hER beta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design or novel hER modulators with improved selectivity.

Morphometric analysis of the urethra of castrated female rats treated with tamoxifen

Objectives: The aim of this study was to evaluate the effects of tamoxifen on the weight and thickness of the urethral epithelium of castrated female rats. Methods: Forty castrated adult female Wistar-Hannover rats were randomly divided into two groups: Group I (n = 20) in which the animals received only the vehicle (propylene glycol) and Group 11 (n = 20) in which the rats received tamoxifen 250 mu g/day by gavage. After 30 days of treatment, all animals were sacrificed and the urethra was immediately removed for weighing. Next, the urethra was divided into the proximal and distal segments, which were fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The data were analyzed using the weighted minimum mean-square error method and Student`s t-test for two independent samples (p < 0.05). Results: There was a significant increase in the mean weight of the urethra in the rats of Group 11 compared to the control group, 32.0 +/- 2.0 mg and 22.0 +/- 1.6 mg, respectively (p < 0.001). The mean thickness of the distal urethral epithelium of the animals treated with tamoxifen was significantly greater than that of the control group, 42.8 +/- 2.0 mu m and 36.6 +/- 1.5 mu m, respectively (p < 0.001). There was no statistically significant difference between the two groups with respect to the epithelial thickness of the proximal urethra (p = 0.514). Conclusion: Treating castrated adult rats with 250 mu g/day of tamoxifen for 30 days may increase the weight of the urethra and the thickness of the distal urethral epithelium. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Effects of Raloxifene on Ki-67 and CD34 Antigen Expression in Breast Cancer

Background: The aim of this study was to evaluate the effect of raloxifene on CD34 and Ki-67 antigen expression in breast cancer specimens from postmenopausal women. Methods: Sixteen postmenopausal patients with operable, stage II (>= 3 cm), estrogen receptor-positive breast cancer, who took 60 mg of raloxifene daily for 28 days, participated in this study. Immunohistochemistry was carried out in tumor samples prior to and following raloxifene treatment to evaluate CD34 and Ki-67 protein expression. Angiogenesis was quantified in 10 randomly selected fields per slide, and Ki-67-stained nuclei were counted in 1,000 cells per slide using an image capture and analysis system with 400 ! magnification. Student`s t test for paired samples was used for the statistical analysis of data. Statistical significance was established at p < 0.05. Results: The mean number of microvessels was 44.44 +/- 3.54 prior to raloxifene therapy and 22.63 +/- 1.61 following therapy (p < 0.001), and the mean percentage of Ki-67-stained nuclei was 19.28 +/- 8 1.61 and 12.13 +/- 8 1.48 prior to and following raloxifene treatment, respectively (p < 0.001). Conclusion: Raloxifene significantly reduces CD34 and Ki-67 protein expression in breast carcinoma in postmenopausal women. Copyright (C) 2008 S. Karger AG, Basel

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.