Medical Library

Placebo response to caffeine improves reaction time performance in sleepy people.

Caffeine is the most widely used stimulant to counteract sleepiness. However, little is known about any placebo effect of caffeine in sleepy people and the effect of suggestibility. Over a 95 min test period, and in a counterbalanced design, 16 young healthy adults underwent 3 x 30 min sessions at the psychomotor vigilance test (PVT), during an early afternoon \’dip\’ enhanced by a prior night\’s sleep restriction (5 h). On both occasions they were given a cup of a decaffeinated coffee; once when the participant was verbally primed to suggest the coffee was caffeinated (Placebo) and on the other under neutral priming (Control). There were significantly fewer lapses and shorter reaction times following Placebo, for the initial two 30 min sessions, indicating that suggestion about consuming caffeine was effective in improving performance in moderately sleepy people. Copyright (c) 2008 John Wiley & Sons, Ltd.

Anderson C, Horne JA.

Sleep Research Centre, Department of Human Sciences, Loughborough University, Loughborough, Leicestershire, LE11 3TU, UK.

Biophysics meets membrane-active peptides.

Castanho MA, Dathe M.

Dark proteins: Effect of inclusion body formation on quantification of protein expression.

Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell\’s dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus \”dark\” to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used calibrated fluorescent intensity measurements to determine the average number of active EGFP present per cell. Both measurements were carried out as a function of cellular doubling time, over a range of 45-75 min. We found that the ratio of inclusion body EGFP to active EGFP varied strongly as a function of the cellular growth rate, and that the number of \”dark\” proteins in the aggregates could in fact be substantial, reaching ratios as high as approximately five proteins locked into inclusion bodies for every active protein (at the fastest growth rate), and dropping to ratios well below 1 (for the slowest growth rate). Our results suggest that efforts to compare computational models to protein numbers derived from fluorescence measurements should take inclusion body loss into account, especially when working with rapidly growing cells. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, McMillen DR.

Department of Chemical and Physical Sciences, and Institute for Optical Sciences, University of Toronto Mississauga, Mississauga ON L5L 1C6, Canada.

Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: Structural evidence for cysteine binding and lack of interactions with serine acetyl transferase.

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

Chinthalapudi K, Kumar M, Kumar S, Jain S, Alam N, Gourinath S.

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

SPARC-induced increase in glioma matrix and decrease in vascularity are associated with reduced VEGF expression and secretion.

Glioblastomas are heterogeneous tumors displaying regions of necrosis, proliferation, angiogenesis, apoptosis and invasion. SPARC, a matricellular protein that negatively regulates angiogenesis and cell proliferation, but enhances cell deadhesion from matrix, is upregulated in gliomas (Grades II-IV). We previously demonstrated that SPARC promotes invasion while concomitantly decreasing tumor growth, in part by decreasing proliferation of the tumor cells. In other cancer types, SPARC has been shown to influence tumor growth by altering matrix production, and by decreasing angiogenesis via interfering with the VEGF-VEGFR1 signaling pathway. We therefore examined whether the SPARC-induced decrease in glioma tumor growth was also, in part, due to alterations in matrix and/or decreased vascularity, and assessed SPARC-VEGF interactions. The data demonstrate that SPARC upregulates glioma matrix, collagen I is a constituent of the matrix and SPARC promotes collagen fibrillogenesis. Furthermore, SPARC suppressed glioma vascularity, and this was accompanied by decreased VEGF expression and secretion, which was, in part, due to reduced VEGF165 transcript abundance. These data indicate that SPARC modulates glioma growth by altering the tumor microenvironment and by suppressing tumor vascularity through suppression of VEGF expression and secretion. These experiments implicate a novel mechanism, whereby SPARC regulates VEGF function by limiting the available growth factor. Because SPARC is considered to be a therapeutic target for gliomas, a further understanding of its complex signaling mechanisms is important, as targeting SPARC to decrease invasion could undesirably lead to the growth of more vascular and proliferative tumors. (c) 2008 Wiley-Liss, Inc.

Yunker CK, Golembieski W, Lemke N, Schultz CR, Cazacu S, Brodie C, Rempel SA.

Barbara Jane Levy Laboratory of Molecular Neuro‐Oncology, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, MI.

Erythropoietin ameliorates chemotherapy-induced fibrosis of the lungs in a preclinical murine model.

Organ toxicity induced by chemotherapeutic drugs is a serious obstacle in the effective treatment of patients suffering from cancer and autoimmune disease. A strong association exists between pulmonary toxicity, particularly fibrosis, and chemotherapeutic drugs. Attempts have been made to identify compounds capable of suppressing fibrosis. In addition to its erythropoietic activity, erythropoietin (EPO) has been shown to have effects on nonhemopoietic cells. Therefore, we postulated that EPO may exert beneficial effects on lung tissue during chemotherapy. To test our hypothesis, we investigated pulmonary changes caused by bleomycin, a fibrosis-inducing agent, in animals treated with the drug alone and in combination with EPO. Fibrosis, cellular alterations and structural changes were assayed by blind analysis of the lung sections. A 6-fold decrease in the number of prominent endothelial cells-suspected to be indicative of cellular activation and inflammatory response-was observed in lung sections derived from mice treated with bleomycin and EPO compared to animals injected with bleomycin alone (p < 0.008). Additionally, there was twice the number of ICAM1-positive endothelial cells in animals treated with bleomycin alone compared with the number in the bleomycin and EPO-treated group (p < 0.05). Alveolar mononuclear phagocytic hyperplasia was reduced by as much as 100% in animals treated with bleomycin and EPO compared to animals treated with bleomycin alone (p < 0.03). Finally, a 5-fold decrease in interstitial fibrosis was observed in lung sections obtained from animals treated with bleomycin and EPO (p < 0.02). We conclude that EPO can ameliorate drug-induced fibrosis and endothelial damage caused by chemotherapeutic agents. (c) 2008 Wiley-Liss, Inc.

Sigounas G, Salleng KJ, Mehlhop PD, Sigounas DG.

Hematology/Oncology Section, Department of Internal Medicine, Brody School of Medicine, East Carolina University, Greenville, NC.

Spotlight.

Kirchweger G.

Next-day cognition, psychomotor function, and driving-related skills following nighttime administration of eszopiclone.

OBJECTIVE: To evaluate next-day driving ability, as assessed by brake reaction time (BRT), and cognitive/psychomotor function following nighttime administration of 3 mg eszopiclone. METHODS: Two randomized, double-blind, placebo-controlled, cross-over studies were performed in healthy volunteers (n = 32) and patients with primary insomnia (n = 32). Study participants received nighttime dosing of 3 mg eszopiclone or placebo. BRT and a psychometric test battery were used to assess the next-day effects of eszopiclone treatment. RESULTS: In both studies, driving ability and measures of cognitive and psychomotor function were not impaired the morning after eszopiclone, as compared to placebo. All eszopiclone subjects reported improved ease in getting to sleep and quality of sleep with no significant changes in behavior upon awakening. A significant increase in next-day feelings of sedation was reported in healthy volunteers, but not in patients with primary insomnia, following eszopiclone treatment relative to placebo. Sleep induction, maintenance, duration, and efficiency, as assessed by PSG, were significantly improved following eszopiclone treatment in patients with insomnia. CONCLUSIONS: Nighttime administration of 3 mg eszopiclone improved objective and subjective sleep measures in patients with insomnia (and subjective sleep measures in healthy patients) and did not impair next-day driving-related skills or measures of cognition in either study population relative to placebo. Copyright (c) 2008 John Wiley & Sons, Ltd.

Boyle J, Trick L, Johnsen S, Roach J, Rubens R.

Clinical Research Centre, University of Surrey, Guildford, Surrey, UK.

Erratum: MRI monitoring of focal cerebral ischemia in peroxisome proliferator-activated receptor (PPAR)-deficient mice.

NMR In Biomedicine: MRI monitoring of focal cerebral ischemia in peroxisome proliferator-activated receptor (PPAR)-deficient mice. Jean-Baptiste Pialat, Tae-Hee Chao, Olivier Beuf, Elisabeth Joye, Samir Moucharaffie, Jean-Baptiste Langlois, Chantal Nemoz, Marc Janier, Yves Berthezene, Norbert Nighoghossian, Béatrice Desvergnre and Marlene Wiart. Published online April 24 2007, Issue 20:3, pp. 335-342; DOI: 10.1002/nbm.1157.Following publication it has been noted that an author\’s name was misspelt. As above, the author was spelt as \”Samir Moucharaffie\”. It should have been spelt as \”Samir Moucharrafie\”.

Pialat JB, Cho TH, Beuf O, Joye E, Moucharaffie S, Langlois JB, Nemoz C, Janier M, Berthezene Y, Nighoghossian N, Desvergne B, Wiart M.

Université Lyon 1, Laboratoire CREATIS, INSA de Lyon, CNRS UMR 5515, INSERM U630, Villeurbanne, France.

Erratum: Effects of AZD2171 and vandetanib (ZD6474, Zactima) on haemodynamic variables in an SW620 human colon tumour model: an investigation using dynamic contrast-enhanced MRI and the rapid clearance blood pool contrast agent, P792 (gadomelitol).

Bradley DP, Tessier JL, Checkley D, Kuribayashi H, Waterton JC, Kendrew J, Wedge SR.

Department of Enabling Capabilities and Sciences, AstraZeneca, Macclesfield, Cheshire, UK.