Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Bio-separation, speciation and determination of chromium in water using partially pyrolyzed olive pomace sorbent

Speciation of Cr(III)/Cr(VI) from water using olive pomace (OP) was improved by partial pyrolysis of OP. The sorbents were characterized by physicochemical techniques. Sorption of Cr(III) on raw and partially pyrolyzed OP sorbents followed Freundlich isotherm and second-order rate kinetics. OP pyrolyzed at 150 °C (sorbent OP-150) exhibited maximum sorption capacity, favorability and the lowest sorption energy. Sorption was exothermic and spontaneous for the raw-OP and OP pyrolyzed at 100 or 150 °C; but endothermic and non-spontaneous for OP pyrolyzed at 200, 250, 300 or 400 °C. A speciation method of chromium was proposed, in which Cr(III) was selectively retained at pH 3 on sorbent OP-150; while total Cr was determined after reduction of Cr(VI). The method was selective with a detection limit for Cr(III) of 1.58 μg L⁻¹. The method was applied on natural and industrial waters (recoveries >97.7%, RSD’s <9%) and on tobacco leaves certified reference material (INCT-PVTL-6).     

Escherichia coli responds with a rapid and large change in growth rate upon a shift from glucose-limited to glucose-excess conditions

Glucose pulse experiments at seconds time scale resolution were performed in aerobic glucose-limited Escherichia coli chemostat cultures. The dynamic responses of oxygen-uptake and growth rate at seconds time scale were determined using a new method based on the dynamic liquid-phase mass balance for oxygen and the pseudo-steady-state ATP balance. Significant fold changes in metabolites (10-1/10) and fluxes (4-1/4) were observed during the short (200 s) period of glucose excess. During glucose excess there was no secretion of by-products and the increased glucose uptake rate led within 40s to a 3.7 fold increase in growth rate. Also within 40-60s a new pseudo-steady-state was reached for both metabolite levels and fluxes. Flux changes of reactions were strongly correlated to the concentrations of involved compounds. Surprisingly the 3.7 fold increase in growth rate and hence protein synthesis rate was not matched by a significant increase in amino acid concentrations. This poses interesting questions for the kinetic factors, which drive protein synthesis by ribosomes.     

Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments

Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans α- and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length.     

Effects of excess vitamin B6 intake on cerebral cortex neurons in rat: an ultrastructural study

The aim of this study was to investigate whether excess of vitamin B6 leads to ultrastructural changes in cerebral cortex of forty-eight healthy albino rats which were included in the study. Saline solution was injected to to the control groups (CG-10, n = 12 for 10 days; CG-15, n = 12 for 15 days; CG-20, n=12 for 20 days). The three experimental groups (EG-10, n = 12; EG-15, n = 12; EG-20, n = 12) were treated with 5 mg/kg vitamin B6 daily for 10 days (EG-10), 15 days (EG-15) and 20 days (EG-20). Brain tissues were prepared by glutaraldehyde-osmium tetroxide double fixation for ultrastructural analysis. No significant changes were observed in the control groups. The ultrastructural analysis revealed that the numbers of damaged mitochondria, lipofuscin granules and vacuoles were significantly higher in all the experimental groups than in the control groups (p < 0.05). However, synaptic density was significantly decreased in the experimental groups as compared to the control groups (p < 0.05). The results suggest that the excess of vitamin B6 intake causes damage to the cerebral cortex due to cellular intoxication and decreased synaptic density. Thus, careful attention should be paid to the time and dose of vitamin B6 recommended for patients who are supplemented with this vitamin.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

Chemical composition effects onto antimicrobial and antioxidant activities of propolis collected from different regions of Turkey

Chrysin, apigenin, flavonoids, flavanones, naringenin, ethyl oleate, 3-4-dimethoxy-cinnamic acid and 9-octadecenoic acid were the predominant components of propolis samples collected from different regions of Turkey. The extracts of P3 from Denizli-Ba?karci, P5 from Denizli and P7 from Tekirda? had effective antibacterial activities on Gram-negatives. Chrysin, which has antibacterial activity, was found to be high concentration. The extracts of P3, P2B from Aydin and P6 from Konya had much more effective antibacterial activities on Gram-positives. The total antioxidant activity increased with the increasing amount of extracts added to linoleic acid emulsion. All doses of propolis ethanol extract displayed antioxidant activity.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

Discovery and Structure Activity Relationship of Small Molecule Inhibitors of Toxic β-Amyloid-42 Fibril Formation

Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.