The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

PREDICTION OF METAL CATION TOXICITY TO THE BIOLUMINESCENT FUNGUS GERRONEMA VIRIDILUCENS

A correlation between the physicochemical properties of mono- [Li(I), K(I), Na(I)] and divalent [Cd(II), Cu(II), Mn(II), Ni(II), Co(II), Zn(II), Mg(II), Ca(II)] metal cations and their toxicity (evaluated by the free ion median effective concentration. EC50(F)) to the naturally bioluminescent fungus Gerronema viridilucens has been studied using the quantitative ion character activity relationship (QICAR) approach. Among the 11 ionic parameters used in the current study, a univariate model based on the covalent index (X(m)(2)r) proved to be the most adequate for prediction of fungal metal toxicity evaluated by the logarithm of free ion median effective concentration (log EC50(F)): log EC50(F) = 4.243 (+/-0.243) -1.268 (+/-0.125).X(m)(2)r (adj-R(2) = 0.9113, Alkaike information criterion [AIC] = 60.42). Additional two- and three-variable models were also tested and proved less suitable to fit the experimental data. These results indicate that covalent bonding is a good indicator of metal inherent toxicity to bioluminescent fungi. Furthermore, the toxicity of additional metal ions [Ag(I), Cs(I), Sr(II), Ba(II), Fe(II), Hg(II), and Pb(II)] to G. viridilucens was predicted, and Pb was found to be the most toxic metal to this bioluminescent fungus (EC50(F)): Pb(II) > Ag(I) > Hg(I) > Cd(II) > Cu(II) > Co(II) Ni(II) > Mn(II) > Fe(II) approximate to Zn(II) > Mg(II) approximate to Ba(II) approximate to Cs(I) > Li(I) > K(I) approximate to Na(I) approximate to Sr(II)> Ca(II). Environ. Toxicol. Chem. 2010;29:2177-2181. (C) 2010 SETAC

Optical properties of red, green and blue emitting rare earth benzenetricarboxylate compounds

Red, blue and green emitting rare earth compounds (RE(3+) = Eu(3+), Gd(3+) and Tb(3+)) containing the benzenetricarboxylate ligands (BTC) [hemimellitic (EMA), trimellitic (TLA) and trimesic (TMA)] were synthesized and characterized by elemental analysis, complexometric titration, X-ray diffraction patterns, thermogravimetric analysis and infrared spectroscopy. The complexes presented the following formula: [RE(EMA)(H(2)O)(2)], [RE(TLA)(H(2)O)(4)] and [RE(TMA)(H(2)O)(G)], except for Tb-TMA compound, which was obtained only as anhydrous. Phosphorescence data of Gd(3+)-(BTC) complexes showed that the triplet states (T) of the BTC(3-) anions have energy higher than the main emitting states of the Eu(3+) ((5)D(0)) and Tb(3+) ((5)D(4)), indicating that BTC ligands can act as intramolecular energy donors for these metal ions. The high values of experimental intensity parameters (Omega(2)) of Eu(3+)-(BTC) complexes indicate that the europium ion is in a highly polarizable chemical environment. Based on the luminescence spectra, the energy transfer from the T state of BTC ligands to the excited (5)D(0) and (5)D(4) levels of the Eu(3+) and Tb(3+) ions is discussed. The emission quantum efficiencies (eta) of the (5)D(0) emitting level of the Eu(3+) ion have been also determined. In the case of the Tb(3+) ion, the photoluminescence data show the high emission intensity of the characteristic transitions (5)D(4) -> (7)F(J) (J=0-6), indicating that the BTC ligands are good sensitizers. The RE(3+)-(BTC) complexes act as efficient light conversion molecular devices (LCMDs) and can be used as tricolor luminescent materials. (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of some metals in Brazilian coffees cultivated during the process of conversion from conventional to organic agriculture

The aim of this study was to evaluate the presence of nutrients and toxic elements in coffees cultivated during the process of conversion, on organic agriculture, in southwest Bahia, Brazil. Levels of the nutrients and toxic elements were determined in samples of soils and coffee tissues from two transitional organic farms by atomic absorption spectrometry (FAAS). The metals in soil samples were extracted by Mehlich1 and USEPA-3050 procedures. Coffee samples from both farms presented relatively high levels of Cd, Zn and Cu (0.75,45.4 and 14.9 mu g g(-1). respectively), but were still below the limits specified by the Brazilian Food Legislation. The application of statistical methods showed that this finding can be attributed to the addition of high amounts of organic matter during the flowering tree period which can act on the bioavailability of metal ions in soils. (C) 2009 Elsevier Ltd. All rights reserved.

Electrostatic layer-by-layer deposition and electrochemical characterization of thin films composed of MnO(2) nanoparticles in a room-temperature ionic liquid

Thin films of MnO(2) nanoparticles were grown using the layer-by-layer method with poly (diallyldimetylammonium) as the intercalated layer. The film growth was followed by UV-vis, electrochemical quartz crystal microbalance (EQCM), and atomic force microscopy. Linear growth due to electrostatic immobilization of layers was observed up to 30 bilayers, but electrical connectivity was maintained only for 12 MnO(2)/PPDA bilayers. The electrochemical characterization of this film in 1-butyl-2,3-dimethyl-imidazolium (BMMI) bis(trifluoromethanesulfonyl)imide (TFSI) (BMMITFSI) with and without addition of a lithium salt indicated a higher electrochemical response of the nanostructured electrode in the lithium-containing electrolyte. On the basis of EQCM experiments, it was possible to confirm that the charge compensation process is achieved mainly by the TFSI anion at short times (<2 s) and by BMMI and lithium cations at longer times. The fact that large ions like TFSI and BMMI participate in the electroneutrality is attributed to the redox reaction that occurs at the superficial sites and to the high concentration of these species compared to that of lithium cations.

Metal transport parameters of a gneiss saprolitic silty soil for liner design

Diffusion coefficients and retardation factors of two metal cations (Cd2+ and Pb2+) were measured for a compacted Brazilian saprolitic soil derived from gneiss, aiming to assess its geoenvironmental performance as a liner for waste disposal sites. This soil occurs extensively all over the country in very thick layers, but has not been used in liners because of its hydraulic conductivity, higher than 10(-9) m/s when compacted at optimum water content of standard Proctor energy, but which can be reduced by means of appropriate compaction techniques or additives. Batch, column, and diffusion tests were carried out with monospecies synthetic solutions at pH 1, 3, and 5.5. Measured diffusion coefficients varied between 0.5 and 4 X 10(-10) m(2)/s. Retardation factors show that cadmium, a very mobile cation, is not adsorbed at pH I but is significantly retained at pH 3 and pH 5.5, whereas lead is retained at all tested pH values though slightly at pH 1. Estimated retardation factors from batch tests were 1.3-2.3 times those resulting from column tests and at its highest when obtained by diffusion tests; whereas batch tests allow a more complete exposure of the soil grains to the solution, time-dependent nonspecific adsorption may take longer to occur. The importance of contact time was observed and should be considered in further investigations. Its significant retention of metals suggests a promising utilization of this soil as a bottom liner for wastes landfills.

The processes involved in the Se electrodeposition and dissolution on Au electrode: the H2Se formation

The processes involved in the Se electrodeposition, mainly the one related to the formation of H2Se species on Au electrode in perchloric acid solutions, have been investigated through cyclic voltammetry, electrochemical quartz crystal microbalance (EQCM), rotating ring-disc electrode (RRDE), and atomic force microscopy (AFM) techniques. In the experiments performed with the EQCM, with the potential sweep in the negative direction, the responses for the mass variation were divided in three well-defined potential regions: A (from 1.55 to 0.35 V), B (from 0.35 to -0.37 V), and C (from -0.37 to -0.49 V). It was verified that the following processes can occur, respectively: the species (AuO)(2)H2SeO3 was desorbed during the AuO reduction, the reduction of Se(IV) to Se(0), and the formation of H2Se. When the potential was swept in the positive direction, the responses for the mass variation were divided in four well-defined potential regions: D (from -0.49 to 0.66 V), E (from 0.66 to 0.99 V), F (from 0.99 to 1.26 V), and G (from 1.26 to 1.55 V), and the described processes in these regions were, respectively: the Se deposition and adsorption of water molecules and/or perchlorate ions, the Se dissolution, the Se incorporating mass in the form of HO-Se, and the Au oxidation (all potentials are referred to the Ag/AgCl electrode). Making use of the RRDE, using the collection technique, the formation of H2Se species during the Se electrodeposition was investigated. Therefore, it was confirmed that this species is formed on the disc electrode between -0.3 and -0.55 V vs the Ag/AgCl potential range (collecting the oxidized compound onto the ring electrode). AFM images also indicated that the surface topography of the Se-massive deposit on Au is different from the images registered after the formation of H2Se species, confirming the cathodic stripping of Se.

The effects of dye dopants on the conductivity and optical absorption properties of polypyrrole

Ten anionic compounds, including four acidic dyes, were used to dope polypyrrole powder. The effects of the dopants on density, optical absorption and conductivity of the polypyrroles were studied. The presence of the dopant in the conducting polymer matrix was verified by ATR-FTIR spectroscopy. Density function theory (DFT) simulation was used to understand the effect of the dopants on the solid structure, optical absorption and energy band structures. Anthraquinone-2-sulfonic acid-doped polypyrrole yielded the highest conductivity. The dye-doped polypyrrole showed an enhancement in its UV–vis optical absorption.

Compressive properties and energy absorption of hollow sphere aluminium

Manufactured cellular aluminums have been developed for a wide range of automotive applications where weight savings, improved safety, crashworthiness and comfort are required. The plateau deformation behavior of cellular aluminums under compressive loading makes this new class of lightweight materials suitable for energy absorption and comes close to ideal impact absorbers. In the present study, aluminum hollow hemispheres were firstly processed by pressing. Hollow sphere aluminum samples with a body-centered cubic (BCC) packing were then fabricated by bonding together single hollow spheres, which were prepared by adhering together hollow hemispheres. Hollow sphere aluminum samples with various kinds of sphere wall thicknesses of 0.1 mm, 0.3 mm and 0.5 mm but the same outside diameter of 4 mm were investigated by compressive tests. The effects of the sphere wall thickness on the mechanical properties and energy absorption characteristics were investigated.

The influence of polymerization time and dopant concentration on the absorption of microwave radiation in conducting polypyrrole coated textiles

Temperature changes in conducting polypyrrole/para-toluene-2-sulphonic acid (PPy/pTSA) coated nylon textiles due to microwave absorption in the 8–9 GHz and 15–16 GHz frequency ranges were obtained by a thermography station during simultaneous irradiation of the samples. The temperature values are compared and related to the amounts of reflection, transmission and absorption obtained with a non-contact free space transmission technique, indicating a relationship between microwave absorption and temperature increase. Non-conductive samples showed no temperature increase upon irradiation irrespective of frequency range. The maximum temperature difference of around 4 °C in the conducting fabrics relative to ambient temperature was observed in samples having 48% absorption and 26.5 ± 4% reflection. Samples polymerized for 60 or 120 min with a dopant concentration of 0.018 mol/l or polymerized for 180 min with a dopant concentration of 0.009 mol/l yielded optimum absorption levels. As the surface resistivity decreased and the reflection levels increased, the temperature increase upon irradiation reduced.

Copper and zinc mediated oligomerisation of Aß peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.

Women's cognitive and emotional processing during sexual arousal : the effects of erotic film and absorption

Sexual arousal is a complex and dynamic element of women’s sexuality. Accounts vary, however most multidimensional models highlight the cognitive, affective and physiological components of the female sexual arousal response. While literature examining the peripheral physiological changes that occur during female sexual arousal abounds, there has been a dearth of literature pertaining to the cognitive and affective components. Thus, while many authors have included cognitive and emotions as independent components of the female sexual arousal response, there has been little empirical research to validate this approach. The aim of the current thesis was to examine the cognitive and affective components of female sexual arousal in more depth, investigating the nature of the relationship between these components under various experimental conditions. In order to do so, two integrated studies were conducted, each highlighting the effects of either external or internal variables on women’s subjective sexual arousal, absorption, positive affect and negative affect. Study One was designed to evaluate women’s emotional and cognitive processing of two elements of erotic film – foreplay and erotic context. 60 women were asked to report their subjective sexual arousal, absorption, positive affect and negative affect in response to one of four erotic film excerpts. The erotic excerpts varied in accordance with the degree of foreplay (low vs. high) depicted and the context in which the sexual activities took place (novel vs. habitual context). Women in the study responded more favourably to the high foreplay erotic film excerpt, subsequently reporting higher degrees of subjective sexual arousal, absorption and positive affect. Women also responded favourably to the erotic excerpt filmed in a novel context, reporting greater subjective sexual arousal as a result. The environment in which the sexual encounters were filmed failed to have an effect however, on women’s absorption or their positive or negative affect. The results of Study One suggest that stimulus specific variables, such as the degree of foreplay depicted, have a significant influence on female cognitive and emotional processing of erotic film. The results also suggest that a relationship exists between absorption, subjective sexual arousal and positive affect, albeit a correlational one. Specifically, there was evidence of parallel processing during sexual arousal, as participant reported sexual arousal, absorption and positive affect all increased and decreased in unison. Based on the results it was suggested that future research attempt to experimentally manipulate one of these variables, to examine its direct effect on the remaining variable. Thus, Study Two aimed to examine the effects of absorption on women’s cognitive and emotional processing of erotica. Study Two manipulated absorption at two levels (high vs. low), examining the impact of these states on participants’ subsequent absorption, subjective sexual arousal and positive and negative affect. 62 women were asked to read one of two sets of test session instructions. The first, participant-oriented instruction set, instructed participants to immerse themselves in the erotic film excerpt, as if they were active participants in the sexual exchange. The second, spectator-oriented instruction set, directed participants to observe and evaluate the erotic film. These instructions were designed to elicit high and low degrees of absorption, respectively. The utility of this approach when manipulating female absorption, was demonstrated by self reported ratings of absorption, given at the conclusion of the film presentation. Participants were also asked to report their subjective sexual arousal and positive and negative affect at the conclusion of the erotic film presentation. The findings of this study suggest that the adoption of a participant-oriented (high absorption) perspective elicits more favourable responses from participants than a spectator-oriented (low absorption) perspective, with participants in the former experimental group reporting greater degrees of subjective sexual arousal and positive affect. Negative affect was equivalent across experimental conditions, with the participants reporting that they experienced little to no aversive feelings during either of the experimental conditions. The results suggest that the degree to which a women immerses and absorbs herself in a sexual stimulus has a significant impact in her subsequent cognitive and affective processing of that stimulus. More specifically, it appears that women respond more favourably when they are highly absorbed and immersed in a stimulus, reporting greater subjective sexual arousal and positive affect. Overall, the results of Studies One and Two highlight the dynamic and complex nature of female sexual arousal. It appears that women have definite cognitive and affective responses to sexual stimuli. The magnitude of these responses may be mediated by a number of factors however, including the intrinsic qualities of the stimulus and the degree to which the woman attends to the stimulus. Both these variables act to either enhance or inhibit the sexual arousal response. There results have important implications for current sexuality literature. While women’s cognitions and emotions in response to erotic film were generally highly correlated, in some instances they differed, warranting their inclusion as separate elements in models of female sexual arousal. Furthermore, it might be suggested that the inclusion of an additional variable – absorption – into current models of female sexual arousal would prove beneficial, aiding researchers to better understand and predict the arousal process. As such, recommendations are made for a revised model of female sexual arousal. In terms of future directions, the results of the present thesis have implications for the treatment of sexual dysfunctions, suggesting that clinicians need to understand the internal and external variables that might contribute to the aetiology and maintenance of their presenting problems.

Surface behaviour and lipid interaction of Alzheimer beta-amyloid peptide 1-42: a membrane-disrupting peptide

Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a β-sheet conformation. One of the most abundant components in amyloid aggregates is the β-amyloid peptide 1–42 (Aβ 1–42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Aβ 1–42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Aβ 1–42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Aβ 1–42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Aβ 1–42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.