Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional H-1 NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta -hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta -sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta -sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.

Hemoglobin-degrading, aspartic proteases of blood-feeding parasites - Substrate specificity revealed by homology models

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH2]Leu-Tyr-Tyr-Ser-NH2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.

Chemistry of stable iminopropadienones, RN=C=C=C=O

The synthesis, spectroscopic properties, and chemical reactions of the stable (neopentylimino)-, (mesitylimino)-, and (o-tert-butylphenylimino)propadienones (6) are reported. Nucleophilic addition of amines affords the malonic amidoamidines 7 and 8. 3,5-Dimethylpyrazole reacts analogously to form 9b. Addition of 1,2-dimethylhydrazine produces pyrazolinones 10-12. Addition of N,Y'-dimethyldiaminoethane, -propane, and -butane gives diazepine, diazocine, and diazonine derivatives 13-15, respectively (X-ray structures of 13c, 14a, and 15a are available). The mesoionic pyridopyrimidinium olates IS are obtained by addition of 2-(methylamino)pyridine (X-ray structure of 18b available). Primary 2-aminopyridines afford the pyridopyrimidininones 20-29 and 31 (X-ray structure of 21a available), and 2-aminopyrimidines and 2-aminopyrazine afford pyrimidopyrimidinones and pyrazinopyrimidinones 33-35. Pyrimidoisoquinolinone 36 results from 1-aminoisoquinoline and pyridoquinolinone 40 from 8-aminoquinoline. 2-Aminothiazoline and 2-aminothiazole afford thiazolopyrimidinone derivatives 41-43 (X-ray structure of 43a available).

Urease inhibitor (NBPT) and efficiency of single or split application of urea in wheat crop

NBPT (N-(n-butyl) thiophosphoric triamide), a urease inhibitor, has been reported as one of the most promising compounds to maximize urea nitrogen use in agricultural systems. The objective of this study was to evaluate the performance of irrigated wheat fertilized with urea or urea + NBPT as single or split application. The experiment was conducted from June to October 2006 in Viçosa, MG, Brazil. The experimental design followed a 2×2 factorial scheme, in which urea or urea + NBPT were combined with two modes of application: full dose at sowing (60kg ha-1) or split (20kg ha-1 at sowing + 40kg ha-1 as topdressing at tillering), in randomized blocks with ten replications. The split application of nitrogen fertilization does not improve the yield wheat under used conditions. The use of urease inhibitor improves the grain yield of wheat crop when urea is applied in topdressing at tillering, but its use does not promote difference when urea is applied in the furrow at planting.

In vivo and in vitro effects of RAD001 on bladder cancer

Objective: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. Methods: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. Results: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P 0.001), was only observed in the 5637 cell line. Conclusion: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Eficiência de antioxidantes em biodiesel

Mestrado em Engenharia Química.Ramo optimização energética na indústria química

Avaliação da influência de antioxidantes na estabilização de biodiesel

O biodiesel é uma fonte de energia renovável, que se pode obter pela transformação dos resíduos domésticos, e é ambientalmente inócuo, e fácil de transportar, pois tem um ponto de fulgor elevado. Atualmente tem-se focado a atenção nos efeitos da oxidação do biodiesel causados pelo contato com o ar ambiente durante o seu armazenamento. Os produtores, fornecedores e consumidores, pretendem garantir que a qualidade do biodiesel e das suas misturas com combustíveis destilados do petróleo, mantém-se durante longos períodos de armazenamento. A maioria dos óleos vegetais e das gorduras animais, usados como matéria – prima, são triacilgliceróis com grupos ácido gordos de cadeia longa (C16 – C18) ligados por ligações éster a uma estrutura de glicerol. O objetivo deste estudo foi avaliar a eficiência do ácido gálhico e seus derivados alquilo ésteres aumentando a resistência relativa à oxidação do ácido linoléico, inibindo a peroxidação ácido gordos de cadeia longa insaturados. Outro objetivo foi estudar a taxa de inibição da auto-oxidação do ácido linoleico. Foi, ainda estudado o efeito que a concentração do antioxidante, tinha na estabilidade do ácido linoleico. No caso do ácido gálhico verificou-se que o aumento para o dobro da concentração do antioxidante utilizada, obtinha-se quase o dobro da taxa de inibição da oxidação do ácido linoleico. A auto-oxidação de ácido linoleico é acompanhada pela formação do seu dieno conjugado, o qual foi medido sua absorvância, durante 7 dias, por espetrofotometria de absorção UV a 234 nm. Uma diminuição da taxa de formação de dieno conjugado, indica o aumento da atividade antioxidante do composto adicionado à micela de ácido linoleico. Os resultados obtidos permitem concluir que de todos os antioxidantes testados o galhato de butilo é o que possibilita uma maior inibição da oxidação do ácido linoleico para as duas concentrações de antioxidantes testadas (0,1 mM e 1mM), obteve-se uma percentagem de inibição do ácido linoleico de 54,0% e 63,6%, respetivamente. O estudo comparativo da estabilização do ácido linoleico com o antioxidante de referência, o butil -hidroxitolueno, mostrou que este composto tem um poder de estabilização inferior a qualquer dos antioxidantes estudados. Os resultados deste estudo demonstraram que a utilização de compostos fenólicos, em especial o galhato de butilo, constitui uma boa alternativa para a estabilização de matrizes lipídicas, nomeadamente de combustíveis como o biodiesel.

Citrate selective electrodes for the flow injection analysis of soft drinks, beers and pharmaceutical products

Aiming the establishment of simple and accurate readings of citric acid (CA) in complex samples, citrate (CIT) selective electrodes with tubular configuration and polymeric membranes plus a quaternary ammonium ion exchanger were constructed. Several selective membranes were prepared for this purpose, having distinct mediator solvents (with quite different polarities) and, in some cases, p-tert-octylphenol (TOP) as additive. The latter was used regarding a possible increase in selectivity. The general working characteristics of all prepared electrodes were evaluated in a low dispersion flow injection analysis (FIA) manifold by injecting 500µl of citrate standard solutions into an ionic strength (IS) adjuster carrier (10−2 mol l−1) flowing at 3ml min−1. Good potentiometric response, with an average slope and a repeatability of 61.9mV per decade and ±0.8%, respectively, resulted from selective membranes comprising additive and bis(2-ethylhexyl)sebacate (bEHS) as mediator solvent. The same membranes conducted as well to the best selectivity characteristics, assessed by the separated solutions method and for several chemical species, such as chloride, nitrate, ascorbate, glucose, fructose and sucrose. Pharmaceutical preparations, soft drinks and beers were analyzed under conditions that enabled simultaneous pH and ionic strength adjustment (pH = 3.2; ionic strength = 10−2 mol l−1), and the attained results agreed well with the used reference method (relative error < 4%). The above experimental conditions promoted a significant increase in sensitivity of the potentiometric response, with a supra-Nernstian slope of 80.2mV per decade, and allowed the analysis of about 90 samples per hour, with a relative standard deviation <1.0%.

Halogen-bonded tris (2,4-Bis(trichloromethyl)-1,3,5,triazapentadienato)-M(III) [M = Mn, Fe, Co] complexes and their catalytic activity in the peroxidative oxidation of 1-phenylethanol to acetophenone

One-pot template condensation of CCl3C=N with ammonia on a metal source [MnCl2 center dot 4H(2)O, FeCl3 center dot 6H(2)O or Co(CH3COO)(2)center dot 4H(2)O] in DMSO led to the formation of tris(2,4-bis(trichloromethyl)-1,3,5-triazapentadienato)-M(III) complexes, [M(NH=C(CCl3)NC(CCl3)=-NH}(3)]center dot n(CH3)(2)SO [M = Mn, n = 1 (1); M = Fe, n = 2 (2); M = Co, n = 2 (3)1, which were characterized using elemental analysis, and IR, ESI-MS and single-crystal X-ray analysis. The role of inter- and intramolecular non-covalent halogen and hydrogen bonds in the synthesis of 1-3 is discussed. It is shown that the crystal ionic radii of the metal ions [68.5 (Co) < 69 (Fe) < 72 (Mn), pm] are related to the corresponding Cl center dot center dot center dot Cl distances [3.178 (3) > 3.155 (2) > 3.133 (1) Al. Compounds 1-3 and the related di(triazapentadienato)-Cu(v) complex [Cu(NH=C(CCl3)NC(CCl3)=NH}2]center dot 2(CH3)(2)SO (4) act as catalyst precursors for the additive-free microwave (MW) assisted homogeneous oxidation of 1-phenylethanol with tert-butylhydroperoxide (TBHP), leading to the formation of acetophenone with yields up to 99% and TONs up to 5.0 x 10(3) after 1 h of low power (10 W) MW irradiation.

Microwave-assisted and solvent-free peroxidative oxidation of 1-phenylethanol to acetophenone with a CuII-TEMPO catalytic system

The water-soluble copper(II) complex [Cu(H2R)(HL)]center dot H2O (1) was prepared by reaction of copper(II) nitrate hydrate with (E)-2-(((1-hydroxynaphthalen-2-yl)methylene)amino) benzenesulfonic acid (H2L) and diethanolamine (H3R). It was characterized by IR and ESI-MS spectroscopies, elemental and X-ray crystal structural analyses. 1 shows a high catalytic activity for the solvent-free microwave (MW) assisted oxidation of 1-phenylethanol with tert-butylhydroperoxide, leading, in the presence of TEMPO, to yields up to 85% (TON = 850) in a remarkably short reaction time (15 min, with the corresponding TOE value of 3.40 x 10(3) h(-1)) under low power (25W) MW irradiation. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

Is it possible to remove polymeric nanoparticles from aqueous paints during the activated sludge treatment?

The market for emulsion polymers (latexes) is large and growing at the expense of other manufacturing processes that emit higher amounts of volatile organic solvents. The paint industry is not an exception and solvent-borne paints have been gradually substituted by aqueous paints. In their life-cycle, much of the aqueous paint used for architectural or decorative purposes will eventually be discharged into wastewater treatment facilities, where its polymeric nanoparticles (mainly acrylic and styrene-acrylic) can work as xenobiotics to the microbial communities present in activated sludge. It is well established that these materials are biocompatible at macroscopic scale. But is their behaviour the same at nanoscale? What happens to the polymeric nanoparticles during the activated sludge process? Do nanoparticles agregate and are discharged together with the sludge or remain in emulsion? How do microorganisms interact with these nanoparticles? Are nanoparticles degradated by them? Are they adsorbed? Are these nanoparticles toxic to the microbial community? To study the influence of these xenobiotics in the activated sludge process, an emulsion of cross-linked poly(butyl methacrylate) nanoparticles of ca. 50 nm diameter was produced and used as model compound. Activated sludge from a wastewater treatment plant was tested by the OCDE’s respiration inhibition test using several concentrations of PBMA nanoparticles. Particle aggregation was followed by Dynamic Light Scattering and microorganism surfaces were observed by Atomic Force Microscopy. Using sequential batch reactors (SBRs) and continuous reactors, both inoculated with activated sludge, the consumption of carbon, ammonia, nitrite and nitrate was monitored and compared, in the presence and absence of nanoparticles. No particles were detected in all treated waters by Dynamic Light Scattering. This can either mean that microorganisms can efficiently remove all polymer nanoparticles or that nanoparticles tend to aggregate and be naturally removed by precipitation. Nevertheless respiration inhibition tests demonstrated that microorganisms consume more oxygen in the presence of nanoparticles, which suggests a stress situation. It was also observed a slight decrease in the efficiency of nitrification in the presence of nanoparticles. AFM images showed that while the morphology of some organisms remained the same both in the presence and absence of nanoparticles, others assumed a rough surface with hilly like shapes of ca. 50 nm when exposed to nanoparticles. Nanoparticles are thus likely to be either incorporated or adsorbed at the surface of some organisms, increasing the overall respiration rate and decreasing nitrification efficiency. Thus, despite its biocompatibility at macroscopic scale, PBMA is likely to be no longer innocuous at nanoscale.

Substituted P-Phenylene Ethynylene trimers as fluorescent sensors for nitroaromatic explosives

New sensory materials based on p-phenylene ethynylene trimers integrating calix[4]arene receptors (CALIX-PET) and tert-butylphenol (TBP-PET) moieties have been synthesized and their sensitivity and selectivity for the detection of nitroaromatic compounds (NACs) such as nitrobenzene (NB), 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT) and picric acid (PA) investigated in fluid phase and solid-state. It was found that both fluorophores displayed high sensitivities toward NACs detection in solution as evaluated by the Stern-Volmer formalism. For all the tested explosives, the ratio of fluorescence intensities (F-0/F) is a linear function of the quencher concentration only after appropriate correction of fluorescence quenching data for inner-filter effects. The quenching efficiencies for CALIX-PET and TBP-PET follow the order PA >> TNT > DNT > NB, which correlate well with the quenchers electron affinities as evaluated from their LUMOs energies thereby suggesting a photoinduced electron transfer as the dominant mechanism of fluorescence quenching. The selectivity of these sensors was checked against exemplar interferents possessing differentiated electronic properties (benzoic acid, 2,4-dichlorophenol and benzoquinone) and reduced quenching activity was detected. The quenching efficiencies and response times of the two fluorophores in the solid-state toward NB, 2,4-DNT and TNT vapors were evaluated through steady-state fluorescence quenching experiments with the materials dispersed in polymeric matrices or as neat films. The most significant fluorescence quenching responses were achieved for drop-casted films of TBP-PET upon exposure to nitroaromatics.

Part I : Comparing derivatization methods for amino acids – one and two-step procedures using gas chromatography – flame ion detector (GC-FID) Part II: Story of an icon from its execution until its conservation

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Conservação e Restauro

Catalytic activity of a benzoyl hydrazone based dimeric dicopper(II) complex in catechol and alcohol oxidation reactions

The benzoyl hydrazone based dimeric dicopper(II) complex [Cu2(R)(CH3O)(NO3)]2(CH3O)2 (R-Cu2+), recently reported by us, catalyzes the aerobic oxidation of catechols (catechol (S1), 3,5- itertiarybutylcatechol (S2) and 3-nitrocatechol (S3)) to the corresponding quinones (catecholase like activity), as shown by UV–Vis absorption spectroscopy in methanol/HEPES buffer (pH 8.2) medium at 25 C. The highest activity is observed for the substituted catechol (S2) with the electron donor tertiary butyl group, resulting in a turnover frequency (TOF) value of 1.13 103 h1. The complex R-Cu2+ also exhibits a good catalytic activity in the oxidation (without added solvent) of 1-phenylethanol to acetophenone by But OOH under low power (10 W) microwave (MW) irradiation. 2014 Elsevier B.V. All rights reserved.