MPO Inhibitors Selected by Virtual Screening

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC(50) values ranging from 1.0 to 2.8 mM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0 +/- 1.1 mu M(-1) s(-1) and 30.0 +/- 0.5 mu M(-1) s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane. (C) 2011 Elsevier Ltd. All rights reserved.

Inhibition Mechanism of Rat alpha(3)beta(4) Nicotinic Acetylcholine Receptor by the Alzheimer Therapeutic Tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer`s disease, on currents elicited by activation of rat alpha(3)beta(4) nAChR heterologously expressed in KX alpha(3)beta(4)R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 +/- 0.2 mu M and 4.3 +/- 1.3 for the channel opening equilibrium constant, Phi(-1). Experiments were performed to investigate whether tacrine is able to activate the alpha(3)beta(4) nAChR. Tacrine did not activate whole-cell currents in KX alpha(3)beta(4)R2 cells but inhibited receptor activity at submicromolar concentration. Dose response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 mu M. The increase of Phi(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance. (C) 2011 Elsevier Inc. All rights reserved.

Irreversible inhibition of human cathepsins B, L, S and K by hypervalent tellurium compounds

The inhibition of human cysteine cathepsins B, L, S and K was evaluated by a set of hypervalent tellurium compounds (telluranes) comprising both organic and inorganic derivatives. All telluranes studied showed a time-and concentration-dependent irreversible inhibition of the cathepsins, and their second-order inactivation rate constants were determined. The organic derivatives were potent inhibitors of the cathepsins and clear specificities were detected, which were parallel to their known substrate specificities. In all cases, the activity of the tellurane-inhibited cathepsins was recovered by treatment of the inactivated enzymes with reducing agents. The maximum stoichiometry of the reaction between cysteine residues and telluranes were also determined. The presented data indicate that it is possible to design organic compounds with a tellurium(IV) moiety as a novel warhead that covalently modifies the catalytic cysteine, and which also form strong interactions with subsites of cathepsins B, L, S and K, resulting in more specific inhibition.

Behaviour of TiO(2)-SiMgO(x) hybrid composites on the solar photocatalytic degradation of polluted air

The photocatalytic performance of TiO(2)-SiMgO(x) ceramic plates for trichloroethylene abatement in gas phase has been evaluated under sun irradiance conditions. A continuous flow Pyrex glass reactor fixed on the focus of a compound parabolic collector has been used. The performance of the hybrid photocatalyst has been evaluated as the variation of TCE conversion and reaction products formation with the solar irradiance at different total gas flow, TCE concentration, and water vapour content. SiMgO(x) not only provides adsorbent properties to the photocatalyst, but it also allows the effective use of the material during low solar irradiance conditions. The adsorption-desorption phenomena play a pivotal role in the behaviour of the system. Thus, TCE conversion curves present two different branches when the sun irradiance increases (sunrise) or decreases (sunset). CO(2), COCl(2) and DCAC were the most relevant products detected. Meanwhile CO(2) concentration was insensitive to the branch analysed, COCl(2) or DCAC were not indicating the ability of these compounds to be adsorbed on the composite. An increase of the UV irradiation at total TCE conversion promotes the CO(2) selectivity. The excess of energy arriving to the reactor favours the direct reaction pathway to produce CO(2). The photonic efficiency, calculated as a function of the rate of CO(2) formation, decreases linearly with the solar irradiance up to around 2 mW cm(-2), where it becomes constant. For decontamination systems high TCE conversion is pursuit and then high solar irradiance values are required, in spite of lower photonic efficiency values. The present photocatalyst configuration, with only 17% of the reactor volume filled with the photoactive material, allows total TCE conversion for 150 ppm and 1 L min(-1) in a wide sun irradiance window from 2 to 4 mW cm(-2). The incorporation of water vapour leads to an increase of the CO(2) selectivity keeping the TCE conversion around 90%, although significant amounts of COCl(2) were observed. (c) 2010 Elsevier B.V. All rights reserved.

Selective photoinactivation of C. albicans and C. dubliniensis with hypericin

The genus Candida includes different species that have the potential to invade and colonize the human body and C. albicans is the most common cause of skin, nail and mucous infections. The increasing resistance against antifungal drugs has renewed the search for new treatment procedures and antimicrobial photodynamic inactivation (PDI) is a propitious candidate. Hypericin (HY) has several wanted properties to be used as a photosensitizer in this technique including a high quantum yield of singlet oxygen generation, a high extinction coefficient near 600 nm, and a relatively low dark toxicity. Although the phototoxicity of HY on several tumor cells has been reported, the data concerning its photoactivity on microorganisms are scarce. The aim of this study was to obtain the experimental parameters to achieve an acceptable selective hypericinphotoinactivation of two species of Candida comparing with fibroblasts and epithelial cells which are the constituents of some potential host tissues, such mucosas, skin and cavities. Microorganisms and cells were incubated with the same HY concentrations and short incubation time followed by irradiation with equal dose of light. The best conditions to kill just Candida were very low HY concentration (0.1-0.4 mu g ml(-1)) incubated by 10 min and irradiated with LED 590 nm with 6 J cm(-2).

Färg och missfärgning hos sågad björk : - Variation samt effekter av avverkningstidpunkt, lagring av timmer och torkning av sågad vara

Variation i björkvirkets färg beror på art, läge i träd, ursprung och lagringstid. Vårtbjörk har en mörkare färg än glasbjörk, men livligare struktur och glans. Timmer hugget under tillväxtsäsongen bör sågas färskt eller lagras högst en månad. Vid brädgårdstorkning är god luftcirkulation och skydd för väta och direkt solljus väsentligt. Strövirkets färskhet är av stor betydelse för att undvika fuktfärgning av virket.Variation in birch colours depend on species, position in tree, origin and the storage period. White birch is darker than hairy birch but with a lively structure and shine. During vegetation period timber storage exceeding one month should be avoided. Air circulation, protection from rain and direct sunlight are essential for yard drying. Freshness of stickers used for stacking is important to avoid water marks on wood surface.

Thermal Evaluation of a Solarus PV-T collector

Low concentrator PV-T hybrid systems produce both electricity and thermal energy; this fact increases the overall efficiency of the system and reduces the cost of solar electricity. These systems use concentrators which are optical devices that concentrate sunlight on to solar cells and reduce expensive solar cell area. This thesis work deals with the thermal evaluation of a PV-T collector from Solarus.Firstly the thermal efficiency of the low concentrator collector was characterized for the thermal-collector without PV cells on the absorber. Only two types of paint were on the absorber, one for each trough of the collector. Both paints are black one is glossy and the other is dull,. The thermal efficiency at no temperature difference between collector and ambient for these two types of paint was 0.65 and 0.64 respectively; the U-value was 8.4 W/m2°C for the trough with the glossy type of paint and 8.6 W/m2°C for the trough with dull type of paint. The annual thermal output of these two paints was calculated for two different geographic locations, Casablanca, Morocco and Älvkarleby, Sweden.Secondly the thermal efficiency was defined for the PV-T collector with PV cells on the absorber. The PV cells cover 85% of the absorber, without any paint on the rest of the absorber area. We also tested how the electrical power output influences the thermal power output of the PV-T collector. The thermal and total performances for the PV-T collector were only characterized with reflector sides, because of the lack of time we could not characterize them with transparent sides also.

Overexpression and Purification of the Oat (Avena Fatua) Protein AFN1 and Construction of a pMAL/AFN1 Expression Plasmid

Abscisic acid (ABA) is an important phytohormone with regulatory roles in many physiological processes. ABA expression is induced by environmental stresses such as drought and it is known to be an inhibitor of seed germination. A wild oat (Avena fatua) called AFN1 has been hypothesized to initiate the early stages of germination as its mRNA accumulates in nondormant seed embryos during imbibition. The polypeptide sequence of AFN1 suggests that it is an ABA glucosyl transferase. Glucosylation by AFN1 and thereby inactivation of ABA could lead to seed germination. In order to understand the role of AFN1 in germination, an ample quantity of AFN1 polypeptide is needed to test for enzymatic ABA glucosylase activity. My work has been to overexpress recombinant AFN1containing a (His)6 tag using a pRSETC E.coli expression system followed by Purification of the AFN1 protein by means of a nickel-affinity column that bind to the (His)6 tag. Due to the insufficient yield of AFN1 fusion protein obtained with this procedure, another method using a pMAL-c2x vector is now being employed. The pMAL expression system provides a method for expressing and purifying protein by tagging proteins with maltose-binding protein (MBP). It is anticipated that MBP tag will be advantageous as it can make the fusion protein more soluble and thereby yield a larger quantity of protein. Currently, work is underway on the construction of pMAL/AFN1 plasmid.

Characterization of AFN1, a Gene Associated with Cereal Grain Germination

The AFN1 gene is transiently expressed in germinating oat grains. As AFN1 is not expressed in dormant oat grains during imbibition, we hypothesize that AFN1 may be involved in stimulating the germination process. Sequence analysis of an AFN1 cDNA clone indicates that the AFN1 polypeptide is similar to a previously identified abscisic acid (ABA) glucosyl transferase. This suggests that AFN1 may be acting to glucosylate ABA, thereby inactivating it. As the hormone ABA is known to inhibit germination, ABA glucosylation/inactivation could lead to germination in grains expressing AFN1. To test this hypothesis, we have constructed an expression plasmid that encodes an MBP::AFN1 (maltose binding protein) fusion protein. E. coli cells carrying the expression plasmid were found to produce the MBP::AFN1 fusion protein as a substantial fraction of total protein. We are currently in the process of purifying the MBP::AFN1 fusion protein by affinity chromatography, so that it can be assayed for ABA glucosyl transferase activity. We also wish to test the effect of AFN1 gene expression during grain imbibition on the germination behavior of the grains. To this end, we have constructed plasmids for the overexpression and RNAi-based suppression of AFN1 in transgenic plants. These plasmids have been introduced into oat cells by particle bombardment and we are in the process of regenerating transgenic plants for study.

Caracterização química da erva mate (Ilex paraguariensis) : aplicação de diferentes processos de extração e influência das condições de plantio sobre a composição química

O objetivo deste trabalho foi estudar a composição química das folhas de erva-mate, sob diferentes condições agronômicas e técnicas de extração. Os métodos de extração usados foram maceração, ultra-som, extração com líquido pressurizado e extração com fluído supercrítico. Foram investigadas as variáveis que podem influenciar no processo de extração, tais como temperatura, pressão, polaridade do solvente, tempo de extração, massa de amostra, entre outras. A identificação dos compostos foi realizada por cromatografia gasosa acoplada à espectrometria de massas. Todos os métodos de extração utilizados mostraram-se eficientes para a obtenção dos extratos, com as diferenças sendo mais quantitativas do que qualitativas . Entre os métodos de extração que utilizam solventes orgânicos, a extração com líquido pressurizado mostrou-se mais eficiente, produzindo maior rendimento em massa de extrato e maior concentração de alguns dos compostos de interesse, com as vantagens de redução de solvente e tempo de extração. A composição química da erva-mate é influenciada pelas condições agronômicas de plantio, bem como pelas condições de extração de suas folhas. A melhor condição agronômica avaliada, ou seja, aquela que produziu maior quantidade de extrato, foi o cultivo das plantas a pleno sol, adubadas com nitrogênio e com idade de poda de 18 meses. A variável mais importante das técnicas de extração utilizadas foi a polaridade do solvente. Solventes de maior polaridade produziram maior rendimento em extrato. A análise cromatográfica dos extratos obtidos permitiu identificar cerca de 50 compostos qualitativamente e 6 quantitativamente, destacando-se a cafeína, fitol, ácido palmítico, ácido esteárico, esqualeno e vitamina E.

Proliferative Defects and Formation of a Double Cortex in Mice Lacking Mltt4 and Cdh2 in the Dorsal Telencephalon

Radial glial cells (RGCs) in the ventricular neuroepithelium of the dorsal telencephalon are the progenitor cells for neocortical projection neurons and astrocytes. Here we showthatthe adherens junction proteins afadin and CDH2 are criticalforthe control of cell proliferation in the dorsal telencephalon and for the formation of its normal laminar structure. Inactivation of afadin or CDH2 in the dorsal telenceph-alon leads to a phenotype resembling subcortical band heterotopia, also known as “double cortex,” a brain malformation in which heterotopic gray matter is interposed between zones of white matter. Adherens junctions between RGCs are disrupted in the mutants, progenitor cells are widely dispersed throughout the developing neocortex, and their proliferation is dramatically increased. Major subtypes of neocortical projection neurons are generated, but their integration into cell layers is disrupted. Our findings suggest that defects in adherens junctions components in mice massively affects progenitor cell proliferation and leads to a double cortex-like phenotype.

Análises de elementos arquitetônicos de proteção solar em edificações institucionais na cidade de Natal/RN Diretrizes projetuais

The present work concerns the use of shade elements as architectural elements to block sunlight in public buildings. In a city like Natal, (5o South) the incidence of sunrays in any type of design should be a constant concern for all the architects. Besides, this habit of avoiding insolation in the environment is not a common practice. Within this context, the present work has the objective to dig deep into the knowledge of solar control, studying some cases and verifying its function according to the orientation and the original design of the building, having in mind if the shade elements usually used in the region have achieved their purpose of providing protection against the incidence of direct sun rays. This study considers the position of the shade element (horizontal and vertical), the angle formed between them and the respective facades, and the local of the buildings in relation to their orientation during the summer, winter and equinox solstice. As supporting instruments the solar map of the city and the protractor, for measuring shade angles, were used. It was concluded that in all the cases studied, it was not possible to obtain the maximum use of the elements. It was verified that the best type of shade element (more efficient) for the city of Natal is the mixed type (horizontal and vertical) and that the vertical shade elements are more efficient in the early mornings and late afternoon. The horizontal shade elements are used more effective at midday. We intend to present the results of this study to the architects in the region in order to show them the correct ways of using the shade elements according to the possible orientation on the facade, as a supporting tool at the time of designing a project as well as a subsidy for further discussions on the elaboration of the new urban standards for the city of Natal/RN

Padronização e flexibilidade: proposta de um edifício tipo para uma escola técnica profissionalizante

Nowadays the acquisition of sustainable elements and concepts in construction has been increasingly discussed, improved and incorporated to buildings, since the sector directly interferes in the urban space and environment, representing environmental impact. In order to make a sustainable building (Green Building) it is vital to incorporate less damaging constructive practice, starting from the project until the operation of the enterprise, that means to consider an integrated process of conception, implementation, construction and operation. The more effective sustainable principles participation in architecture happens at the projecting step through minimal environment impact solutions. Among the issues varieties that goes with sustainability proposal of the buildings project, there were added to this work the elements that are directly attached to bioclimatic architecture, more specifically the climate variation, ventilation, lighting and sunlight, that directly affect the project conception. It is important to put in evidence that architecture role goes far beyond the simple activity of building spaces; it is the sequence of political, economic, social and cultural elements, having the users as the main apparatus to its materialization. Thereby this professional dissertation consists of an architecture draft for a professional and technological school in the Rio Grande do Norte State, this dissertation is based on the analysis of previous experience and the bioclimatic principles that implicate in building on hot and dry, hot and humid climates, and the use of strategic solutions that aim the optimization of natural light and ventilation