Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.

Free tools and strategies for the generation of 3D finite element meshes: Modeling of the cardiac structures

The Finite Element Method is a well-known technique, being extensively applied in different areas. Studies using the Finite Element Method (FEM) are targeted to improve cardiac ablation procedures. For such simulations, the finite element meshes should consider the size and histological features of the target structures. However, it is possible to verify that some methods or tools used to generate meshes of human body structures are still limited, due to nondetailed models, nontrivial preprocessing, or mainly limitation in the use condition. In this paper, alternatives are demonstrated to solid modeling and automatic generation of highly refined tetrahedral meshes, with quality compatible with other studies focused on mesh generation. The innovations presented here are strategies to integrate Open Source Software (OSS). The chosen techniques and strategies are presented and discussed, considering cardiac structures as a first application context. © 2013 E. Pavarino et al.

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis (Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Bacterial cellulose/poly(3-hydroxybutyrate) composite membranes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients. (c) 2007 Elsevier Ltd. All rights reserved.

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chrornatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and C-13 NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q(1A) that was a beta-(1 -> 6)-D-glucan with the following structure:[GRAPHICS]The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: KIP (insoluble) that comprised a beta-(1 -> 3)-D-glucan with beta-D-glucose branches at C-6 with the structure:[GRAPHICS]and K1SA (soluble) consisting of a backbone chain of alpha-(1 -> 4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues:[GRAPHICS](c) 2008 Elsevier Ltd. All rights reserved.

Including the sub-grid scale plume rise of vegetation fires in low resolution atmospheric transport models

We describe and begin to evaluate a parameterization to include the vertical transport of hot gases and particles emitted from biomass burning in low resolution atmospheric-chemistry transport models. This sub-grid transport mechanism is simulated by embedding a 1-D cloud-resolving model with appropriate lower boundary conditions in each column of the 3-D host model. Through assimilation of remote sensing fire products, we recognize which columns have fires. Using a land use dataset appropriate fire properties are selected. The host model provides the environmental conditions, allowing the plume rise to be simulated explicitly. The derived height of the plume is then used in the source emission field of the host model to determine the effective injection height, releasing the material emitted during the flaming phase at this height. Model results are compared with CO aircraft profiles from an Amazon basin field campaign and with satellite data, showing the huge impact that this mechanism has on model performance. We also show the relative role of each main vertical transport mechanisms, shallow and deep moist convection and the pyro-convection (dry or moist) induced by vegetation fires, on the distribution of biomass burning CO emissions in the troposphere.

The characterization of a thermostable and cambialistic superoxide dismutase from thermus filiformis

The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0·41, 0·56 and 13·73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis. © 2013 The Society for Applied Microbiology.

Estudo e modelagem 3D de uma turbina Francis

Most of the hydraulic turbines that are running in the world are the Francis type, because they are able to fit a wide range of head and flow. We also can declare that most of the installed hydraulic energy up to this date is generated by this type of turbine. On the other hand, if we pay attention to mechanical details of a Francis turbine we will see that among the three most used types it is the one that presents the more complex design. And, the simplest one is the Pelton type, whereas the Kaplan is the one less utilized. This work aims to develop the hydrodynamic calculation of a Francis turbine as well as to obtain its assembly drawing and 3D modeling drawing. In this way all details must be shown therefore needing a complete knowledge about this issue. Both 2D and 3D drawings help much if one wants the machine to be running in its power house. It is well noted that most of the entities that design and manufacture the Francis turbines are not brazilian. In this way the more we study how to design this kind of machine the more is our contribution to our country in order to conquer technology and manufacture Francis turbines

Production of β-(1,3)-glucanases by Trichoderma harzianum Rifai: Optimization and Application to Produce Gluco-oligosaccharides from Paramylon and Pustulan

β-(1→3)-Glucanases were produced by Trichoderma harzianum Rifai PAMB-86 cultivated on botryosphaeran in a bench-fermenter and optimised by the response surface method. Maximal enzyme titres occurred at 5 days, initial pH 5.5 and aeration of 1.5vvm. β-(1→3)-The β-glucanolytic enzyme complex produced by T. harzianum Rifai PAMB- 86 was fractionated by gel filtration into 2 fractions (F-I, F-II), and employed to produce gluco-oligosaccharides from algal paramylon ((1→3)-β-D-glucan) and lichen pustulan ((1→6)-β-D-glucan). Both enzymes attacked paramylon to the extent of ~15-20% in 30 min releasing glucose and laminaribiose as major end-products, and laminarioligosaccharides of degree of polymerization (DP) ≥3. Only F-I degraded pustulan resulting in ~2% degradation at 30 min, with glucose, gentiobiose and gentio-oligosaccharides of DP ≥4 as major products. The difference in the nature of the hydrolysis products can be explained by the substrate specificities of each enzyme fraction, and the structural differences of the β-D-glucans attacked.

Semiautomatic object-space road extraction combining a stereoscopic image pair and a TIN-Based DTM

In this letter, a semiautomatic method for road extraction in object space is proposed that combines a stereoscopic pair of low-resolution aerial images with a digital terrain model (DTM) structured as a triangulated irregular network (TIN). First, we formulate an objective function in the object space to allow the modeling of roads in 3-D. In this model, the TIN-based DTM allows the search for the optimal polyline to be restricted along a narrow band that is overlaid upon it. Finally, the optimal polyline for each road is obtained by optimizing the objective function using the dynamic programming optimization algorithm. A few seed points need to be supplied by an operator. To evaluate the performance of the proposed method, a set of experiments was designed using two stereoscopic pairs of low-resolution aerial images and a TIN-based DTM with an average resolution of 1 m. The experimental results showed that the proposed method worked properly, even when faced with anomalies along roads, such as obstructions caused by shadows and trees.

Esforços em ferramentas rompedoras de solo de semeadoras de plantio direto

The individual evaluation of mechanisms for opening furrows to drop the seeds in a no-tillage seeders cultivation assists the companies in the sizing of opening tools of the ground, that search for minor energy petition and for diminishing the ground area mobilized. Therefore this work had the aim of evaluate the efforts of five different mechanisms of opening of ridge of seeding of direct plantation varyng the vertical load. The experiment was installed in one ground classified as Dystroferric Red Nitosol. The used experimental delineation was of the one randomized blocks in a factorial project 5 x 4, constituted a the following factors: five mechanisms of furrow opening (even disk - DL, corrugated disk - DC, ondulated disk - DO, double disk - DD and furrow rod - F) and four vertical loads (750, 1500, 2250 and 3000 N). To measure the forces in the studied treatments a dynamometer with sensitivity in the directions was used horizontal, lateral and vertical direction. The evaluation showed that the interaction between mechanisms of opening of ridge and vertical load was not significant, however the factors were significant when analyzed individually. The connecting rod promoted mobilization of the ground with smaller draft of horizontal effort reaching depths of work. The work showed that the vertical load influenced the depth of work of the mechanisms of ridge opening. The mechanisms of ridge opening of record type need vertical force to penetrate into the ground.

Growth regulator losses from cotton plants due to rainfall

Reguladores de crescimento aplicados às plantas de algodoeiro (Gossypium hirsutum L.) podem ser lavados em função da ocorrência de chuvas. Chuvas que ocorrem próximas à época de aplicação podem ocasionar elevada perda e necessidade de reaplicação dos produtos visando à taxa de crescimento desejada. Avaliou-se o efeito do intervalo de tempo entre a ocorrência de chuva simulada e a aplicação de cloreto de mepiquat e cloreto de chlormequat no algodoeiro no crescimento das plantas, além de estimar a necessidade de reaplicação dos reguladores. Plantas de algodão foram cultivadas em vasos de 12 L que permaneceram em casa de vegetação. Os reguladores de crescimento foram aplicados 40 dias após a emergência, quando 50% das plantas apresentavam botão floral. A chuva foi simulada 1, 2, 4, 6 e 24 horas após a aplicação dos reguladores. Determinou-se a altura das plantas antes da aplicação dos produtos e a cada 3 dias até o 30º dia. Na colheita, foi avaliado o número de ramos e estruturas reprodutivas, com posterior determinação da massa da matéria seca. Também foi determinado o crescimento acumulado e taxa de crescimento das plantas. Os dois reguladores reduziram a massa da matéria seca das plantas, independente do intervalo para ocorrência da chuva. O crescimento excessivo das plantas foi controlado, porém, com eficiência reduzida quanto menor o intervalo para simulação de chuva. em todos os períodos avaliados houve perda de produtos, com necessidade de reaplicação. A taxa de reaplicação de cloreto de mepiquat para os diferentes intervalos de chuva foi, em média, 17% maior.

Integration of a regional GPS network within ITRF using precise point positioning

GPS precise point positioning (PPP) can provide high precision 3-D coordinates. Combined pseudorange and carrier phase observables, precise ephemeris and satellite clock corrections, together with data from dual frequency receivers, are the key factors for providing such levels of precision (few centimeters). In general, results obtained from PPP are referenced to an arbitrary reference frame, realized from a previous free network adjustment, in which satellite state vectors, station coordinates and other biases are estimated together. In order to obtain consistent results, the coordinates have to be transformed to the relevant reference frame and the appropriate daily transformation parameters must be available. Furthermore, the coordinates have to be mapped to a chosen reference epoch. If a velocity field is not available, an appropriated model, such as NNR-NUVEL-IA, has to be used. The quality of the results provided by this approach was evaluated using data from the Brazilian Network for Continuous Monitoring of the Global Positioning System (RBMC), which was processed using GIPSY-OASIS 11 software. The results obtained were compared to SIRGAS 1995.4 and ITRF2000, and reached precision better than 2cm. A description of the fundamentals of the PPP approach and its application in the integration of regional GPS networks with ITRF is the main purpose of this paper.

Object-Space Road Extraction in Rural Areas Using Stereoscopic Aerial Images

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)