Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

O presente estudo consiste em uma caracterização preliminar da atividade proteolítica de frações de proteínas purificadas a partir de lisados de trofozoítos de cepa isolada e axenizada no Brasil. Frações obtidas por cromatografia líquida (FPLC) foram analisadas quanto ao perfil eletroforético em géis de poliacrilamida (SDS-PAGE) e a atividade proteolítica foi avaliada em géis contendo gelatina como substrato. A caracterização das enzimas foi realizada a partir da análise do efeito de inibidores sintéticos de cisteína-proteases (E-64, IAA), serina-proteases (PMSF), serina e cisteína-proteases (TPCK, TLCK, elastatinal), metalo-proteases (EDTA) e aspartil proteases (pepstatina) sobre a degradação do substrato. Entre 30 frações eluídas, bandas de proteínas foram observadas em oito delas, entretanto, atividade proteolítica foi detectada apenas nas frações 23, 24, 25 e 26. O perfil eletroforético das proteínas revelou poucas bandas distribuídas na faixa de 45 a 18 kDa. Os zimogramas revelaram zonas de proteólise na faixa de aproximadamente 62 a 35 kDa, entretanto destacaram-se as bandas de hidrólise de 62, 55, 53, 50, 46 e 40 kDa. Nos ensaios de inibição, a proteólise foi marcantemente inibida por E-64, TPCK, TLCK e elastatinal. Redução discreta da proteólise foi observada com IAA e PMSF, enquanto que EDTA e pepstatina não promoveram alteração dos perfis de hidrólise. Estas observações são relevantes, especialmente se considerarmos que para elucidar o envolvimento das proteases na relação parasita-hospedeiro, a purificação dessas moléculas é um requisito importante.

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

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

Partial purification and properties of cellulase-free alkaline xylanase produced by Rhizopus stolonifer in solid-state fermentation

Rhizopus stolonifer was cultivated in wheat bran to produce a cellulase-free alkaline xylanase. The purified enzyme obtained after molecular exclusion chromatography in Sephacryl S-200 HR showed optimum temperature as 45 degrees C and hydrolysis pHs optima as pH 6.0 and 9.0. Xylanase presented higher Vmax at pH 9.0 (0.87 mu mol/mg protein) than at pH 6.0 and minor Km at pH 6.0 (7.42 mg/mL)than at pH 9.0.

Genetic variability of porcine parvovirus isolates revealed by analysis of partial sequences of the structural coding gene VP2

The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.

Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Adaptation of the reservoir fish assemblage index (RFAI) for assessing the Barra Bonita Reservoir (São Paulo, Brazil)

In order to evaluate the environmental condition of the Barra Bonita Reservoir, we adapted the Index of Biotic Integrity (IBI). We chose 24 sampling sites in which three types of habitats were sampled: the mouths of tributaries, the central reservoir and the lateral reservoir. Fish were caught in two seasons (dry and rainy) using 10 gillnet gangs, with meshes ranging from 3 to 12 cm between opposite knots, and funnel traps. Abiotic and biotic variables were measured. Due to the artificial nature of the reservoir, the term biotic integrity was considered inappropriate and the term RFAI (Reservoir Fish Assemblage Index) was adopted. Twelve metrics from sixteen possibles were selected using the Pearson correlation coefficient. The reference conditions were set up based on the criteria of the best condition observed. For each metric, a score of 1, 3 or 5 was assigned as it strongly departs (1), slightly departs (3) or approaches (5) the reference condition. The index value is the sum of the metrics partial scores and ranges from 12 to 60. To evaluate the importance of the unit of measurement of the metric, the index was calculated in fish number (RFAI(N)) and in weight (RFAI(w)). The correlation between RFAIN and RFAIw was very high (r = 0.90, n = 46) indicating that the unit of measurement does not influence the final result of the index. Most of the sampling sites were classified in the 'reasonable' RFAI category. Only the central sites were classified as 'poor'. To validate the RFAI, another index, the Habitat Quality Index (HQI), was built starting from the physiochemical and habitat variables collected. The correlation of the RFAI with the HQI was highly significant (RFAIN, r = 0.37; RFAI(w), r = 0.47; n = 46), indicating that they respond in the same way to environmental degradation. The HQI metrics which most affect RFAI were depth, surrounding landscape and macrophyte presence/absence. Copyright C) 2007 John Wiley & Sons, Ltd.

Gait training with partial body weight support during overground walking for individuals with chronic stroke: a pilot study

Background: It is not yet established if the use of body weight support (BWS) systems for gait training is effective per se or if it is the combination of BWS and treadmill that improves the locomotion of individuals with gait impairment. This study investigated the effects of gait training on ground level with partial BWS in individuals with stroke during overground walking with no BWS.Methods: Twelve individuals with chronic stroke (53.17 +/- 7.52 years old) participated of a gait training program with BWS during overground walking, and were evaluated before and after the gait training period. In both evaluations, individuals were videotaped walking at a self-selected comfortable speed with no BWS. Measurements were obtained for mean walking speed, step length, stride length and speed, toe-clearance, durations of total double stance and single-limb support, and minimum and maximum foot, shank, thigh, and trunk segmental angles.Results: After gait training, individuals walked faster, with symmetrical steps, longer and faster strides, and increased toe-clearance. Also, they displayed increased rotation of foot, shank, thigh, and trunk segmental angles on both sides of the body. However, the duration of single-limb support remained asymmetrical between each side of the body after gait training.Conclusions: Gait training individuals with chronic stroke with BWS during overground walking improved walking in terms of temporal-spatial parameters and segmental angles. This training strategy might be adopted as a safe, specific and promising strategy for gait rehabilitation after stroke.

Microsatellite loci isolation from river buffalo using enriched partial genomic libraries

The extensive use of buffalo in agriculture, especially in developing countries, begs for genetic resources to evaluate and improve traits important to local and regional economies. Brazil presents the largest water buffalo populations in the New World, with 1 1 million heads including swamp and river types. To design rational breeding strategies for optimum utilization and conservation of available genetic variability in the Brazilian buffalo's population, it is essential to understand their genetic architecture and relationship among various breeds. This depends, in part, on the knowledge of their genetic structure based on molecular markers like microsatellites. In the present study, we developed six enriched partial genomic libraries for river buffalo using selective hybridization methods. Genomic DNA was hybridized with six different arrays of repeat motif, 5' biotinylated - (CA)(15), (CT)(15), (AGG)(8), (GAAA)(8), (GATA)(8), (AAAAC)(8) - and bound to streptavidin coated beads. The cloning process generated a total of 1920 recombinant clones. Up to date, 487 were directly sequenced for the presence of repeats, from which 13 have been positive for presence of repeats as follows: 9 for di-nucleotide repeats, 3 for tri-nucleotide repeats and 1 for tetra-nucleotide repeat. PCR primer pairs for the isolated microsatellites are under construction to determine optimum annealing temperature. These microsatellites will be useful for studies involving phylogenetic relationships, genome mapping and genetic diversity analysis within buffalo populations worldwide.

Localization and partial characterization of thermostable glucoamylase produced by newly isolated Thermomyces lanuginosus TO3 in submerged fermentation

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

Edge detection and noise removal by use of a partial differential equation with automatic selection of parameters

This work deals with noise removal by the use of an edge preserving method whose parameters are automatically estimated, for any application, by simply providing information about the standard deviation noise level we wish to eliminate. The desired noiseless image u(x), in a Partial Differential Equation based model, can be viewed as the solution of an evolutionary differential equation u t(x) = F(u xx, u x, u, x, t) which means that the true solution will be reached when t ® ¥. In practical applications we should stop the time ''t'' at some moment during this evolutionary process. This work presents a sufficient condition, related to time t and to the standard deviation s of the noise we desire to remove, which gives a constant T such that u(x, T) is a good approximation of u(x). The approach here focused on edge preservation during the noise elimination process as its main characteristic. The balance between edge points and interior points is carried out by a function g which depends on the initial noisy image u(x, t0), the standard deviation of the noise we want to eliminate and a constant k. The k parameter estimation is also presented in this work therefore making, the proposed model automatic. The model's feasibility and the choice of the optimal time scale is evident through out the various experimental results.

Multiscale Fractal Descriptors and Polynomial Classifier for Partial Pixels Identification in Regions of Interest of Mammographic Images

Computer systems are used to support breast cancer diagnosis, with decisions taken from measurements carried out in regions of interest (ROIs). We show that support decisions obtained from square or rectangular ROIs can to include background regions with different behavior of healthy or diseased tissues. In this study, the background regions were identified as Partial Pixels (PP), obtained with a multilevel method of segmentation based on maximum entropy. The behaviors of healthy, diseased and partial tissues were quantified by fractal dimension and multiscale lacunarity, calculated through signatures of textures. The separability of groups was achieved using a polynomial classifier. The polynomials have powerful approximation properties as classifiers to treat characteristics linearly separable or not. This proposed method allowed quantifying the ROIs investigated and demonstrated that different behaviors are obtained, with distinctions of 90% for images obtained in the Cranio-caudal (CC) and Mediolateral Oblique (MLO) views.

Partial characterization of protease from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein

Proteases are one of the most important groups of industrial enzymes, with considerable application in the food industry. The aim of this work was to study a novel protease produced by the thermophilic fungus, Thermoascus aurantiacus, through solid-state fermentation (SSF). The enzyme acted optimally at pH 5.5 and 60 degrees C it was stable up to 60 degrees C for 1 h and in the pH range 3.0-9.5. To elucidate the enzyme's proteolytic activity, its hydrolytic profile on bovine casein, an important protein in the food industry, was studied by enzymatic hydrolysis on skim milk, analyzed by gel electrophoresis (UREA-PAGE), which clearly showed that the protease does not have the same specificity as bovine chymosin. (c) 2006 Elsevier Ltd. All rights reserved.

Production and partial characterization of polygalacturonases produced by thermophilic Monascus sp N8 and by thermotolerant Aspergillus sp N12 on solid-state fermentation

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

Detection and partial characterization of a carlavirus causing stem necrosis of soybean in Brazil

No ano agrícola de 2000/2001, ocorreu um surto de nanismo e necrose da haste em soja (Glycine max) plantada em duas áreas do Brasil Central e na safra seguinte, esta anomalia foi constatada em outras regiões produtoras, mesmo distantes mais de 2.000 km de onde fora inicialmente constatada. Estudos envolvendo ensaios de transmissão (enxertia, mecânica e insetos vetores), microscopia eletrônica, purificação, sorologia e ensaios moleculares indicaram que a enfermidade foi causada por um carlavirus transmitido por mosca branca, possivelmente relacionado ao Cowpea mild mottle virus (CpMMV).

Effect of axial loads on implanted-supported partial fixed prostheses by strain gauge analysis

Objectives: The present study used strain gauge analysis to perform an in vitro evaluation of the effect of axial loading on 3 elements of implant-supported partial fixed prostheses, varying the type of prosthetic cylinder and the loading points. Material and methods: Three internal hexagon implants were linearly embedded in a polyurethane block. Microunit abutments were connected to the implants applying a torque of 20 Ncm, and prefabricated Co-Cr cylinders and plastic prosthetic cylinders were screwed onto the abutments, which received standard patterns cast in Co-Cr alloy (n=5). Four strain gauges (SG) were bonded onto the surface of the block tangentially to the implants, SG 01 mesially to implant 1, SG 02 and SG 03 mesially and distally to implant 2, respectively, and SG 04 distally to implant 3. Each metallic structure was screwed onto the abutments with a 10 Ncm torque and an axial load of 30 kg was applied at five predetermined points (A, B, C, D, E). The data obtained from the strain gauge analyses were analyzed statistically by RM ANOVA and Tukey's test, with a level of significance of p<0.05. Results: There was a significant difference for the loading point (p=0.0001), with point B generating the smallest microdeformation (239.49 mu epsilon) and point D the highest (442.77 mu epsilon). No significant difference was found for the cylinder type (p=0.748). Conclusions: It was concluded that the type of cylinder did not affect in the magnitude of microdeformation, but the axial loading location influenced this magnitude.