Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus

An extracellular alpha-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-d-glucopyranoside and presented an optimum temperature and pH of 65A degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60A degrees C, presenting t (50) of 90 min at 65A degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an alpha-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

Visible and near-infrared luminescent Eu(3+) or Er(3+) doped laponite-derived xerogels and thick films: Structural and spectroscopic properties

Laponite-derived materials represent promising materials for optical applications. In this work, Eu(3+)- or Er(3+)-doped laponite xerogels and films were prepared from colloidal dispersion. Homogeneous, crack-free and transparent single layers were deposited on soda-lime substrates with a thickness of 10 mu m. Structural and spectroscopic properties were analyzed by thermal analyses, X-ray diffractometry, transmission electron microscopy, infrared spectroscopy, and luminescence spectroscopy. The addition of a rare earth ion to the laponite does not promote any changes in thermal stability or phase transition. Laponite clay was identified after annealing up to 500 degrees C, with a decrease in basal spacing when the annealing temperature is changed from 100 degrees C to 500 degrees C. Enstatite polymorphs and amorphous silicate phases were observed after heat treatment at 700 degrees C and 900 degrees C. Stationary and time-dependent luminescence spectra in the visible region for Eu(3+), and (5)D(0) lifetime are discussed in terms of thermal treatment and structural evolution. In the layered host, the Eu(3+) ions are distributed in many different local environments. However, Eu(3+) ions were found to occupy at least two symmetry sites, and the ions are preferentially incorporated into the crystalline enstatite for the materials annealed at 700 degrees C and 900 degrees C. A (5)D(0) lifetime of 1.3 ms and 3.1 ms was obtained for Eu(3+) ions in an amorphous silicate and crystalline MgSiO(3) local environment, respectively. Strong Er(3+) emission at the 1550 nm region was observed for the materials annealed at 900 degrees C, with a bandwidth of 44 nm. (C) 2008 Elsevier B.V. All rights reserved.

Er(3+) doped phosphoniobate glasses and planar waveguides: structural and optical properties

Phosphoniobate glasses with composition (mol%) (100-x) NaPO(3)-xNb(2)O(5) ( x varying from 11 to 33) were prepared and characterized by means of thermal analysis, Fourier transform infrared spectroscopy, Raman scattering and (31)P nuclear magnetic resonance. The addition of Nb(2)O(5) to the polyphosphate base glass leads to depolymerization of the metaphosphate structure. Different colors were observed and assigned as indicating the presence of Nb(4+) ions, as confirmed by electron paramagnetic resonance measurements. The color was observed to depend on the glass composition and melting temperature as well. Er(3+) containing samples were also prepared. Strong emission in the 1550 nm region was observed. The Er(3+4)I(15/2) emission quantum efficiency was observed to be 90% and the quenching concentration was observed to be 1.1 mol%( 1.45 x 10(20) ions cm(-3)). Planar waveguides were prepared by Na(+)-K(+)-Ag(+) ion exchange with Er(3+) containing samples. Optical parameters of the waveguides were measured at 632.8, 543.5 and 1550 nm by the prism coupling technique as a function of the ion exchange time and Ag(+) concentration. The optimized planar waveguides show a diffusion depth of 5.9 mu m and one propagating mode at 1550 nm.

Potential of Amaranthus cruentus BRS Alegria in the production of flour, starch and protein concentrate: chemical, thermal and rheological characterization

BACKGROUND: Amaranth is a little-known culture in Brazilian agriculture. Amaranthus cruentus BRS Alegria was the first cultivar recommended by Embrapa for the soil of the Brazilian scrubland. In order to evaluate the potential of this species in the production of flour, starch and protein concentrates, the latter products were obtained from A. cruentus BRS Alegria seeds, characterized and compared with the products obtained from the A. caudatus species cultivated in its soil of origin. RESULTS: The seeds of A. cruentus BRS Alegria furnished high-purity starch and flour with significant content of starch, proteins, and lipids. The starch and flour of this species presented higher gelatinization temperatures and formed stronger gels upon cooling compared with those obtained from the A. caudatus species. This is due to their greater amylose content and a difference in the composition of the more important fatty acids, such as stearic, oleic and linoleic acids, which indicates that they have greater heat stability. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and differential scanning calorimetry revealed the presence of albumins, globulins, glutelins and prolamins in the protein concentrate, which was obtained as a byproduct of starch production. CONCLUSION: Amaranthus cruentus BRS Alegria has potential application in the production of flour, starch and protein concentrates, with interesting characteristics for use as food ingredients. (C) 2010 Society of Chemical Industry

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K (m) and V (max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 A mu mol/min/mg, respectively. PG was found to have temperature optimum at 65 A degrees C and was totally stable at 45 A degrees C for 90 min. Half-life at 55 A degrees C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Relationship between induced sputum cytology and inflammatory status with lung structural and functional abnormalities in asbestosis

Background Asbestosis is associated with lung cellular and immunological abnormalities. Induced sputum cytology and local and systemic markers of inflammation may be helpful to characterize disease status and progression in these patients. Methods Thirty-nine ex-workers with asbestosis on high-resolution CT (HRCT) and 21 non-exposed controls were evaluated. Sputum cytology and IL-8 in serum and sputum were related to lung function impairment. Results Subjects with asbestosis had reduced sputum cellularity but higher macrophagel neutrophil ratio and % macrophage as compared with controls. Sputum and serum IL-8 were also higher in patients with asbestosis (P < 0.05). In addition, evidence of lung architectural distorption on HRCT was associated with increased levels of serum IL-8. Interestingly, absolute macrophage number was negatively correlated with total lung capacity (r = -0.40; P = 0.04) and serum IL-8 to lung diffiusing capacity (r = -0.45; P = 0.01). Conclusions Occupationally exposed subjects with asbestosis on HRCT have cytologic abnormalities in induced sputum and increased local and systemic pro-inflammatory status which are correlated to functional impairment.

Comparative neuroanatomical and temporal characterization of FluoroJade-positive neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine in Wistar rats

The aims of this study were to characterize the spatial distribution of neurodegeneration after status epilepticus (SE) induced by either systemic (S) or intrahippocampal (H) injection of pilocarpine (PILO), two models of temporal lobe epilepsy (TLE), using FluoroJade (FJ) histochemistry, and to evaluate the kinetics of FJ staining in the H-PILO model. Therefore, we measured the severity of behavioral seizures during both types of SE and also evaluated the FJ staining pattern at 12, 24, and 168 h (7 days) after the H-PILO insult. We found that the amount of FJ-positive (FJ+) area was greater in SE induced by S-PILO as compared to SE induced by H-PILO. After SE induced by H-PILO, we found more FJ+ cells in the hilus of the dentate gyrus (DG) at 12 h, in CA3 at 24 h, and in CA1 at 168 h. We found also no correlation between seizure severity and the number of FJ+ cells in the hippocampus. Co-localization studies of FJ+ cells with either neuronal-specific nuclear protein (NeuN) or glial fibrillary acidic protein (GFAP) labeling 24 h after H-PILO demonstrated spatially selective neurodegeneration. Double labeling with FJ and parvalbumin (PV) showed both FJ+/PV+ and FJ+/PV- cells in hippocampus and entorhinal cortex, among other areas. The current data indicate that FJ+ areas are differentially distributed in the two TLE models and that these areas are greater in the S-PILO than in the H-PILO model. There is also a selective kinetics of FJ+ cells in the hippocampus after SE induced by H-PILO, with no association with the severity of seizures, probably as a consequence of the extra-hippocampal damage. These data point to SE induced by H-PILO as a low-mortality model of TLE, with regional spatial and temporal patterns of FJ staining. (C) 2010 Elsevier B.V. All rights reserved.

Clinical and molecular characterization of Brazilian families with von Hippel-Lindau disease: a need for delineating genotype-phenotype correlation

von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumours, especially cerebellar haemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). The etiology and manifestations are due to germline and somatic mutations in the VHL tumour suppressor gene. VHL disease is classified into type 1 and type 2, showing a clear genotype-phenotype correlation, as type 2 is associated with phaeochromocytoma and essentially caused by missense mutations. The aim of this study is to characterize the phenotype and genotype of families with VHL disease. Eighteen of twenty patients from ten unrelated families underwent genetic testing, nine of them fulfilled VHL disease criteria and one had an apparently sporadic cerebellar haemangioblastoma. Four different germline mutations in the VHL gene were identified: c.226_228delTTC (p.Phe76del); c.217C > T (p.Gln73X); IVS1-1 G > A and IVS2-1 G > C. The first three mutations were associated with type 1 disease and the last one with type 2B, which had never been identified in the germline. The transcriptional processing of a novel splice-site mutation was characterised. Three type 1 VHL families showed large deletions of the VHL gene, two of them encompassed the FANCD2/C3orf10 genes and were not associated with renal lesions. We also suggest that such families should be subclassified according to the risk of RCC and the extent of the VHL gene deletions. This study highlights the need for a through clinical and molecular characterisation of families with VHL disease to better delineate its genotype-phenotype correlation.

Ageing and surface EMG activity patterns of masticatory muscles

P>The purpose of this study was to evaluate the influence of age on the electromyographic activity of masticatory muscles. All volunteers were Brazilian, fully dentate (except for Group I - mixed dentition), Caucasian, aged 7-80, and divided into five groups: I (7-12 years), II (13-20 years), III (21-40 years), IV (41-60 years) and V (61-80 years). Except for Group V, which comprised nine women and eight men, all groups were equally divided with respect to gender (20 M/20 F). Surface electromyographic records of masticatory muscles were obtained at rest and during maximal voluntary contraction, right and left laterality, maximal jaw protrusion and maximal clenching in the intercuspal position. Statistically significant differences (P < 0 center dot 05) were found in all clinical conditions among the different age groups. Considerably different patterns of muscle activation were found across ages, with greater electromyographic activity in children and youth, and decreasing from adults to aged people.

Characteristics of Filiform, Fungiform and Vallate Papillae and Surface of Interface Epithelium-Connective Tissue of the Maned Sloth Tongue Mucosa (Bradypus torquatus, Iliger, 1811): Light and Scanning Electron Microscopy Study

The study of lingual surfaces and the surface of interface epithelium-connective tissue of the tongue of Bradypus torquatus was performed by employing the light and scanning electron microscopy (SEM) techniques. The results revealed that the rostral part of the tongue presents a round apex and covered by filiform and fungiform lingual papillae and a ventral smooth surface. It was observed that the epithelial layer of the dorsal surface possesses the basal, spinosum, granular and cornified epithelial cells. The lamina propria is characterized by a dense connective tissue forming the long, short and round papillae. Numerous typical filiform papillae are located especially in the rostral part intermingled for few fungiform papillae, which were revealed in three-dimensional SEM images. Usually, the fungiform papillae are located in the border of rostral apex of the tongue exhibiting the rounded form. They are covered by keratinized epithelial cells. In the fungiform papillae, several taste pores were observed on the surface. The vallate papillae presented numerous taste buds in the wall of epithelial cells, being that the major number of taste buds is located on the superior half of vallate papilla. The taste pores are surrounded by several laminae of keratinized epithelial cells. The samples treated with NaOH solution and examined by SEM revealed, after removal of the epithelial layer, the dense connective core in original disposition, presenting different sizes and shapes. The specimens stained with Picrosirius and examined by polarized light microscopy revealed the connective tissue, indicating the collagen fibres type I and type III.

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica. (C) 2010 Elsevier Ltd. All rights reserved.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in Sao Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline x human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of Fly. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. (C) 2011 Elsevier B.V. All rights reserved.

Phenotypic and molecular characterization of recent and archived Erysipelothrix spp. isolated from Brazilian swine

One hundred fifty-one Erysipelothrix spp. isolates from Brazilian swine were characterized by serotyping, determination of antimicrobial susceptibility, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). Among all isolates, 139 were classified in 18 different serotypes and serotype 2b was the most frequent. The susceptibility profiles of the isolates were very similar among each other, which did not permit subtyping Erysipelothrix spp. isolates by the antimicrobial susceptibility testing. Despite the fact that AFLP and PFGE provided the same discriminatory index (0.98), PFGE was more discriminatory than AFLP, given the types of groups it generates. Regardless the technique employed (AFLP or PFGE), no discrimination between recent and historical isolates was established, neither a fixed epidemiologic pattern for their grouping was observed. Nevertheless, AFLP could be an interesting alternative for discriminating the Erysipelothrix species, while PFGE could be an indication for discerning this bacterium according to the serotypes. (C) 2011 Elsevier Inc. All rights reserved.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.