Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Isolation and characterization of microsatellite loci for white-lipped peccaries (Tayassu pecari) and cross-amplification in collared peccaries (Pecari tajacu)

White-lipped peccaries, Tayassu pecari, are neotropical ungulates whose populations have been declining in numerous locations within their geographical distribution. Here we describe 16 microsatellite loci isolated from T. pecari and their cross-amplification in collared peccaries, Pecari tajacu. In 30 individuals of T. pecari, a total of 32 alleles were found in ten polymorphic loci, ranging from 2 to 8 alleles per locus with a mean of 3.2. The expected and observed heterozygosity ranged from 0.143 to 0.802 and from 0 to 0.704, respectively. Two loci deviated from Hardy-Weinberg equilibrium. In P. tajacu, nine loci were polymorphic with a mean of 3.2 alleles per locus. These molecular markers will be useful to study the genetic status of peccary populations and, consequently, to help their conservation.

Isolation and characterization of 15 microsatellite loci in the stingless bee Plebeia remota (Apidae: Meliponini)

The destruction of Brazilian natural habitats has reduced bee populations and negative impacts of native flora pollination have been noticed. This work describes the isolation and characterization of microsatellite loci and evaluates them as molecular markers to study genetic variability of the stingless bee Plebeia remota. A microsatellite enriched genomic library was constructed and 15 primer pairs were designed for this species. The survey was conducted by analyzing 21 unrelated individuals. Genetic diversity indexes were calculated. The mean allelic richness was 6.3, the observed heterozygosity was 0.568, and the percentage of polymorphic loci was 93.33%. Also the primers were tested in cross-species amplification and showed promising results for P. droryana, P. emerina, P. lucii, P. meridionalis, P. pugnax, and P. saiqui. The microsatellite loci described here will be useful to evaluate genetic variability of stingless bees, and certainly will improve our knowledge about population dynamics especially in threatened environments.

Isolation and characterization of microsatellite loci in the Brazilian orchid Epidendrum fulgens

Epidendrum fulgens has a patchy distribution along the Atlantic Rainforest in the Brazilian coast, due to the destruction of its native habitat. Here, we report on both the development of nine new microsatellite markers isolated from this species and the characterization of their allele variability in two distant and unrelated populations. The number of alleles observed for each locus ranged from 2 to 17 with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying the effect of habitat fragmentation on the genetic structure of E. fulgens populations.

Isolation and characterization of microsatellite loci in Epidendrum puniceoluteum, an endemic orchid from the Atlantic Rainforest

Epidendrum puniceoluteum is an endemic orchid of Atlantic Rainforest, restricted to few populations only due to the destruction and fragmentation of its native habitat. Here, we report on the development of 10 microsatellite markers isolated from this orchid species. Genetic variability was characterized in two distant populations from Brazil coast. The number of alleles observed for each locus ranged from two to 12 and with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying both fine-scale genetic structure of scattered E. puniceoluteum population and patterns will be useful genetic markers for other closely related taxa.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Isolation and genotype analysis of rubella virus from a case of Guillain-Barre syndrome

Rubella virus (RV) infection has sporadically been linked to Guillain-Barre syndrome (GBS), but the association with RV has been based only on clinical and/or serological backgrounds. In the present case it was possible to isolate RV (genotype 1a) from cerebrospinal fluid and peripheral blood mononuclear cells of an 18-year-old woman diagnosed with GBS after clinical manifestations of rubella. This report contributes to confirm RV as one of the triggering pathogens of this peripheral nervous system disease. (C) 2008 Elsevier B.V. All rights reserved.

Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system

Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright (C) 2008 S. Karger AG, Basel.

Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO(2))-Phe-Val-Leu-O(4)MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (>= 68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (>= 80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation. (c) 2009 Elsevier Inc. All rights reserved.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Isolation, chemical composition and anti-inflammatory activity of Copaifera langsdorffii desf. fruit peels essential oil according to successive hydrodistillations

In this study, the isolation and characterization of chemical composition of C. langsdorffii (copaiba) fruit peel volatile oil from Alfenas - MG was carried out according to successive hydrodistillations, likewise the anti-inflammatory activity was evaluated by rat paw edema model. The chemical composition of the oil was determined by GC-MS analysis. The major components were (E)-caryophyllene, germacrene B, 1,5-epoxysalvial-4(14)-ene, caryophyllene oxide and isospathulenol. The inflammatory process was inhibited in 39 +/- 6 % (p<0.05, Tukey-Kramer) by one sample of essential oil. The C. langsdorffii fruit peel is a rich source of essential oil (3.8% w/w), and due to potential industrial interest of its components this ecotype presents feasible crop seeking productivity and it could constitutes in a good alternative to the sustainable use of this specie.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.

Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37 degrees C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference (p> 0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus. (C) 2010 Elsevier GmbH. All rights reserved.

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in Sao Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A. allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviare, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.

The influence of halo assembly on galaxies and galaxy groups

In this paper, we study the variations of groups (galaxy properties according to the assembly history in Sloan Digital Sky Survey Data Release 6 (SDSS-DR6) selected groups. Using mock SDSS group catalogues, we find two suitable indicators of group formation time: (i) the isolation of the group, defined as the distance to the nearest neighbour ill terms of its virial radius and 00 the concentration. measured as the groups inner density calculated using the fifth nearest bright galaxy to the groups centre. Groups Within narrow ranges of Mass ill the mock catalO-Lie show increasing Ifl-OLIP alle With isolation and concentration. However, in the observational data the stellar age, as indicated by the spectral type, only shows a correlation with concentration. We study groups of similar mass and different assembly history. finding important differences ill their galaxy population. Particularly, ill high-mass SDSS groups. the number of members. mass-to-light ratios, red galaxy fractions and the magnitude difference between the brightest and second-brightest group galaxies, show different trends as a function of isolation and concentration, even when it is expected that the latter two quantities correlate with group age. Conversely. low-mass SDSS groups appear to be less sensitive to their assembly history. The correlations detected in the SDSS are not consistent with the trends measured in the mock catalogues. However, discrepancies can he explained in terms of the disagreement found in the a-e-isolation trends, suggesting that the model might be overestimating the effects of, environment, We discuss how the modelling of the cold gas ill satellite galaxies could be responsible for this problem. These results call be Used to improve our Understanding of the evolution of galaxies ill high-density environments.