Platelet hyperaggregability in high-fat fed rats: A role for intraplatelet reactive-oxygen species production

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

Zoogeography of Chiropotes albinasus (Platyrrhini, Atelidae) in southwestern Amazonia

White-nosed bearded sakis (Chiropotes albinasus) are endemic to the Madeira-Xingu interfluvium in southern Amazonia, though recent fieldwork has produced conflicting data on the southwestern limits of the species's geographic range. We reevaluated the distribution of bearded sakis on the basis of surveys from 34 sites throughout the Brazilian state of Rondonia. Chiropotes albinasus occurred at seven sites in the eastern part of the state, including two west of the Jiparana-Pimenta Bueno river system in the extreme south, but there is no record of their presence further north and west in the Jiparana-Guapore interfluvium and they were absent from the Jiparana-Mamore interfluvium. The data suggest that ecological, rather than geographic barriers restrict the distribution of Chiropotes albinasus in southern Rondonia, but are contradictory with regard to the possible determining factors. Chiropotes albinasus appears able to thrive in transitional, savanna-like ecosystems in southern Rondonia, but is unexpectedly absent from adjacent areas of terra firme forest. Syntopy with the only other pitheciine found in the state (Pithecia irrorata) appears to have a negative effect on the abundance of Chiropotes albinasus which implies that interspecific competition may reinforce a complex of limiting factors, such as the availability of key plant species. Despite showing that Chiropotes albinasus is widespread in southern Rondonia, we also confirm its absence from the western two thirds of the state, a significant reduction in the known range of the species.

Quasispecies of hepatitis C virus genotype 1 and treatment outcome with Peginterferon and Ribavirin

The majority of patients with chronic hepatitis C fail to respond to antiviral therapy. The genetic basis of this resistance is unknown. The quasispecies nature of HCV may have an important implication concerning viral persistence and response to therapy. The HCV nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy. To evaluate whether the NS5A quasispecies pre-treatment composition of HCV 1a/1b is related to responsiveness to combined pegylated interferon (PEG-IFN) and Ribavirin therapy, detailed analyses of the complete NS5A were performed. Fifteen full-length NS5A clones were sequenced from 11 pretreatment samples of patients infected with genotype 1 HCV (3 virological sustained responders, 4 non-responders, and 4 end-of-treatment responders). Our study could not show a significant correlation between the mean number of mutations in HCV NS5A before treatment and treatment outcome, and the phylogenetic construction of complete NS5A sequences obtained from all patients failed to show any clustering associated with a specific response pattern. No single amino acid position was associated with different responses to therapy in any of the NS5A regions analyzed, and mutations were clustered downstream the ISDR, primarily in the V3 region. We observed that the CRS and NLS regions of the NS5A protein were conflicting for some variables analyzed, although no significant differences were found. If these two regions can have antagonistic functions, it seems viable that they present different mutation profiles when compared with treatment response. The patient sample that presented the lowest genetic distance values also presented the smallest number of variants, and the most heterogeneous pattern was seen in the end-of-treatment patients. These results suggest that a detailed molecular analysis of the NS5A region on a larger sample size may be necessary for understanding its role in the therapy outcome of HCV 1a/1b infection. (C) 2008 Elsevier B.V. All rights reserved.

Crystallization and preliminary diffraction data of BaP1, a haemorrhagic metalloproteinase from Bothrops asper snake venom

BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 Angstrom. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 Angstrom.

Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A(2) (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region pert-nits quaternary structural transitions between open and closed membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure [1]. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78Angstrom, (ii) MjTX-II/STE complex at a resolution of 1.8 Angstrom and (W) BthTX-I/DMPC complex at 2.72Angstrom. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (W) and using using a Synchrotron Radiation Source (Laboratorio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).

Effect of surfactants and polyethylene glycol on the activity and stability of a lipase from oilseeds of Pachira aquatica

Lipases from oilseeds have a great potential for commercial exploration as industrial enzymes. Lipases are used mixed with surfactants in cleaning and other formulated products, and accordingly, both components must be compatible with each other. This work presents the results of the effects of anionic, cationic and nonionic surfactants, polyethylene glycol and urea on the activity and stability of a lipase extracted of oilseeds from Pachira aquatica. The enzyme was purified and the spectrophotometric assays were done using p-nitrophenyl acetate (p-NPA) as substrate pH 7.5 and 25 degrees C. The activity was significantly enhanced by the cationic surfactant CTAB. Bile salts increased the lipase activity in the tested concentration range, whereas anionic and nonionic surfactants showed an inhibitory effect. Aqueous solutions of PEG activated the lipase and maximum activation (161%) occurred in PEG 12,000. This effect on lipase that can be due to exposition of some hydrophobic residues located in the vicinity of the active site or aggregation.

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coil can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coil homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH. (C) 2012 Elsevier B.V. All rights reserved.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phospate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na2SO4, pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Phase Diagrams of the Aqueous Two-Phase Systems of Poly(ethylene glycol)/Sodium Polyacrylate/Salts

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

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

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

Synthesis, characterization and solution properties of amphiphilic N-isopropylacrylamide-poly(ethylene glycol)-dodecyl methacrylate thermosensitive polymers

Terpolymers of N-isopropylacrylamide, dodecyl methacrylate (DOMA) and poly(ethylene glycol) (PEG) methacrylate, were synthesized by random copolymerization, and the composition was controlled to achieve systems having different thermosensitivities. H-1 NMR spectra and gel permeation chromatography (GPC) were employed to characterize the different samples obtained. The solution properties were studied by employing spectrophotometry, fluorescence, and dynamic light scattering techniques. The chemical compositions in the final terpolymers are close to those in the feed. The polymers exhibited cloud point temperatures (T-es) varying from 17 to 52 degrees C. Micropolarity studies using I-1/I-3 ratio of the vibronic bands of pyrene show the formation of amphiphilic aggregates capable of incorporating hydrophobic drugs as the polymer concentration is increased. The critical aggregation concentration (CAC) increases from 3.6 x 10(-3) to 1 x 10(-2) g/l with the PEG content varying from 5 to 35 mol%. Anisotropy measurements confirm the results obtained by pyrene fluorescence and show that the aggregates resulting from intermolecular interactions present different organizations. The hydrodynamic diameters (Dh) of the aggregates determined by dynamic light scattering (DLS) vary from 40 to 150 nm depending on the terpolymer composition. The T-cs and Dh values decreased with the ionic strength, and this behavior was attributed to the dehydration of the polymeric micelles. The capacity of solubilization of the aggregates was evaluated by employing pyrene, and the obtained results confirm the ability to incorporate hydrophobic molecules. (c) 2005 Elsevier B.V All rights reserved.

Effect of Static and Cyclic Loading on Ceramic Laminate Veneers Adhered to Teeth with and Without Aged Composite Restorations

Purpose: Existing composite restorations on teeth are often remade prior to the cementation of fixed dental prostheses. The aim of this study was to evaluate the effect of static and cyclic loading on ceramic laminate veneers adhered to aged resin composite restorations.Materials and Methods: Eighty sound maxillary incisors were collected and randomly divided into four groups: group 1: control group, no restorations; group 2: two Class III restorations; group 3: two Class IV restorations; group 4: complete composite substrate. Standard composite restorations were made using a microhybrid resin composite (Anterior Shine). Restored teeth were subjected to thermocycling (6000 cycles). Window preparations were made on the labial surface of the teeth for ceramic laminate fabrication (Empress II). Teeth were conditioned using an etch-and-rinse system. Existing composite restorations representing the aged composites were silica coated (CoJet) and silanized (ESPE-Sil). Ceramic laminates were cemented using a bis-GMA-based cement (Variolink Veneer). The specimens were randomly divided into two groups and were subjected to either static (groups 1a, 2a, 3a, 4a) or cyclic loading (groups 1b, 2b, 3b, 4b). Failure type and location after loading were classified. Data were analyzed using one-way ANOVA and Tukey's test.Results: Significantly higher fracture strength was obtained in group 4 (330 +/- 81 N) compared to the controls in group 1 (179 +/- 120 N) (one-way ANOVA, p < 0.05). Group lb survived a lower mean number of cyclic loads (672,820 cycles) than teeth of groups 2b to 4b (846x10(3) to 873x10(3) cycles). Failure type evaluation after the fracture test showed predominantly adhesive failures between dentin and cement, but after cyclic loading, more cohesive fractures in the ceramic were seen.Conclusion: Ceramic laminate veneers bonded to conditioned aged composite restorations provided favorable results. Surface conditioning of existing restorations may eliminate the necessity of removing aged composite restorations.

Evaluation of interface characterization and adhesion of glass ceramics to commercially pure titanium and gold alloy after thermal- and mechanical-loading

Objectives. This study evaluated the effect of thermal- and mechanical-cycling on the shear bond strength of three low-fusing glassy matrix dental ceramics to commercial pure titanium (cpTi) when compared to conventional feldspathic ceramic fused to gold alloy.Methods. Metallic frameworks (diameter: 5 min, thickness: 4 mm) (N = 96, n = 12 per group) were cast in cpTi and gold alloy, airborne particle abraded with 150 mu m aluminum oxide. Low-fusing glassy matrix ceramics and a conventional feldspathic ceramic were fired onto the alloys (thickness: 4mm). Four experimental groups were formed; Gr1 (control group): Vita Omega 900-Au-Pd alloy; Gr2: Ticeram-cpTi; Gr3: Super Porcelain Ti-22-cpTi and G4: Vita Titankeramik-cpTi. While half of the specimens from each ceramic-metal combination were randomly tested without aging (water storage at 37 C for 24h only), the other half were first thermocycled (6000 cycles, between 5 and 55 C, dwell time: 13 s) and then mechanically loaded (20,000 cycles under SON load, immersion in distilled water at 37 C). The ceramic-alloy interfaces were loaded under shear in a universal test machine (cross-head speed: 0.5 mm/min) until failure occur-red. Failure types were noted and the interfaces of the representative fractured specimens from each group were examined with stereo microscope and scanning electron microscope (SEM). in an additional study (N = 16, n = 2 per group), energy dispersive X-ray spectroscopy (EDS) analysis was performed from ceramic-alloy interfaces. Data were analyzed using ANOVA and Tukey's test.Results. Both ceramic-metal combinations (p < 0.001) and aging conditions (p < 0,001) significantly affected the mean bond strength values. Thermal- and mechanical-cycling decreased the bond strength (MPa) results significantly for Gr3 (33.4 +/- 4.2) and Gr4 (32.1 +/- 4.8) when compared to the non-aged groups (42.9 +/- 8.9, 42.4 +/- 5.2, respectively). Gr1 was not affected significantly from aging conditions (61.3 +/- 8.4 for control, 60.7 +/- 13.7 after aging) (p > 0.05). Stereomicroscope images showed exclusively adhesive failure types at the opaque ceramic-cpTi interfacial zone with no presence of ceramic on the substrate surface but with a visible dark titanium oxide layer in Groups 2-4 except Gr1 where remnants of bonder ceramic was visible. EDS analysis from the interfacial zone for cpTi-ceramic groups showed predominantly 34.5-85.1% O(2) followed by 1.1-36.7% Aland 0-36.3% Si except for Super Porcelain Ti-22 where a small quantity of Ba (1.4-8.3%), S (0.7%) and Sn (35.3%) was found. In the Au-Pd alloy-ceramic interface, 56.4-69.9% O(2) followed by 15.6-26.2% Si, 3.9-10.9% K, 2.8-6% Na, 4.4-9.6% Al and 0-0.04% Mg was observed.Significance. After thermal-cycling for 6000 times and mechanical-cycling for 20,000 times, Triceram-cpTi combination presented the least decrease among other ceramic-alloy combinations when compared to the mean bond strength results with Au-Pd alloy-Vita Omega 900 combination. (c) 2008 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Fractal character of the SAXS correlation volume in poly(ethylene glycol)/silica hybrid wet gels

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