Purification and characterization of GlcNAc-6-P 2-epimerase from Escherichia coli K92

N-Acetylmannosamine (ManNAc) is the first committed intermediate in sialic acid metabolism. Thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. In prokaryotic organisms, UDP-N-acetylglucosamine (GlcNAc) 2-epimerase and GlcNAc-6-P 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc-6-P, respectively. We have purified for the first time native GlcNAc-6-P 2-epimerase from bacterial source to apparent homogeneity (1 200 fold) using Butyl-agarose, DEAE-FPLC and Mannose-6-P-agarose chromatography. By SDS/PAGE the pure enzyme showed a molecular mass of 38.4 +/- 0.2 kDa. The maximum activity was achieved at pH 7.8 and 37 degrees C. Under these conditions, the K(m) calculated for GlcNAc-6-P was 1.5 mM. The 2-epimerase activity was activated by Na(+) and inhibited by mannose-6-P but not mannose-1-P. Genetic analysis revealed high homology with bacterial isomerases. GlcNAc-6-P 2-epimerase from E. coli K92 is a ManNAc-inducible protein and is detected from the early logarithmic phase of growth. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes the biosynthesis of sialic acid, GlcNAc-6-P 2-epimerase plays a catabolic role. When E. coli grows using ManNAc as a carbon source, this enzyme converts the intracellular ManNAc-6-P generated into GlcNAc-6-P, diverting the metabolic flux of ManNAc to GlcNAc.     

Homology assessment in parsimony and model‐based analyses: two sides of the same coin

The present paper is mainly concerned with homology assessment through phylogenetic analyses. It raises a fundamental question: What are the epistemological differences between modern parsimony and model‐based analyses in relation to homology assessment and phylogenetic inference? Although these methods usually achieve concordant topological results, they may generate discordant inferences of character evolution from the same datasets. This indicates that method selection has serious implications for evolutionary scenarios and classificatory arrangements. Notwithstanding that parsimony and model‐based approaches use the Hennigian concepts of monophyly and synapomorphy, they employ different epistemological ways of dealing with the monophyly/synapomorphy relationship. Independently of their differences, these analyses should take into account all relevant evidence in support of the phylogenetic inferences. A focus on morphological homologues means that they must be included in data matrices, evaluated as part of the phylogenetic analysis, and cannot be ignored in calculation of the tree(s) length (parsimony), maximum‐likelihood (maximum‐likelihood), and posterior probabilities (Bayes).     

Optimization of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology: the case of human interferon beta

A human interferon beta (hINF-beta) synthetic gene was optimized and expressed in Escherichia coli BL21-SI using a vector with the T7 promoter. To determine the best culture conditions such as culture medium, temperature, cell density and inducer concentration, we used the response surface methodology and a Box-Behnken design to get the highest hINF-beta production. The maximum hINF-beta production of 61 mg l(-1) was attained using minimum medium and the following predicted optimal conditions: temperature of 32.5 degrees C, cell density of 0.64, and inducer concentration of 0.30 M NaCl. This is the first report showing the successful performance of the BL21-SI system in a minimum medium. The response surface methodology is effective for the optimization of recombinant protein production using synthetic genes.     

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction,oxidative stress and proteases

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca²⁺ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψ_m). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψ_m and intracellular free Ca²⁺, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects. D. Cervia and M. Garcia-Gil equally contributed to the work.     

Effects of reduced‐impact logging on physical habitat and fish assemblages in streams of Eastern Amazonia

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Exploratory analysis of diffusion tensor imaging in children with attention deficit hyperactivity disorder: evidence of abnormal white matter structure

Abnormalities in the white matter microstructure of the attentional system have been implicated in the aetiology of attention deficit hyperactivity disorder (ADHD). Diffusion tensor imaging (DTI) is a promising magnetic resonance imaging (MRI) technology that has increasingly been used in studies of white matter microstructure in the brain. The main objective of this work was to perform an exploratory analysis of white matter tracts in a sample of children with ADHD versus typically developing children (TDC). For this purpose, 13 drug-naive children with ADHD of both genders underwent MRI using DTI acquisition methodology and tract-based spatial statistics. The results were compared to those of a sample of 14 age- and gender-matched TDC. Lower fractional anisotropy was observed in the splenium of the corpus callosum, right superior longitudinal fasciculus, bilateral retrolenticular part of the internal capsule, bilateral inferior fronto-occipital fasciculus, left external capsule and posterior thalamic radiation (including right optic radiation). We conclude that white matter tracts in attentional and motor control systems exhibited signs of abnormal microstructure in this sample of drug-naive children with ADHD.     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative, 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene, we evaluated membrane fluidity in living polymorphonuclear leukocytes and in erythrocytes of psoriatic patients. Our results have shown that erythrocyte membranes of psoriatic patients exhibit a decrease of fluidity. These changes were not associated with any relevant modifications of the cholesterol to phospholipid molar ratio. Moreover, we observed a decrease in polymorphonuclear leukocytes membrane fluidity associated with changes in chemotactic migration. Our results indicate changes of membrane fluidity involving membranes different from the epidermal cells and suggest the hypothesis of a defective membrane-cytoskeleton interaction in psoriasis.     

A study on the environmental aspects of WEEE plastic recycling in a Brazilian company

Purpose

The high consumption of electrical and electronic equipment motivated by the rapid technological advances seen over the years has lead to an increase in the generation of waste electrical and electronic equipment (WEEE). Such residues contain various dangerous substances and therefore deserve special attention. To that end, the Brazilian Policy on Solid Waste has provided guidelines on integrated and solid waste management, such as consumer electronics, aiming at their appropriate disposal and treatment through reverse logistics. In this context, the present work focuses on studying the recycling of some WEEE plastics.

Methods

This study was conducted using the methodological framework presented in the International Standard ISO 14040:2006 and aimed to determine the life cycle inventory (LCI) of a WEEE plastic recycling process in a company in Brazil. Having collected the data, it was possible to identify and quantify the environmental aspects caused by the recycling process of major plastics (acrylonitrile-butadiene-styrene (ABS) and high impact polystyrene (HIPS). The study was conducted in the only company in Brazil that operates WEEE plastic recycling in large scale.

Results and discussion

Some of the environmental aspects caused during the recycling process of the plastics under study were identified and quantified. As a result, besides presenting the inventory, it was also possible to determine a reduction in the consumption of energy and in CO₂ emissions. When compared to the production of virgin ABS and HIPS, the recycling processes for such plastics showed a reduction in energy consumption by approximately 90% for both plastics and a reduction in CO₂ emissions by approximately 84% for HIPS and 87% for ABS. The plastics recycled by the company retain over 90% of their virgin mechanical properties.

Conclusions

The study shows that recycling is highly relevant and that components present in WEEE received appropriate destination and treatment. Recycling avoids environmental impacts as it prevents WEEE from being disposed of in landfills and as the pellets of recycled plastics can re-enter the supply chain as raw materials. Considering the legislation in Brazil, the stage of collection/transport/treatment of WEEE conducted by the company under study presents strong indications of contributions to the environment, society, and economy of the country.