Flexibility and inhibitor binding in Cdc25 phosphatases

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

Pseudomonas aeruginosa PA14 cupD transcription is activated by the RcsB response regulator, but repressed by its putative cognate sensor RcsC

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Electrochemical studies of the interface Fe/0.5 mol L(-1) H(2)SO(4) in the presence of benzotriazole and tolytriazole

The effect of benzotriazole (BTAH) and tolytriazole (TTAH) on the electrochemical behaviour of the Fe/0.5 mol L(-1) H(2)SO(4) interface at 25 degrees C was studied using cronopotentiometry, anodic and cathodic polarization curves and electrochemical impedance spectroscopy. BTAH and TTAH are inhibitors of anodic iron dissolution and the subsequent hydrogen evolution in 0.5 mol L(-1) H(2)SO(4) medium. Mass transport is an important step in the anodic process of inhibitive film formation. Electrochemical impedance spectroscopy was used to investigate the iron dissolution mechanism in the presence of the inhibitors and showed that BTAH and TTAH are adsorbed on the iron surface, thereby changing its dissolution mechanism in sulfate media. Starting from an iron dissolution model, it was possible to suggest two different mechanisms for iron dissolution in 0.5 mol L(-1) H(2)SO(4) containing BTAH or TTAH that involve a complex Fe(II)-inhibitor. (C) 2009 Elsevier B.V. All rights reserved

The corrosion of austenitic 304 stainless steel in biodiesel

Fuel distribution uses 304 stainless steel containers for the storage of biofuels, however there are few reports in the literature about the corrosive aspects this. steel in biodiesel. The objective of this research is to study the corrosive behavior of 304 austenitic stainless steel in the presence of biodiesel, unwashed and washed, with aqueous solutions of citric, oxalic, acetic and ascorbic acids 0,01 mol L(-1), and compare with results obtained for the copper (ASTM D130). The employedtechniques were: atomic absorption spectrometry (AAS) and optical microscopy (OM). The results of EA A showed a low rate of corrosion for the stainless steel, the alloys elements studied were Cr, Ni and Fe, the highest rate was observed for the chrome, 1.78 ppm / day in biodiesel with or without washing. The OM of the 304 steel, when compared with that of copper has a low corrosion rate in the 304 steel/biodiesel system. Not with standing, this demonstrates that not only the 304 steel, but also the copper corrodes in biodiesel

Wettability of Aqueous Rhamnolipids Solutions Produced by Pseudomonas aeruginosa LBI

The wetting behavior of rhamnolipids produced by Pseudomonas aeruginosa LBI strain grown on waste oil substrate and sodium dodecyl sulfate (SDS) on glass, polyethylene terephthalate (PET), poly(vinyl chloride) (PVC), poly(epsilon-caprolactone) (PCL) and polymer blend (PVC-PCL) was investigated by the measuring contact angle of sessile drops, to determine the wetting characteristics of rhamnolipids. The comparison of the wetting profiles showed that at low SDS and rhamnolipid concentrations, the contact angle increased and when the concentration of the surfactant increased further, the contact angle decreased. The blend surface (PVC-PCL) showed better wettability than the homopolymers themselves and the blend changed the surface hydrophobicity of the polymer, making it more hydrophilic. The rhamnolipids produced by the LBI strain exhibited superior wetting abilities than the chemical surfactant SDS one. This is the first work that evaluates the wetting properties of rhamnolipids on polymer blends.

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography-mass spectrophotometry (MS) and by high-performance liquid chromatography-MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l(-1). Under these conditions, the surface tension of the culture decreased to 30 mN m(-1), and the critical micelle concentration was 26.5 mg l(-1). It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.

Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases

Sunflower trypsin inhibitor-1 (SFI-1), a natural 14-residue cyclic peptide, and some of its synthetic acyclic variants are potent protease inhibitors displaying peculiar inhibitory profiles. Here we describe the synthesis and use of affinity sorbents prepared by coupling SFTI-1 analogues to agarose resin. Chymotrypsinand trypsin-like proteases could then be selectively isolated from pancreatin; similarly, other proteases were obtained from distinct biological sources. The binding capacity of [Lys5]-SFTI-1-agarose for trypsin was estimated at over 10 mg/mL of packed gel. SFTI-1-based resins could find application either to improve the performance of current purification protocols or as novel protease-discovery tools in different areas of biological investigation. (C) 2009 Elsevier B.V. All rights reserved.

A study of slag corrosion of oxides and oxide-carbon refractories during steel refining

The use of ceramic material as refractories in the manufacturing industry is a common practice worldwide. During usage, for example in the production of steel, these materials do experience severe working conditions including high temperatures, low pressures and corrosive environments. This results in lowered service lives and high consumptions of these materials. This, in turn, affects the productivity of the whole steel plant and thereby the cost. In order to investigate how the service life can be improved, studies have been carried out for refractories used in the inner lining of the steel ladles. More specifically, from the slag zone, where the corrosion is most severe. By combining thermodynamic simulations, plant trails and post-mortem studies of the refractories after service, vital information about the behaviour of the slagline refractories during steel refining and the causes of the accelerated wear in this ladle area has been achieved. The results from these studies show that the wear of the slagline refractories of the ladle is initiated at the preheating station, through reduction-oxidation reactions. The degree of the decarburization process is mostly dependent on the preheating fuel or the environment. For refractories without antioxidants, refractory decarburization is slower when coal gas is used in ladle preheating than when a mixture of oil and air is used. In addition, ladle preheating of the refractories without antioxidants leads to direct wear of the slagline refractories. This is due to the total loss of the matrix strength, which results in a sand-like product. Thermal chemical changes that take place in the slagline refractories are due to the MgO-C reaction as well as the formation of liquid phases from impurity oxides. In addition, the decrease in the system pressure during steel refining makes the MgO-C reaction take place at the steel refining temperatures. This reduces the refractory’s resistance to corrosion. This is a serious problem for both the magnesia-carbon and dolomite-carbon refractories. The studies of the reactions between the slagline refractories and the different slag compositions showed that slags rich in iron oxide lead mostly to the oxidation of carbon/graphite in the carbon-containing refractories. This leads to an increased porosity and wettability and therefore an enhanced penetration of slag into the refractory structure. If the slag contains high contents of alumina and or silica (such as the steel refining slag), reactions between the slag components and the dolomite-carbon refractory are promoted. This leads to the formation of low-temperature melting phases such as calcium-aluminates and silicates. The state of these reaction products during steel refining leads to an accelerated wear of the dolomite-carbon refractory. The main products of the reactions between the magnesia-carbon refractory and the steel refining slag are MgAl2O4 spinels, and calcium-aluminates, and silicates. Due to the good refractory properties of MgAl2O4 spinels, the slag corrosion resistance of the magnesiacarbon refractory is promoted.