An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

ASSOCIATION OF A MYOSIN MOTOR WITH MEMBRANE-BOUNDED PIGMENT GRANULES IN FRESHWATER SHRIMP CHROMATOPHORES: EVIDENCE FROM THE NITELLA ACTIN-CABLE ASSAY

We have adapted an actin-mosin motility assay to examine the interactions in vitro between actin cables isolated from the giant internodal cells of the freshwater alga, Nitella, and pigment granules extracted from red ovarian chromatophores of the freshwater palaemonid shrimp, Macrobrachium olfersi. The chromatophore pigment mass consists of large (0.5-1.0-mu m diameter) membrane-bounded granules, and small (140-nm diameter), a membranous granules, both structurally continuous with the abundant smooth endoplasmic reticulum. Our previous immunocytochemical studies show a myosin motor to be stably associated with the pigment mass; however, to which granule type or membrane the myosin motor is attached is unclear. Here, we show that sodium vanadate, a myosin ATPase inhibitor, markedly increases the affinity of isolated, large, membrane-bounded granules for Nitella actin cables to which they become permanently attached. This interaction does not occur in granule preparations containing ATP with uninhibited, active myosin without vanadate. We propose that a stable state of elevated affinity is established between the granule-located myosin motor and the Nitella actin cables, resulting from a vanadate-inhibited acto-myosin-ADP complex. This finding provides further evidence for a myosin motor positioned on the surface of the membrane-bounded pigment granules in shrimp ovarian chromatophores.

FBXO25-associated nuclear domains: A novel subnuclear structure

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Electronic properties of liquid ammonia: A sequential molecular dynamics/quantum mechanics approach

The electronic properties of liquid ammonia are investigated by a sequential molecular dynamics/quantum mechanics approach. Quantum mechanics calculations for the liquid phase are based on a reparametrized hybrid exchange-correlation functional that reproduces the electronic properties of ammonia clusters [(NH(3))(n); n=1-5]. For these small clusters, electron binding energies based on Green's function or electron propagator theory, coupled cluster with single, double, and perturbative triple excitations, and density functional theory (DFT) are compared. Reparametrized DFT results for the dipole moment, electron binding energies, and electronic density of states of liquid ammonia are reported. The calculated average dipole moment of liquid ammonia (2.05 +/- 0.09 D) corresponds to an increase of 27% compared to the gas phase value and it is 0.23 D above a prediction based on a polarizable model of liquid ammonia [Deng , J. Chem. Phys. 100, 7590 (1994)]. Our estimate for the ionization potential of liquid ammonia is 9.74 +/- 0.73 eV, which is approximately 1.0 eV below the gas phase value for the isolated molecule. The theoretical vertical electron affinity of liquid ammonia is predicted as 0.16 +/- 0.22 eV, in good agreement with the experimental result for the location of the bottom of the conduction band (-V(0)=0.2 eV). Vertical ionization potentials and electron affinities correlate with the total dipole moment of ammonia aggregates. (c) 2008 American Institute of Physics.

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 angstrom, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 angstrom resolution on a rotating-anode X-ray source.

Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive ""Lid""

Background: The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Methodology: Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 angstrom resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. Conclusion: The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six alpha-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this ""lid"" may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Nickel sorption capacity of ground xylem of Quercus ilex trees and effects of selected ligands present in the xylem sap

In this work the influence of four different ligands present in the xylem sap of Quercus ilex (histidine, citric, oxalic and aspartic acids) on Ni(II) adsorption by xylem was investigated. Grinded xylem was trapped in acrylic columns and solutions of Ni(II), in the absence and presence of the four ligands prepared in KNO(3) 0-1 mol L(-1) at pH 5.5, were percolated through the column. Aliquots of solutions were recovered in the column end for Ni determination by graphite furnace atomic absorption spectrometry (GFAAS). The experimental. data to describe Ni sorption by xylem in both the presence and absence of ligands was better explained by the Freundlich isotherm model. The decreasing affinity order of ligands for Ni was: oxalic acid > citric acid > histidine > aspartic acid. On the other hand, the Ni(II) adsorption by xylem increased following the inverse sequence of ligands. Potentiometric titrations of acidic groups were carried out to elucidate the sorption site groups available in Q. ilex xylem. The potentiometric titration has shown three sorption sites: pK(a) 2.6 (57.7% of the sorption sites), related to monobasic aliphatic carboxylic acids or nitrogen aromatic bases, pK(a) 8.1 (9.6%) and pK(a) 9.9 (32.7%), related to phenolic groups. (C) 2008 Elsevier GmbH. All rights reserved.

Cardiac anti-remodelling effect of aerobic training is associated with a reduction in the calcineurin/NFAT signalling pathway in heart failure mice

Cardiomyocyte hypertrophy occurs in response to a variety of physiological and pathological stimuli. While pathological hypertrophy in heart failure is usually coupled with depressed contractile function, physiological hypertrophy associates with increased contractility. In the present study, we explored whether 8 weeks of moderate intensity exercise training would lead to a cardiac anti-remodelling effect in an experimental model of heart failure associated with a deactivation of a pathological (calcineurin/NFAT, CaMKII/HDAC) or activation of a physiological (Akt-mTOR) hypertrophy signalling pathway. The cardiac dysfunction, exercise intolerance, left ventricle dilatation, increased heart weight and cardiomyocyte hypertrophy from mice lacking alpha(2A) and alpha(2C) adrenoceptors (alpha(2A)/alpha(2C)ARKO mice) were associated with sympathetic hyperactivity induced heart failure. The relative contribution of Ca(2+)-calmodulin high-affinity (calcineurin/NFAT) and low-affinity (CaMKII/HDAC) targets to pathological hypertrophy of alpha(2A)/alpha(2C)ARKO mice was verified. While nuclear calcineurin B, NFATc3 and GATA-4 translocation were significantly increased in alpha(2A)/alpha(2C)ARKO mice, no changes were observed in CaMKII/HDAC activation. As expected, cyclosporine treatment decreased nuclear translocation of calcineurin/NFAT in alpha(2A)/alpha(2C)ARKO mice, which was associated with improved ventricular function and a pronounced anti-remodelling effect. The Akt/mTOR signalling pathway was not activated in alpha(2A)/alpha(2C)ARKO mice. Exercise training improved cardiac function and exercise capacity in alpha(2A)/alpha(2C)ARKO mice and decreased heart weight and cardiomyocyte width paralleled by diminished nuclear NFATc3 and GATA-4 translocation as well as GATA-4 expression levels. When combined, these findings support the notion that deactivation of calcineurin/NFAT pathway-induced pathological hypertrophy is a preferential mechanism by which exercise training leads to the cardiac anti-remodelling effect in heart failure.

Pseudomonas fluorescens lipase immobilization on polysiloxane-polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol-gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane-polyvinyl alcohol (POS-PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS-PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS-PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS-PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis-Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS-PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis-Menten constant value (K-m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities. (C) 2007 Elsevier B.V. All rights reserved.

Effect of pH and oxalic acid on the reduction of Fe(3+) by a biomimetic chelator and on Fe(3+) desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe mono-oxalate (Fe(C(2)O(4))(+)). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe(3+) molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5. it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe-oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2-4.5, iron from Fe-oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe(3+) molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6-5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes. (C) 2009 Elsevier Ltd. All rights reserved.

Thermoplastic starch modified during melt processing with organic acids: The effect of molar mass on thermal and mechanical properties

Thermoplastic starch (TPS) was modified with ascorbic acid and citric acid by melt processing of native starch with glycerol as plasticizer in an intensive batch mixer at 160 degrees C. It was found that the molar mass decreases with acid content and processing time causing the reduction in melting temperature (T(m)). As observed by the results of X-ray diffraction and DSC measurements, crystallinity was not changed by the reaction with organic acids. T(m) depression with falling molar mass was interpreted on the basis of the effect of concentration of end-chain units, which act as diluents. FTIR did not show any appreciable change in starch chemical compositions, leading to the conclusion that the main changes observed were produced by the variation in molar mass of the material. We demonstrated that it is possible to decrease melt viscosity without the need for more plasticizer thus avoiding side-effects such as an increase in water affinity or relevant changes in the dynamic mechanical properties. (C) 2010 Elsevier B.V. All rights reserved.

Computer-Assisted Numerical Analysis for Oxygen and Carbon Dioxide Mass Transfer in Blood Oxygenators

A two-dimensional numeric simulator is developed to predict the nonlinear, convective-reactive, oxygen mass exchange in a cross-flow hollow fiber blood oxygenator. The numeric simulator also calculates the carbon dioxide mass exchange, as hemoglobin affinity to oxygen is affected by the local pH value, which depends mostly on the local carbon dioxide content in blood. Blood pH calculation inside the oxygenator is made by the simultaneous solution of an equation that takes into account the blood buffering capacity and the classical Henderson-Hasselbach equation. The modeling of the mass transfer conductance in the blood comprises a global factor, which is a function of the Reynolds number, and a local factor, which takes into account the amount of oxygen reacted to hemoglobin. The simulator is calibrated against experimental data for an in-line fiber bundle. The results are: (i) the calibration process allows the precise determination of the mass transfer conductance for both oxygen and carbon dioxide; (ii) very alkaline pH values occur in the blood path at the gas inlet side of the fiber bundle; (iii) the parametric analysis of the effect of the blood base excess (BE) shows that V(CO2) is similar in the case of blood metabolic alkalosis, metabolic acidosis, or normal BE, for a similar blood inlet P(CO2), although the condition of metabolic alkalosis is the worst case, as the pH in the vicinity of the gas inlet is the most alkaline; (iv) the parametric analysis of the effect of the gas flow to blood flow ratio (Q(G)/Q(B)) shows that V(CO2) variation with the gas flow is almost linear up to Q(G)/Q(B) = 2.0. V(O2) is not affected by the gas flow as it was observed that by increasing the gas flow up to eight times, the V(O2) grows only 1%. The mass exchange of carbon dioxide uses the full length of the hollow-fiber only if Q(G)/Q(B) > 2.0, as it was observed that only in this condition does the local variation of pH and blood P(CO2) comprise the whole fiber bundle.

Influence of the Type of Quaternary Ammonium Salt Used in the Organic Treatment of Montmorillonite on the Properties of Poly(Styrene-co-Butyl Acrylate)/Layered Silicate Nanocomposites Prepared by In Situ Miniemulsion Polymerization

Hybrid latices of poly(styrene-co-butyl acrylate) were synthesized via in situ miniemulsion polymerization in the presence of 3 and 6 wt % organically modified montmorillonite (OMMT). Three different ammonium salts: cetyl trimethyl ammonium chloride (CTAC), alkyl dimethyl benzyl ammonium chloride (Dodigen), and distearyl dimethyl ammonium chloride (Praepagen), were investigated as organic modifiers. Increased affinity for organic liquids was observed after organic modification of the MMT. Stable hybrid latices were obtained even though miniemulsion stability was disturbed to some extent by the presence of the OMMTs during the synthesis. Highly intercalated and exfoliated polymer-MMT nanocomposites films were produced with good MMT dispersion throughout the polymeric matrix. Materials containing MMT modified with the 16 carbons alkyl chain salt (CTAC) resulted in the largest increments of storage modulus, indicating that single chain quaternary salts provide higher increments on mechanical properties. Films presenting exfoliated structure resulted in the largest increments in the onset temperature of decomposition. For the range of OMMT loading studied, the nanocomposite structure influenced more significantly the thermal stability properties of the hybrid material than did the OMMT loading. The film containing 3 wt % MMT modified with the two 18 carbons alkyl chains salt (Praepagen) provided the highest increment of onset temperature of decomposition. (C) 2010 Wiley Periodicals, Inc. J Appl Polym Sci 119: 3658-3669, 2011