Expression, crystallization and preliminary crystallographic analysis of PilZ(XAC1133) from Xanthomonas axonopodis pv. citri

Proteins containing PilZ domains are widespread in Gram-negative bacteria and have recently been shown to be involved in the control of biofilm formation, adherence, aggregation, virulence-factor production and motility. Furthermore, some PilZ domains have recently been shown to bind the second messenger bis(3'-> 5') cyclic diGMP. Here, the cloning, expression, purification and crystallization of PilZ(XAC1133), a protein consisting of a single PilZ domain from Xanthomonas axonopodis pv. citri, is reported. The closest PilZ(XAC1133) homologues in Pseudomonas aeruginosa and Neisseria meningitidis control type IV pilus function. Recombinant PilZ(XAC1133) containing selenomethionine was crystallized in space group P6(1). The unit-cell parameters were a = 62.125, b = 62.125, c = 83.543 angstrom. These crystals diffracted to 1.85 angstrom resolution and a MAD data set was collected at a synchrotron source. The calculated Matthews coefficient suggested the presence of two PilZ(XAC1133) molecules in the asymmetric unit.

Dynamic transitions in a three dimensional associating lattice gas model

The aggregation of interacting Brownian particles in sheared concentrated suspensions is an important issue in colloid and soft matter science per se. Also, it serves as a model to understand biochemical reactions occurring in vivo where both crowding and shear play an important role. We present an effective medium approach within the Smoluchowski equation with shear which allows one to calculate the encounter kinetics through a potential barrier under shear at arbitrary colloid concentrations. Experiments on a model colloidal system in simple shear flow support the validity of the model in the concentration range considered. By generalizing Kramers' rate theory to the presence of shear and collective hydrodynamics, our model explains the significant increase in the shear-induced reaction-limited aggregation kinetics upon increasing the colloid concentration.

KeaA, a Dictyostelium kelch-domain protein that regulates the response to stress and development

Background: The protein kinase YakA is responsible for the growth arrest and induction of developmental processes that occur upon starvation of Dictyostelium cells. yakA-cells are aggregation deficient, have a faster cell cycle and are hypersensitive to oxidative and nitrosoative stress. With the aim of isolating members of the YakA pathway, suppressors of the death induced by nitrosoative stress in the yakA-cells were identified. One of the suppressor mutations occurred in keaA, a gene identical to DG1106 and similar to Keap1 from mice and the Kelch protein from Drosophila, among others that contain Kelch domains. Results: A mutation in keaA suppresses the hypersensitivity to oxidative and nitrosoative stresses but not the faster growth phenotype of yakA-cells. The growth profile of keaA deficient cells indicates that this gene is necessary for growth. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. A morphological analysis of keaA deficient cells during multicellular development indicated that, although the mutant is not absolutely deficient in aggregation, cells do not efficiently participate in the process. Gene expression analysis using cDNA microarrays of wild-type and keaA deficient cells indicated a role for KeaA in the regulation of the cell cycle and pre-starvation responses. Conclusions: KeaA is required for cAMP signaling following stress. Our studies indicate a role for kelch proteins in the signaling that regulates the cell cycle and development in response to changes in the environmental conditions.

Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

Enhancement of hematoporphyrin IX potential for photodynamic therapy by entrapment in silica nanospheres

The entrapment of hematoporphyrin IX (Hp IX) in silica by means of a microemulsion resulted in silica spheres of 33 +/- 6 nm. The small size, narrow size distribution and lack of aggregation maintain Hp IX silica nanospheres stable in aqueous solutions for long periods and permit a detailed study of the entrapped drug by different techniques. Hp IX entrapped in the silica matrix is accessed by oxygen and upon irradiation generates singlet oxygen which diffuses very efficiently to the outside solution. The Hp IX entrapped in the silica matrix is also reached by iron(II) ions, which causes quenching of the porphyrin fluorescence emission. The silica matrix also provides extra protection to the photosensitizer against interaction with BSA and ascorbic acid, which are known to cause suppression of singlet oxygen generation by the Hp IX free in solution. Therefore, the incorporation of Hp IX molecules into silica nanospheres increased the potential of the photosensitizer to perform photodynamic therapy.

Continuous monitoring of ascorbate transport through neuroblastoma cells with a ruthenium oxide hexacyanoferrate modified microelectrode

The uptake of ascorbate by neuroblastoma cells using a ruthenium oxide hexacyanoferrate (RuOHCF)-modified carbon fiber disc (CFD) microelectrode (r = 14.5 mu m) was investigated. By use of the proposed electrochemical sensor the amperometric determination of ascorbate was performed at 0.0 V in minimum essential medium (MEM, pH = 7.2) with a limit of detection of 25 mu mol L(-1). Under the optimum experimental conditions, no interference from MEM constituents and reduced glutathione (used to prevent the oxidation of ascorbate during the experiments) was noticed. The stability of the RuOHCF-modified electrode response was studied by measuring the sensitivity over an extended period of time (120 h), a decrease of around 10% being noticed at the end of the experiment. The rate of ascorbate uptake by control human neuroblastoma SH-SY5Y cells, and cells transfected with wild-type Cu,Zn-superoxide dismutase (SOD WT) or with a mutant typical of familial amyotrophic lateral sclerosis (SOD G93A), was in agreement with the level of oxidative stress in these cells. The usefulness of the RuOHCF-modified microelectrode for in vivo monitoring of ascorbate inside neuroblastoma cells was also demonstrated.

A flexible consensus scheme for multicriteria group decision making under linguistic assessments

The present paper proposes a flexible consensus scheme for group decision making, which allows one to obtain a consistent collective opinion, from information provided by each expert in terms of multigranular fuzzy estimates. It is based on a linguistic hierarchical model with multigranular sets of linguistic terms, and the choice of the most suitable set is a prerogative of each expert. From the human viewpoint, using such model is advantageous, since it permits each expert to utilize linguistic terms that reflect more adequately the level of uncertainty intrinsic to his evaluation. From the operational viewpoint, the advantage of using such model lies in the fact that it allows one to express the linguistic information in a unique domain, without losses of information, during the discussion process. The proposed consensus scheme supposes that the moderator can interfere in the discussion process in different ways. The intervention can be a request to any expert to update his opinion or can be the adjustment of the weight of each expert`s opinion. An optimal adjustment can be achieved through the execution of an optimization procedure that searches for the weights that maximize a corresponding soft consensus index. In order to demonstrate the usefulness of the presented consensus scheme, a technique for multicriteria analysis, based on fuzzy preference relation modeling, is utilized for solving a hypothetical enterprise strategy planning problem, generated with the use of the Balanced Scorecard methodology. (C) 2009 Elsevier Inc. All rights reserved.

HYBRID AND INCREMENTAL FUZZY LEARNING FOR HUMAN SKIN DETECTION

In this paper, a framework for detection of human skin in digital images is proposed. This framework is composed of a training phase and a detection phase. A skin class model is learned during the training phase by processing several training images in a hybrid and incremental fuzzy learning scheme. This scheme combines unsupervised-and supervised-learning: unsupervised, by fuzzy clustering, to obtain clusters of color groups from training images; and supervised to select groups that represent skin color. At the end of the training phase, aggregation operators are used to provide combinations of selected groups into a skin model. In the detection phase, the learned skin model is used to detect human skin in an efficient way. Experimental results show robust and accurate human skin detection performed by the proposed framework.

Spatial pattern of black spot incidence within citrus trees related to disease severity and pathogen dispersal

Guignardia citricarpa, the causal agent of citrus black spot, forms airborne ascospores on decomposing citrus leaves and water-spread conidia on fruits, leaves and twigs. The spatial pattern of diseased fruit in citrus tree canopies was used to assess the importance of ascospores and conidia in citrus black spot epidemics in Sao Paulo State, Brazil. The aggregation of diseased fruit in the citrus tree canopy was quantified by the binomial dispersion index (D) and the binary form of Taylor`s Power Law for 303 trees in six groves. D was significantly greater than 1 in 251 trees. The intercept of the regression line of Taylor`s Power Law was significantly greater than 0 and the slope was not different from 1, implying that diseased fruit was aggregated in the canopy independent of disease incidence. Disease incidence (p) and severity (S) were assessed in 2875 citrus trees. The incidence-severity relationship was described (R-2 = 88.7%) by the model ln(S) = ln(a) + bCLL(p) where CLL = complementary log-log transformation. The high severity at low incidence observed in many cases is also indicative of low distance spread of G. citricarpa spores. For the same level of disease incidence, some trees had most of the diseased fruit with many lesions and high disease severity, whereas other trees had most of the fruit with few lesions and low disease severity. Aggregation of diseased fruit in the trees suggests that splash-dispersed conidia have an important role in increasing the disease in citrus trees in Brazil.

Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell-free supernatants containing bacteriocins, added to 3-h-old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto-aggregation was strain-specific, and values ranged from 7 center dot 2% for ET35 to 12 center dot 1% for ET05. Various degrees of co-aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61 center dot 9-64 center dot 6%), Lact. fermentum (78 center dot 9%), Lact. delbrueckii (43 center dot 7%) and Ped. acidilactici (51 center dot 3%), which are higher than the one recorded for Lact. rhamnosus GG (53 center dot 3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain-dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco-2 cells was within the range reported for Lact. rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.

Cu and Fe metallic ions-mediated oxidation of low-density lipoproteins studied by NMR, TEM and Z-scan technique

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu(2+) and Fe(3+)). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Virulence features of atypical enteropathogenic Escherichia coli identified by the eae(+) EAF-negative stx(-) genetic profile

This study characterized 76 atypical enteropathogenic Escherichia coli (aEPEC) strains, previously classified by the eae(+) EAF-negative stx(-) genotype, isolated from children with diarrhea in Brazil. Presence of bfpA and bfpA/perA was detected in 2 and 6 strains, respectively. The expression of bundle-forming pilus (BFP), however, was observed by immunofluorescence in 1 bfpA and 3 bfpA/perA strains, classifying them as typical EPEC (tEPEC). The remaining 72 aEPEC strains were characterized by serotyping, intimin typing, adherence patterns to HEp-2 cells, capacity to induce actin aggregation (fluorescent actin staining test), and antimicrobial resistance. Our results show that aEPEC comprise a very heterogeneous group that does not present any prevalence or association regarding the studied characteristics. It also suggest that tEPEC and aEPEC must not be classified only by the reactivity with the EAF probe, and that the search of other markers present in pEAF, as well as the BFP expression, must be considered for this matter. (C) 2009 Elsevier Inc. All rights reserved.

Acute ingestion of yerba mate infusion (Ilex paraguariensis) inhibits plasma and lipoprotein oxidation

Yerba mate extract (Ilex paraguariensis) is a Source of phenolic compounds that possesses in vitro antioxidant activities and may contribute to a reduction in the risk of cardiovascular disease. In this Study we examined the acute effects of the consumption of mate infusion on ex vivo plasma and low-density lipoprotein (LDL) oxidation, plasma antioxidant capacity, and platelet aggregation. Twelve healthy fasted subjects ingested 500 mL. of mate infusion and blood samples were collected before and I h after mate intake. Lipid peroxidation of plasma and LDL was monitored by the measurement of cholesteryl-ester hydroperoxides (CE-OOH) and cholesterol oxides. The plasma antioxidant capacity was measured as ferric-reducing antioxidant potential (FRAP). Platelet aggregation was evaluated in platelet-rich plasma Stimulated with adenosine diphosphate and coagulation was tested in platelet-poor plasma. Ingestion of mate infusion diminished the ex vivo oxidizability of both plasma and LDL particles. After mate intake, the CE-OOH levels were around 50% lower in plasma oxidized with copper or 2,2`-azobis[-2-amidine-propane-hydrochloride] (AAPH) and the lag time to plasma oxidation increased 2-fold (P < 0.05). Copper- and AAPH-induced LDL peroxidation were also inhibited by around 50% and 20%, respectively, after mate Consumption (P < 0.05). The levels of various oxysterols were significantly reduced in oxidized-plasma and LDL (P < 0.05) and FRAP increased by 7.7% after mate intake (P < 0.01). However. mate consumption did not inhibit platelet aggregation or blood coagulation. In summary, intake of yerba mate infusion improved the antioxidant capacity and the resistance of plasma and LDL particles to ex vivo lipid peroxidation. (C) 2008 Elsevier Ltd. All rights reserved.

A new acidic myotoxic, anti-platelet and prostaglandin I(2) inductor phospholipase A(2) isolated from Bothrops moojeni snake venom

Phospholipase A(2) (PLA(2), EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A(2) from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA(2)S from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-1 with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA(2)S from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities. (C) 2008 Elsevier Ltd. All rights reserved.

Further sesquiterpene lactones from viguiera robusta and the potential anti-inflammatory activity of a heliangolide: Inhibition of human neutrophil elastase release

In addition to known heliangolides, a new eudesmanolide was isolated from the leaf rinse extract of Viguiera robusta (Asteraceae). Structural elucidation was based oil spectral analysis. It is the first report on eudesmanolides in members of the subgenus Calanticaria of Viguiera. In this work, the main isolated compound, the furanoheliangolide budlein A, besides its previously, reported in vitro and in vivo anti-inflammatory activities, inhibited human neutrophil elastase release. The inhibition was at the concentration of (16.83 +/- 1.96) mu M for formylated bacterial tripeptide (fMLP) stimulation and (11.84 +/- 1.62) mu M for platelet aggregation factor (PAF) stimulation, being slightly less active than the reference drug parthenolide. The results are important to demonstrate the potential anti-inflammatory activities of sesquiterpene lactones and corroborate the previous studies using other targets.