Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM AND HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Catalytic mechanism of inulinase from Arthrobacter sp S37

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K-m) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K-m, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6b(rd1)). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin 1, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Lack of effects of isoflavones on the lipid profile of Brazilian postmenopausal women

Objective: To evaluate the effects of soy isoflavone supplementation on profile lipid and endogenous hormone levels. Methods: In this double-blind, placebo-controlled Study, 47 post menopausal women 47-66 v of age received 40 mg of isoflavone (n = 25) or 40 mg of casein placebo (11 = 22). Cardiovascular risk factors were assessed by evaluating lipid profile at baseline and after 6 mo of treatment. To examine the effects of this regime on endogenous hormone levels, follicle-stimulating hormone and beta-estradiol were measured. Urinary isoflavone concentrations (genistein and daidzein) were measured as markers of both compliance and absorption using high performance liquid chromatography. Baseline characteristics were compared by the unpaired Student`s t-test. Within-group changes were determined by paired Student`s t-test and comparison between the isoflavone and casein placebo groups were determined by analysis of variance. Results: Lipid levels (low-density lipoprotein and total cholesterol) similarly decreased in both,groups. High-density lipoprotein increased significantly in both groups and cannot thus be attributable to treatment: the reason for Such variation is unknown and can be attributed to chance or to bias (even that of a real placebo effect in both groups or perhaps in spontaneous changes in exercise and dietary habits of patients after their inclusion). Furthermore, in both groups very low-density lipoprotein and triacylglycerol levels increased in a non-significant manner. Conclusion: The results of the present Study do not support any biologically significant estrogenic effects of isoflavone on the parameters assessed. Further research will he necessary to definitively assess the safety and efficacy of isoflavone. (C) 2008 Elsevier Inc. All rights reserved.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide`s primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Disruption of Saccharomyces cerevisiae by Plantaricin 149 and investigation of its mechanism of action with biomembrane model systems

The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity data, we determined that 2.5 mu M is the critical concentration at which Pln149a will disrupt a negative DPPG monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain in the membrane, promoting its disruption. (C) 2009 Elsevier B.V. All rights reserved.

In Situ Infrared (FTIR) Study of the Mechanism of the Borohydride Oxidation Reaction on Smooth Pt Electrode

Although Pt has been thoroughly studied regarding its activity for the borohydride oxidation reaction (BOR), the BOR mechanism at Pt remains unclear: Depending on the applied potential, spontaneous BH(4)(-) hydrolysis can compete with the direct BOR. The goal of the present work is to provide more insight into the behavior of smooth Pt electrodes toward the BOR, by coupling in situ infrared reflectance spectroscopy with electrochemistry. The measurements were performed on a Pt electrode in 1 M NaOH/1 M NaBH(4), so as to detect the reaction intermediate species generated as a function of the applied potential. Several bands were monitored in the B-H ((v) over bar approximate to 1180, 1080, and 972 cm(-1)) and B-O ((v) over bar = 1325 and similar to 1425 cm(-1)) bond regions upon increased electrode polarization. These absorption bands, which appear sequentially and were already detected for similar measurements on Au electrodes, are assigned to BH(3), BH(2), and BO(2)(-) species. In light of these experimental data and previous results obtained in our group for Pt- or Au-based electrodes, possible initial elementary steps of the BOR on platinum electrodes are proposed and discussed according to the relevant literature data.

On the apparent lack of preferential site occupancy and electrooxidation of CO adsorbed at low coverage onto stepped platinum surfaces

We report time evolution studies of low coverage CO adsorption (surface hydrogen site blocking < 40%) and oxidative stripping on stepped Pt(776) and Pt(554) surfaces. It was observed that there is no preferential site occupancy for CO adsorption on step or terrace. It is proposed that CO adsorption onto these surfaces is a random process, and after CO adsorption there is no appreciable shift from CO-(111) to CO-(110) sites. This implies that after adsorption, CO molecules either have a very long residence time, or that the diffusion coefficient is much lower than previously thought. After CO electrooxidation the sites released included both terrace (111) and step (110) orientations. For surface hydrogen site blocking > 40%, the lateral interactions might play a role in the preferential CO site occupancy. (C) 2011 Elsevier B.V. All rights reserved.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midguit cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3. (C) 2009 Elsevier Ltd. All rights reserved.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Emplacement mechanism of the main granite pluton of the Lavras do Sul intrusive complex, South Brazil, determined by magnetic anisotropies

Magnetic fabric and rock magnetism studies were performed on apparently isotropic granite facies from the main intrusion of the Lavras do Sul Intrusive Complex pluton (LSIC, Rio Grande do Sul, South Brazil). This intrusion is roughly circular (similar to 12 x 13.5 km), composed of alkali-calcic and alkaline granitoids, with the latter occupying the margin of the pluton. Magnetic fabrics were determined by applying both anisotropy of low-field magnetic susceptibility (AMS) and anisotropy of anhysteretic remanent magnetization (AARM). The two fabrics are coaxial. The parallelism between AMS and AARM tensors excludes the presence of a single domain (SD) effect on the AMS fabric of the granites. Several rock-magnetism experiments performed in one specimen from each sampled site show that for all sites the magnetic susceptibility is dominantly carried by ferromagnetic minerals, while mainly magnetite carries the magnetic fabrics. Lineations and foliations in the granite facies were successful determined by applying magnetic methods. Magnetic lineations are gently plunging and roughly parallel to the boundaries of the pluton facies, except at the few sites in the central facies which have a radial orientation pattern. In contrast, the magnetic foliations tend to follow the contacts between the different granite facies. They are gently outerward-dipping inside the pluton, and become either steeply southwesterly dipping or vertical towards its margin. The lack of solid-state and subsolidus deformations at outcrop scale and in thin sections precludes deformation after full crystallization of the pluton. This evidence allows us to interpret the observed magnetic fabrics as primary in origin (magmatic) acquired when the rocks were solidified as a result of processes reflecting magma flow. The foliation pattern displays a dome-shaped form for the main LSIC-pluton. However, the alkaline granites which outcrop in the southern part of the studied area have an inward-dipping foliation, and the steeply plunging magnetic lineation suggests that this area could be part of a feeder zone. The magma ascent probably occurred due to ring-diking. (C) 2008 Elsevier B.V. All rights reserved.

The Catalytic Acid in the Dephosphorylation of the Cdk2-pTpY/CycA Protein Complex by Cdc25B Phosphatase

The development of anticancer therapeutics that target Cdc25 phosphatases is now an active area of research. A complete understanding of the Cdc25 catalytic mechanism would certainly allow a more rational inhibitor design. However, the identity of the catalytic acid used by Cdc25 has been debated and not established unambiguously. Results of molecular dynamics simulations with a calibrated hybrid potential for the first reaction step catalyzed by Cdc25B in complex with its natural substrate, the Cdk2-pTpY/CycA protein complex, are presented here. The calculated reaction free-energy profiles are in very good agreement with experimental measurements and are used to discern between different proposals for the general acid. In addition, the simulations give useful insight on interactions that can be explored for the design of inhibitors specific to Cdc25.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W, Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525-532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, VI., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66,331-341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1` in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high beta sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. (c) 2007 Elsevier Inc. All rights reserved.

On the catalytic hydrogenation of polycyclic aromatic hydrocarbons into less toxic compounds by a facile recoverable catalyst

Here we present the catalytic hydrogenation of polycyclic aromatic hydrocarbons (PAHs) to less toxic mixtures of saturated and partial unsaturated polycyclic hydrocarbons under mild reaction conditions using a magnetically recoverable rhodium catalyst and molecular hydrogen as the exclusive H source. The catalyst is easily recovered after each reaction by placing a permanent magnet on the reactor wall and it can be reused in successive runs without any significant loss of catalytic activity. As an example, anthracene was totally converted into the saturated polycyclic hydrocarbon form (ca. 60%) and the partially hydrogenated form, 1,2,3,4,5,6,7,8-octahydroanthracene (ca. 40%). The catalyst operates in a broad range of temperature and H(2) pressure in both organic and aqueous/organic solutions of anthracene and it also exhibits significant activity at low substrate concentrations (20 ppm). This can be an efficient recycling process for hydrogenation of PAHs present in contaminated fluid waste streams. (C) 2009 Elsevier B.V. All rights reserved.