The Intriguing Phospholipases A(2) Homologues: Relevant Structural Features on Myotoxicity and Catalytic Inactivity

Phospholipases A(2) homologues are found in the venom of Crotalinae snakes, being their main action related to myonecrosis induction. Although many studies on these toxins had already been performed, their mechanism of action remains unclear. Here, important aspects about these toxins are reviewed, including their correct biological assembly and how essential is the natural substitution D49K for their catalytic inactivity.

Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom

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Bacterial biofilms with emphasis on coagulase-negative staphylococci

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of pyraclostrobin and acibenzolar-S-methyl on common bacterial blight of snap bean

Assays were done under greenhouse conditions in order to evaluate the effect of pyraclostrobin (0.0375, 0.0750 and 0.150 mL.L-1) and acibenzolar-S-methyl (ASM) (0.025 g.L-1) in common bacterial blight on leaves of snap beans cultivar Braganca. These chemicals were sprayed at three different times: five days before; five days before + five days after; and five days after leaf inoculation with an isolate of Xanthomonas axonopodis pv. phaseoli. They were determinate the levels of polyphenoloxidase, peroxidase and total soluble proteins on inoculated and non-inoculated leaves of snap beans sprayed with pyraclostrobin (0.075 g.L-1) and ASM (0.025 g.L-1). All concentration of pyraclostrobin and ASM reduced the area under the disease progress curve (AUDPC) on leaves of snap beans, and the least AUDPC value was observed when this products were sprayed five days before + five days after inoculation. Higher levels of polyphenoloxidase, peroxidase and the total soluble proteins were observed on leaves sprayed with pyraclostrobin or ASM.

Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Screening of bacterial strains for pectinolytic activity: characterization of the polygalacturonase produced by Bacillus sp

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Insights into metal ion binding in phospholipases A(2): ultra high-resolution crystal structures of an acidic phospholipase A(2) in the Ca2+ free and bound states

The electrophile Ca2+ is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca2+ free and bound states at 0.97 and 1.60 angstrom resolutions, respectively. In the Ca2+ bound state, the Ca2+ ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca2+, a water molecule occupies the position of the Ca2+ ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation. (c) 2005 Elsevier SAS. All rights reserved.

At the interface: Crystal structures of phospholipases A(2)

The protein content of many snake venoms often includes one or more phospholipases A(2) (PLA(2)). In recent years a growing number of venoms from snakes of Agkistrodon, Bothrops and Trimeresurus species have been shown to contain a catalytically inactive PLA(2)-homologue in which the highly conserved aspartic acid at position 49 (Asp49) is substituted by lysine (Lys49). Although demonstrating little or no catalytic activity, these Lys49-PLA(2)s disrupt membranes by a Ca2+-independent mechanism of action. In addition, this family of PLA(2)s demonstrates myotoxic and cytolytic pharmacological activities, however the structural bases underlying these functional properties are poorly understood. Through the application of X-ray crystallography in combination with biophysical and bioinformatics techniques, we are studying structure/function relationships of Lys49-PLA(2)s. We here present results of a systematic X-ray crystallographic and amino acid sequence analysis study of Lys49-PLA(2)s and propose a model to explain the Ca2+ independent membrane damaging activity. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

A micelle nucleation model for the interaction of dodecyl sulphate with Lys49-phospholipases A(2)

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of Bothrops jararacussu that lacks detectable catalytic activity, yet causes rapid Ca2+-independent membrane damage. With the aim of understanding the interaction between BthTx-I and amphiphilic molecules, we have studied the interaction of sodium dodecyl sulphate (SDS) with the protein. Circular dichroism and attenuated total reflection Fourier-transform infrared spectra of BthTx-I reveal changes in the alpha-helical organization of the protein at an SDS/BthTx-I molar ratio of 20-25. At SDS/BthTx-I ratios of 40-45 the alpha-helices return to a native-like conformation, although fluorescence emission anisotropy measurements of 2-amino-N-hexadecyl-benzamide (AHBA) demonstrate that the total SDS is below the critical micelle concentration when this transition occurs. These results may be interpreted as the result of SDS accumulation by the BthTx-I homodimer and the formation of a pre-micelle SDS/BthTx-I complex, which may subsequently be released from the protein surface as a free micelle. Similar changes in the alpha-helical organization of BthTx-I were observed in the presence of dipalmitoylphosphatidylcholine liposomes, suggesting that protein structure transitions coupled to organization changes of bound amphiphiles may play a role in the Ca2+-independent membrane damage by Lys49-PLA(2)s. (c) 2006 Elsevier B.V. All rights reserved.

Surface Roughness and Bacterial Adherence to Resin Composites and Ceramics

Purpose: The aim of this study was to evaluate the surface roughness and the in vitro adherence of Streptococcus mutans to indirect aesthetic restorative materials that are uncoated with saliva.Materials and Methods: Four groups of restorative materials were evaluated according to material type: (1) microparticulate feldspathic ceramic; (2) leucite-reinforced feldspathic ceramic; (3) microhybrid resin composite and (4) microfilled resin composite. Twenty standardised samples of each material were produced. Roughness analysis (Ra, n = 10) was performed using a roughness analyser. Adhesion tests (n = 10) were carried out in 24-well plates; colony-forming units (CFU/mL) were evaluated. The mean values of roughness (mu m) and adherence (CFU/mL) for each group were subjected to an analysis of variance and a Tukey test.Results: The leucite-reinforced feldspathic ceramic was rougher and presented higher bacterial adherence than the microparticulate feldspathic ceramic. The resin composites were similar with regard to surface roughness and bacterial adherence.Conclusions: The microhybrid and microfilled resin composites were similar and the leucite-reinforced feldspathic ceramic was rougher and presented higher bacterial adherence than the microparticulate feldspathic ceramic.

Morphology and bacterial colonisation of tooth/ceramic restoration interface after different cement excess removal techniques

Objectives: To evaluate the influence of different protocols for resin cement removal during cementation on biofilm formation.Methods: Twenty-eight ceramic blocks, which were injected under pressure, were placed over enamel blocks obtained from freshly extracted bovine incisors. The ceramic blocks were cemented to the enamel blocks using a dual-cured resin cement and the excess resin was removed according to the experimental group: TS: Teflon spatula; BR: brush; BR+: brush and polishing; SB+: scalpel blade and polishing. After autoclaving, the samples were colonised by incubation in a sucrose broth suspension standardised with Streptococcus mutans in microaerophilic stove. Specimens were quantitatively analysed for bacterial adherence at the adhesive interface using confocal laser scanning microscopy and counting the colony forming units, and qualitatively analysed using SEM. The roughness (Ra/Rz/RSm) was also analysed. Data were analysed by 1-way ANOVA and Tukey's test (5%).Results: The roughness values ranged from 0.96 to 1.69 mu m for Ra (p > 0.05), from 11.59 to 22.80 mu m for Rz (p = 0.02 < 0.05) and from 293.2 to 534.3 mu m for RSm (p = 0.00). Bacterial adhesion varied between 1,974,000 and 2,814,000 CFU/ml (p = 0.00). Biofilm mean thickness ranged from 0.477 and 0.556 mu m (p > 0.05), whilst the biovolume values were between 0.388 and 0.547 mu m(3)/mu m(2) (p = 0.04). Lower values for roughness, bacterial adhesion, biofilm thickness and biovolume were found with BR, whilst TS presented the highest values for most of the parameters. SEM images confirmed the quantitative values.Conclusions: The restoration margin morphology and interface roughness affects bacterial accumulation. The brush technique promoted less bacterial colonisation at the adhesive interface than did the other removal methods.Clinical significance: The brush technique seems to be a good option for removing the excess resin cement after adhesive cementation in clinical practice, as indicated by its better results with lower bacterial colonisation. (C) 2012 Elsevier Ltd. All rights reserved.

Peptides based on CcdB protein as novel inhibitors of bacterial topoisomerases

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