The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Colloidal gas aphrons based separation process for the purification and fractionation of natural phenolic extracts

Following previous studies, the aim of this work is to further investigate the application of colloidal gas aphrons (CGA) to the recovery of polyphenols from a grape marc ethanolic extract with particular focus on exploring the use of a non-ionic food grade surfactant (Tween 20) as an alternative to the more toxic cationic surfactant CTAB. Different batch separation trials in a flotation column were carried out to evaluate the influence of surfactant type and concentration and processing parameters (such as pH, drainage time, CGA/extract volumetric and molar ratio) on the recovery of total and specific phenolic compounds. The possibility of achieving selective separation and concentration of different classes of phenolic compounds and non-phenolic compounds was also assessed, together with the influence of the process on the antioxidant capacity of the recovered compounds. The process led to good recovery, limited loss of antioxidant capacity, but low selectivity under the tested conditions. Results showed the possibility of using Tween 20 with a separation mechanism mainly driven by hydrophobic interactions. Volumetric ratio rather than the molar ratio was the key operating parameter in the recovery of polyphenols by CGA.

Separation of natural colorants from the fermented broth of filamentous fungi using colloidal gas aphrons

There is a worldwide interest in the development of processes for producing colorants from natural sources. Microorganisms provide an alternative source of natural colorants produced by cultivation technology and extracted from the fermented broth. The aim of the present work was to study the recovery of red colorants from the fermented broth of Talaromyces amestolkiae using the technique of colloidal gas aphrons (CGA) comprising surfactant-stabilized microbubbles. Preliminary experiments were performed to evaluate the red colorants’ solubility in different organic solvents, octanol/water partitioning, and their stability in surfactant solutions, namely hexadecyl trimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and polyoxyethylenesorbitan monolaurate (Tween 20), which are cationic, anionic and nonionic surfactants, respectively. The first recovery experiments were carried out using CGA generated by these surfactants at different volumetric ratios (VR, 3–18). Subsequently, two different approaches to generate CGA were investigated at VR values of 6 and 12: the first involved the use of CTAB at pH 6.9–10.0, and the second involved the use of Tween 20 using red colorants partially dissolved in ethanol and Tween 20. The characterization results showed that red colorants have a hydrophilic nature. The highest recoveries were obtained with Tween 20 (78%) and CTAB (70%). These results demonstrated that the recovery of the colorants was driven by both electrostatic and hydrophobic interactions. The VR was found to be an important operating parameter and at VR 12 with CTAB (at pH 9) maximum recovery, partitioning coefficient (K = 5.39) and selectivity in relation to protein and sugar (SP = 3.75 and SS = 7.20 respectively) were achieved. Furthermore, with Tween 20, the separation was driven mainly by hydrophobic interactions. Overall CGA show promise for the recovery of red colorants from a fermented broth. Although better results were obtained with CTAB than with Tween 20 the latter may be more suitable for some application due to its lower toxicity.

Capillary assay device with internal hydrophilic coating

The present invention provides assay devices having a unitary body with an exterior surface, the unitary body being substantially transparent to visible light and formed from a material having a refractive index in the range 1.26 to 1.40, the refractive index being measured at 20 °C with light of wavelength 589 nm, and wherein the unitary body is formed from a hydrophobic material, and at least two capillary bores extending internally along the unitary body, wherein at least a portion of the surface of each capillary bore includes a hydrophilic layer for retaining an assay reagent, and wherein the hydrophilic layer is also substantially transparent to visible light to allow optical interrogation of the capillary bores through the capillary wall. The present invention also provides assay systems including such assay devices, methods of performing an assay using such assay devices and method of method for manufacturing such assay devices.

Effect of temperature and strain rate on the compressive behaviour of supramolecular polyurethane

Supramolecular polyurethanes (SPUs) possess thermoresponsive and thermoreversible properties, and those characteristics are highly desirable in both bulk commodity and value-added applications such as adhesives, shape-memory materials, healable coatings and lightweight, impact-resistant structures (e.g. protection for mobile electronics). A better understanding of the mechanical properties, especially the rate and temperature sensitivity, of these materials are required to assess their suitability for different applications. In this paper, a newly developed SPU with tuneable thermal properties was studied, and the response of this SPU to compressive loading over strain rates from 10−3 to 104 s−1 was presented. Furthermore, the effect of temperature on the mechanical response was also demonstrated. The sample was tested using an Instron mechanical testing machine for quasi-static loading, a home-made hydraulic system for moderate rates and a traditional split Hopkinson pressure bars (SHPBs) for high strain rates. Results showed that the compression stress-strain behaviour was affected significantly by the thermoresponsive nature of SPU, but that, as expected for polymeric materials, the general trends of the temperature and the rate dependence mirror each other. However, this behaviour is more complicated than observed for many other polymeric materials, as a result of the richer range of transitions that influence the behaviour over the range of temperatures and strain rates tested.

Self-assembly of telechelic tyrosine end-capped PEO and poly(alanine) polymers in aqueous solution

The self-assembly in aqueous solution of three novel telechelic conjugates comprising a central hydrophilic polymer and short (trimeric or pentameric) tyrosine end-caps has been investigated. Two of the conjugates have a central poly(oxyethylene) (polyethylene oxide, PEO) central block with different molar masses. The other conjugate has a central poly(l-alanine) (PAla) sequence in a purely amino-acid based conjugate. All three conjugates self-assemble into β-sheet based fibrillar structures, although the fibrillar morphology revealed by cryogenic-TEM is distinct for the three polymers—in particular the Tyr5-PEO6k-Tyr5 forms a population of short straight fibrils in contrast to the more diffuse fibril aggregates observed for Tyr5-PEO2k-Tyr5 and Tyr3-PAla-Tyr3. Hydrogel formation was not observed for these samples (in contrast to prior work on related systems) up to quite high concentrations, showing that it is possible to prepare solutions of peptide–polymer-peptide conjugates with hydrophobic end-caps without conformational constraints associated with hydrogelation. The Tyr5-PEO6k-Tyr5 shows significant PEO crystallization upon drying in contrast to the Tyr5-PEO2k-Tyr5 conjugate. Our findings point to the remarkable ability of short hydrophobic peptide end groups to modulate the self-assembly properties of polymers in solution in model peptide-capped “associative polymers”. Retention of fluidity at high conjugate concentration may be valuable in potential future applications of these conjugates as bioresponsive or biocompatible materials, for example exploiting the enzyme-responsiveness of the tyrosine end-groups

Effect of blood contamination on the shear bond strength at resin/dentin interface in primary teeth

Purpose: To assess in vitro the shear bond strength at the resin/dentin interface in primary teeth after contamination with fresh human blood. Methods: 75 crowns of primary molars were embedded in acrylic resin and mechanically ground to expose a flat dentin surface. The specimens were randomly assigned to five groups (n=15), according to the surface treatment. Group I (control) had no blood contamination. The other groups were blood-contaminated and subjected to different post-contamination protocols: in Group 2, the surfaces were rinsed with water; in Group 3, the surfaces were air-dried; in Group 4, the surfaces were rinsed and air-dried; and in Group 5, no post-contamination treatment was done. In all groups, a 3-mm dentin bonding site was demarcated, Single Bond adhesive system was applied and resin composite cylinders were bonded. After 24 hours in distilled water, shear bond strength was tested at a crosshead speed of 0.5 mm/minute. Results: Means (in MPa) were: Group 1: 7.1 (+/- 4.2); Group 2: 4.0 (+/- 1.8); Group 3: 0.9 (+/- 0.7); Group 4: 3.9 (+/- 2.2) and Group 5: 1.3 (+/- 1.5). Data were analyzed statistically by the Kruskal-Wallis test at 5% significance level. Groups 2 and 4 were similar to each other (P > 0.05) and both ware similar to Group 1 (P > 0.05). These groups (2, 3 and 4) had statistically significantly higher bond strengths than Groups 3 and 5 (P < 0.05). Blood contamination negatively affected the shear bond strength to primary tooth dentin. Among the blood-contaminated groups, water-rinsed specimens had higher bond strengths than those that were exclusively air-dried or not submitted to any post-contamination protocol before adhesive application.

Detection of a new mumps virus genotype during parotitis epidemic of 2006-2007 in the state of Sao Paulo, Brazil

Nucleoticle sequence analyses of the SH gene of 18 mumps virus isolates collected in the 2006-2007 parotitis epidemic in the state of Sao Paulo identified a new genotype, designated genotype M. This new designation fulfills all the parameters required to define a new mumps virus genotype. The parameters were established by an expert panel in collaboration with the World Health Organization (WHO) in 2005. This information will enhance the mumps virus surveillance program both at the national and global levels.

Recovery and upgrading bovine rumen protein by extrusion: Effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9% or 3.8%) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein Solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9% residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8% in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of surfactants on the functional properties of gelatin-polysaccharide-based films

The aim of this study was to evaluate the effects of the addition of surfactants sodium stearoyl lactate (SSL) and sucrose ester (SE) on the functional properties of films produced with polysaccharides mixtures (methylcellulose/glucomannan/pectin in 1/4/1 ratio, respectively) and gelatin. The films were produced by the casting method and characterized for their water vapor permeability (WVP), mechanical (tensile strength and elongation to break point), morphological and optical properties. Films with low WVP were obtained with surfactants. Addition of SE to the films with polysaccharide/gelatin ratio of 90/10 showed improved mechanical properties. Films presented smooth surfaces with micro voids and lumpiness, depending on the surfactant tested. Surfactants increased the opacity of the films by a factor of 1-3%. All film properties were dependent on the surfactant affinity for the biopolymer matrix. SE presented more affinity for biopolymer matrix containing high polysaccharide proportion, and SSL presented more affinity for polymer matrix containing high gelatin proportion. The addition of surfactants decreased the water vapor permeability of the films, increasing their hydrophobic character.

Peptidomic Analysis of Human Cell Lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEIC293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the PI position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

Site-directed mutagenesis and structural modeling of Coq10p indicate the presence of a tunnel for coenzyme Q6 binding

Coq10p is a protein required for coenzyme Q function, but its specific role is still unknown. It is a member of the START domain superfamily that contains a hydrophobic tunnel implicated in the binding of lipophilic molecules. We used site-directed mutagenesis, statistical coupling analysis and molecular modeling to probe structural determinants in the Coq10p putative tunnel. Four point mutations were generated (coq10-K50E, coq10-L96S, coq10-E105K and coq10-K162D) and their biochemical properties analysed, as well as structural consequences. Our results show that all mutations impaired Coq10p function and together with molecular modeling indicate an important role for the Coq10p putative tunnel. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Vitellin-binding proteins in the nematode Oscheius tipulae (Nematoda, Rhabditida)

We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm`s vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of alpha-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions. (C) 2008 Elsevier Inc. 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.

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.