Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: structural basis for the high specificity of exfoliative toxins

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

Dextran sulfate/Triton X two-phase micellar systems as an alternative first purification step for clavulanic acid

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Nanopartículas magnéticas como suporte para imobilização de lipases

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise estrutural de halogenases encontradas em clusters gênicos da biossíntese de antibióticos glicopeptídicos

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

High-Pressure Micellar Solutions of Polystyrene-block-polybutadiene and Polystyrene-block-polyisoprene in Propane Exhibit Cloud-Pressure Reduction and Distinct Micellization End Points

Micellar solutions of polystyrene-block-polybutadiene and polystyrene-block-polyisoprene in propane are found to exhibit significantly lower cloud pressures than the corresponding hypothetical nonmicellar solutions. Such a cloud-pressure reduction indicates the extent to which micelle formation enhances the apparent diblock solubility in near-critical and hence compressible propane. Concentration-dependent pressure-temperature points beyond which no micelles can be formed, referred to as the micellization end points, are found to depend on the block type, size, and ratio. The cloud-pressure reduction and the micellization end point measured for styrene-diene diblocks in propane should be characteristic of all amphiphilic diblock copolymer solutions that form micelles in compressible solvents.

What can light scattering spectroscopy do for membrane-active peptide studies

Highly charged peptides are important components of the immune system and belong to an important family of antibiotics. Although their therapeutic activity is known, most of the molecular level mechanisms are controversial. A wide variety of different approaches are usually applied to understand their mechanisms, but light scattering techniques are frequently overlooked. Yet, light scattering is a noninvasive technique that allows insights both on the peptide mechanism of action as well as on the development of new antibiotics. Dynamic light scattering (DLS) and static light scattering (SLS) are used to measure the aggregation process of lipid vesicles upon addition of peptides and molecular properties (shape, molecular weight). The high charge of these peptides allows electrostatic attraction toward charged lipid vesicles, which is studied by zeta potential (zeta-potential) measurements. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Interaction of the meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50 degrees C) and below (25 degrees C) the main phase transition temperature of the LUVs (similar to 41 degrees C). The binding constant values, K-b, estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the K-b value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies, where a partial neutralization of the LUVs surface charge, upon porphyrin titration, was observed. Dynamic light scattering (DLS) results showed that, for some phospholipid systems, this partial neutralization leads to the LUVs flocculation. (C) 2012 Elsevier Inc. All rights reserved.

Light scattering on the structural characterization of DMPG vesicles along the bilayer anomalous phase transition

Highly charged vesicles of the saturated anionic lipid dimyristoyl phosphatidylglycerol (DMPG) in low ionic strength medium exhibit a very peculiar thermo-structural behavior. Along a wide gel-fluid transition region, DMPG dispersions display several anomalous characteristics, like low turbidity, high electrical conductivity and viscosity. Here, static and dynamic light scattering (SLS and DLS) were used to characterize DMPG vesicles at different temperatures. Similar experiments were performed with the largely studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). SLS and DLS data yielded similar dimensions for DMPC vesicles at all studied temperatures. However, for DMPG, along the gel-fluid transition region, SLS indicated a threefold increase in the vesicle radius of gyration, whereas the hydrodynamic radius, as obtained from DLS, increased 30% only. Despite the anomalous increase in the radius of gyration, DMPG lipid vesicles maintain isotropy, since no light depolarization was detected. Hence, SLS data are interpreted regarding the presence of isotropic vesicles within the DMPG anomalous transition, but highly perforated vesicles, with large holes. DLS/SLS discrepancy along the DMPG transition region is discussed in terms of the interpretation of the Einstein-Stokes relation for porous vesicles. Therefore, SLS data are shown to be much more appropriate for measuring porous vesicle dimensions than the vesicle diffusion coefficient. The underlying nanoscopic process which leads to the opening of pores in charged DMPG bilayer is very intriguing and deserves further investigation. One could envisage biotechnological applications, with vesicles being produced to enlarge and perforate in a chosen temperature and/or pH value. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

The Chameleon-Like Nature of Zwitterionic Micelles: Effect of Cation Binding

Addition of salts, especially perchlorates, to zwitterionic micelles of SB3-14, C(14)H(29)NMe(2)(+)(CH(2))(3)SO(3)(-), induces anionic character and uptake of H(3)O(+) by SB3-14 micelles. Thus, the addition of alkali metal perchlorates accelerates the acid hydrolysis of 2-(p-heptoxypheny1)-1,3-dioxolane, HPD, in the presence of SB3-14 micelles, which depends on the local proton concentration at the micelle surface. The addition of metal chlorides to solutions of such perchlorate-modified SB3-14 micelles decreases both the negative zeta potential of the micelles and the observed rate constant for acid hydrolysis of HPD. The effect of the monovalent cations Li(+), Na(+), and K(+) is smaller than that of the divalent cations Be(2+), Mg(2+), and Ca(2+), and much smaller than that of the trivalent cations Al(3+), La(3+), and Er(3+). The major factor responsible for this cation valence dependence of these effects is shown to be electrostatic in nature, reflecting the strong dependence of the micellar surface potential on the cation valence. The fact that the salt effects are not identical after correction for the electrostatic effects indicates that additional secondary nonelectrostatic effects may contribute as well.

Effect of alkyl chain length of alcohols on nematic uniaxial-to-biaxial phase transitions in a potassium laurate/alcohol/K2SO4/water lyotropic mixture

Lyotropic liquid crystalline quaternary mixtures of potassium laurate (KL), potassium sulphate (K2SO4)/alcohol (n-OH)/water, with the alcohols having different numbers of carbon atoms in the alkyl chain (n), from 1-octanol to 1-hexadecanol, were investigated by optical techniques (optical microscopy and laser conoscopy). The biaxial nematic phase domain is present in a window of values of n = n(KL) +/- 2, where n(KL) = 11 is the number of carbon atoms in the alkyl chain of KL. The biaxial phase domain became smaller and the uniaxial-to-biaxial phase transition temperatures shifted to relatively higher temperatures upon going from 1-nonanol to 1-tridecanol. Moreover, compared with other lyotropic mixtures these new mixtures present high birefringence values, which we expect to be related to the micellar shape anisotropy. Our results are interpreted assuming that alcohol molecules tend to segregate in the micelles in a way that depends on the relative value of n with respect to nKL. The larger the value of n, the more alcohol molecules tend to be located in the curved parts of the micelle, favoring the uniaxial nematic calamitic phase with respect to the biaxial and uniaxial discotic nematic phases.

An environmentally friendly analytical procedure for nickel determination by atomic and molecular spectrometry after cloud point extraction in different samples

Cloud point extraction (CPE) was employed for separation and preconcentration prior to the determination of nickel by graphite furnace atomic absorption spectrometry (GFAAS), flame atomic absorption spectrometry (FAAS) or UV-Vis spectrophotometry. Di-2-pyridyl ketone salicyloylhydrazone (DPKSH) was used for the first time as a complexing agent in CPE. The nickel complex was extracted from the aqueous phase using the Triton X-114 surfactant. Under optimized conditions, limits of detection obtained with GFAAS, FAAS and UV-Vis spectrophotometry were 0.14, 0.76 and 1.5 mu g L-1, respectively. The extraction was quantitative and the enrichment factor was estimated to be 27. The method was applied to natural waters, hemodialysis concentrates, urine and honey samples. Accuracy was evaluated by analysis of the NIST 1643e Water standard reference material.

Photophysical properties and interactions of xanthene dyes in aqueous micelles

Photosensitizers (PS) photodynamic activities are regulated by their location in the biological target, which modulates their photophysical and photochemical features. In this work the PS partition for the Xanthene Dyes Fluorescein (FSC), Eosin Y(EOS), Erythrosin B (ERY) and Rose Bengal B (RBB) in biomimetic models (SDS, CTAB and Pluronic P-123 micelles) and the effects on their photophysical characteristics are evaluated. The hydrophobic and electrostatic forces that govern the PS-micelle interaction are analyzed. At physiological pH (7.25), the ability of the dianionic protolytic form of the dyes to be positioned into the micelle palisade and its micelle interaction depends not only on the hydrophobicity of the dye but also on the micellar surface charge. The Binding Constants obey exactly the same order of the Partition Coefficients for the dyes in P-123 and CTAB micelles. The Stern-Volmer treatment pointed out that dyes are located inside the micelle, especially ERY and RBB. The magnitude of the dye-micelle interaction increased from SDS, P-123 and finally CTAB micelles due to the charges between dye and micelle, and among the xanthenes, their hydrophobic characteristics. Within the micelle pseudo phase, ERY and RBB are still very efficient photosensitizers exhibiting high quantum yield of singlet oxygen, which turns them very attractive especially with P-123 polymeric system as drug delivery systems in photodynamic therapy. (C) 2012 Elsevier B.V. All rights reserved.

Photo-activated phase separation in giant vesicles made from different lipid mixtures

Using giant unilamellar vesicles (GUVs) made from POPC. DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo-Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo-Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance. (C) 2011 Elsevier B.V. All rights reserved.

Polymeric micelles and molecular modeling applied to the development of radiopharmaceuticals

Micelles composed of amphiphilic copolymers linked to a radioactive element are used in nuclear medicine predominantly as a diagnostic application. A relevant advantage of polymeric micelles in aqueous solution is their resulting particle size, which can vary from 10 to 100 nm in diameter. In this review, polymeric micelles labeled with radioisotopes including technetium (99mTc) and indium (111In), and their clinical applications for several diagnostic techniques, such as single photon emission computed tomography (SPECT), gamma-scintigraphy, and nuclear magnetic resonance (NMR), were discussed. Also, micelle use primarily for the diagnosis of lymphatic ducts and sentinel lymph nodes received special attention. Notably, the employment of these diagnostic techniques can be considered a significant tool for functionally exploring body systems as well as investigating molecular pathways involved in the disease process. The use of molecular modeling methodologies and computer-aided drug design strategies can also yield valuable information for the rational design and development of novel radiopharmaceuticals.