The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.

Tunicamycin inhibition of N-glycosylation of alpha-glucosidase from Aspergillus niveus: partial influence on biochemical properties

Treatment of Aspergillus niveus with 30 mu g tunicamycin/ml did not interfere with alpha-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, similar to 56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65A degrees C). The K(m) and V(max) values of under- and fully-glycosylated forms of alpha-glucosidase were similar when assessed for hydrolysis of starch (similar to 0.6 mg/ml, similar to 350 mu mol glucose per min per ml), maltose (similar to 0.54 mu mol, similar to 330 mu mol glucose per min per ml) and p-nitrophenyl-alpha-d-glucopyranoside (similar to 0.54 mu mol, similar to 8.28 mu mol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.

The relation between action, predictability and temporal contiguity in temporal binding

Previous studies have documented a subjective temporal attraction between actions and their effects. This finding, named intentional binding, is thought to be the result of a cognitive function that links actions to their consequences. Although several studies have tried to outline the necessary and sufficient conditions for intentional binding, a quantitative comparison between the roles of temporal contiguity, predictability and voluntary action and the evaluation of their interactions is difficult due to the high variability of the temporal binding measurements. In the present study, we used a novel methodology to investigate the properties of intentional binding. Subjects judged whether an auditory stimulus, which could either be triggered by a voluntary finger lift or be presented after a visual temporal marker unrelated to any action, was presented synchronously with a reference stimulus. In three experiments, the predictability, the interval between action and consequence and the presence of action itself were manipulated. The results indicate that (1) action is a necessary condition for temporal binding; (2) a fixed interval between the two events is not sufficient to cause the effect and (3) only in the presence of voluntary action do temporal predictability and contiguity play a significant role in modulating the effect.These findings are discussed in the context of the relationship between intentional binding and temporal expectation. (C) 2010 Elsevier B.V. All rights reserved.

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His(600). In the present work, the role of His(600) of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S(1) and S(1)`, specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His(600) residue makes important interactions with the substrate, supporting the prediction that His(600) moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K(m) and k(cat), showing the importance of His(600) for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His(600) in TOP catalysis, transferring a proton to the newly generated NH(2)-terminus or helping Tyr(605) and/or Tyr(612) in the intermediate oxyanion stabilization. (C) 2010 Elsevier Inc. All rights reserved.

Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) is a dimorphic fungal pathogen that causes paracoccidioidomycosis the most severe deep mycosis from South America Although cell mediated immunity is considered the most efficient protective mechanism against Pb infection mechanisms of innate immunity are poorly defined Herein we investigated the interaction of the complement system with high and low virulence isolates of Pb We demonstrated that Pb18 a high virulence Pb Isolate when incubated with normal human serum (NHS) induces consumption of hemolytic complement and when immobilized promotes binding of C4b C3b and C5b-C9 Both low virulence (Pb265) and high virulence (Pb18) isolates consumed C4 C3 and mannose-binding learn (MBL) of MBL-sufficient but not of MBL-deficient serum as revealed by deposition of residual C4 C3 and MBL on immune complexes and mannan However higher complement components consumption was observed with Pb265 as compared with Pb18 The suggested relationship between low virulence and significant complement activation properties of Pb isolates was confirmed by the demonstration that virulence attenuation of Pb 18 results in acquisition of the ability to activate complement Conversely reactivation of attenuated Pb18 results in loss of the ability to activate complement Our results demonstrate for the first time that Pb yeasts activate the complement system by the lectin pathway and there is an Inverse correlation between complement activating ability and Pb virulence These differences could exert an influence on Innate immunity and severity of the disease developed by infected hosts (C) 2010 Elsevier Ltd All rights reserved

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.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5), respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.

Termination of diamond surfaces with hydrogen, oxygen and fluorine using a small, simple plasma gun

We have formed and characterized polycrystalline diamond films with surfaces having hydrogen terminations, oxygen terminations, or fluorine terminations, using a small, simple and novel plasma gun to bombard the diamond surface, formed by plasma assisted CVD in a prior step, with ions of the wanted terminating species. The potential differences between surface regions with different terminations were measured by Kelvin Force Microscopy (KFM). The highest potential occurred for oxygen termination regions and the lowest for fluorine. The potential difference between regions with oxygen terminations and hydrogen terminations was about 80 mV, and between regions with hydrogen terminations and fluorine terminations about 150 mV. Regions with different terminations were identified and imaged using the secondary electron signal provided by scanning electron microscopy (SEM). since this signal presents contrast for surfaces with different electrical properties. The wettability of the surfaces with different terminations was evaluated, measuring contact angles. The sample with oxygen termination was the most hydrophilic, with a contact angle of 75 degrees. hydrogen-terminated regions with 83 degrees, and fluorine regions 93 degrees, the most hydrophobic sample. (C) 2010 Elsevier B.V. All rights reserved.

Electronic and magnetic properties of Ti and Fe on graphene

The structural, electronic and magnetic properties of Fe and Ti atomic wires and the complete covering when adsorbed on graphene are presented through ab initio calculations based on density functional theory. The most stable configurations are investigated for Fe and Ti in different concentrations adsorbed on the graphene surface, and the corresponding binding energies are calculated. The results show a tendency of the Ti atoms to cover uniformly the graphene surface, whereas the Fe atoms form clusters. The adsorption of the transition metal on the graphene surface changes significantly the electronic density of states near the graphene Fermi region. In all arrangements studied, a charge transfer is observed from the adsorbed species to the graphene surface due to the high hybridizations between the systems.

Pressure-induced electrical and structural anomalies in Pb(1-x)Ca(x)TiO(3) thin films grown at various oxygen pressures by chemical solution route

Lead calcium titanate (Pb(1-x)Ca(x)TiO(3) or PCT) thin films have been thermally treated under different oxygen pressures, 10, 40 and 80 bar, by using the so-called chemical solution deposition method. The structural, morphological, dielectric and ferroelectric properties were characterized by x-ray diffraction, FT-infrared and Raman spectroscopy, atomic force microscopy and polarization-electric-field hysteresis loop measurements. By annealing at a controlled pressure of around 10 and 40 bar, well-crystallized PCT thin films were successfully prepared. For the sample submitted to 80 bar, the x-ray diffraction, Fourier transformed-infrared and Raman data indicated deviation from the tetragonal symmetry. The most interesting feature in the Raman spectra is the occurrence of intense vibrational modes at frequencies of around 747 and 820 cm(-1), whose presence depends strongly on the amount of the pyrochlore phase. In addition, the Raman spectrum indicates the presence of symmetry-breaking disorder, which would be expected for an amorphous (disorder) and mixed pyrochlore-perovskite phase. During the high-pressure annealing process, the crystallinity and the grain size of the annealed film decreased. This process effectively suppressed both the dielectric and ferroelectric behaviour. Ferroelectric hysteresis loop measurements performed on these PCT films exhibited a clear decrease in the remanent polarization with increasing oxygen pressure.

Substitutional Impurities in PPV Crystals: An Intrinsic Donor-Acceptor System for High V(oc) Photovoltaic Devices

Defects are usually present in organic polymer films and are commonly invoked to explain the low efficiency obtained in organic-based optoelectronic devices. We propose that controlled insertion of substitutional impurities may, on the contrary, tune the optoelectronic properties of the underivatized organic material and, in the case studied here, maximize the efficiency of a solar cell. We investigate a specific oxygen-impurity substitution, the keto-defect -(CH(2)-C=O)- in underivatized crystalline poly(p-phenylenevinylene) (PPV), and its impact on the electronic structure of the bulk film, through a combined classical (force-field) and quantum mechanical (DFT) approach. We find defect states which suggest a spontaneous electron hole separation typical of a donor acceptor interface, optimal for photovoltaic devices. Furthermore, the inclusion of oxygen impurities does not introduce defect states in the gap and thus, contrary to standard donor-acceptor systems, should preserve the intrinsic high open circuit voltage (V(oc)) that may be extracted from PPV-based devices.

Tight-binding model for carbon nanotubes from ab initio calculations

Here we present a parametrized tight-binding (TB) model to calculate the band structure of single-wall carbon nanotubes (SWNTs). On the basis of ab initio calculations we fit the band structure of nanotubes of different radii with results obtained with an orthogonal TB model to third neighbors, which includes the effects of orbital hybridization by means of a reduced set of parameters. The functional form for the dependence of these parameters on the radius of the tubes can be used to interpolate appropriate TB parameters for different SWNTs and to study the effects of curvature on their electronic properties. Additionally, we have shown that the model gives an appropriate description of the optical spectra of SWNTs, which can be useful for a proper assignation of SWNTs` specific chirality from optical absorption experiments.

Oxygen-mediated electron transport through hybrid silicon-organic interfaces

We investigate from first principles the electronic and transport properties of hybrid organic/silicon interfaces of relevance to molecular electronics. We focus on conjugated molecules bonded to hydrogenated Si through hydroxyl or thiol groups. The electronic structure of the systems is addressed within density functional theory, and the electron transport across the interface is directly evaluated within the Landauer approach. The microscopic effects of molecule-substrate bonding on the transport efficiency are explicitly analyzed, and the oxygen-bonded interface is identified as a candidate system when preferential hole transfer is needed.

Thermoelectric transport properties through a T-shaped single quantum dot

We study the thermopower, thermal conductance, electric conductance and the thermoelectric figure of merit for a gate-defined T-shaped single quantum dot (QD). The QD is solved in the limit of strong Coulombian repulsion U -> infinity, inside the dot, and the quantum wire is modeled on a tight-binding linear chain. We employ the X-boson approach for the Anderson impurity model to describe the localized level within the quantum dot. Our results are in qualitative agreement with recent experimental reports and other theoretical researches for the case of a quantum dot embedded into a conduction channel, employing analogies between the two systems. The results for the thermopower sign as a function of the gate voltage (associated with the quantum dot energy) are in agreement with a recent experimental result obtained for a suspended quantum dot. The thermoelectric figure of merit times temperature results indicates that, at low temperatures and in the crossover between the intermediate valence and Kondo regimes, the system might have practical applicability in the development of thermoelectric devices. (c) 2010 Elsevier B.V. All rights reserved.