Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers

A series of photosensitizers (PS), which are meso-substituted tetra-cationic porphyrins, was synthesized in order to study the role of amphiphilicity and zinc insertion in photodynamic therapy (PDT) efficacy. Several properties of the PS were evaluated and compared within the series including photophysical properties (absorption spectra, fluorescence quantum yield Phi(f), and singlet oxygen quantum yield Phi(Delta)), uptake by vesicles, mitochondria and HeLa cells, dark and phototoxicity in HeLa cells. The photophysical properties of all compounds are quite similar (Phi(f) <= 0.02; Phi(Delta) similar to 0.8). An increase in lipophilicity and the presence of zinc in the porphyrin ring result in higher vesicle and cell uptake. Binding in mitochondria is dependent on the PS lipophilicity and on the electrochemical membrane potential, i.e., in uncoupled mitochondria PS binding decreases by up to 53%. The porphyrin substituted with octyl groups (TC8PyP) is the compound that is most enriched in mitochondria, and its zinc derivative (ZnTC8PyP) has the highest global uptake. The stronger membrane interaction of the zinc-substituted porphyrins is attributed to a complexing effect with phosphate groups of the phospholipids. Zinc insertion was also shown to decrease the interaction with isolated mitochondria and with the mitochondria of HeLa cells, an effect that has been explained by the particular characteristics of the mitochondrial internal membrane. Phototoxicity was shown to increase proportionally with membrane binding efficiency, which is attributed to favorable membrane interactions which allow more efficient membrane photooxidation. For this series of compounds, photodynamic efficiency is directly proportional to the membrane binding and cell uptake, but it is not totally related to mitochondrial targeting.

Deletion of the Basement Membrane Heparan Sulfate Proteoglycan Type XVIII Collagen Causes Hypertriglyceridemia in Mice and Humans

Background: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. Methods and Findings: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. Conclusions: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

A novel electrocatalyst support with proton conductive properties for polymer electrolyte membrane fuel cell applications

The objective of this study is to graft the Surface of carbon black, by chemically introducing polymeric chains (Nafion (R) like) with proton-conducting properties. This procedure aims for a better interaction of the proton-conducting phase with the metallic catalyst particles, as well as hinders posterior support particle agglomeration. Also loss of active surface call be prevented. The proton conduction between the active electrocatalyst site and the Nafion (R) ionomer membrane should be enhanced, thus diminishing the ohmic drop ill the polymer electrolyte membrane fuel cell (PEMFC). PtRu nanoparticles were supported on different carbon materials by the impregnation method and direct reduction with ethylene glycol and characterized using amongst others FTIR, XRD and TEM. The screen printing technique was used to produce membrane electrode assemblies (MEA) for single cell tests in H(2)/air(PEMFC) and methanol operation (DMFC). In the PEMFC experiments, PtRu supported on grafted carbon shows 550 mW cm(-2) gmetal(-1) power density, which represents at least 78% improvement in performance, compared to the power density of commercial PtRu/C ETEK. The DMFC results of the grafted electrocatalyst achieve around 100% improvement. The polarization Curves results clearly show that the main Cause of the observed effect is the reduction in ohmic drop, caused by the grafted polymer. (C) 2009 Elsevier B.V. All rights reserved.

On-line fault diagnostic system for proton exchange membrane fuel cells

In this paper, a supervisor system, able to diagnose different types of faults during the operation of a proton exchange membrane fuel cell is introduced. The diagnosis is developed by applying Bayesian networks, which qualify and quantify the cause-effect relationship among the variables of the process. The fault diagnosis is based on the on-line monitoring of variables easy to measure in the machine such as voltage, electric current, and temperature. The equipment is a fuel cell system which can operate even when a fault occurs. The fault effects are based on experiments on the fault tolerant fuel cell, which are reproduced in a fuel cell model. A database of fault records is constructed from the fuel cell model, improving the generation time and avoiding permanent damage to the equipment. (C) 2007 Elsevier B.V. All rights reserved.

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved beta-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

NMR imaging of fluid pathways during drainage of softwood in a pressure membrane chamber

An experiment was implemented to study fluid flow in a pressure media. This procedure successfully combines nuclear magnetic resonance imaging with a pressure membrane chamber in order to visualize the non-wetting and wetting fluid flows with controlled boundary conditions. A specially designed pressure membrane chamber, made of non-magnetic materials and able to withstand 4 MPa, was designed and built for this purpose. These two techniques were applied to the drainage of Douglas fir sapwood. In the study of the longitudinal flow, narrow drainage fingers are formed in the latewood zones. They follow the longitudinal direction of wood and spread throughout the sample length. These fingers then enlarge in the cross-section plane and coalesce until drainage reaches the whole latewood part. At the end of the experiments, when the drainage of liquid water in latewood is completed, just a few sites of percolation appear in earlywood zones. This difference is a result of the wood anatomical structure, where pits, the apertures that allow the sap to flow between wood cells, are more easily aspirated in earlywood than in latewood. (C) 2007 Elsevier Ltd. All rights reserved.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria. (C) 2008 Elsevier Masson SAS. All rights reserved.

Study of sunless tanning formulas using molted snake skin as an alternative membrane model

Sunless tanning formulas have become increasingly popular in recent years for their ability to give people convincing tans without the dangers of skin cancer. Most sunless tanners currently on the market contain dihydroxyacetone (DHA), a keto sugar with three carbons. The temporary pigment provided by these formulasis designed to resemble a UV-induced tan. This study evaluated the effectiveness of carbomer gels and cold process self emulsifying bases on skin pigmentation, using different concentrations of a chemical system composed of DHA and N-acetyl tyrosine, which are found in moulted snake skins and their effectiveness was tested by Mexameter (R) MX 18. Eight different sunless tanning formulas were developed, four of which were gels and four of which were emulsions (base, base plus 4.0%, 5.0% and 6.0% (w/w) of a system of DHA and N-acetyl tyrosine). Tests to determine the extent of artificial tanning were done by applying 30 mg cm(-2) of each formula onto standard sizes of moulted snake skin (2.0 cm x 3.0 cm). A Mexameter (R) MX 18 was used to evaluate the extent of coloration in the moulted snake skin at T(0) (before the application) and after 24, 48, 72, 168, 192 and 216 h. The moulted snake skins can be used as an alternative membrane model for in vitro sunless tanning efficacy tests due to their similarity to the human stratum corneum. The DHA concentration was found to influence the initiation of the pigmentation in both sunless tanning systems (emulsion and gel) as well as the time required to increases by a given amount on the tanning index. In the emulsion system, the DHA concentration also influenced the final value on the tanning index. The type of system (emulsion or gel) has no influence on the final value in the tanning index after 216 h for samples with the same DHA concentration.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, A beta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles, In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of A beta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6.0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for A beta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of A beta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the Literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.