Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5 +/- 1.8 angstrom, in contrast to only 2 +/- 1.5 angstrom usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca2+ the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na+ was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors. (C) 2011 Elsevier B.V. All rights reserved.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Sciatic nerve regeneration in rats by a nerve conduit engineering with a membrane derived from natural latex

PURPOSE: To evaluate the capacity of natural latex membrane to accelerate and improve the regeneration quality of the of rat sciatic nerves. METHODS: Forty male adult Wistar rats were used, anesthetized and operated to cut the sciatic nerve and receive an autograft or a conduit made with a membrane derived from natural latex (Hevea brasiliensis). Four or eight weeks after surgery, to investigate motor nerve recovery, we analyzed the neurological function by walking pattern (footprints analysis and computerized treadmill), electrophysiological evaluation and histological analysis of regenerated nerve (autologous nerve graft or tissue cables between the nerve stumps), and anterior tibial and gastrocnemius muscles. RESULTS: All functional and morphological analysis showed that the rats transplanted with latex conduit had a better neurological recovery than those operated with autologous nerve: quality of footprints, performance on treadmill (p<0.01), electrophysiological response (p<0.05), and quality of histological aspects on neural regeneration. CONCLUSION: The data reported showed behavioral and functional recovery in rats implanted with latex conduit for sciatic nerve repair, supporting a complete morphological and physiological regeneration of the nerve.

Performance of a poly(2,5-benzimidazole)-based polymer electrolyte membrane fuel cell

The performance of an ABPBI-based High Temperature H-2/O-2 PEMFC system was studied under different experimental conditions. Increasing the temperature from 130 to 170 degrees C improved the cell performance, even though further increase was not beneficial for the system. Humidification of the H-2 stream ameliorated this behaviour, even though operating above 170 degrees C is not advisable in terms of cell performance. A significant electrolyte dehydration seems to negatively affect the fuel cell performance, especially in the case of the anode. In the presence of 2% vol. CO in the H-2 stream, the temperature exerted a positive effect on the cell performance, reducing the strong adsorption of this poison on the platinum sites. Moreover, humidification of the H-2 + CO stream increased the maximum power densities of the cell, further alleviating the CO poisoning effects. Actual CO-O-2 fuel cell results confirmed the significant beneficial effect of the relative humidity on the kinetics of the CO oxidation process. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Phos has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma. Published by Elsevier Masson SAS.

Leishmanicidal activity of an alkenylphenol from Piper malacophyllum is related to plasma membrane disruption

Leishmaniasis and Chagas disease are parasitic protozoan infections that affect the poorest population in the world, causing high mortality and morbidity. As a result of highly toxic and long-duration treatments, novel, safe and more efficacious drugs are essential. In this work, the methanol (MeOH) extract from the leaves of Piper malacophyllum (Piperaceae) was fractioned to afford one alkenylphenol, which was characterized as 4-[(3'E)-decenyl]phenol (gibbilimbol B) by spectroscopic methods. Anti-protozoan in vitro assays demonstrated for the first time that Leishmania (L.) infantum chagasi was susceptible to gibbilimbol B. with an in vitro EC50 of 23 mu g/mL against axenic promastigotes and an EC50 of 22 mu g/mL against intracellular amastigotes. Gibbilimbol B was also tested for anti-trypanosomal activity (Trypanosoma cruzi) and showed an EC50 value of 17 mu g/mL against trypomastigotes. To evaluate the cytotoxic parameters, this alkenylphenol was tested in vitro against NCTC cells, showing a CC50 of 59 mu g/mL and absent hemolytic activity at the highest concentration of 75 mu g/mL. Using the fluorescent probe SYTOX Green suggested that the alkenylphenol disrupted the Leishmania plasma membrane upon initial incubation. Further drug design studies aiming at derivatives could be a promising tool for the development of new therapeutic agents for leishmaniasis and Chagas disease. (C) 2012 Elsevier Inc. All rights reserved.

Upregulation of soluble and membrane-bound human leukocyte antigen G expression is primarily observed in the milder histopathological stages of chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a worldwide health problem that may evolve to cirrhosis and hepatocellular carcinoma. Incompletely understood immune system mechanisms have been associated with impaired viral clearance. The nonclassical class I human leukocyte antigen G (HLA-G) molecule may downregulate immune system cell functions exhibiting well-recognized tolerogenic properties. HCV genotype was analyzed in chronic HCV-infected patients. Because HLA-G expression may be induced by certain viruses, we evaluated the presence of HLA-G in the liver microenvironment obtained from 89 biopsies of patients harboring chronic HCV infection and stratified according to clinical and histopathological features. Overall, data indicated that HCV genotype 1 was predominant, especially subgenotype 1a, with a prevalence of 87%. HLA-G expression was observed in 45(51%) liver specimens, and it was more frequent in milder stages of chronic hepatitis (67.4%) than in moderate (27.8%; p = 0.009) and severe (36.0%; p = 0.021) stages of the disease. Altogether, these results suggest that the expression of HLA-G in the context of HCV is a complex process modulated by many factors, which may contribute to an immunologic environment favoring viral persistence. However, because the milder forms predominantly expressed HLA-G, a protective role of this molecule may not be excluded. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Effect of topical clay application on the synthesis of collagen in skin: an experimental study

Background. Clay is often used in cosmetic treatments, although little is known about its action. Aim. To evaluate the effect of topical clay application on the histoarchitecture of collagen fibres in rat skin. Methods. Animals received a daily application of clay and retinoic acid (RA) 0.025% to the dorsal skin over 7 and 14 days, under vaporization at 37 degrees C for 40 min. Control skin was not vaporized. Samples from each region were excised, and stained with picrosirius red for collagen evaluation. Results. Seven days after clay treatment, an increase in the number of collagen fibres was observed in treated skin compared with control skin (51.74 +/- 1.28 vs. 43.39 +/- 1.79%, respectively, P < 0.01), whereas RA did not alter the collagen level (45.66 +/- 1.10%). Clay application over 14 days did not induce a further increase in skin collagen, whereas treatment with RA did (58.07 +/- 1.59%; P = 0.001 vs. control). Conclusion. Clay application promotes an increase in the number of collagen fibres, which may account for its beneficial effects.

Staining of the Internal Limiting Membrane with the Use of Heavy Brilliant Blue G

Background: Brilliant blue G (BBG) is frequently used in chromovitrectomy to facilitate internal limiting membrane (ILM) peeling. A study was initiated to evaluate if heavy BBG is safe and effective in staining the ILM. Methods: We studied 30 eyes, 23 with idiopathic macular holes and 7 of patients with diabetic macular edema. Removal of the ILMs was assisted by heavy BBG staining. In cases with histopathological correlation the ILMs were evaluated with hematoxylin and eosin, Masson's trichrome, periodic acid-Schiff and glial fibrillary acidic protein staining. In addition, immunohistochemistry was also performed using specific antibodies for vimentin, neuron-specific enolase, factor VIII and CD68. Using the Image-Pro Plus software of Media Cybernetics Co. we found an average thickness in ILMs. Results: Of the ILM specimens sent, 19/30(63.33%) could not be processed properly because of the limited sample material, recognizing only fragments of dispersed fibrillar material. In macular hole ILMs we found an average thickness of 1.3 +/- 0.65 mu m, and in diabetic macular edema ILMs an average thickness of 6.2 +/- 1.4 mu m. Conclusions: In heavy BBG-assisted ILM peeling we observed no intraoperative or postoperative complications after a mean follow-up of 12 months. Heavy BBG could be an effective and safe vehicle for staining the ILM. Copyright (C) 2012 S. Karger AG, Basel

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

Cassava starch biodegradable films: Influence of glycerol and clay nanoparticles content on tensile and barrier properties and glass transition temperature

In this this study, glycerol content and its incorporation method on tensile and barrier properties of biodegradable films (BF) based on cassava starch were analyzed. ANOVA showed that the glycerol incorporation method did not influence the results (P > 0.05), however the glycerol content influenced significantly the tensile and barrier properties of the films (P < 0.05). Films prepared with lower glycerol content presented better tensile and barrier properties than films with higher content. Films were then prepared with addition of clay nanoparticles and their tensile and barrier properties and glass transition temperature were measured. ANOVA indicated that both glycerol and clay nanoparticles influenced significantly the tensile and barrier properties (P < 0.05), diminishing film permeability when clay nanoparticles were present, while the glass transition temperature was not influenced (P > 0.05). (C) 2011 Elsevier Ltd. All rights reserved.

Cu(GlyGlyHis) effects on MCF7 cells: Copper uptake, reactive oxygen species generation and membrane topography changes

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness. (C) 2012 Elsevier Inc. All rights reserved.

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Tip indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

Qualitative study of young, adult, and aged wistar rats temporomandibular synovial membrane employing light, scanning, and transmission electron microscopy

The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. Microsc. Res. Tech. (c) 2012 Wiley Periodicals, Inc.