Interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)6(Arg), A6R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A6R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A6R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A6R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A6R exhibits antimicrobial activity without membrane lysis.

Efficient masking of corrosion and fission products such as Ni(II) and Pd(II) in the presence of the minor actinide Am(III) using hydrophilic anionic or cationic bis-triazines

Water soluble anionic and cationic bis-triazine ligands are able to suppress (mask) the extraction of corrosion and fission products such as Ni(II) and Pd(II) that are found in PUREX raffinates. Thus it is possible to separate these elements from the minor actinide Am(III). Although some masking agents have previously been developed that retard the extraction of Pd(II), this is the first time a masking agent has been developed for Ni(II).

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique's potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell's physiological state changes over time.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: Surface charge and pore formation activity

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G(1) = 118.00 +/- 3.67 pS or G(2) = 160.00 +/- 7.07 pS) and DPhPC membranes (G = 61.13 +/- 7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K (+)) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstrom in azolectin membranes and about 4.3 angstrom in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity. (c) 2007 Elsevier Ltd. All rights reserved.

Modifications and Alternatives to the Tests of Levene and Brown & Forsythe for Equality of Variances and Means

The usual tests to compare variances and means (e. g. Bartlett`s test and F-test) assume that the sample comes from a normal distribution. In addition, the test for equality of means requires the assumption of homogeneity of variances. In some situation those assumptions are not satisfied, hence we may face problems like excessive size and low power. In this paper, we describe two tests, namely the Levene`s test for equality of variances, which is robust under nonnormality; and the Brown and Forsythe`s test for equality of means. We also present some modifications of the Levene`s test and Brown and Forsythe`s test, proposed by different authors. We analyzed and applied one modified form of Brown and Forsythe`s test to a real data set. This test is a robust alternative under nonnormality, heteroscedasticity and also when the data set has influential observations. The equality of variance can be well tested by Levene`s test with centering at the sample median.

Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed its one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Protein Assembly onto Cationic Supported Bilayers

Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB(5) hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.

Interactions between Bacteriophage DNA and Cationic Biomimetic Particles

The interaction between giant bacteriophage DNA and cationic biomimetic particles was characterized from sizing by dynamic light-scattering, zeta-potential analysis, turbidimetry, determination of colloid stability, visualization from atomic force microscopy (AFM), and determination of cytotoxicity against E. coli from colony forming unities counting. First, polystyrene sulfate (PSS) particles with different sizes were covered by a dioctadecyldimethylammonium bromide (DODAB) bilayer yielding the so-called cationic biomimetic particles (PSS/DODAB). These cationic particles are highly organized, present a narrow size distribution and were obtained over a range of particle sizes. Thereafter, upon adding lambda, T5 or T2-DNA to PSS/DODAB particles, supramolecular assemblies PSS/DODAB/DNA were obtained and characterized over a range of DNA concentrations and particle sizes (80-700 nm). Over the low DNA concentration range, PSS/DODAB/DNA assemblies were cationic, colloidally stable with moderate polydispersity and highly cytotoxic against E. coli. From DNA concentration corresponding to charge neutralization, neutral or anionic supramolecular assemblies PSS/DODAB/DNA exhibited low colloid stability, high polydispersity and moderate cytotoxicity. Some nucleosome mimetic assemblies were observed by AFM at charge neutralization (zeta-potential equal to zero).

Hybrid materials from intermolecular associations between cationic lipid and polymers

Intermolecular associations between a cationic lipid and two model polymers were evaluated from preparation and characterization of hybrid thin films cast on silicon wafers. The novel materials were prepared by spin-coating of a chloroformic solution of lipid and polymer on silicon wafer. Polymers tested for miscibility with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) were polystyrene (PS) and poly(methyl methacrylate) (PMMA). The films thus obtained were characterized by ellipsometry, wettability, optical and atomic force microscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and activity against Escherichia coli. Whereas intermolecular ion-dipole interactions were available for the PMMA-DODAB interacting pair producing smooth PMMA-DODAB films, the absence of such interactions for PS-DODAB films caused lipid segregation, poor film stability (detachment from the silicon wafer) and large rugosity. In addition, the well-established but still remarkable antimicrobial DODAB properties were transferred to the novel hybrid PMMA/DODAB coating, which is demonstrated to be highly effective against E. coli.

Interaction of cationic bilayer fragments with a model oligonucleotide

The interaction between cationic bilayer fragments and a model oligonucleotide was investigated by differential scanning calorimetry, turbidimetry, determination of excimer to monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidyl-choline in bilayer fragment dispersions and dynamic light scattering for sizing and zeta-potential analysis. Salt (Na(2)HPO(4)), mononucleotide (2`-deoxyadenosine-5`-monophosphate) or poly (dA) oligonucleotide (3`-AAA AAA AAA A-5`) affected structure and stability of dioctadecyldimethylammonium bromide bilayer fragments. Oligonucleotide and salt increased bilayer packing due to bilayer fragment fusion. Mononucleotide did not reduce colloid stability or did not cause bilayer fragment fusion. Charge neutralization of bilayer fragments by poly (dA) at 1:10 poly (dA):dioctadecyldimethylammonium bromide molar ratio caused extensive aggregation, maximal size and zero of zeta-potential for the assemblies. Above charge neutralization, assemblies recovered colloid stability due to charge overcompensation. For bilayer fragments/poly (dA), the nonmonotonic behavior of colloid stability as a function of poly (dA) concentration was unique for the oligonucleotide and was not observed for Na(2)HPO(4) or 2`-deoxyadenosine-5`-monophosphate. For the first time, such interactions between cationic bilayer fragments and mono- or oligonucleotide were described in the literature. Bilayer fragments/oligonucleotide assemblies may find interesting applications in drug delivery. (c) 2010 Elsevier B.V. All rights reserved.

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) after the incubation of 2`-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.

Antimicrobial Particles from Cationic Lipid and Polyelectrolytes

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CM C, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta= 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of p. aeruginosa viability (10(7) CFU/mL) at 1 or 2 mu g/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 mu g/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.