Exercise-induced cardiac mitochondrial reorganization and enhancement in spontaneously hypertensive rats.

The myocardium is a highly oxidative tissue in which mitochondria are essential to supply the energy required to maintain pump function. When pathological hypertrophy develops, energy consumption augments and jeopardizes mitochondrial capacity. We explored the cardiac consequences of chronic swimming training, focusing on the mitochondrial network, in spontaneously hypertensive rats (SHR). Male adult SHR were randomized to sedentary or trained (T: 8-week swimming protocol). Blood pressure and echocardiograms were recorded, and hearts were removed at the end of the training period to perform molecular, imaging, or isolated mitochondria studies. Swimming improved cardiac midventricular shortening and decreased the pathological hypertrophic marker atrial natriuretic peptide. Oxidative stress was reduced, and even more interesting, mitochondrial spatial distribution, dynamics, function, and ATP were significantly improved in the myocardium of T rats. In the signaling pathway triggered by training, we detected an increase in the phosphorylation level of both AKT and glycogen synthase kinase-3 ß, key downstream targets of insulin-like growth factor 1 signaling that are crucially involved in mitochondria biogenesis and integrity. Aerobic exercise training emerges as an effective approach to improve pathological cardiac hypertrophy and bioenergetics in hypertension-induced cardiac hypertrophy.

Biophysical Analysis to Assess the Interaction of CRAC and CARC Motif Peptides of Alpha Hemolysin of Escherichia coli with Membranes.

Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is a prototype of the "Repeat in Toxins" (RTX) family. It was demonstrated that HlyA-cholesterol interaction facilitates the insertion of the toxin into membranes. Putative cholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC), and CARC (analogous to CRAC but with the opposite orientation) were identified in the HlyA sequence. In this context, two peptides were synthesized, one derived from a CARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other one from a CRAC site from the domain between the acylated lysines (residues 639-644) (PEP 2), to study their role in the interaction of HlyA with membranes. The interaction of peptides with membranes of different lipid compositions (pure POPC and POPC/Cho of 4:1 and 2:1 molar ratios) was analyzed by surface plasmon resonance and molecular dynamics simulations. Results demonstrate that both peptides interact preferentially with Cho-containing membranes, although PEP 2 presents a lower KD than PEP 1. Molecular dynamics simulation results indicate that the insertion and interaction of PEP 2 with Cho-containing membranes are more prominent than those caused by PEP 1. The hemolytic activity of HlyA in the presence of peptides indicates that PEP 2 was the only one that inhibits HlyA activity, interfering in the binding between the toxin and cholesterol.

Histidine 19 Residue Is Essential for Cell Internalization of Antifungal Peptide SmAPα1-21 Derived from the α-Core of the Silybum marianum Defensin DefSm2-D in Fusarium graminearum.

The synthetic peptide SmAPα1-21 (KLCEKPSKTWFGNCGNPRHCG) derived from DefSm2-D defensin α-core is active at micromolar concentrations against the phytopathogenic fungus Fusarium graminearum and has a multistep mechanism of action that includes alteration of the fungal cell wall and membrane permeabilization. Here, we continued the study of this peptide's mode of action and explored the correlation between the biological activity and its primary structure. Transmission electron microscopy was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. New peptides were designed by modifying the parent peptide SmAPα1-21 (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Membrane permeability and subcellular localization in conidia were studied by confocal laser scanning microscopy (CLSM). Reactive oxygen species (ROS) production was assessed by fluorescence spectroscopy and CLSM. SmAPα1-21 induced peroxisome biogenesis and oxidative stress through ROS production in F. graminearum and was internalized into the conidial cells' cytoplasm. SmAPH19R and SmAPH19A were active against F. graminearum with minimal inhibitory concentrations (MICs) of 38 and 100 µM for SmAPH19R and SmAPH19A, respectively. The replacement of His19 by Ala produced a decrease in the net charge with a significant increase in the MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. Like SmAPα1-21, SmAP2H19A and SmAP2H19R produced the permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, SmAPH19R and SmAPH19A were localized in the conidia cell wall. The replacement of His19 by Ala turned all the processes slower. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His19 residue in the internalization.

Formation and Nanoscale Characterization of Asymmetric Supported Lipid Bilayers Containing Raft-Like Domains.

The development of new strategies for achieving stable asymmetric membrane models has turned interleaflet lipid asymmetry into a topic of major interest. Cyclodextrin-mediated lipid exchange constitutes a simple and versatile method for preparing asymmetric membrane models without the need for sophisticated equipment. Here we describe a protocol for preparing asymmetric supported lipid bilayers mimicking membrane rafts by cyclodextrin-mediated lipid exchange and the main guidelines for obtaining structural information and quantitative measures of their mechanical properties using Atomic force microscopy and Force spectroscopy; two powerful techniques that allow membrane characterization at the nanoscale.

Anandamide Exerts a Differential Effect on Human Placenta Before and After the Onset of Labor.

The onset of labor involves the action of multiple factors and recent reports have postulated the endocannabinoid system as a new regulator of this process. Our objective was to study the role of anandamide, one of the main endocannabinoids, on the regulation of placental molecules that contribute to the onset of labor at term. Placental samples were obtained from patients with laboring vaginal deliveries and from non-laboring elective cesarean sections. Vaginal delivery placentas produced higher prostaglandins levels than cesarean section samples. Besides, no differences were observed in NOS basal activity between groups. Incubation of vaginal delivery placentas with anandamide increased prostaglandins concentration and decreased NOS activity. Antagonism of type-1cannabinoid receptor (CB1) did not alter the effect observed on NOS activity. Conversely, incubation of cesarean section placentas with anandamide reduced prostaglandins levels and enhanced NOS activity, the latter involving the participation of CB1. Furthermore, we observed a differential expression of the main components of the endocannabinoid system between placental samples, being the change in CB1 localization the most relevant finding. Our results suggest that anandamide acts as a modulator of the signals that regulate labor, exerting differential actions depending on CB1 localization in laboring or non-laboring term placentas.

Interaction of cationic surfactants with DPPC membranes: effect of a novel Nα-benzoylated arginine-based compound.

Cationic amino acid-based surfactants are known to interact with the lipid bilayer of microorganism resulting in cell death through a disruption of the membrane topology. To elucidate the interaction of a cationic surfactant synthesized in our lab, investigations involving Nα-benzoyl-arginine decyl amide (Bz-Arg-NHC10), and model membranes composed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were done. Bz-Arg-NHC10was able to penetrate into DPPC monolayers up to a critical pressure of 59.6 mN m-1. Differential scanning calorimetry revealed that as the concentration of Bz-Arg-NHC10 increased, the main transition temperature of DPPC slightly decreased. Atomic force microscopy (AFM) in situ experiments performed on supported DPPC bilayers on mica allowed monitoring the changes induced by Bz-Arg-NHC10. DPPC bilayer patches were partially removed, mainly in borders and bilayer defects for 50 µM Bz-Arg-NHC10 solution. Increasing the concentration to 100 µM resulted in a complete depletion of the supported bilayers. Surface plasmon resonance (SPR) experiments, carried out with fully DPPC bilayers covered chips, showed a net increase of the SPR signal, which can be explained by Bz-Arg-NHC10 adsorption. When patchy DPPC bilayers were formed on the substrate, a SPR signal net decrease was obtained, which is consistent with the phospholipids' removal observed in the AFM images. The results obtained suggest that the presence of the benzoyl group attached to the polar head of our compound would be the responsible of the increased antimicrobial activity against gram-negative bacteria when compared with other arginine-based surfactants.

Asymmetric bilayers mimicking membrane rafts prepared by lipid exchange: Nanoscale characterization using AFM-Force spectroscopy.

Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate important cellular functions. Notwithstanding, the asymmetric transbilayer distribution of phospholipids that exists in the membrane confers the two leaflets different potentials to form lateral domains as next to no sphingolipids are present in the inner leaflet. How the physical properties of one leaflet can influence the properties of the other and its importance on signal transduction across the membrane are questions still unresolved. In this work, we combined AFM imaging and Force spectroscopy measurements to assess domain formation and to study the nanomechanical properties of asymmetric supported lipid bilayers (SLBs) mimicking membrane rafts. Asymmetric SLBs were formed by incorporating N-palmitoyl-sphingomyelin (16:0SM) into the outer leaflet of preformed 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Cholesterol SLBs through methyl-ß-cyclodextrin-mediated lipid exchange. Lipid domains were detected after incorporation of 16:0SM though their phase state varied from gel to liquid ordered (Lo) phase if the procedure was performed at 24 or 37 °C, respectively. When comparing symmetric and asymmetric Lo domains, differences in size and morphology were observed, with asymmetric domains being smaller and more interconnected. Both types of Lo domains showed similar mechanical stability in terms of rupture forces and Young's moduli. Notably, force curves in asymmetric domains presented two rupture events that could be attributed to the sequential rupture of a liquid disordered (Ld) and a Lo phase. Interleaflet coupling in asymmetric Lo domains could also be inferred from those measurements. The experimental approach outlined here would significantly enhance the applicability of membrane models.

Anandamide regulates oxytocin/oxytocin receptor system in human placenta at term.

Oxytocin plays a pivotal role in the regulation of human parturition, however its role and modulation in the placenta is not fully understood. Non-labour cesarean section placentas were cultured with the endocannabinoid anandamide. We observed an increase in placental oxytocin receptor expression and oxytocin release. We postulate anandamide as a relevant modulator of oxytocin system in the placenta at term.

Induction of erythrocyte microvesicles by Escherichia Coli Alpha hemolysin.

Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains - which proteolyse cytoskeleton proteins - and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.

Impact of sphingomyelin acyl chain (16:0 vs 24:1) on the interfacial properties of Langmuir monolayers: A PM-IRRAS study.

Membrane structure is a key factor for the cell`s physiology, pathology, and therapy. Evaluating the importance of lipid species such as N-nervonoyl sphingomyelin (24:1-SM) -able to prevent phase separation- to membrane structuring remains a formidable challenge. This is the first report in which polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) is applied to investigate the lipid-lipid interactions in 16:0 vs 24:1-SM monolayers and their mixtures with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol) (DOPC/SM/Chol 2:1:1). From the results we inferred that the cis double bond (Δ15) in 24:1-SM molecule diminishes intermolecular H-bonding and chain packing density compared to that of 16:0-SM. In ternary mixtures containing 16:0-SM, the relative intensity of the two components of the Amide I band reflected changes in the H-bonding network due to SM-Chol interactions. In contrast, the contribution of the main components of the Amide I band in DOPC/24:1-SM/Chol remained as in 24:1-SM monolayers, with a larger contribution of the non-H-bonded component. The most interesting feature in these ternary films is that the CO stretching mode of DOPC appeared with an intensity similar to that of SM Amide I band in DOPC/16:0-SM/Chol monolayers (a two-phase [Lo/Le] system), whereas an extremely low intensity of the CO band was detected in DOPC/24:1-SM/Chol monolayers (single Le phase). This is evidence that the unsaturation in 24:1-SM affected not only the conformational properties of acyl chains but also the orientation of the chemical groups at the air/water interface. The physical properties and overall H-bonding ability conferred by 24:1-SM may have implications in cell signaling and binding of biomolecules.

Phase-segregated Membrane Model assessed by a combined SPR-AFM Approach.

Model biomembranes can provide valuable insights into the properties of complex biological membranes. Among several techniques, Surface Plasmon Resonance (SPR) provides a label-free analysis of the interactions of bioactive molecules with biomembranes with an experimental setup that allows mimicking biological environments. Nevertheless, protocols that enable the preparation of stable supported membrane systems with reproducible structural and functional properties on the biosensor chip are still needed. In this work, we present a simple protocol to modify SPR substrates that allows the formation of a phase-segregated supported lipid bilayer (SLB). SLBs are formed by fusion of lipid vesicles of pure phospholipids (DMPC, DPPC and DOPC) and of a ternary mixture (DOPC/16:0 SM/Cho in 2:1:1 molar ratio) on a SPR gold sensor chip covered with a dithiothreitol monolayer. The formation of a SLB on the SPR sensing surface in a reproducible way was assessed by the combined use of the SPR technique with AFM. The interaction of a cholesterol-extracting drug with SLBs was studied as a model of membrane-lipophilic biomolecule interaction. The proposed strategy allowed us to obtain a membrane model where phase coexistence is present and where Cho depletion from ternary mixtures was comparable to the extraction results reported for human erythrocytes.

Volume expansion of erythrocytes is not the only mechanism responsible for the protection by arginine-based surfactants against hypotonic hemolysis.

A novel arginine-based cationic surfactant Nα-benzoyl-arginine dodecylamide (Bz-Arg-NHC12) was synthesized in our laboratory. In this paper we study the interaction of Bz-Arg-NHC12 with sheep and human red blood cells (SRBC and HRBC respectively) due to their different membrane physicochemical/biophysical properties. SRBC demonstrated to be slightly more resistant than HRBC to the hemolytic effect of the surfactant, being the micellar structure responsible for the hemolytic effect in both cases. Moreover, besides the hemolytic effect, a dual behavior was observed for the surfactant studied: Bz-Arg-NHC12 was also able to protect red blood cells against hypotonic lysis for HRBC in a wide range of surfactant concentrations. However, the degree of protection showed for SRBC was about 50% lower than for HBRC. In this regard, a remarkable volume expansion was evidenced only for SRBC treated with Bz-Arg-NHC12, although no correlation with the antihemolytic potency (pAH) was found. On the contrary, our surfactant showed a greater pAH when human erythrocytes were submitted to hypotonic stress, with a low volume expansion, showing a higher amount of solubilized phospholipids in the supernatant when compared with SRBC behavior. Surface plasmon resonance measurements show the molecular interaction of the surfactant with lipid bilayers from HRBC and SRBC lipids, demonstrating that in the latter neither microvesicle release or lipid extraction occurred. Our results demonstrate that the volume expansion of erythrocytes is not the only mechanism responsible for the protection by surfactants against hypotonic hemolysis: volume expansion could be compensated via microvesicle release or by the extraction of membrane components upon collisions between red blood cells and surfactant aggregates depending on the membrane composition.

Interaction of acylated and unacylated forms of E. coli alpha-hemolysin with lipid monolayers: a PM-IRRAS study.

Uropathogenic strains of Escherichia coli produce virulence factors, such as the protein toxin alpha-hemolysin (HlyA), that enable the bacteria to colonize the host and establish an infection. HlyA is synthetized as a protoxin (ProHlyA) that is transformed into the active form in the bacterial cytosol by the covalent linkage of two fatty-acyl moieties to the polypeptide chain before the secretion of HlyA into the extracellular medium. The aim of this work was to investigate the effect of the fatty acylation of HlyA on protein conformation and protein-membrane interactions. Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) experiments were performed at the air-water interface, and lipid monolayers mimicking the outer leaflet of red-blood-cell membranes were used as model systems for the study of protein-membrane interaction. According to surface-pressure measurements, incorporation of the acylated protein into the lipid films was faster than that of the nonacylated form. PM-IRRAS measurements revealed that the adsorption of the proteins to the lipid monolayers induced disorder in the lipid acyl chains and also changed the elastic properties of the films independently of protein acylation. No significant difference was observed between HlyA and ProHlyA in the interaction with the model lipid monolayers; but when these proteins became adsorbed on a bare air-water interface, they adopted different secondary structures. The assumption of the correct protein conformation at a hydrophobic-hydrophilic interface could constitute a critical condition for biologic activity.

Dynamic regulation of extracellular ATP in Escherichia coli.

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

Induction of eryptosis by low concentrations of E. coli alpha-hemolysin.

Uropathogenic strains of Escherichia coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations have appeared to interfere with host-cell-signaling pathways causing cell death by apoptosis. Nevertheless, what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the course of an infection. In the present investigation, we demonstrate that a low toxin concentration induces the suicidal death of erythrocytes (eryptosis), the major cell type present in blood. Eryptosis is triggered both by an increment in intracellular calcium and by ceramide. Since we have previously demonstrated that a low concentration of HlyA induces an increase in intraerythrocyte calcium, in the present experiments we have shown that this ion activates calpains, which hydrolyze skeleton proteins such as spectrin, ankyrin, protein 4.1 and the electrophoretic Band-3 species, thus resulting in morphologic changes in the erythrocytes. We furthermore observed that a low toxin concentration induced the activation of endogenous sphingomyelinases that in turn increased the amount of ceramide in erythrocyte membranes. Both spectrin proteolysis and ceramide formation may cause the exposure of phosphatidylserine on the membrane so as to trigger a macrophage engulfment of the erythrocyte. By this means eryptosis may be an advantageous mechanism for removing defective erythrocytes before hemolysis.

N-nervonoylsphingomyelin (C24:1) prevents lateral heterogeneity in cholesterol-containing membranes.

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: a real-time study.

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale-both in situ and in real-time-the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.

Novel evidence for the specific interaction between cholesterol and α-haemolysin of Escherichia coli.

Several toxins that act on animal cells present different, but specific, interactions with cholesterol or sphingomyelin. In the present study we demonstrate that HlyA (α-haemolysin) of Escherichia coli interacts directly with cholesterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in cholesterol and sphingomyelin; moreover, toxin oligomerization, and hence haemolytic activity, diminishes in cholesterol-depleted erythrocytes. Considering these results, we studied the insertion process, an essential step in the lytic mechanism, by the monolayer technique, finding that HlyA insertion is favoured in cholesterol- and sphingomyelin-containing membranes. On the basis of this result, we studied the direct interaction with either of the lipids by lipid dot blotting, lysis inhibition and SPR (surface plasmon resonance) assays. The results of the present study demonstrated that an interaction between cholesterol and HlyA exists that seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.