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.

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.

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.

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.

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.

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.

Arachidonic acid pools of rat kidney cell nuclei.

We have assessed that nuclear lipids from rat kidney cells are not only membrane components, but they are also found within the nucleus. The most abundant nuclear and endonuclear lipids have a high proportion of unsaturated fatty acids (n-6 series: arachidonic > linoleic), mainly esterified to PtdCho. Nuclear most abundant molecular species are 16:0-20:4, 16:0-18:2, 18:0-20:4, 18:0-18:2, and 16:0-18:1. Arachidonic acid is esterified at the sn-2 position of PtdCho: 16:0-20:4(25%), 18:0-20:4(15%), 18:2-20:4(3%), 18:1-20:4(2%). Exogenous [1-(14)C]20:4n-6-CoA is esterified in vitro in GP (glycerophospholipids) > > TAG and DAG. Five PtdCho molecular species were labeled: 16:0-20:4, 18:0-20:4, 18:1-20:4, 18:2-20:4, and 20:4-20:4. In conclusion, these results demonstrated that: (1) there is an important lipid pool within kidney cell nuclei; (2) main nuclear and endonuclear lipid pools were PtdCho molecular species which contained a high proportion of unsaturated fatty acids (20:4n-6 and 18:2n-6) esterified at sn-2 position and 16:0 esterified at sn-1 position; (3) kidney cell nuclei also contained the necessary enzymes to esterify exogenous 20:4n-6-CoA to glycerolipids and to GP; (4) exogenous 20:4n-6-CoA was esterified in five PtdCho molecular species with 20:4n-6 at the sn-2 position, although the most actively synthesized PtdCho contained 20:4n-6 at both the sn-1 and sn-2 positions of the molecule; (5) we can infer that by a remodeling process, the unsaturated fatty acids at the sn-1 position of PtdCho molecular species could be replaced by 16:0 and 18:0, and thus PtdCho would achieve the physiological profile characteristic of the organ.

Incorporation of arachidonic and stearic acids bound to L-FABP into nuclear and endonuclear lipids from rat liver cells.

The incorporation of exogenous fatty acids bound to L-FABP into nuclei was studied. Rat liver cell nuclei and nuclear matrices (membrane depleted nuclei) were incubated in vitro with [1-(14)C]18:0 and 20:4n-6 either free or bound to L-FABP, ATP and CoA. FA esterification in whole nuclei and endonuclear lipids was ATP-CoA-dependent, and with specificity regarding fatty acid type and lipid class. 18:0 and 20:4n-6, free or L-FABP bound, showed the same incorporation and esterification pattern in lipids of whole nuclei. Only 20:4n-6 L-FABP bound was less incorporated into TAG with respect to free 20:4n-6. In the nuclear matrix, 18:0 free or L-FABP bound was esterified with a higher specific activity (SA) into: PtdEtn > PtdIns, PtdSer > PtdCho. 20:4n-6 free or L-FABP bound was esterified into: PtdIns > PtdEtn > PtdCho. 20:4n-6:L-FABP was esterified in endonuclear total-PL and PtdIns with a greater SA with respect to free 20:4n-6 and with a minor one as FFA. To summarize, trafficking of FA to nuclei includes esterification of 18:0 and 20:4n-6 either free or L-FABP-bound, into nuclear and endonuclear lipids by an ATP-CoA-dependent pathway. Endonuclear fatty acid esterification was more active than that in whole nuclei, and independent of the nuclear membrane. Esterification patterns of fatty acids L-FABP-bound or free into whole nuclear lipids were the same whereas in the nuclear matrix, L-FABP could play an important role in the mobilization of 20:4n-6 into specific sites of utilization such as the PtdIns pools.

Endonuclear lipids in liver cells.

Lipids are not only components of cell nucleus membranes, but are also found in the membrane-depleted nuclei where they fulfill special functions. We have investigated the lipid composition of membrane-depleted rat liver nuclei obtained by incubation with low Triton X-100 concentrations of 0.04% and 0.08%, which rendered them unaltered or hardly altered. Under these conditions, 26% of proteins and 22% of phospholipids were recovered. The main phospholipids were phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol = or > phosphatidylserine and sphingomyelin (in decreasing concentrations). The fatty acid components of total lipids and phosphatidylcholine were mainly unsaturated. Over 40% belonged to the n-6 series (arachidonic > or = 25% and linoleic 15%); approximately 40% corresponded to saturated acids and <10% were monoenoic. Endonuclear phosphatidylcholine was built up by 16 molecular species, the most abundant being 18:0-20:4 (32%), 16:0-20:4 (19%), 16:0-18:2 (13%), and 18:0-18:2 (11%). The fatty acid composition and phosphatidylcholine molecular species distribution in the membrane-depleted nucleus of rat liver showed patterns similar to the whole nucleus, mitochondria, microsomes, and homogenate of the parent liver cells, suggesting that endonuclear lipid pool composition is mainly determined by a liver organ profile.