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In this study, poly( e -caprolactone)-co-poly(ethylene glycol) copolymers grafted with a cationic ligand, propargyltrimethyl ammonium iodide (PTA), to fabricate the cationized triblock (P(CatCLCL) 2 -PEG) and diblock (P(CatCLCL)-mPEG) copolymers were investigated their potential use for oral delivery of enoxaparin (ENX). Influences of various PTA contents and different structures of the copolymers on molecular characteristics, ENX encapsulation, particle characteristics, and capability of drug transport across Caco-2 cells were elucidated. The results showed that P(CatCLCL) 2 -PEG and P(CatCLCL)-mPEG copolymers self-aggregated and encapsulated ENX into spherical particles of ∼200–450 nm. The increasing amount of PTA on the copolymers increased encapsulation efficiency of over 90%. The ENX release from both types of the cationized copolymer particles was pH-dependent which was retarded at pH 1.2 and accelerated at pH 7.4, supporting the drug protection in the acidic environment and possible release in the blood circulation. The toxicity of ENX-loaded particles on Caco-2 cells decreased when decreasing the amount of PTA. The triblock and diblock particles dramatically enhanced ENX uptake and transport across Caco-2 cells as compared to the ENX solution. However, the different structures of the copolymers slightly affected ENX transport. These results suggested that P(CatCLCL) 2 -PEG and P(CatCLCL)-mPEG copolymers would be potential carriers for oral delivery of ENX. Statement of Significance The anionic drugs such as proteins, peptides or polysaccharides are generally administered via invasive route causing patient incompliance and high cost of hospitalization. The development of biomaterials for non-invasive delivery of those drugs has gained much attention, especially for oral delivery. However, they have limitation due to non-biocompatibility and poor drug bioavailability. In this study, the novel poly( e -caprolactone)-co-poly(ethylene glycol) copolymers grafted with propargyltrimethyl ammonium iodide, a small cationic ligand, were introduced to use as a carrier for oral delivery of enoxaparin, a highly negatively charged drug. The study showed that these cationized copolymers could achieve high enoxaparin entrapment efficiency, protect drug release in an acidic environment and enhance enoxaparin permeability across Caco-2 cells, the intestinal cell model. These characteristics of the cationized copolymers make them a potential candidate for oral delivery of anionic drugs for biomaterial applications.

International audience; The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

International audience; Clinical data and experimental studies suggest that bronchial epithelium could serve as a portal of entry for invasive fungal infections. We therefore analyzed the interactions between molds and the bronchial/bronchiolar epithelium at the early steps after inhalation. We developed invasive aspergillosis (Aspergillus fumigatus) and mucormycosis (Lichtheimia corymbifera) murine models that mimic the main clinical risk factors for these infections. Histopathology studies were completed with a specific computer-assisted morphometric method to quantify bronchial and alveolar spores and with transmission electron microscopy. Morphometric analysis revealed a higher number of bronchial/bronchiolar spores for A. fumigatus than L. corymbifera. The bronchial/bronchiolar spores decreased between 1 and 18 hours after inoculation for both fungi, except in corticosteroid-treated mice infected with A. fumigatus, suggesting an effect of cortisone on bronchial spore clearance. No increase in the number of spores of any species was observed over time at the basal pole of the epithelium, suggesting the lack of transepithelial crossing. Transmission electron microscopy did not show spore internalization by bronchial epithelial cells. Instead, spores were phagocytized by mononuclear cells on the apical pole of epithelial cells. Early epithelial internalization of fungal spores in vivo cannot explain the bronchial/bronchiolar epithelium invasion observed in some invasive mold infections. The bioimaging approach provides a useful means to accurately enumerate and localize the fungal spores in the pulmonary tissues.

International audience; Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum-Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore-forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore-forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol-dependent cytolysins.

International audience; We have recently characterized bicarinalin as the most abundant peptide from the venom of the ant Tetramorium bicarinatum. This antimicrobial peptide is active against Staphylococcus and Enterobacteriaceae. To further investigate the antimicrobial properties of this cationic and cysteine-free peptide, we have studied its antibacterial, antifungal and antiparasitic activities on a large array of microorganisms. Bicarinalin was active against fifteen microorganisms with minimal inhibitory concentrations ranging from 2 and 25μmolL(-1). Cronobacter sakazakii, Salmonella enterica, Candida albicans, Aspergilus niger and Saccharomyces cerevisiae were particularly susceptible to this novel antimicrobial peptide. Resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and C. albicans were as susceptible as the canonical strains. Interestingly, bicarinalin was also active against the parasite Leishmania infantum with a minimal inhibitory concentrations of 2μmolL(-1). The bicarinalin pre-propeptide cDNA sequence has been determined using a combination of degenerated primers with RACE PCR strategy. Interestingly, the N-terminal domain of bicarinalin pre-propeptide exhibited sequence similarity with the pilosulin antimicrobial peptide family previously described in the Myrmecia venoms. Moreover, using SYTOX green uptake assay, we showed that, for all the tested microorganisms, bicarinalin acted through a membrane permeabilization mechanism. Two dimensional-NMR experiments showed that bicarinalin displayed a 10 residue-long α-helical structure flanked by two N- and C-terminal disordered regions. This partially amphipathic helix may explain the membrane permeabilization mechanism of bicarinalin observed in this study. Finally, therapeutic value of bicarinalin was highlighted by its low cytotoxicity against human lymphocytes at bactericidal concentrations and its long half-life in human serum which was around 15h.

The family of plant membrane transporters named HKT (for high-affinity K+ transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na+-selective transporters and transporters able to function as Na+-K+ symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K+ channels or transporters permeable to Ca2+ when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca2+ permeability through OsHKT2;4 was not detected, even at very low external K+ concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca2+ conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca2+. The Ca2+ permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K+ compared with that to Na+ (permeability sequence: K+ > Rb+ ≈ Cs+ > Na+ ≈ Li+ ≈ NH4 +). Examination of OsHKT2;4 current sensitivity to external pH suggested that H+ is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na+ and K+ indicated that OsHKT2;4 functions as K+-selective transporter at low external Na+, but transports also Na+ at high (>10 mm) Na+ concentrations. These data identify OsHKT2;4 as a new functional type in the K+ and Na+-permeable HKT transporter subfamily. Furthermore, the high permeability to K+ in OsHKT2;4 supports the hypothesis that this system is dedicated to K+ transport in the plant.

International audience; The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.

Introduction The aquaporins (AQP1, 3, 8, 9 and 11) are known to be expressed, and involved in the transport of water and small molecules through fetal membranes. To exert these crucial functions, these AQPs have to be finely regulated. All -trans -retinoic acid (atRA) was previously found to regulate some genes in this environment, raising the question of whether these AQPs were regulated by atRA. Methods Explants, and primary and established amniotic cells were cultured to determine which AQP were transcriptionally modified by atRA, using the qRT-PCR strategy. Immunohistochemistry and glycerol uptake tests were used to determine the impact of atRA on AQP protein expression and function. Specific agonists of retinoic acid receptors were used to identify the molecular mechanisms of AQP promoter activation. A classical gene AQP promoter study was also used to identify DR5 retinoic acid receptor elements (RAREs). Results Beyond these AQPs, only one specific atRA-dependent increase in AQP3 transcripts and proteins level was established in amnion (not in chorion) and in related primary and established cells. We found three DR5-RAREs essential for inducing this transcriptional AQP3 through RARα. This transactivation of the AQP3 coding gene was functionally related to an increase of AQP3 permeability tests by a glycerol uptake assay. Discussion Our data support an atRA regulatory model of AQP3 expression leading to an increased cellular permeability in the epithelial amniotic environment. We cast new light on AF regulation in healthy pregnancy, and advance new hypotheses for obstetrical complications linked to impairment of the retinoic signaling pathway.

International audience; Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (Pf) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the Pf of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.

International audience; Introduction: Ectopic activity arising from pulmonary veins (PV) plays a prominent role in the onset of atrial fibrillation in humans. Rat PV cardiac muscle cells have a lower resting membrane potential (RMP) than the left atria (LA) and presents in the presence of norepinephrine an automatic activity, which occurs in bursts. This study investigated the role of Na channels upon the RMP and the catecholaminergic automatic activity (CAA) in PV cardiac muscle.Methods and results: RMP and CAA experiments were performed in male Wistar rat PV. Whole-cell INa was recorded in isolated PV and LA cardiomyocytes. PV has a higher tetrodotoxin (TTX)-sensitive basal Na(+) permeability than the LA, due to a ∼ 5 mV more negative Na window current in the former tissue. TTX, quinidine, and ranolazine (1 to 10 μM each) decreased CAA incidence and arrhythmias by increasing burst intervals because of a reduction of the slope of slow depolarization between bursts. TTX and ranolazine also reduced burst duration. At 1 Hz, 10 μM quinidine, ranolazine, and TTX inhibited peak INa by 33%, 28%, and 98%, respectively. Each reduced the Na window current. There was no evidence for a TTX- or ranolazine-sensitive late Na current.Conclusion: Na channels confer a TTX-sensitive basal Na(+) permeability to rat PV cardiac muscle cells and contribute to the slope of slow depolarization between bursts of CAA. Na channel blockers act mostly via reduction of the Na window current. Ranolazine also has an anti-α1 adrenergic effect, which contributed to its antiarrhythmic effect.

International audience; In the cell, homo- and hetero-associations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide for the selection of cell membrane permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.

The accurate identification and thorough characterization of tumorigenic cells in glioblastomas are essential to enhance our understanding of their malignant behavior and for the design of strategies that target this important cell population. We report here that, in rat brain, the scaffolding protein IQGAP1 is a marker of brain nestin+ amplifying neural progenitor cells. In a rat model of glioma, IQGAP1 also characterizes a subpopulation of nestin+ amplifying tumor cells in glioblastoma-like tumors but not in tumors with oligodendroglioma features. We next confirmed that IQGAP1 represents a new marker that may help to discriminate human glioblastoma from oligodendrogliomas. In human glioblastoma exclusively, IQGAP1 specifies a subpopulation of amplifying nestin+ cancer cells. Neoplastic IQGAP1+ cells from glioblastoma can be expanded in culture and possess all the characteristics of cancer stem-like progenitors. The similarities between amplifying neural progenitors and glioblastoma amplifying cancer cells may have significant implications for understanding the biology of glioblastoma. (Cancer Res 2006; 66(18): 9074-82)

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.

Electroporation is a method to introduce molecules, such as gene constructs or small drugs, into cells by temporarily permeating the cell membrane with electric pulses. In molecular medicine and biotechnology, tissue electroporation is performed with electrodes placed in the target area of the body. Currently, tissue electroporation, as with all other methods of molecular medicine, is performed without real-time control or near-term information regarding the extent and degree of electroporation. This paper expands the work from our previous study by implementing new ex vivo experimental data with "front-tracking" analysis for the image reconstruction algorithm. The experimental data is incorporated into numerical simulations of electroporation procedures and images are generated using the new reconstruction algorithm to demonstrate that electrical impedance tomography (EIT) can produce an image of the electroporated area. Combining EIT with electroporation could become an important biotechnological and medical technique to introduce therapeutic molecules into cells in tissue at predetermined areas of the body.

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze–thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze–thawing conditions, high rates of adenosine 5 ′ -diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5 ′ -triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze–thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.

International audience; L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)μl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

International audience; Biological membranes represent a physical barrier that most viruses have to cross for replication. While enveloped viruses cross membranes through a well-characterized membrane fusion mechanism, non-enveloped viruses, such as rotaviruses, require the destabilization of the host cell membrane by processes that are still poorly understood. We have identified, in the C-terminal region of the rotavirus glycoprotein VP7, a peptide that was predicted to contain a membrane domain and to fold into an amphipathic α-helix. Its structure was confirmed by circular dichroism in media mimicking the hydrophobic environment of the membrane at both acidic and neutral pHs. The helical folding of the peptide was corroborated by ATR-FTIR spectroscopy, which suggested a transmembrane orientation of the peptide. The interaction of this peptide with artificial membranes and its affinity were assessed by plasmon waveguide resonance. We have found that the peptide was able to insert into membranes and permeabilize them while the native protein VP7 did not. Finally, NMR studies revealed that in a hydrophobic environment, this helix has amphipathic properties characteristic of membrane-perforating peptides. Surprisingly, its structure varies from that of its counterpart in the structure of the native protein VP7, as was determined by X-ray. All together, our results show that a peptide released from VP7 is capable of changing its conformation and destabilizing artificial membranes. Such peptides could play an important role by facilitating membrane crossing by non-enveloped viruses during cell infection.

International audience; Maturation of newly formed vessels is a multistep phenomenon during which functional endothelial barriers are established. Disruption of vessel integrity is an important feature in many physiological and pathological processes. We previously reported that lymphoblastic leukemia-derived sequence 1 (LYL1) is required for the late stages of postnatal angiogenesis to limit the formation of new blood vessels, notably by regulating the activity of the small GTPase Rap1. In this study, we show that LYL1 is also required during the formation of the mature endothelial barrier in the lungs of adult mice. Specifically, LYL1 knockdown in human endothelial cells downregulated the expression of ARHGAP21 and ARHGAP24, which encode two Rho GTPase-activating proteins, and this was correlated with increased RhoA activity and reorganization of the actin cytoskeleton into stress fibers. Importantly, in lungs of Lyl1-deficient mice, both vascular endothelial (VE)-cadherin and p120-catenin were poorly recruited to endothelial adherens junctions, indicative of defective cell-cell junctions. Consistent with this, higher Evans blue dye extravasation, edema, and leukocyte infiltration in the lung parenchyma of Lyl1-/- mice than in wild-type littermates confirmed that lung vascular permeability is constitutively elevated in Lyl1-/- adult mice. Our data show that LYL1 acts as a stabilizing signal for adherens junction formation by operating upstream of VE-cadherin and of the two GTPases Rap1 and RhoA. As increased vascular permeability is a key feature and a major mechanism of acute respiratory distress syndrome, molecules that regulate LYL1 activity could represent additional tools to modify the endothelial barrier permeability.

International audience; Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open. We have evaluated the effects of various combinations of square wave electric pulses of variable field strength and duration, on cell permeabilization and on DNA transfection in the skeletal muscle in vivo. One high voltage pulse of 800 V/cm, 0.1 ms duration (short high pulse) or a series of four low voltage pulses of 80 V/cm, 83 ms duration (long low pulses) slightly amplified transfection efficacy, while no significant permeabilization was detected using the (51)Cr-EDTA uptake test. By contrast, the combination of one short high pulse followed by four long low pulses led to optimal gene transfer efficiency, while inducing muscle fibers permeabilization. These results are consistent with additive effects of electropermeabilization and DNA electrophoresis on electrotransfer efficiency. Finally, the described new combination, as compared to the previously reported use of repeated identical pulses of intermediate voltage, leads to similar gene transfer efficiency, while causing less permeabilization and thus being likely less deleterious. Thus, combination of pulses of various strengths and durations is a new procedure for skeletal muscle gene transfer that may represents a clear improvement in view of further clinical development.

International audience; Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble--cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

International audience; On the basis of our previous identification of aberrant phosphatidylinositol-3-kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235-induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235-induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase-dependent apoptosis. Lysosome-mediated apoptosis occurs in a ROS-dependent manner, as ROS scavengers significantly reduce BEZ235/CQ-induced loss of MMP, LMP and apoptosis. There is a mitochondrial-lysosomal cross-talk, since lysosomal enzyme inhibitors significantly decrease BEZ235- and CQ-induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome-mediated apoptosis via a mitochondrial-lysosomal cross-talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor-based combination therapies.

L'article original est publié par The American Society of Plant Biologists; International audience; Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH*. In root cells, extracellular OH* activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH*-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH*. An OH*-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii .

International audience; Manganese is an essential trace element, and a contrast agent of potential interest for brain magnetic resonance imaging. Brain overexposure to manganese, however induces a neurodegenerative syndrome. Imaging data suggest that manganese appearance into the CSF precedes its accumulation into the cerebral parenchyma. We therefore investigated manganese uptake and transport at the blood-CSF barrier. Like lead, the non protein-bound divalent manganese accumulated into the rat choroid plexus. The metal accumulation was especially high in developing animals. Using a differentiated cellular model of the blood-CSF barrier, we demonstrated that manganese crosses the choroid plexus epithelium by a concentrating, unidirectional blood-to-CSF transport mechanism. This transport was inhibited by calcium, which is also transported into the CSF against its concentration gradient. The permeability barrier function towards lipid-insoluble compound and the organic anion transport property of the blood-brain interface were affected by exposure of the blood-facing membrane of choroidal cells to micromolar concentrations of manganese, but its antioxidant capacity was not. The unidirectional transport of manganese across the choroid plexus provides the anatomo-functional basis linking the systemic exposure to manganese with the spreading pattern of manganese accumulation observed in brain imaging, and explains the polarized sensitivity of choroidal epithelial cells to manganese toxicity.

International audience; Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Here, we analyzed channel formation of the recombinant septicum alpha-toxin and characterized its activity on living cells. Recombinant septicum alpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175pS in 0.1M KCl in lipid bilayer membranes, which is typical for a large diffusion pore. Septicum alpha-toxin channels remained mostly in the open configuration, displayed no lipid specificity, and exhibited slight anion selectivity. Septicum alpha-toxin caused a rapid decrease in the transepithelial electrical resistance of MDCK cell monolayers grown on filters, and induced a rapid cell necrosis in a variety of cell lines, characterized by cell permeabilization to propidium iodide without DNA fragmentation and activation of caspase-3. Septicum alpha-toxin also induced a rapid K(+) efflux and ATP depletion. Incubation of the cells in K(+)-enriched medium delayed cell death caused by septicum alpha-toxin or epsilon-toxin, another potent pore-forming toxin, suggesting that the rapid loss of intracellular K(+) represents an early signal of pore-forming toxins-mediated cell necrosis.

International audience; This study aimed at combining an iron-based, steady-state, vessel size index magnetic resonance imaging (VSI MRI) approach, and a gadolinium (Gd)-based, dynamic contrast-enhanced MRI approach (DCE MRI) to characterize tumoral microvasculature. Rats bearing an orthotopic glioma (C6, n=14 and RG2, n=6) underwent DCE MRI and combined VSI and DCE MRI 4 h later, at 2.35 T. Gd-DOTA (200 mumol of Gd per kg) and ultrasmall superparamagnetic iron oxide (USPIO) (200 micromol of iron per kg) were used for DCE and VSI MRI, respectively. C6 and RG2 gliomas were equally permeable to Gd-DOTA but presented different blood volume fractions and VSI, in good agreement with histologic data. The presence of USPIO yielded reduced K(trans) values. The K(trans) values obtained with Gd-DOTA in the absence and in the presence of USPIO were well correlated for the C6 glioma but not for the RG2 glioma. It was also observed that, within the time frame of DCE MRI, USPIO remained intravascular in the C6 glioma whereas it extravasated in the RG2 glioma. In conclusion, VSI and DCE MRI can be combined provided that USPIO does not extravasate with the time frame of the DCE MRI experiment. The mechanisms at the origin of USPIO extravasation remain to be elucidated.

International audience; This study aimed at combining an iron-based, steady-state, vessel size index magnetic resonance imaging (VSI MRI) approach, and a gadolinium (Gd)-based, dynamic contrast-enhanced MRI approach (DCE MRI) to characterize tumoral microvasculature. Rats bearing an orthotopic glioma (C6, n=14 and RG2, n=6) underwent DCE MRI and combined VSI and DCE MRI 4 h later, at 2.35 T. Gd-DOTA (200 mumol of Gd per kg) and ultrasmall superparamagnetic iron oxide (USPIO) (200 micromol of iron per kg) were used for DCE and VSI MRI, respectively. C6 and RG2 gliomas were equally permeable to Gd-DOTA but presented different blood volume fractions and VSI, in good agreement with histologic data. The presence of USPIO yielded reduced K(trans) values. The K(trans) values obtained with Gd-DOTA in the absence and in the presence of USPIO were well correlated for the C6 glioma but not for the RG2 glioma. It was also observed that, within the time frame of DCE MRI, USPIO remained intravascular in the C6 glioma whereas it extravasated in the RG2 glioma. In conclusion, VSI and DCE MRI can be combined provided that USPIO does not extravasate with the time frame of the DCE MRI experiment. The mechanisms at the origin of USPIO extravasation remain to be elucidated.

International audience; BACKGROUND : Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. METHODOLOGY/PRINCIPAL FINDINGS : To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the latest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step CONCLUSIONS/SIGNIFICANCE : Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound.

International audience; Background: Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide.Methodology/Principal Findings: Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide.Conclusions/Significance: Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide.

Twenty-five years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulse delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a crossroad in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here as an introduction to the large range of applications described in this book. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. The feasibility of electric pulse for gene transfer in humans is discussed taking into account that electric pulse delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments have made DNA electrotransfer more efficient and safer, this nonviral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and a respect for safe procedures will be key elements for the successful future transition of DNA electrotransfer to the clinics.

International audience; The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs), which have been successfully applied for in vivo delivery of biomolecules and constitute very promising tools. Distinct families of CPPs have been described; some require chemical linkage between the drug and the carrier for cellular drug internalization while others like Pep-and MPG-families, form stable complexes with drugs depending on their chemical nature. Pep and MPG are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG and Pep based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes in a fully biologically active form into a large variety of cell lines as well as in animal models. This review will focus on the mechanisms of non-covalent MPG and Pep-1 strategies and their applications in cultured cells and animal models.

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation., Author Summary By replicating within red blood cells malaria parasites are largely hidden from immune recognition, but within mature erythrocytes nutrients are limiting and accumulation of potentially hazardous metabolic end products can rapidly become critical. In order to survive within red blood cells malaria parasites, therefore, alter the permeability of the erythrocyte plasma membrane either by up-regulating existing carriers, or by creating new permeation pathways. Recent electrophysiological studies of Plasmodium-infected erythrocytes have demonstrated that these changes reflect trans-membrane transport through ion channels in the infected erythrocyte plasma membrane. Protein phosphorylation has been documented in protozoan parasites for a number of years and is implicated in key processes of both parasites and parasitized host cells. It has been established that cAMP-dependent regulated pathways are able to activate ion channels in the red cell membrane and a better understanding of how the parasite manipulates cAMP-dependent signaling to activate anion channels could be important in developing novel strategies for future anti-malarial chemotherapies.

The effect of the application of pulsed electric fields to potato tissue on the diffusion of the fluorescent dye FM1-43 through the cell wall was studied. Potato tissue was subjected to field strengths ranging from 30 to 500 V/cm, with one 1 ms rectangular pulse, before application of FM1-43 and microscopic examination. Our results show a slower diffusion of FM1-43 in the electropulsed tissue when compared with that in the non-pulsed tissue, suggesting that the electric field decreased the cell wall permeability. This is a fast response that is already detected within 30 s after the delivery of the electric field. This response was mimicked by exogenous H2O2 and blocked by sodium azide, an inhibitor of the production of H2O2 by peroxidases.

A cDNA sequence with homologies to members of the LPS-binding protein and bactericidal/permeability-increasing protein (BPI) family was identified in the oysterCrassostrea gigas. The recombinant protein was found to bind LPS, to display bactericidal activity againstEscherichia coli, and to increase the permeability of the bacterial cytoplasmic membrane. This indicated that it is a BPI rather than an LPS-binding protein. Byin situhybridization, the expression of theC. gigasBPI (Cg-bpi) was found to be induced in hemocytes after oyster bacterial challenge and to be constitutive in various epithelia of unchallenged oysters. Thus,Cg-bpitranscripts were detected in the epithelial cells of tissues/organs in contact with the external environment (mantle, gills, digestive tract, digestive gland diverticula, and gonad follicles). Therefore,Cg-BPI, whose expression profile and biological properties are reminiscent of mammalian BPIs, may provide a first line of defense against potential bacterial invasion. To our knowledge, this is the first characterization of a BPI in an invertebrate.

Specific control of gene expression by synthetic oligonucleotides (ON) is now widely used for target validation but clinical applications are limited by ON bioavailability. Moreover, most currently used strategies for physical and chemical delivery cannot be easily implemented in vivo. This article reviews new strategies which appear promising for ON delivery. The first part deals with ON chemical modifications aiming at improving cellular uptake as for instance the grafting of cationic groups on the ON backbone. The second part concerns ON conjugation to cell penetrating peptides.

International audience; Electropermeabilisation is a well established physical method, based on the application of electric pulses, which induces the transient permeabilisation of the cell membrane. External molecules, otherwise nonpermeant, can enter the cell. Electropermeabilisation is now in use for the delivery of a large variety of molecules, as drugs and nucleic acids. Therefore, the method has great potential in the fields of cancer treatment and gene therapy. However many open questions about the underlying physical mechanisms involved remain to be answered or fully elucidated. In particular, the induced changes by the effects of the applied field on the membrane structure are still far from being fully understood. The present review focuses on questions related to the current theories, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses recent findings using molecular dynamics simulations as well as experimental studies of the effect of the field on membrane components.

International audience; Tumor necrosis factor-alpha (TNF-alpha) promotes lipolysis in mammal adipocytes via the mitogen-activated protein kinase (MAPK) family, resulting in reduced expression/function of perilipin (PLIN). The role of another pivotal intracellular messenger activated by TNF-alpha, nuclear factor-kappaB (NF-kappaB), has not been recognized. We explored the role of NF-kappaB in TNF-alpha-induced lipolysis of human fat cells. Primary cultures of human adipocytes were incubated in the presence of a cell-permeable peptide that inhibits NF-kappaB signaling (WP). Incubation with WP, but not with a biologically inactive peptide (MP), abolished the nuclear translocation of NF-kappaB and effectively abrogated TNF-alpha-induced lipolysis in a concentration-dependent manner. Western blot analysis demonstrated that although TNF-alpha per se reduced mainly PLIN protein expression, TNF-alpha in the presence of WP resulted in a pronounced combined reduction of both hormone-sensitive lipase (HSL) and PLIN protein. The expression of a set of other lipolytic or adipocyte-specific proteins was not affected. The regulation was presumably at the transcriptional level, because mRNA expression for HSL and PLIN was markedly reduced with TNF-alpha in the presence of NF-kappaB inhibition. This was confirmed in gene reporter assays using human PLIN and HSL promoter constructs. We conclude that in the presence of NF-kappaB inhibition, TNF-alpha-mediated lipolysis is reduced, which suggests that NF-kappaB is essential for retained human fat cell lipolysis.

International audience; Type III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation.

Genomic signaling mechanisms require a relatively long time to get into action and represent the main way through which steroid hormones affect target cells. In addition, steroids may rapidly activate cellular functions by non-genomic signaling mechanisms involving membrane sites. Understanding in depth the molecular mechanisms of the non-genomic action represents an important frontier for developing new and more selective pharmacologic tools for endocrine therapies. In the present study, we report that membrane-impermeable testosterone-bovine serum albumin (BSA) acts synergistically with paclitaxel in modifying actin and tubulin cytoskeleton dynamics in LNCaP (androgen sensitive) and DU-145 (androgen insensitive) human prostate cancer cell lines. In addition, coincubation of either cell line with testosterone-BSA and paclitaxel induced inhibition of cell proliferation and apoptosis. Finally, in vivo experiments in LNCaP and DU-145 tumor xenografts in nude mice showed that both agents decrease tumor mass, whereas testosterone-BSA enhances the effect of paclitaxel. Our findings suggest that chronic activation of membrane androgen receptors in vitro and in vivo facilitates and sustains for a longer time the antitumoral action of cytoskeletal acting agents. [Mol Cancer Ther 2006;5(5):1342–51]

International audience; Staphylococcal γ-haemolysin HlgA–HlgB forms a β-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues – placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor–acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA–HlgB or HlgB–HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA–HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB–HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.

International audience; N'-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye.

Neurons regulate the propagation of chemoelectric signals throughout the nervous system by opening and closing ion channels, a process known as gating. Here, histidine-based metal-binding sites were engineered along the intrinsic pore of a chimeric Cys-loop receptor to probe state-dependent Zn 2+ -channel interactions. Patterns of Zn 2+ ion binding within the pore reveal that, in the closed state, the five pore-lining segments adopt an oblique orientation relative to the axis of ion conduction and constrict into a physical gate at their intracellular end. The interactions of Zn 2+ with the open state indicate that the five pore-lining segments should rigidly tilt to enable the movement of their intracellular ends away from the axis of ion conduction, so as to open the constriction (i.e., the gate). Alignment of the functional results with the 3D structure of an acetylcholine receptor allowed us to generate structural models accounting for the closed and open pore conformations and for a gating mechanism of a Cys-loop receptor.

International audience; The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.

Permeabilization, when observed on a tissue level, is a dynamic process resulting from changes in membrane permeability when exposing biological cells to external electric field (E). In this paper we present a sequential finite element model of E distribution in tissue which considers local changes in tissue conductivity due to permeabilization. These changes affect the pattern of the field distribution during the high voltage pulse application. The presented model consists of a sequence of static models (steps), which describe E distribution at discrete time intervals during tissue permeabilization and in this way present the dynamics of electropermeabilization. The tissue conductivity for each static model in a sequence is determined based on E distribution from the previous step by considering a sigmoid dependency between specific conductivity and E intensity. Such a dependency was determined by parameter estimation on a set of current measurements, obtained by in vivo experiments. Another set of measurements was used for model validation. All experiments were performed on rabbit liver tissue with inserted needle electrodes. Model validation was carried out in four different ways: 1) by comparing reversibly permeabilized tissue computed by the model and the reversibly permeabilized area of tissue as obtained in the experiments; 2) by comparing the area of irreversibly permeabilized tissue computed by the model and the area where tissue necrosis was observed in experiments; 3) through the comparison of total current at the end of pulse and computed current in the last step of sequential electropermeabilization model; 4) by comparing total current during the first pulse and current computed in consecutive steps of a modeling sequence. The presented permeabilization model presents the first approach of describing the course of permeabilization on tissue level. Despite some approximations (ohmic tissue behavior) the model can predict the permeabilized volume of tissue, when exposed to electrical treatment. Therefore, the most important contribution and novelty of the model is its potentiality to be used as a tool for determining parameters for effective tissue permeabilization.

International audience; Recently, we described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. We have examined the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD analysis revealed a transition from a nonstructured to a helical conformation upon increase of the concentration. Determination of the structure by NMR showed that in water, its alpha-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, we observe a partial conformational transition when the complex encounters phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not associated with complexation with its cargo but is induced upon association with the cell membrane.

The role of the amplitude, number, and duration of unipolar rectangular electric pulses in cell membrane electropermeabilization in vitro has been the subject of several studies. With respect to unipolar rectangular pulses, an improved efficiency has been reported for several modifications of the pulse shape: separate bipolar pulses, continuous bipolar waveforms, and sine-modulated pulses. In this paper, we present the results of a systematic study of the role of pulse shape in permeabilization, cell death, and molecular uptake. We have first compared the efficiency of 1-ms unipolar pulses with rise- and falltimes ranging from 2 to 100 μs, observing no statistically significant difference. We then compared the efficiency of triangular, sine, and rectangular bipolar pulses, and finally the efficiency of sine-modulated unipolar pulses with different percentages of modulation. We show that the results of these experiments can be explained on the basis of the time during which the pulse amplitude exceeds a certain critical value.

Electropermeabilization of the cell membrane is a phenomenon caused by exposure of the cell to electric pulses. Permeabilization depends on pulse duration, pulse amplitude, the number of pulses delivered, and also on other experimental conditions. With these parameters properly chosen, the process of permeabilization is reversible and cells return to their normal physiological state. This article describes the development of a model of diffusion-driven transmembrane transport of small molecules caused by electropermeabilization. The process of permeabilization is divided into a short permeabilizing phase that takes place during the pulse, and a longer resealing phase that begins after the end of the pulse. Because both phases of permeabilization are important for uptake of molecules into cells, most of the effort is focused on the optimization of parameters that influence the flow between intracellular and extracellular space. The model describes well the transmembrane transport caused by electropermeabilization, allowing to study the uptake of molecules as a function of elapsed time, voltage and pulse duration. In addition, our results show that the shapes of the curves of cell permeabilization and survival as functions of pulse amplitude can to a large extent be explained by cell size distribution.

Using reversible electropermeabilization of cells and spheroplasts, we show that the cell wall and plasma membrane partly account for bleomycin resistance by acting as two independent barriers. We also report on the presence of a membrane protein that may be responsible for bleomycin internalization and toxicity in Saccharomyces cerevisiae .

International audience; The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety problems connected with viral vectors, and it allows the control of the amount of cytokines delivered with the vaccine.

Cryosurgery employs freezing to destroy solid tumours. However, frozen cells can survive and cause cancer recurrence. Bleomycin, an anticancer drug with a huge intrinsic cytotoxicity is normally not very effective because it is nonpermeant. We report that freezing facilitates bleomycin penetration into cells making it toxic to cryosurgery surviving cells at concentrations that are non-toxic systemically. British Journal of Cancer (2002) 86, 1658–1660. DOI: 10.1038/sj/bjc/6600306 www.bjcancer.com © 2002 Cancer Research UK