Transmembrane Electrical Potential Research Articles

Identification of the Ion Conduction Pathway in a TMEM16 Scramblase

The TMEM16 family constitutes a class of widespread membrane proteins that is functionally split into ion channels and phospholipid scramblases. The recent crystallization of the fungal TMEM16 (nhTMEM16) has provided the first glimpse into the structural properties of TMEM16 proteins. This structure revealed a membrane-spanning hydrophilic crevice on the protein surface which is shown by our atomistic simulations to serve as the pathway for lipid movement. However, it is still puzzling how the same structure can also transport ions, because the structure does not reveal an obvious alternative pathway for ion permeation through the bilayer other than the path taken by phospholipids. In order to investigate the pathway and mechanism of ion permeation, and the weak ionic selectivity, we have performed multiple molecular dynamic (MD) simulations with nhTMEM16 embedded in asymmetric lipid bilayers under different levels of transmembrane electric potentials. The simulations reveal the formation of a “proteolipidic” pore along the hydrophilic crevice, where lipids play a structural role by lining the hydrophilic ion conduction pathway with their headgroups. Externalization of the anionic phospholipid PS along the pore wall was observed as water penetrates into the bilayer and extends across the membrane. Notably, ion permeation events through the aqueous pore formed between the protein and the lipid headgroups were also captured frequently for both cations (Na+) and anions (Cl-). Moreover, the inclusion of various lipid compositions in the simulated systems allowed us to characterize the interaction between the ions and the pore-lining headgroups as they transverse the membrane. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s.

Evaluation the sensitivity of diffusion and perfusion weighted imaging in therapeutic timing of stroke

The concept of ischemic penumbra was defined as an area of reduced in cerebral blood flow [CBF] with electrical failure but preserved ion homeostasis and trans-membrane electrical potentials. Then, some other definitions for the ischemic penumbra have been proposed based on energy metabolism, CBF thresholds and protein synthesis. Hypothermia reduced ischemic lesion volume on diffusion weighted imaging [DWI] and may improve functional outcome. Since, the DWI show initial reduction with no abnormal change on T2W or fluid attenuated inversion-recovery [FLAIR] images in hyper acute stroke, a DWI-T2W mismatch was proposed to represent the ischemic penumbra. The DWI-T2W mismatch was defined as a hyper intense lesion on DWI [lowed] with no hyper intense lesion on T2WI or FLAIR, and no hypo intense lesion on T1WI. Perfusion weighted imaging [PWI] of the entire brain is one of the main advantages of PWI, in which MMT or TTP perfusion maps are generated for the entire brain. Like CT perfusion, it can identify the ischemic penumbra. The ischemic penumbra is the difference between the DWI defect [cytotoxic edema-irreversible ischemia- the ischemic core] and the perfusion defect-analogous to MTT or TTP]. The penumbra is the DWI-PWI mismatch. The accurate identification of this ischemic penumbra will help ischemic stroke therapy and potentially aide in extending the time window for treatment in the future.

Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of gram-positive and gram-negative foodborne bacteria. The objective of this study was to investigate the antibacterial activity and mechanism of action of bifidocin A against Listeria monocytogenes, one of the most susceptible bacteria to this bacteriocin. The minimum inhibitory concentration (MIC) of bifidocin A for L. monocytogenes 35152 was 0.029 mg/mL. Time-kill assays showed that bifidocin A effectively inhibited the growth of L. monocytogenes in a time-and concentration-dependent manner. The mechanism of action of bifidocin A was studied by analyzing its effects at a MIC on the cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of L. monocytogenes. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of L. monocytogenes cells. Confocal laser scanning microscopy images showed that L. monocytogenes cells treated with bifidocin A took up propidium iodide. Bifidocin A treatment also induced the leakage of K+ and inorganic phosphate, the hydrolysis and release of ATP, and a collapse of the transmembrane electrical potential and pH gradient in L. monocytogenes cells. These results suggested that bifidocin A exerted its anti-Listeria monocytogenes effect through the dissipation of the cytoplasmic membrane PMF, increased membrane permeability, cell membrane pore formation, destruction of membrane integrity, and ultimately complete disintegration of the cells.

Candidate probiotic strains, with immunomodulatory and antioxidant effects, isolated from traditional Italian dairy products

Cancer is a multifactorial disease and inflammation could be the 10th hallmark of cancer. Increased systemic inflammation is almost, if not always, a sign of dysbiosis and increased translocation of of bacterial origin, such as endotoxin. Therefore, therapeutic manipulations of intestinal bacteria could reverse the inflammatory responses and restore mucosal homeostasis. Thus, we evaluated the role of a fresh Pasta Filata cheese of the Alto Molise area called “Stracciata”, made from milk of cows fed by grazing and with local dry forages, as a source of potentially probiotic microorganisms. Molecular characterization by sequencing of the 16S rRNA gene were carried out on all bacteria isolated from fresh Pasta Filata cheese manufactured with natural starter cultures. Transmembrane electrical potential (ΔΨ) assay, antioxidant system activity, Glutathione disulphide (GSSG)/reduced Glutathione (GSH) ratio and nucleotides pool were measured by fluorescence techniques and HPLC. Moreover, the ability of bacterial isolates to stimulate IL-10 and TNF-α production by human peripheral blood mononuclear cells (PBMC) was determined in vitro by ELISA kits. We demonstrate how bacteria isolate by Stracciata cheese or their metabolites regulate immunomodulatory effects. Further studies are opportune for conferring therapeutic strategies to traditional dairy products in bacteria-metabolite-immune system axis contest.

Integral equation methods for vesicle electrohydrodynamics in three dimensions

In this paper, we develop a new boundary integral equation formulation that describes the coupled electro- and hydro-dynamics of a vesicle suspended in a viscous fluid and subjected to external flow and electric fields. The dynamics of the vesicle are characterized by a competition between the elastic, electric and viscous forces on its membrane. The classical Taylor–Melcher leaky-dielectric model is employed for the electric response of the vesicle and the Helfrich energy model combined with local inextensibility is employed for its elastic response. The coupled governing equations for the vesicle position and its transmembrane electric potential are solved using a numerical method that is spectrally accurate in space and first-order in time. The method uses a semi-implicit time-stepping scheme to overcome the numerical stiffness associated with the governing equations.

Quantifying the Relationship between Curvature and Electric Potential in Lipid Bilayers

Cellular membranes mediate vital cellular processes by being subject to curvature and transmembrane electrical potentials. Here we build upon the existing theory for flexoelectricity in liquid crystals to quantify the coupling between lipid bilayer curvature and membrane potentials. Using molecular dynamics simulations, we show that headgroup dipole moments, the lateral pressure profile across the bilayer, and spontaneous curvature all systematically change with increasing membrane potentials. In particular, there is a linear dependence between the bending moment (the product of bending rigidity and spontaneous curvature) and the applied membrane potentials. We show that biologically relevant membrane potentials can induce biologically relevant curvatures corresponding to radii of around 500 nm. The implications of flexoelectricity in lipid bilayers are thus likely to be of considerable consequence both in biology and in model lipid bilayer systems.

Mode of action of leucocin K7 produced by Leuconostoc mesenteroides K7 against Listeria monocytogenes and its potential in milk preservation.

To investigate the mode of action of leucocin K7 against Listeria monocytogenes and to assess its inhibitory effect on Lis. monocytogenes in refrigerated milk. A bacteriocin-producing strain, Leuconostoc mesenteroides K7, was isolated from a fermented pickle. The bacteriocin, leucocin K7, exhibited antagonistic activity against Lis. monocytogenes with an MIC of 28µg/ml. It was sensitive to proteaseS and displayed good thermal stability and broad active pH range. Leucocin K7 had no effect on the efflux of ATP from Lis. monocytogenes but triggered the efflux of K(+) and the intracellular hydrolysis of ATP. It also dissipated the transmembrane electrical potential completely and transmembrane pH gradient partially. It 80 AU/ml inhibited the growth of Lis. monocytogenes by 2.3-3.9 log units in milk; when combined with glycine (5mg/ml), it completely eliminated viable Lis. monocytogenes over 7days Leucocin K7 shows different mode of action from nisin and may have potential application in milk preservation.

Geometric phase and quantum interference in photosynthetic reaction center: Regulation of electron transfer

Photosynthesis is driven by electron transfer in reaction centers in which the functional unit is composed of several simple molecules C2-symmetrically arranged into two branches. In view of quantum mechanism, both branches are possible pathways traversed by the transferred electron. Due to different evolution of spin state along two pathways in transmembrane electric potential (TEP), quantum state of the transferred electron at the bridged site acquires a geometric phase difference dependent on TEP, the most efficient electron transport takes place in a specific range of TEP beyond which electron transfer is dramatically suppressed. What’s more, reaction center acts like elaborately designed quantum device preparing polarized spin dependent on TEP for the transferred electron to regulate the reduction potential at bridged site. In brief, electron transfer generates the TEP, reversely, TEP modulates the efficiency of electron transfer. This may be an important approach to maintaining an appreciable pH environment in photosynthesis.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

Impact of nickel on grapevine (Vitis vinifera L.) root plasma membrane, ROS generation, and cell viability

Abstract The present study investigated the impact of nickel (Ni2+) on trans-membrane electrical potential (EM) and permeability properties of plasma membrane (PM) in epidermal cells of adventitious grapevine roots. The relationship between disturbances of membrane functionality and the production of superoxide anion, hydrogen peroxide and cell viability after the exposure of roots to Ni2+ was also studied. Treatments with 0.1-5 mmol L-1 NiCl2 induced a concentration-dependent transient PM depolarization, which was recovered to the initial resting potential within 50-70 min in the presence of Ni2+. Longer (up to 24 h) exposure of roots to 1 mmol L-1 of Ni2+ hyperpolarized the EM by approximately 17 mV. Application of the highest 5 mmol L-1 concentration of Ni2+ during longer treatments (up to 48 h) resulted in the increase of membrane permeability; however the EM, cell viability, and superoxide content remained unaffected. The increase in the formation of hydrogen peroxide was time- and concentration- dependent and maximum production was recorded after 180 min of Ni2+ treatment. We can conclude that oxidative stress resulting from an imbalance in the generation and/ or removal of hydrogen peroxide in the root tissues of grapevine was the major cause of Ni2+ toxicity.

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells.

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

Synergistic Antibacterial Effect of the Combination of ɛ-Polylysine and Nisin against Enterococcus faecalis

This study evaluated the antibacterial effect of the combination of ε-polylysine (ε-PL) and nisin against Enterococcus faecalis strains. The combination of ε-PL and nisin showed synergistic antibacterial activity against three Enterococcus strains. Scanning electron microscopy and a membrane permeability assay revealed that the combined treatment with ε-PL and nisin synergistically damaged the cell morphology of E. faecalis strain R612Z1 cells. Both ε-PL and nisin can dissipate the transmembrane electric potential of E. faecalis R612Z1 cells, but these peptides did not affect the transmembrane pH gradient. The combination of ε-PL and nisin can produce a high reactive oxygen species level in E. faecalis R612Z1 cells. The results indicated that the uptake of ε-PL into cells was promoted through nisin and that the combination of ε-PL and nisin could produce a high reactive oxygen species level in E. faecalis R612Z1 cells, leading to cell growth inhibition.

Two-peptide bacteriocin PlnEF causes cell membrane damage to Lactobacillus plantarum

Biologically active, artificially synthesized two-peptide bacteriocin PlnEF was used to study its mode of action on sensitive bacteria Lactobacillus plantarum pl2. The data obtained showed that PlnEF induced membrane permeabilization, allowing for the efflux of electrolytes, which was evidenced by the increased extracellular conductivity, the dissipation of transmembrane electrical potential and pH gradient, and rapid intracellular ATP depletion after L. plantarum pl2 cells were treated with PlnEF for minutes. Laser confocal microscopy showed that PlnEF accumulated very quickly in L. plantarum pl2 cells and the accumulation of PlnEF caused damage to cell membrane. Scanning electron microscopy and transmission electron microscopy further showed that PlnEF induced morphological changes and structure disruption to L. plantarum pl2 cells, such as the formation of blebs, microspheres, membrane deformation and cell lysis. In summary, the data obtained show that PlnEF caused cell membrane damage to L. plantarum pl2 cells. Our study reveals the antimicrobial mechanism of two-peptide bacteriocin PlnEF against L. plantarum.

Antibacterial mechanism of bifidocin A, a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04

Bifidocin A, a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04, was isolated from the feces of a healthy centenarian. To understand the mechanism of the antibacterial action of bifidocin A against gram-negative bacteria, its effects at a minimum inhibitory concentration on cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of Escherichia coli 1.90 were investigated. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of E. coli cells. The intracellular organization was more susceptible to changes induced by bifidocin A than the morphology. Bifidocin A treatment caused the leakage of K+ and inorganic phosphate, the release of ATP and UV-absorbing materials, and a collapse of the transmembrane electrical potential and pH gradient in E. coli cells. Confocal laser scanning microscopy images showed that E. coli cells treated with bifidocin A took up propidium iodide. These results suggested that the mechanism of action bifidocin A against E. coli involved dissipation of the PMF of the cytoplasmic membrane, an increase in membrane permeability, pore formation in the cell membrane, a change in membrane integrity, and complete cell disintegration.

From Quantum Photosynthesis to the Sentient Brain

Energy harvesting by photosynthesis in “brainless” plants and green algae is identified as the root non-trivially quantum process powering neural correlates of consciousness in humans and other “brainy” animals. Thermofield attributes of solar energy flow through the biosphere’s food chain are suggested as a “bottom up” mediator between quantum-coherent aspects of photosynthesis and emergent dynamical architectonics of transmembrane electrical potentials in neurons. This quantum-ecological approach to energetics of brain function as part of an open dissipative world system offers a segue, experimentally grounded by empirical evidence for photosynthetic coherence, into qualitatively gauged links between quantum tunneling and the Hard Problem of consciousness.

Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium-Selective Electrode.

Mitochondrial bioenergetics is based on the generation of the protonmotive force by the electron transport chain. The protonmotive force is used by mitochondria for different critical aspects of its normal function, ranging from calcium accumulation to the synthesis of ATP. The transmembrane electric potential (ΔΨ) is the major component of the protonmotive force and is also the main responsible for ATP synthesis by mitochondrial ATP synthase. Although several methods can be used to measure the ΔΨ, the use of the tetraphenylphosphonium cation (TPP(+))-selective electrode is still a method of election due to its sensitivity. The method is based on the accumulation of TPP(+) by energized mitochondria, which develop a negative charge in the matrix due to the ejection of protons. This unit describes how to build a custom-made TPP(+)-selective electrode and how to establish the necessary set-up to follow ΔΨ fluctuations in isolated mitochondrial fractions.

Acidity-Mediated, Electrostatic Tuning of Asymmetrically Charged Peptides Interactions with Protein Nanopores.

Despite success in probing chemical reactions and dynamics of macromolecules on submillisecond time and nanometer length scales, a major impasse faced by nanopore technology is the need to cheaply and controllably modulate macromolecule capture and trafficking across the nanopore. We demonstrate herein that tunable charge separation engineered at the both ends of a macromolecule very efficiently modulates the dynamics of macromolecules capture and traffic through a nanometer-size pore. In the proof-of-principle approach, we employed a 36 amino acids long peptide containing at the N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and we studied its capture by the α-hemolysin (α-HL) and the mean residence time inside the pore in the presence of a pH gradient across the protein. We propose a solution to effectively control the dynamics of peptide interaction with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide addition side and the transmembrane electric potential, while preserving the amplitude of the blockade current signature.

28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

Characterization of bacteriocin produced by Enterococcus faecium CN-25 isolated from traditionally Thai fermented fish roe

Enterococcus faecium CN-25 was isolated from a traditionally Thai fermented fish roe, som-kai-pla, and was found to possess enterocin A and B genes. Among the 30 microorganisms tested, the produced bacteriocin exhibited antagonistic activity against Listeria monocytogenes TISTR 1327. The bacterium maximally produced bacteriocin at a concentration of 1828 AU/ml at the beginning of the stationary phase (at 18 h) in the inexpensive rice bran medium. The antilisterial activity of the compound was not affected by heating at 60–100 °C for up to 30 min. High antilisterial activity remained at pH 2. Partially purified CN-25 bacteriocin derived from E. faecium CN-25 completely inhibited the growth of L. monocytogenes at the minimum concentration of 2.38 μg/ml. The compound had no effect on the efflux of ATP from L. monocytogenes but triggered the intracellular hydrolysis of ATP, as evidenced by a decrease in the luminescence of total ATP. The bacteriocin also depleted the transmembrane electrical potential (ΔΨ) but had no effect on the transmembrane pH gradient (ΔpH). Therefore, the partially purified CN-25 peptide may be useful for improving the product safety of fermented fish products.

BPX preconditioners for the Bidomain model of electrocardiology

The aim of this work is to develop a BPX preconditioner for the Bidomain model of electrocardiology. This model describes the bioelectrical activity of the cardiac tissue and consists of a system of a non-linear parabolic reaction–diffusion partial differential equation (PDE) and an elliptic linear PDE, modeling at macroscopic level the evolution of the transmembrane and extracellular electric potentials of the anisotropic cardiac tissue. The evolution equation is coupled through the non-linear reaction term with a stiff system of ordinary differential equations, the so-called membrane model, describing the ionic currents through the cellular membrane. The discretization of the coupled system by finite elements in space and semi-implicit finite differences in time yields at each time step the solution of an ill-conditioned linear system. The goal of the present study is to construct, analyze and numerically test a BPX preconditioner for the linear system arising from the discretization of the Bidomain model. Optimal convergence rate estimates are established and verified by two- and three-dimensional numerical tests on both structured and unstructured meshes. Moreover, in a full heartbeat simulation on a three-dimensional wedge of ventricular tissue, the BPX preconditioner is about 35% faster in terms of CPU times than ILU(0) and an Algebraic Multigrid preconditioner.